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Molecular Biology of Adenosine Triphosphate-Sensitive Potassium Channels¹

Departments of Medicine (L.A.-B.) and Cell Biology (J.B.), Baylor College of Medicine, Houston, Texas 77030

	Abstract

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 

I. Introduction

II. How Are K_ATP Channels Defined?

III. How Do K_ATP Channels Affect the Membrane Potential of Pancreatic ß-Cells?

IV. K_ATP Channel Subunits

A. The K_IR family of inwardly rectifying K+ channels

B. Sulfonylurea receptors

V. Reconstitution of K_ATP Channel Activity from SUR1 and K_IR6.2

A. The question of "promiscuous coupling" of SUR1 with other inward rectifiers

VI. K_ATP Channel Structure

A. K_IR6.2 forms the pore of a K_ATP channel

B. SUR1 and K_IR6.x are physically associated

C. Coexpression with K_IR6.2 affects the maturation of SUR1

D. Complex glycosylated SUR1 and K_IR6.2 assemble a large multimer

E. A 1:1 stoichiometry of SUR to K_IR is both necessary and sufficient to make K_ATP channels

F. Other K_IR channels are tetramers

G. The stoichiometry of active ß-cell K_ATP channels is (SUR1/K_IR6.2)₄

VII. Regulation of K_ATP Channel Activity

A. How do ATP and ADP exert their effects on K_ATP channels?

B. Where are the nucleotide binding sites located?

C. C-terminally truncated K_IR6.2 channels show abnormal kinetics

D. Coexpression of K_IR6.2C subunits with SUR restores normal K_ATP channel activity

E. Why are K_IR6.2 channels silent?

F. The N terminus of K_IR6.2 limits burst duration

G. Where do the openers bind and how do they work?

H. Do SURs have adenosine triphosphatase (ATPase) activity?

I. Do SURs have transport activity?

J. Is there an endogenous substrate?

VIII. Human SUR1 and K_IR6.2 Genes

IX. K_ATP Channels and Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI)

A. HI-GK

B. HI-GlnDH

C. HI-"unknown"

D. HI-K_IR6.2

E. HI-SUR1

X. Linking PHHI to Defects in K_ATP Channel Activity

A. ß-Cells from newborns diagnosed with "sporadic" PHHI lack K_ATP channel activity

B. PHHI ß-cells with the SUR1 exon 35 mutation lack K_ATP channel activity

C. Why is there a lack of dominant negative mutations?

D. Development of mouse models

XI. Other Issues

A. Nesidioblastosis does not cause PHHI

B. "Diffuse" vs. "focal" forms of PHHI

XII. K_ATP and Non-Insulin-Dependent Diabetes Mellitus (NIDDM)

A. ß-Cell type K_ATP channels in the brain

XIII. The Leptin Connection

XIV. Summary and Conclusions

	I. Introduction

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
ION CHANNELS are present in the plasma membrane and intracellular organelles of all cells, where they coordinate such diverse functions as neurotransmission, contraction, secretion, and control of cell volume. Over the past decade it has become increasingly clear that mutations in the genes that encode the subunits of ion channels can result in pathological states in a wide variety of tissues, including those not generally thought of as "excitable." The number of ion channels that give rise to "channelopathies" is increasing. The list currently spans all of the major channel types, including the following nonexhaustive survey. Perhaps the most prevalent ion channelopathy is cystic fibrosis (Online Inheritance in Man, OMIM 219700), which is due to reduced chloride ion transport resulting from mutations in the cystic fibrosis transconductance regulator, CFTR, a member of the large ATP-binding cassette (ABC) superfamily of proteins. A dominant form of myotonia congenita (OMIM 160800), Thomsen’s disease, is associated with mutations in CLCN1, a voltage-gated chloride channel. Mutations in the gene encoding another member of this family, CLCN5, give rise to hypercalciuric nephrolithiasis (Dent’s disease; OMIM 300009), an X-linked recessive disorder. Mutations in the genes encoding the ß- and -subunits of the amiloride-sensitive, non-voltage-gated epithelial Na⁺ channels, ENac, activate channel activity resulting in Liddle’s syndrome (OMIM 177200), which is associated with hypertension and hypokalemia. Various episodic disorders have been shown to arise from mutations of voltage-dependent ion channels in nerve and muscle. Periodic paralysis I, or hypokalemic periodic paralysis (OMIM 170400), results from mutations in the gene encoding the 1-subunit of the muscle dihydropyridine-sensitive calcium channel, while periodic paralysis II, hyperkalemic periodic paralysis (OMIM 170500), is associated with mutations in the gene encoding a voltage-gated Na⁺ channel subunit. Mutations and CAG repeat expansions in the gene encoding a second Ca²⁺ channel 1-subunit isoform, termed isoform 4, have been identified in familial hemiplegic migraine 1 (OMIM 141500), episodic ataxia type 2 (OMIM 108500), and spinocerebellar ataxia 6 (OMIM 183086). Mutations in the genes that encode potassium channels, perhaps the most diverse class of ion channels, give rise to a number of disorders; for example, variants of the long QT syndrome, LQT1 (OMIM 192500) and LQT2 (OMIM 152427) are caused by mutations in a voltage-gated K⁺ channel, KVLQT1, and in HERG (human ether-a-go-go related gene), a Ca²⁺-modulated K⁺ channel, respectively. Mutations in the KCNJ1 gene encoding ROMK1, the first discovered potassium inward rectifier (1) give rise to the antenatal variant of Bartter’s syndrome with renal tubular hypokalemic alkalosis (OMIM 601678). Finally, mutations in the gene encoding a water channel, aquaporin 2, cause the renal form of diabetes insipidus (OMIM 125800). Ion channelopathies have been reviewed recently (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17). The reader is referred to the extensive bibliographies cited in the Online Mendelian Inheritance in Man database (http://www3.ncbi.nlm.nih.gov/Omim/) for references to each of the channelopathies cited above and for current information on others.

Our objective is to review the molecular biology of ATP-sensitive K⁺ channels, or K_ATP channels, which are present in pancreatic ß-cells in the islets of Langerhans where they play a key role in stimulus-secretion coupling by providing a link between changes in metabolism and membrane electrical activity. Evidence suggests this channel is regulated by changes in adenine nucleotide levels, an increase in the ATP/ADP ratio, resulting from changes in glucose metabolism, and is the target for a class of drugs called sulfonylureas used in the treatment of non-insulin-dependent diabetes mellitus. Closure of K_ATP channels as a result of increased glucose metabolism or by sulfonylureas leads to the release of insulin. Nearly 50 mutations in either of the two subunits that make up the ß-cell K_ATP channel, namely, the high-affinity sulfonylurea receptor, SUR1, and the K⁺ inward rectifier, K_IR6.2, are responsible for a channelopathy, the recessive genetic form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI), characterized by the uncoupling of glucose metabolism from ß-cell electrical activity (see Refs. 18, 19, 20, 21 for reviews). Investigation of this recessive form of PHHI has led to the identification of at least two other causes of dominant forms of persistent neonatal hypoglycemia and should provide insight into the regulation of the insulin-secretory pathway in both normal and in diabetic individuals.

The objective of this article is to review developments in the molecular biology of ATP-sensitive potassium channels. We will focus mainly on recent studies of the molecular biology of the "classic" ß-cell type K_ATP channels composed of the high-affinity sulfonylurea receptor, SUR1 (OMIM 600509), and K_IR6.2, a potassium inward rectifier subunit (OMIM 600937), but will draw comparison with the striated and smooth muscle type channels where appropriate. Over the last several years, the subunits of the ß-cell K_ATP channel have been cloned, expressed, and reconstituted into functional nucleotide-sensitive channels and have been used to investigate their overall structure and regulation by nucleotides, potassium channel openers, and sulfonylureas. The availability of cDNAs for SUR1 (22) allowed the isolation of the low-affinity sulfonylurea receptors, SUR2A and SUR2B (23, 24). In humans, the mRNAs encoding the SUR2A and SUR2B receptors originate from a single SUR2 gene by differential splicing of the last exon (19). Two K_IR6.x isoforms have been cloned, K_IR6.1 (uKATP-1) (25) and K_IR6.2 (BIR) (26). Interestingly, the SUR and K_IR genes are paired on human chromosomes, with SUR1 and K_IR6.2 adjacent on chromosome 11, and SUR2 and K_IR6.1 near each other on chromosome 12. When the low-affinity receptors are reconstituted with K_IR6.1 or K_IR6.2, they produce K_ATP channels with distinctive pharmacologies which, along with their tissue distribution, suggest they make up the cardiac-skeletal and vascular smooth muscle K_ATP channels (23, 24, 27, 28, 29).

	II. How Are KATP Channels Defined?

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
The application of ATP to the intracellular face of an excised patch was initially used to identify K_ATP channels. These observations were made by Cook and Hales (30) in the United States, by Noma (31) in Japan, and by Trube and Hescheler (32) in Germany. The first reference to ATP-sensitive K⁺ channels that we can uncover is an abstract by Trube and Hescheler (33) outlining the properties of the cardiac K_ATP channel from isolated patches of cardiac cell membranes. Historically, a large number of studies defined the electrophysiological properties and pharmacology of these channels, including their single-channel current properties, pattern of regulation by nucleotides, and response to channel openers and blockers. It is now possible to begin to define these channels in terms of their molecular composition. K_ATP channels are heteromultimers of two types of subunits, inward rectifiers, K_IR6.x, and sulfonylurea receptors, SURs, members of the ABC superfamily. The available evidence, discussed in more detail below, indicates functional channels are assembled as tetramers (SUR/K_IR)₄. Table 1 relates the known subunits to channel types identified by tissue type. The list is provisional and is not meant to be exhaustive, as it is not known whether all the subunit isoforms have been identified. This classification is based largely on pharmacological criteria, and there remains a strong need for tissue and cellular localization work to confirm this classification and to look for overlapping expression of subunit types.

View larger version (32K): [in this window] [in a new window]   Figure 1. Electrical activity of human SUR1/KIR6.2 ATP-sensitive K+ channels. A, Recordings from excised membrane patches of COSm6 cells transfected with human SUR1 and human KIR6.2 illustrate channel activity during perfusion of the cytoplasmic face of the patch with the indicated reagents. Panels B and C are traces plotted at shorter time intervals chosen to show open and closed states of a single channel within a burst of activity. D, A current vs. voltage plot to show the inward rectification (reduced current at more positive potentials) of KATP channels and the effect of an added polyamine. The single-channel conductance is approximately 75 pS. E, The ATP inhibition curve is the average of five experiments, and the bars are the ±SE. The activity in the absence of ATP was taken as 100%; the IC50 was 14.7 µM with a Hill coefficient of 1.1.

View larger version (20K): [in this window] [in a new window]   Figure 2. Chemical structures of compounds that activate and block KATP channels.

	III. How Do KATP Channels Affect the Membrane Potential of Pancreatic ß-Cells?

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
The function of K_ATP channels is best understood in pancreatic ß-cells, the membrane potential of which is responsive to external glucose concentration. ß-Cells show a remarkably complex electrical bursting behavior (see Fig. 3) in response to an increase in glucose level. It is generally agreed that the oscillatory electrical activity leads to oscillatory changes in [Ca²⁺]_i, which drive pulsatile insulin release in isolated islets (see for example Ref. 52). Various suggestions on how K_ATP channels may participate in this response have been put forward; these range from maintaining the resting membrane potential when glucose is low (53) to playing a major role in control of bursting when glucose is high (54, 55) or terminating individual bursts (56). The origin and control of the electrical bursting activity in pancreatic ß-cells remain controversial, and a detailed discussion of this area is beyond the scope of this review. The reader is directed to recent articles for detailed discussions (57, 58, 59, 60, 61, 62, 63). Our intention is to provide a framework for understanding the molecular biology and structure of K_ATP channels, how they affect membrane potential and thus how they can affect bursting and insulin release, and finally, how their absence leads to familial hyperinsulinism.

View larger version (16K): [in this window] [in a new window]   Figure 3. Illustration of electrical activity and [Ca2+]i in a pancreatic ß-cell. Islets, loaded with fura-2, were stimulated with 12 mM glucose, and simultaneous recordings were made from the same ß-cell using a microelectrode and a microfluorometer (modified from Fig. 1 in Ref. 60). The earliest change, marked phase 0, is a gradual depolarization of the membrane and a decrease in [Ca2+]i. An abrupt initiation of a sustained period of depolarization with superimposed Ca2+-dependent action potentials or spikes marks phase 1. [Ca2+]i rises abruptly during phase 1, the result of Ca2+ mobilization from intracellular stores and from Ca2+ influx through voltage-dependent Ca2+ channels. Phase 1 can be correlated temporally with first-phase insulin release. Phase 2 is marked by the initiation of steady-state oscillations in both membrane potential and [Ca2+]i, commonly called "bursting." The frequency of bursting is dependent on the glucose level (see for example Ref. 88). F340/F380 is the fluorescence ratio for fura-2 used as a measure of [Ca2+]i. [Adapted with permission from I. D. Dukes et al.: Curr Opin Endocrinol Diab 4:262–271, 1997 (60 ). © Lippincott Williams and Wilkins.]

As illustrated in Fig. 3, V_M in the ß-cell is regulated by glucose metabolism. When the external glucose concentration is less than 3 mM, V_M is near -65 to -70 mV. Increasing the external glucose causes gradual depolarization to a new steady-state level. Further increases cause the membrane to depolarize to a threshold potential, near -50 mV, where electrical activity, due in part to Ca²⁺ currents through voltage-dependent Ca²⁺ channels, is initiated. Several types of pumps and ion channels have been identified in ß-cells, which could or have been suggested to contribute to this observed electrical bursting behavior, including the following.

1. Na⁺/K⁺ ATPase. The asymmetric distribution of sodium and potassium ions is maintained by the ß-cell Na⁺/K⁺ ATPase, which has been identified and subjected to limited study (65, 66). The activity of this enzyme is generally considered insensitive to K_ATP channel blockers and openers, although high concentrations of glibenclamide have been reported to be inhibitory (67). Inhibition of the Na⁺/K⁺ ATPase with ouabain results in membrane depolarization and release of insulin. In a recent report, Ding et al. (56) used manipulations of the activity of the Na⁺/K⁺ ATPase to change the ATP/ADP ratio and affect K_ATP channel activity and suggest that a fall in [ATP]_i (and presumably an increase in [ADP]_i), terminates a burst of electrical activity.

2. Na⁺ channels. Early studies on the effects of veratridine and tetrodotoxin suggested that Na⁺ channels were functionally important for insulin release from pancreatic islets (68, 69), although Donatsch et al. (70) found tetrodotoxin did not affect insulin release from mouse islets. Several reports have described a transient, voltage-dependent, inward Na⁺ current that is blocked by tetrodotoxin (71, 72). The canine channel shows steep activation and inactivation between -50 and -40 mV (73, 74). Philipson et al. (75) identified Na⁺ channel subunit cDNAs in canine, human, and rodent islets, and in hamster and mouse insulinoma cell lines, which are most closely related to the rat brain III 1 isoform of Na⁺ channel subunits. This conductance could play a role in the initial depolarization, but its role is usually discounted in rodent ß-cells because the voltage dependence of inactivation of the rodent channel suggests it will be largely inactive (71, 76). Na⁺ channels may contribute to the action potential in canine and human ß-cells (73, 74), but their role is generally believed to be minimal.

3. Chloride channels. Several reports have suggested chloride ions can affect insulin release and ß-cell electrical activity (77, 78). The mechanism(s) of these effects are not known, and it is unclear whether or not chloride ions are passively distributed across the ß-cell membrane. Kinard and Satin (79) have identified an ATP-sensitive chloride channel in ß-cells, termed I_Cl,islet, which is activated by cAMP, glibenclamide (1–10 µM), and cell swelling. Decreasing [ATP]_i reduces the amplitude of the current; thus, Kinard and Satin suggest this channel may be under metabolic control. I_Cl,islet mediates a large inward current that would depolarize the ß-cell membrane. A second, Ca²⁺-dependent Cl^- conductance has been described recently in the ßTC-3 ß-cell line (80), but its physiological significance is uncertain.

4. Ca²⁺ channels. The importance of Ca²⁺ in insulin release has been reviewed by many others (81, 82, 83, 84, 85, 86, 87, 88). Insulin release is attenuated in Ca²⁺-deficient media and by the action of Ca²⁺ channel blockers. The early reports describing Ca²⁺ currents in ß-cells (89, 90, 91) were followed by others identifying at least two types of Ca²⁺ channels in ß-cells, which could be distinguished by their kinetics and pharmacology (71, 72, 92, 93, 94, 95, 96). The larger conductance channel has the properties of a fast deactivating, dihydropyridine-sensitive L-type Ca²⁺ channel with an activation threshold near -30 mV. The smaller conductance is similar to the T-type Ca²⁺ channel, is slowly deactivated, and has a lower activation threshold, near -50 mV. The T-type channel is not sensitive to dihydropyridines, but is inhibited by NiCl₂, a selective inhibitor of other T-type calcium channels (96). It has been suggested that the slow T-type channel could be responsible for the slow Ca²⁺ waves, while the L-type channels contribute the spikes. The evidence for and against this idea has been reviewed by Satin and Smolen (58). The possible involvement of T-type channels in control of insulin secretion has become more accessible with the cloning of these channels (97, 98) and the finding of T-type channel mRNAs in the pancreas. In addition to the voltage-dependent Ca²⁺ channels, a glucose-activated calcium channel has been reported in ß-cells (99).

5. Ca²⁺-release-activated nonselective cation channels (I_CRAN). Several lines of evidence indicate the importance of a nonselective cation channel, which is activated by depletion of internal Ca²⁺ stores, in the regulation of ß-cell electrical bursting. This current has been termed I_CRAN (60, 100, 101) or I_CRAC for Ca²⁺ release-activated currents (102, 103, 104, 105). The use of the term I_CRAC in this context appears to be somewhat unfortunate and this channel, which has a conductance in the 20–40 picosiemens (pS) range, should not be confused with the low-conductance Ca²⁺ release-activated Ca²⁺ channel, I_CRAC, described previously (106, 107, 108).

Dukes et al. (60) have reviewed the evidence for the role of I_CRAN in bursting. This model underscores the importance of Ca²⁺ mobilization from the endoplasmic reticulum through activation of the inositol triphosphate (IP₃) receptor by IP₃ which is generated by membrane depolarization. Emptying of internal Ca²⁺ stores signals I_CRAN to open. Under physiological conditions Na⁺ is the primary current carrier; thus, as described above, the increased Na⁺ permeability depolarizes the ß-cell causing L-type Ca²⁺ channels to open, which provides Ca²⁺ to help refill internal calcium stores. Replenishment reduces the signal to and activity of I_CRAN, allowing the cell to repolarize and continue cycling. Since the extent of repolarization will be determined by the K⁺ permeability and will determine whether bursting continues, this is one potential control point for K_ATP channels. Coupling the control of I_CRAN to emptying of internal Ca²⁺ stores is able to explain the effects of compounds such as maitotoxin (109, 110, 111), which activates I_CRAN, and thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺ pump [sarcoplasmic or endoplasmic reticulum Ca-ATPase (SERCA)] (112, 113), on ß-cell membrane potential and insulin release (105, 114, 115). How the filling level of the internal Ca²⁺ store is determined and the nature of the chemical signal that passes to I_CRAN remains to be elucidated.

6. Potassium channels. In addition to K_ATP channels, other K⁺ channels have been identified in pancreatic ß-cells. This area has been reviewed recently by Dukes and Philipson (87) and need not be repeated beyond indicating that delayed rectifier channels (Kv1.x), Ca²⁺-activated K⁺ channels (maxi-K), an -adrenoreceptor-activated K⁺ channel, and G protein-gated K⁺ channels (K_IR3.1, 3.2 and 3.4) have all been identified in ß-cells either by electrical recording or molecular biological methods. The Ca²⁺-activated K⁺ channel was proposed to play a role in bursting, but inhibitors of this channel have little effect on electrical activity.

What role do K_ATP channels play in bursting? There is general agreement that K_ATP channels are the predominant conductance in a ß-cell in low glucose, and that the initial depolarization, which starts a train of bursts, results from an increase in glucose metabolism, which reduces their activity. The action of sulfonylureas is readily understood within this context. Sulfonylureas close K_ATP channels, in effect mimicking the signal from glucose metabolism, thus depolarizing the ß-cell membrane. Titration of K_ATP channel activity with tolbutamide will lead to bursting at intermediate glucose concentrations (116, 117). The action of a KCO is also readily understood. The application of diazoxide inhibits insulin release by hyperpolarizing ß-cells by opening more K_ATP channels, thus shifting V_M more toward E_K. The question of how K_ATP channels participate in the actual dynamics of bursting is controversial. Using whole-cell recording, Smith et al. (118) were unable to see changes in conductance in K_ATP channels during bursting, indicating they are not involved in the dynamics of bursting. Larsson et al. (119) have reported the reverse: that the conductance of K_ATP channels oscillates during slow bursting in single mouse ß-cells and in small clusters of ß-cells. Rosario et al. (120) have demonstrated bursting in ß-cells, incubated with high extracellular Ca²⁺, when K_ATP channels were blocked by sulfonylureas. These results indicate K_ATP channels are not essential for bursting to proceed, but that residual channel activity may play a role in normal repolarization. Finally, if K_ATP channels are stimulated by diazoxide during an episode of bursting, the increase in K⁺ permeability hyperpolarizes the ß-cell membrane and thereby terminates bursting. These results have led to the idea that there are both K_ATP channel-dependent and independent mechanisms for controlling insulin secretion (see for example Refs. 121, 122, 123).

The physiological ligand(s) or mechanism(s) controlling K_ATP channels remains somewhat controversial. In excised, inside-out patches, the application of ATP results in channels closing (Fig. 1), which led to the early suggestion that changes in the intracellular concentration of ATP might regulate ß-cell K_ATP channels (30, 124). However, the IC₅₀ for inhibition of channel activity was determined to be in the 10–50 µM range, well below the millimolar concentrations estimated for intracellular ATP levels (125, 126, 127); when millimolar concentrations of ATP were applied to the intracellular face of excised patches, K_ATP channel activity was strongly inhibited. Subsequent studies showed that MgADP could open K_ATP channels inhibited by ATP, implying that the ADP/ATP ratio was the important variable (43, 44, 128, 129, 130). As described below, studies on channels reconstituted from a mutant SUR1 identified in a PHHI patient and wild-type K_IR6.2 are consistent with this idea and suggest further that fluctuations in the intracellular ADP concentration that occur as a result of glucose metabolism (126, 127, 131) are a critical regulator in ß-cells.

	IV. KATP Channel Subunits

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
A. The K_IR family of inwardly rectifying K⁺ channels
The observations (30, 44, 124, 132, 133, 134) that ATP-sensitive K⁺ channels are both potassium selective and inwardly rectifying suggested they would have some relationship to the inwardly rectifying potassium channel superfamily, the definition of which began with the cloning of three distinct inwardly rectifying K⁺ channels: K_IR1.1 (ROMK1), a weak inward rectifier found in rat kidney (1), K_IR2.1 (IRK1), a strong inward rectifier cloned from a macrophage cell line (135), and K_IR3.1 (KGA, GIRK1), a G protein-gated, strong inward rectifier isolated from rat heart (136, 137). The descriptive terms "weak" and "strong" in this context are indicators of the amount of current the channels can pass in the outward direction when the membrane potential is greater than E_K; weak rectifiers conduct more current. These proteins are all smaller than the voltage-gated K⁺ channels and, based on hydropathy plots, were predicted to have two transmembrane domains (TMDs) or helices, termed M1 and M2. These segments flanked a sequence with a high degree of similarity to the P (pore) or H5 loop first identified in the voltage-gated K⁺ channel (K_V) family (138, 139) and shown to be part of the potassium selectivity "filter." Insight into how the pore is probably formed has been provided by the determination of the structure of the pore of the KcsA channel (34, 35) [1BL8 in the Protein Data Bank (140, 141)] which is depicted in Fig. 4. Although the KcsA channel is a pH-regulated, bacterial K⁺ channel, the general similarity between the pore regions of the Kv channels, the inward rectifiers, and KcsA, all of which share an architecture with two transmembrane helices flanking a GYG or GFG sequence responsible for the K⁺ selectivity, indicates their structures will be similar. As expected from earlier studies, the M2, or inner pore helix, actually forms the permeation pathway with the signature GYG sequence positioned to act as a selectivity filter. The structure and studies on the dynamics of KcsA suggest the actual gate is formed by amino acids at the cytoplasmic ends of the M2 helices. Lateral or twisting motions of these helices could control access or gating of internal K⁺ ions into the water- filled vestibule immediately below the selectivity filter. The availability of a general structural model, even one missing the cytoplasmic domains that presumably regulate motion of the M2 helices, should allow real progress toward understanding gating mechanisms.

View larger version (64K): [in this window] [in a new window]   Figure 4. Three-dimensional structure of the Streptomyces lividans K+ channel. The top view looks down into the conduction pathway through the selectivity filter assumed to be composed of the backbone carbonyl oxygen atoms of the gly-phe-gly motif found in other potassium channels. The side view conveys the overall structure, including the vestibule below the selectivity filter and the convergence of the four M2 helices, which form the conduction path at the cytoplasmic face of the channel, to form a gate which can open and close (35 ). The figure was generated using the coordinates, 1BL8, obtained from the Protein Data Bank (140 141 ) using RasMol (373 ). [Derived from D. A. Doyle et al.: Science 280:69–77, 1998 (34 ).]

1. Cloning of K_IR6.x cDNAs. Using a fragment of K_IR3.1 (GIRK1) cDNA as a probe, Inagaki et al. (144) cloned K_IR6.1 (ruK_ATP-1) from a rat library. The tissue distribution of K_IR6.1 was broad; hence the designation "u" K_ATP-1 for ubiquitous. Expression of K_IR6.1 in HEK293 cells suggested it formed a K⁺ channel whose activity was blocked by 1 mM ATP, and activated by diazoxide, but was not inhibited by sulfonylureas. Subsequent expression of K_IR6.1 in other cell types has failed to generate novel K⁺ channel activity with similar properties (145, 146), and it is unclear whether the original observation was due to coupling of K_IR6.1 to an unidentified SUR-like protein in HEK293 cells (21), to mischaracterization of an endogenous channel, or to having a sufficiently high level of expression to overcome a K_IR retention signal. This report further added to the confusion surrounding the cloning and identification of K_ATP channels since K_IR1.1 (1), K_IR3.4 (147), and K_IR6.1 (144) were all reported initially to be ATP-sensitive potassium channels.

K_IR6.1 was not detected in ß-cell lines, and Inagaki et al. (144) used the K_IR6.1 cDNA to clone the cDNA for K_IR6.2 (originally designated BIR for ß-cell inward rectifier). Subsequent work showed that K_IR6.2 mRNA was relatively abundant in pancreatic islets and ß-cell lines and was expressed in brain, heart, and skeletal muscle (see Ref. 21 for a review). The K_IR6.2 gene was found to contain no introns and is therefore relatively easy to obtain using the PCR.

K_IR6.1 and K_IR6.2 have significant blocks of identical sequence. Seventy percent, 276 of the 390 residues in human K_IR6.2, are identical to those of K_IR6.1 after conservative alignment (Fig. 5). We have marked the proposed transmembrane helices and the P loop by comparison with the KcsA structure and have marked the approximate positions of the PHHI mutations described below. K_IR6.1 has several intriguing features, including a triple repeat at its C-terminal end, S(M/I)RRNN.

View larger version (59K): [in this window] [in a new window]   Figure 5. Schematic representation of an inward rectifier potassium channel. The diagram is based on the Streptomyces lividans K+ channel described by Doyle et al. (34 ) and on data described in the text. M1 and M2 identify the two TMDs; the region determining the K+ selectivity contains the gly-phe-gly motif. The asterisks mark the positions of mutations in KIR6.2 that have been identified in patients with PHHI. The dotted circles identify segments of the KATP channel referred to in the text. The alignment compares the sequences of human KIR6.1 with KIR6.2. Identical residues are shaded. The M1 and M2 segments are boxed. Note the repeated sequence in the C terminus of KIR6.1 marked by the bar.

A significant advance in the biochemistry of these receptors came with the realization that [³H]glibenclamide could function as a photoaffinity probe and would label a polypeptide with an apparent molecular mass of 140 kDa in rat ß-cell tumor membranes (151). A ¹²⁵I-labeled derivative of glibenclamide, [¹²⁵I]iodoglibenclamide (152) (Fig. 2), identified a similar protein in membranes isolated from HIT T15 cells and had the advantage of higher specific activity, which made studies of the receptor and its purification feasible. Nelson et al. (154) investigated the photolabeling of membrane proteins by this reagent and showed that the 140-kDa protein accounted for most of its high-affinity binding activity, K_D 5 nM, in HIT T15 cell membranes. Rajan et al. (134) and Ronner et al. (156) used [¹²⁵I]iodoglibenclamide and [³H]gli-benclamide, respectively, to identify the high-affinity receptor in TC-6 cells, a glucagon-secreting cell line. Both groups characterized the sulfonylurea-sensitive K_ATP channels in these cells by rubidium efflux and single-channel recording and concluded they were equivalent to the ß-cell channel.

Schwanstecher et al. (162) synthesized a 4-azidosalicyloyl analog of glibenclamide, ¹²⁵I-iodo-azidoglibenclamide (Fig. 2), and demonstrated that this derivative would photolabel a 38- to 40-kDa protein in addition to SUR1 (163). Competition binding experiments indicated that both the novel 38-to 40-kDa species and SUR1 were labeled with ¹²⁵I-iodo-azidoglibenclamide with the same apparent K_D. As described below, the 38- to 40-kDa species is K_IR6.2.

2. SUR1 is differentially glycosylated. Labeling of TC-6 cells with [¹²⁵I]iodoglibenclamide showed two labeled species with estimated molecular masses of 140 kDa and 150–170 kDa, the latter being a diffuse band (134). The higher molecular mass receptor accounted for approximately half of the high-affinity labeling in TC-6 cells. Competition binding studies demonstrated that the two receptors had the same affinity for iodoglibenclamide, K_D 3.5 nM. Rajan et al. (134) suggested the 150- to 170-kDa receptor resulted from differential glycosylation, but at the time the possibility of an additional gene product could not be eliminated. Nelson et al. (164) demonstrated that the 140 and 150- to 170-kDa receptors were present in several other cell types. In RINm5F and TC-6 cells the two glycosylated species are present in about equal amounts, while in HIT cells the predominant species was the 140-kDa receptor, with the 150- to 170-kDa species accounting for less than 10% of the high-affinity binding activity. Ozanne et al. (165) used [³H]glibenclamide to label receptors in rat pancreatic islets, insulinomas, and CRI-G1 cells and were able to identify both forms of the receptor. The 150- to 170-kDa species was the major form in islets and insulinomas, while the 140-kDa species was predominant in CRI-G1 cells.

Biochemical studies indicate the mass difference between the two forms of the receptor is the result of differential glycosylation (164). Photolabeling of membranes isolated from RINm5F cells grown in tunicamycin, which blocks N-linked glycosylation, produced a single photolabeled band with an estimated mass of 137 kDa, distinct from the 140-kDa species. A 137-kDa species was also generated by digestion with endoglycosidases. The 140 and 150- to 170-kDa receptors could be separated using concanavalin A and wheat germ agglutinin, the 140 kDa receptors binding to concanavalin A-agarose, while the 150- to 170-kDa receptors bound to wheat germ agglutinin-agarose. The results indicate that the 140-kDa receptors are "core" glycosylated with mannose-containing glycosyl groups that bind to concanavalin A, while the 150- to 170-kDa receptors are "complex" glycosylated with terminal sialic acid residues that bind to wheat germ agglutinin (164). Since core glycosylation takes place in the endoplasmic reticulum after protein synthesis, while trimming of the mannose chains and addition of sialic acid groups takes place in the medial Golgi, the data indicate that the 150- to 170-kDa species is the "mature" form of the receptor and is either in, or in transit to, the plasma membrane. This is consistent with the results of Ozanne et al. (165) showing that the "immature" 140-kDa receptors in insulinomas were found in internal membranes, whereas the 150- to 170-kDa receptors were in granules and plasma membranes. The results raise questions about how glycosylation of SUR1 is regulated, whether the core glycosylated receptors on internal membranes might have a function apart from K_ATP channels, and whether the subunits can traffic independently to the plasma membrane. For example, Eliasson et al. (166) have suggested that sulfonylureas can stimulate insulin secretion independently of their blockage of K_ATP channels, perhaps acting through SUR1 in granule membranes.

The available sequence and site-directed mutagenesis has confirmed there are two N-glycosylation sites in SUR1 at residues 10 and 1050. The N10Q or N1050Q mutations partially eliminate glycosylation, while the double mutant is not glycosylated. All three of the glycosylation mutants generate channels when expressed with K_IR6.2, although the number of channels, estimated by ⁸⁶Rb⁺ efflux, is reduced (J. P. Clement IV and J. Bryan, unpublished data). Interestingly, the glycosylation patterns of SUR1 appear to differ between endocrine and neuronal tissues as well as - and ß-cell lines (164). The SUR1 in brain, for example, appears to be only the highly glycosylated form (162).

3. Linking the sulfonylurea receptor with K_ATP channel activity. The link between sulfonylurea receptors and K_ATP channels was the subject of some early controversy. The simple view that receptor and channel might be a single entity (167) was questioned by Khan et al. (168), who reported the dissociation of K_ATP channel activity and sulfonylurea receptors in a rat insulin-secreting clonal cell line, CRI-D11. Aguilar-Bryan et al. (169) produced evidence that receptor and channel activity were lost in parallel during serial passage of HIT cells at approximately the same passage number at which glucose sensitivity for insulin release was lost (170). These studies have not been reexamined using the molecular probes now available, and it is unclear, for example, whether the CRI-D11 cells actually have active K_IR6.2 channels, without receptors, or whether high passage HIT cells have lost K_IR6.2 in addition to SUR1.

4. Receptor purification. Hamster SUR1, prelabeled with [¹²⁵I]iodoglibenclamide, was purified from HIT cell membranes using chromatographic and electrophoretic methods (22, 164). The general strategy was to prelabel the receptor with [¹²⁵I]glibenclamide, then purify the iodinated protein. The end point was obtaining sufficient pure receptor for N-terminal peptide sequencing. The purification has been described in detail (164, 171) and will not be discussed further. The availability of cloned cDNAs has allowed introduction of specific markers, for example hexa-histidine tags, to facilitate purification of expressed recombinant receptors (146, 172) and K_IR6.x subunits.

5. Cloning strategy. The N-terminal protein sequence was used to design oligonucleotide primers for amplification of a small segment of the SUR1 cDNA by the PCR (22, 164). The resulting cDNA fragment was used to screen hamster, rat, mouse, and human pancreatic cDNA libraries to obtain full-length clones that encode homologous proteins with molecular masses of approximately 176 kDa (1582 amino acids for the rodent receptors and 1581 or 1582 amino acids for the human receptor). The cDNA and gene sequences for SUR1 and SUR2 are readily available through the National Library of Medicine (http://www.ncbi.nlm.nih.gov/Web/Search/index.html).

6. SUR1 is a member of the ATP-binding cassette superfamily. The rodent and human SUR1 sequences are quite similar with absolute identities of >90%. Blast searches (173) of the Genbank database show similarities between SUR’s and a large number of ABC proteins including multidrug resistance proteins (174), multidrug resistance associated proteins (MRPs) (175), CFTRs (176), canalicular multispecific organic anion transporter (177, 178), and the cadmium-binding protein YCF1 (179). Dendrograms and the overall topology of SURs indicate they can be placed within the MRP subfamily of ABC proteins (180) with two potential nucleotide-binding folds (NBFs), multiple TMDs, and a very hydrophobic segment at their N termini. The greatest sequence similarities are in the NBFs that have the Walker A (-GlyXXGlyXGlyLysSer/Thr-, where X is any amino acid) and B (-YYYYAsp-, where Y is a hydrophobic amino acid) consensus motifs (181) and a conserved -LeuSerGlyGlyGln- sequence in the segment linking the two Walker motifs. NBF1 matches this motif exactly, while NBF2 has a somewhat degenerate sequence, -PheSerGlnGlyGln- (SUR1), -PheSerValGlyGln- (SUR2). Manavalan et al. (182) have noted the similarity of the NBF sequence between CFTR and G proteins. X-ray crystal structures for the three major classes of GTPases, the small p21^ras-like proteins, the heterotrimeric G proteins, and EF-Tu, indicate that the Walker A motif interacts with Mg²⁺ and with the oxygen atoms of the - and ß- phosphates (see Ref. 183 for a review). The Gln in the -LeuSerGlyGlyGln- linker sequence has been proposed to act as a general base during nucleotide hydrolysis, but this has been difficult to establish (184). The lack of a structure for the NBFs of the ABC cassette proteins has hindered progress. The recent preliminary report on the determination of the structure of an NBF from the ribose transporter by Armstrong et al. (185) should allow major advances in this area. A major question with regard to the sulfonylurea receptors is whether they can hydrolyze ATP, and, if so, how this hydrolysis is coupled to the regulation of channel activity. The purification and ATPase activity of the closely related MRP1 protein have been described (186). Interestingly, the ATPase activity of this ABC protein is stimulated several fold by low concentrations of nucleoside diphosphates (187).

7. Proposed membrane topology for SUR1. Aguilar-Bryan et al. (22) predicted a topology for SUR1 based on plots of hydrophobicity and hydrophobic moments using the analysis of Eisenberg and colleagues (188). The constraints on the proposed topology were that the N terminus, glycosylated at N10 (22), was extracellular and the two NBFs were intracellular. The resulting model consisted of nine TMDs followed by a nucleotide-binding fold, NBF1, a second transmembrane region with four spanning helices followed by a second nucleotide-binding fold, NBF2 (i.e., 9 TMD–NBF1–4 TMD–NBF2). This topology resembled the model originally put forward for MRP1 (175). Tusnády et al. (180) have revised the model of the MRP subfamily using multisequence alignments of recently cloned ABC proteins. Their model, 11 TMD–NBF1–6 TMD–NBF2, emphasizes the existence within the MRP subfamily proteins of an multidrug resistance protein-like core consisting of 6 TMD–NBF1–6 TMD–NBF2. This core is preceded, in the MRP subfamily, by a highly hydrophobic N-terminal extension with 5 or more TMDs. Work is needed to establish the topology experimentally and to determine what function the additional TMDs might have. We have discussed the topology of the receptors in more detail elsewhere (19).

8. Expression of SUR1 cDNAs. Transfection of SUR1 cDNAs into COSm6 cells lacking endogenous sulfonylurea receptors led to the expression of proteins that could be photolabeled with [¹²⁵I]glibenclamide and had an apparent mobility on SDS polyacrylamide gels equivalent to the native receptor (22). The discrepancy in mass between 176 kDa predicted from the cDNA sequence and 140 kDa estimated by gel electrophoresis is artifactual. Purification of receptors tagged with hexa-histidine at either the N- or C termini yield the same size protein. Membranes isolated from COS cells expressing the recombinant hamster and rat receptors contained high-affinity [¹²⁵I]glibenclamide-binding activity with K_Ds of 10 and 2 nM, respectively (22). These values were in good agreement with a value of 5 nM determined for the native hamster receptor in isolated HIT cell membranes (19, 154, 169). Efforts to show that COS cells expressing SUR1 alone had novel K⁺ channel activity were unsuccessful, suggesting other components were required to form a K_ATP channel. A puzzling result, from these early studies, was that only the 140-kDa core glycosylated protein was expressed, while the 150- to 170-kDa receptors were not detected.

	V. Reconstitution of KATP Channel Activity from SUR1 and KIR6.2

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
Coexpression of SUR1 and K_IR6.2 in COS cells and in Xenopus oocytes produced a novel K⁺ conductance that was characterized by ⁸⁶Rb⁺ efflux and single-channel recording (26). Metabolic poisoning with 2-deoxyglucose and oligomycin revealed a novel ⁸⁶Rb⁺ efflux pathway not detected in cells transfected with ß-galactosidase, or with either SUR1 or K_IR6.2 alone. Efflux was blocked by tolbutamide and gli-benclamide and was activated by diazoxide, but was poorly activated by cromakalim or pinacidil (23). Single-channel recordings characterized a moderate, inwardly rectifying potassium-selective channel with a conductance of about 65 pS in 140 mM KCl at -60 mV (26). The extent of inward rectification was dependent upon the presence of Mg²⁺ or polyamines on the intracellular side of the channel (19, 146). Channel activity was half-maximally inhibited by ATP at approximately 10 µM (26). Later measurements indicated the recombinant channels were activated by MgADP in the presence of ATP (189, 190). Table 2 provides a comparison of the properties of recombinant and native K_ATP channels. The recombinant channels appear to mimic all of the basic electrophysiological and pharmacological properties of native ß-cell ATP-sensitive potassium channels. These results have been confirmed by studies in oocytes (145) and HEK cells (191). Additional work will be required to determine whether and by what means the recombinant K_ATP channels are coupled to the receptors for the various peptides, such as galanin, reported to affect their activity (Ref. 192 but see Ref. 193), somatostatin (194), GLP-1 (195), CGRP (196), and leptin (197, 198, 199, 200, 201).

A. The question of "promiscuous coupling" of SUR1 with other inward rectifiers
The full range of inward rectifiers that SUR1 will partner with has not been established. Ämmälä et al. (202) showed that K_IR6.1 and SUR1 would form K_ATP channels, but these were not characterized. These authors also argued for greater promiscuity and pairing of the receptor with K_IR1.1 (ROMK1) and K_IR3.4 (rcKATP-1/CIR/GIRK4). Clement et al. (146) have confirmed that K_IR6.1 and SUR1 produce metabolically activated K_ATP channels, while coexpressing SUR1, and K_IR1.1 (ROMK1) or K_IR3.4 (CIR, rcKATP) failed to show association or K_ATP channel activity. Using Xenopus oocytes, Gribble et al. (145) were unable to reproduce the original observations on coupling of SUR1 to ROMK1 and showed that K_IR2.1 also failed to couple to SUR1. We infer that, although all the potential inward rectifier candidate subunits have not been screened, the degree of promiscuity involved in K_ATP channel formation will not be large. Wellman et al. (203) have reached a similar conclusion using coronary arterial myocytes. Interestingly, the reports on the SUR1/K_IR6.1 channel have all been with intact cells, since observations on excised patches have been hampered by rapid channel rundown (145).

	VI. KATP Channel Structure

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
The reconstitution experiments demonstrated that both SUR and K_IR subunits were required to form K_ATP channels, but raised a number of questions about the interactions, stoichiometry, and functional role(s) of each subunit within a channel and about the trafficking and activity of individual subunits. It was not at all clear, for example, why K_IR6.2 was silent in the absence of SUR. Some of these questions have been addressed, and it is becoming clear that essentially all of the kinetic, pharmacological, and regulatory properties of K_ATP channels including their sensitivity to inhibitory ATP and trafficking to the plasma membrane are highly dependent on interactions between SUR and K_IR subunits.

A. K_IR6.2 forms the pore of a K_ATP channel
An early question was which subunit formed the pore or permeation pathway of the channel and whether a part of SUR was involved. Two lines of evidence have been developed that show that K_IR6.2 subunits are sufficient to form the permeation pathway. Clement et al. (146) and Shyng et al. (204) showed that substitution of an aspartate for the asparagine at position 160, N160D, produced strongly rectifying channels; whereas asparagine, in the wild-type channel produced weaker rectification. The result did not eliminate the possibility that SUR1 could contribute directly to the permeation pathway, but did indicate that the M2 segments of K_IR6.2, like those of other inward rectifiers, were part of the permeation pathway. The discovery that truncated K_IR6.2 subunits, missing 16–35 amino acids from their C-terminal ends, can assemble a K⁺ channel provided a direct demonstration that SUR1 was not required to form the pore (51). These findings suggested that the underlying architecture of the pore of K_ATP channels would resemble that of the other members of the K_IR channel family.

B. SUR1 and K_IR6.x are physically associated
The requirement for coexpression of both SUR and K_IR to make an ATP-sensitive K⁺ channel implied these subunits were associated, but other alternatives were possible. Al-Awqati (205), for example, proposed that SUR might regulate a physically separate potassium channel by releasing ATP, as CFTR has been proposed to regulate the outwardly rectifying chloride channel (206, 207). The first direct evidence that SUR1 was associated with a second subunit came from the work of Schwanstecher, Panten, and colleagues (162, 163) as mentioned above. These authors developed an azido derivative of glibenclamide, [¹²⁵I]azidoglibenclamide (Fig. 2), and demonstrated that in addition to labeling SUR1 in brain membranes, an additional 38-kDa protein displayed high-affinity labeling. The identification of the 38-kDa component as K_IR6.2 became clear from [¹²⁵I]azidoglibenclamide labeling studies on COS cells expressing the cloned subunits (146). In these experiments SUR1 photolabeled independently of K_IR6.2, but K_IR6.2 did not bind glibenclamide directly and was only labeled when coexpressed with SUR1. These findings implied that ¹²⁵I-iodo-azidoglibenclamide was bound to SUR1 and that K_IR6.2 must be in close proximity, since the lifetime of the photo-activated species is brief.

C. Coexpression with K_IR6.2 affects the maturation of SUR1
The finding that coexpression of SUR1 and K_IR6.2 affected the maturation or glycosylation state of the receptor provided additional support for subunit association, provided a means to demonstrate complex formation directly, and was the first indication that SUR-K_IR assembly was needed for SUR to traffick to the plasma membrane. Expression of SUR1 alone generated the immature 140-kDa core glycosylated receptor, while coexpression with K_IR6.2 also generated mature complex glycosylated receptors (146). Chromatography on wheat germ agglutinin was used to show that K_IR6.2 was preferentially associated with mature SUR1 and was assembled into a stable multimer that could be isolated after solubilization in digitonin (146). These findings suggested a plausible assembly pathway in which the immature receptor associates with K_IR6.2 in the endoplasmic reticulum and maturation to the complex glycosylated form occurs in the medial Golgi as the assembled channel moves to the plasma membrane. These results agree with the observations of Ozanne et al. (165) that the 150-kDa species of SUR1 is in plasma membranes. It was not clear whether K_IR6.2 could traffic independently to the plasma membrane, but the result indicated SUR1 did not move efficiently through the medial Golgi in the absence of K_IR. The later observation that C-terminally truncated K_IR6.2 subunits could generate K⁺ channels raised the question of whether K_IR subunits could traffic independently to the plasma membrane (51).

D. Complex glycosylated SUR1 and K_IR6.2 assemble a large multimer
The finding that maturation of SUR1 was coupled with expression of K_IR6.2 and that these subunits assembled a stable complex argued that the multimer might be the channel itself. The mass of the stable multimer, estimated by sucrose gradient sedimentation, was approximately 950 kDa, consistent with a large channel composed of four complex glycosylated SUR1 subunits plus four K_IR6.2 subunits (an expected protein mass of 885 kDa = 4 x 176 kDa + 4 x 45 kDa, plus an unspecified carbohydrate mass) (146).

E. A 1:1 stoichiometry of SUR to K_IR is both necessary and sufficient to make K_ATP channels
The simplest model for the assembly of SUR1 with K_IR6.2 was a 1:1 association. Therefore, cDNAs encoding fusion proteins with defined stoichiometries were engineered to determine whether a 1:1 SUR/K_IR stoichiometry was both sufficient and necessary for the formation of functional K_ATP channels (146). Expression of SUR1K_IR6.2, a fusion of the N terminus of K_IR6.2 with the C terminus of SUR1 through a 6-glycine linker produced potassium-selective channels that were activated by metabolic poisoning and inhibited by sulfonylureas. Single-channel recording showed that the SUR1K_IR6.2 channels were weak inward rectifiers, which had the same conductance as unfused channels, although the sensitivity to both ATP and glibenclamide were reduced. Sucrose gradient analysis showed that SUR1K_IR6.2, like the wild-type subunits, produced a 950-kDa multimer. The results showed that a 1:1 stoichiometry was sufficient for formation of K_ATP channels.

Expression of a "triple" fusion construct, SUR1(K_IR6.2)₂, with a defined 1:2 stoichiometry, did not generate ATP-sensitive potassium currents. However, the triple fusion could be rescued by coexpression with monomeric SUR1, implying that the additional receptor(s) reestablished the required 1:1 stoichiometry. The SUR1 + SUR1(K_IR6.2)₂ channels had the same conductance properties as unfused channels, but were less sensitive to both ATP and sulfonylureas. The results showed that a 1:1 stoichiometry was necessary for assembly of K_ATP channels and, together with the mass estimates, argued for a tetrameric organization similar to other members of the K_IR channel family.

F. Other K_IR channels are tetramers
Several reports (208, 209) indicate that the K_IR channels, like the K_V channels (138, 139, 210, 211, 212), are tetrameric. Glowatzki et al. (208) coexpressed weak (K_IR1.1, ROMK1) and strong (K_IR4.1, BIR10) inward rectifiers and observed intermediate rectification properties expected from the assembly of heterologous channels. The differential sensitivity of these channels to voltage-dependent block by spermine was used to study the distribution of channel types assembled in cotransfected cells. Five types were identified that approximated the binomial distribution expected for a pore composed of four subunits. The polyamine binding site, in the pore, is therefore assumed to be assembled from amino acids contributed by four M2 segments. Yang et al. (209) studied the subunit stoichiometry of the strongly rectifying K_IR2.1 channel (IRK1) by fusing subunits to form multimers, a strategy that had been used earlier by Liman et al. (211) on K_V1.1 (RCK1) channels. The trimeric and tetrameric constructs, (K_IR2.1)₃ and (K_IR2.1)₄, both produced channels. Coexpression of the strongly rectifying wild-type trimers with a weakly rectifying mutant monomer produced a heterologous channel with intermediate rectification and weaker affinity for spermidine, while coexpression of the mutant monomer with the wild-type tetramer did not affect rectification or polyamine binding. The results were consistent with a tetrameric channel.

G. The stoichiometry of active ß-cell K_ATP channels is (SUR1/K_IR6.2)₄
A strategy based on the formation of heterologous channels was used to show that functional K_ATP channels are tetramers (146). cDNAs were engineered to express triple-fusion proteins, SUR1(K_IR6.2)₂ and SUR1(K_IR6.2_N160D)₂, which generated moderate or strongly rectifying channels, respectively, when rescued by SUR1 monomers. When these constructs were coexpressed, three classes of channels could be identified: the parental weak and strong rectifiers plus a third species with intermediate rectification. The presence of a species with intermediate rectification implies the pore is constructed from two triple-fusion proteins, one with wild-type K_IR6.2, the other with K_IR6.2_N160D. Shyng and Nichols (213) used SUR1K_IR6.2 constructs engineered with and without the N160D mutation to reach the same conclusion, and similar results with triple-fusion constructs were obtained by Inagaki et al. (214).

Clement et al. (146) specifically proposed that active channels are composed of four complex glycosylated receptors interacting with four K_IR6.x subunits, which form the K⁺ selective pore (SUR1/K_IR6.2)₄. The observation that the fusion channels were active suggested that the N terminus of K_IR6.2 may be near the C terminus of SUR1 in the ß-cell channel, where it may serve a role in the transduction of nucleotide-induced conformational changes from the receptor to the pore. This model, illustrated in Fig. 6, further suggests the possibility of extensive cooperative interactions between the eight nucleotide-binding domains, four sulfonylurea-binding sites, and four potassium channel opener (diazoxide)-binding sites.

View larger version (47K): [in this window] [in a new window]   Figure 6. Topology and assembly of KATP channels. The topologies of SUR, based on the model of Tusnády et al. (180 ), and KIR6.x are illustrated. The wild-type channel is assembled from SUR1 and KIR6.2 subunits as an octameric complex as described by Clement et al. (146 ). Truncation of the C-terminal end of KIR6.2 allows trafficking of the inward rectifier to the cell surface to form homomeric K+ channels (KIR6.2C)4 with abnormal properties (51 ). Truncated KIR subunits can also assemble with SUR. These channels are compared in Fig. 8.

	VII. Regulation of KATP Channel Activity

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

1. Refreshment. When a patch containing K_ATP channels is excised into an ATP-free solution, channels open, as was first described by Noma for cardiac myocytes (31) and by Cook and Hales (30) and Trube and Hescheler (32) for pancreatic ß-cells. These channels lose their activity or "rundown" as a function of time. Activity can be restored, or "refreshed," by brief application of MgATP at millimolar concentrations. Refreshment can be thought of as switching the channel from a nonoperational to an operational state. The transition to an operational state requires hydrolysable nucleotides and is not supported by nonhydrolysable ATP analogs or by hydrolysable nucleotides in the absence of Mg²⁺ or Mn²⁺. Several possible explanations have been put forward to explain the biochemical basis for this switching, including phosphorylation/dephosphorylation reactions (see Refs. 218, 219 but also Refs. 220, 221, 222, 223 for another view), uncoupling of K_ATP channels from the actin cytoskeleton (223), and hydrolysis of anionic phospholipids, which are proposed to stabilize the channel in the operational state (224, 225). It is unclear whether one or all of these mechanisms is a critical factor in rundown and refreshment of channels in excised patches. Perhaps more importantly, it is not certain whether there is a physiological analog of rundown in intact cells where the level of MgATP is in the millimolar range. One could propose a coupling between metabolism and, for example, a phosphorylation/dephosphorylation cycle that controls K_ATP channel openings. This idea has been explored by Ribalet et al. (226) but has not received general support.

2. Inhibition. The application of ATP, with or without Mg²⁺, to the intracellular face of an excised patch inhibits K_ATP channels. The IC₅₀ or K_i values for this inhibition are quite low, estimated at 5 to 10 µM for the pancreatic ß-cell channel and for reconstituted SUR1/K_IR6.2 channels (26), and in the range of 8 to >500 µM for the native cardiac channel and from approximately 20 (28) to 100 µM or more (23, 29) for reconstituted SUR2A/K_IR6.2 channels. These values predict approximately 99% inhibition of the ß-cell channel, assuming a cytosolic [ATP]_i level near 1 mM. This exquisite sensitivity to ATP has been a source of confusion when coupled with the idea that ATP is the regulator of channel activity. It is worth noting that several studies have suggested, and continue to suggest, that fluctuations in [ATP]_i regulate K_ATP channels either through large changes in the concentration of ATP (40) or through compartmentalization models that propose significant local changes in [ATP] (see for example Ref. 227).

Various mechanisms have been proposed to explain the discrepancy and render K_ATP channels less sensitive to ATP. For example, the inhibitory effect of MgATP has been reported to be weaker than ATP^4-, for the ß-cell channel, although Findlay (41) has reported the reverse for the cardiac channel. An early proposal by Ashcroft and Kakei (40), therefore, suggested the idea that a significant fraction of the total ATP would be complexed with Mg²⁺ at the intracellular free Mg²⁺ concentration (0.5–1 mM) (see Refs. 228, 229 for reviews); therefore, the concentration of ATP^4- would be closer to the IC₅₀ determined for K_ATP channels, thus allowing variations in ATP to regulate channel activity directly. Our own studies on the Mg²⁺ dependence of the ATP inhibition of K_ATP channel activity have failed to show significant effects. In a different vein, Cook et al. (230) pointed out, in their spare-channel hypothesis, that the high sensitivity of K_ATP channels to ATP is required to inhibit the large number of channels (assumed in the model to be 10,000 per ß-cell). Thus while approximately 99% of the channels were inhibited, the remaining channels were sufficient to maintain the resting membrane potential in ß-cells, which are small and have a high membrane impedance. According to the spare-channel hypothesis, the exquisite sensitivity of the ß-cell K_ATP channel to ATP is a necessity rather than a problem.

Studies on cells expressing a mutant of SUR1, identified in a patient with familial hyperinsulinism, argue against ATP as the physiological regulator of K_ATP channels. This mutation, a glyarg at position 1479 in NBF2 (189), uncouples the inhibition of K_ATP channels by ATP from their activation by MgADP and thus provides a tool to distinguish between regulation by changes in ATP vs. ADP. Coexpression of SUR1_G1479R with wild-type K_IR6.2 produces K⁺ channels that are inhibited by ATP^4-, or MgATP, in excised patches with nearly the same IC₅₀ as native or wild-type reconstituted channels, but are not activated by MgADP (189). The SUR1_G1479R/K_IR6.2 channels are not activated by metabolic inhibition in which [ATP]_i is reduced and [ADP]_i increases, conditions that strongly activate wild-type channels. Inhibition by 2-deoxyglucose and oligomycin in the absence of glucose cannot lower the [ATP]_i sufficiently to activate these K_ATP channels with an altered response to MgADP. Similarly, in PHHI ß-cells the G1479R channels are blocked by ATP and apparently fail to respond to fluctuations in ADP. The result implies that fluctuations of [ATP]_i alone are not sufficient to trigger insulin secretion in these patients and indicate ADP is a critical factor.

3. Stimulation. While ATP inhibits channel activity, the application of MgADP was shown to stimulate channel openings in the absence of added ATP and could activate channels inhibited by ATP (43, 44). The presence of Mg²⁺ is critical: ADP and other nucleoside diphosphates, in the absence of Mg²⁺, inhibit K_ATP channel activity. These observations, on excised patches, led to the idea that the ADP/ATP ratio was critical for regulation of K_ATP channel activity (43, 44). In ß-cells, for example, increased glucose metabolism was proposed to reduce [ADP]_i and increase [ATP]_i (see, for example, Ref. 216 for a recent review). The expectation was that the decrease in [ADP]_i would reduce channel activity (and any increase in [ATP]_i would do the same) and thus lead to membrane depolarization, activation of voltage-gated Ca²⁺ channels, increased [Ca²⁺]_i, and initiation of the exocytosis of insulin. These studies pointed to the importance of ADP as a potential regulator of K_ATP channels in ß-cells, while similar studies have shown the importance of ADP for regulation of cardiac K_ATP channels (231, 232).

What is the evidence that the ADP/ATP ratio changes? Direct measurements of the ADP/ATP ratio under different metabolic conditions are difficult to make, but several reports in ß-cells (126, 127, 216, 233) and cardiomyocytes (234) are in accord with this general model. The specific experiments support the idea that [ATP]_i increases, while [ADP]_i is reduced during periods of increased glucose metabolism. The magnitude of the measured increase in the ATP/ADP ratio depends on the method of assay. Ghosh et al. (126) reported an increase, measured by microchemical methods, but questioned its statistical significance. Detimary et al. (235) used cultured islets to show that degranulation of ß-cells, by preincubation in glucose, reduced the level of a nucleotide compartment, presumably insulin granules, with an ATP/ADP ratio near 1. Cultured islets showed an increase in the ATP/ADP ratio of 2.4 to >8 when incubated with 2 and 20 mM glucose, respectively. Detimary et al. (127) also developed a lysed cell protocol, which distinguished a diffusible cytosolic pool of nucleotides from a nondiffusible pool located within the insulin-containing granules. Incubation of islets in 20 mM glucose did not affect the nondiffusible pool but caused a change in the ATP/ADP ratio from 3.8 to 12.1, over a range of 2–20 mM glucose, in the diffusible pool. Matschinsky and colleagues used ³¹P-nuclear magnetic resonance techniques on ßTC3 and ßHC9 ß-cell lines and estimated that the concentration of free ADP dropped from approximately 35 µM to 20 µM when cells were perfused with a high concentration, 25 mM, of glucose (216).

Cook and colleagues (128) developed a semiquantitative model to explain the regulation of K_ATP channel activity in ß-cells. In their model, K_ATP channels were tonically inhibited by [ATP]_i at concentrations estimated to be in the millimolar range, well above the values (10 µM) required to half-maximally inhibit activity of the ß-cell channel. In resting or fasting ß-cells in a low glucose milieu, inhibition by ATP is relieved by elevated MgADP, which opens a small, but sufficient, number of K_ATP channels to hold V_M near -60 mV, below the threshold for activation of voltage-gated Ca²⁺ channels. Increased glucose metabolism causes [ADP]_i to fall; thus K_ATP channels close and V_M shifts to more positive values, which triggers insulin release through activation of Ca²⁺ channels. Cooperativity built into the model increases the steepness of the response curve, thus reducing the magnitude of the change in [ADP]_i required for regulation. This model relies heavily on the spare channel hypothesis (230) and the idea that maintaining the resting membrane potential of a ß-cell requires openings of only a small number of K⁺ channels.

The general wisdom is that ß-cells have several thousand K_ATP channels, but the evidence in support of this figure is somewhat difficult to track down. Rorsman and Trube (124) estimated an average of 500 channels per mouse ß-cell from whole-cell recordings during dialysis with ATP-free solution. Ohno-Shosaku et al. (236) estimated the channel density at 1.5 per µm² and calculated an average of 750 channels per ß-cell based on a spherical cell of 13 µm diameter. Misler et al. (44) reported a greater density, 8 channels per µm², although it is not clear whether this was an average value. The number of sulfonylurea receptors in RINm5f cells was estimated from [³H]glibenclamide binding studies at approximately 5120 ± 500 per ß-cell (237). This would yield a maximum of about 1250 K_ATP channels per ß-cell, assuming all the receptors are assembled as tetramers and are surface accessible.

It is interesting to note that, during the nearly 20 yr K_ATP channels have been studied, there are numerous published examples of ATP inhibition curves, but the number of studies aimed at providing a quantitative basis for understanding stimulation by MgADP are remarkably few. It is essentially impossible to draw quantitative conclusions about this model, although it appears qualitatively correct. The experiments with the SUR1_G1479R channels, for example, provide strong support, and other SUR1 mutations produce a similar phenotype. Mutations in the conserved lysine residues in the Walker A motifs in both NBFs of SUR1 have been made (lys₇₁₉ala and lys₁₃₈₄met) and shown to result in loss of activation by ADP (190, 238) while maintaining sensitivity to ATP. It seems reasonable to conclude that, in the ß-cell, ATP acts on K_ATP channels to maintain an operational state, i.e., minimize "rundown," and to inhibit channel activity, while fluctuations in ADP regulate opening of the channel when the rate of glucose metabolism changes. What is needed to validate this model for the ß-cell is quantitative data on the actual physiological variation in [ADP]_i with glucose metabolism and data indicating that ß-cell K_ATP channels respond to variations in the ADP/ATP ratio within this physiological range.

A. How do ATP and ADP exert their effects on K_ATP channels?
It is not understood, at the molecular level, how ATP and ADP exert their effects on K_ATP channels. Several factors need to be investigated and integrated into a coherent regulatory scheme, including the number and location of the nucleotide binding sites, a determination of whether sulfonylurea receptors, like other members of the ABC superfamily, have ATPase activity and, if so, how hydrolysis is involved in gating of these channels. It will be necessary to understand how potassium channel openers exert their effects and what relationship their action has to activation of K_ATP channels by MgADP.

B. Where are the nucleotide-binding sites located?
1. SUR binds ATP. Amino acid sequence similarities placed the sulfonylurea receptors in the ATP-binding cassette superfamily and predicted that SURs have two NBFs, while K_IR6.1 and K_IR6.2 were members of the inwardly rectifying K⁺ channel family with no obvious nucleotide-binding motif. Thus family lineage, plus the observation that the SUR2A/K_IR6.2 channels displayed a somewhat higher IC₅₀ for ATP inhibition (23) than the SUR1/K_IR6.2 channels, suggested the receptor was the nucleotide sensor. As described below, the situation has become more complicated.

Ueda et al. (239) have shown that [³²P] 8-azido ATP can be used to photolabel SUR1. Based on the pattern of labeling of receptors with mutations in the conserved Walker A motifs of NBF1 and NBF2, high- and low- affinity ATP-binding sites were distinguished, and NBF1 was identified as the high-affinity site. MgADP reduced labeling and was proposed to antagonize binding of ATP at NBF1 by its interaction with NBF2. Ueda et al. (239) further suggested that incubation of SUR1 with 8-azido ATP at 37 C induced a conformational change that made nucleotide binding tighter and more stable. The results demonstrate SUR1 can bind ATP as anticipated and appeared to identify a stable, long-lived ATP-liganded state of SUR1 with no apparent relationship to inhibition of K_ATP channels.

Figure 7 illustrates several properties of the photolabeling of SUR1 with [³²P] 8-azido ATP and competition with unlabeled 8-azido ATP and ATP. Qualitatively similar results were obtained with ATP^4-, ADP^3-, MgATP, MgADP, and several nonhydrolysable analogs. It is difficult to interpret the results of the experiments done in the presence of Mg²⁺ since these membrane preparations contain adenylate kinase, which readily converts ADP to ATP plus AMP. In addition, it is uncertain whether SURs have an intrinsic ATPase activity; thus we have not tried to determine an apparent IC₅₀ for nucleotides in the presence of divalent cations. The estimated IC₅₀ for 8-azido ATP^4- is approximately 1 µM and approximately 10–15 µM for ATP^4-, in general agreement with the data of Ueda et al. (239). Interestingly, this is the range where ATP^4- half-maximally inhibits K_ATP channels (Fig. 1 for example) and would be consistent with binding of ATP^4- to SUR1, serving to inhibit channel activity. On the other hand, we estimate the off rate for 8-azido ATP bound to SUR1 to be approximately 10 min at 25 C (Fig. 7), several orders of magnitude slower than would be expected from patch-clamp experiments. Thus, while the affinity of SUR1 for ATP^4- appears to be consistent with that observed for inhibition of K_ATP channels, the kinetics of dissociation are markedly slower than expected.

View larger version (47K): [in this window] [in a new window]   Figure 7. Labeling of SUR1 with [32P] 8-azido ATP. A, Loss of 8-azido ATP from SUR1 at 25 C. Membranes were incubated with 2 µM [32P]8-azido ATP in 150 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5 for 10 min, diluted with 20 volumes of the same buffer (ice cold), pelleted in a microfuge for 15 min at 4 C, and resuspended in 50 µl aliquots of ice-cold buffer. Dissociation of the complex was initiated by transfer to a water bath at 25 C. Time points were taken by addition of 1 ml of ice-cold buffer and irradiation at 254 nm (total energy = 1 J/cm2) on ice. Previous experiments indicated the loss of label at 0–4 C is negligible of the time course of the experiment (J. Bryan, unpublished data). The estimated half-life of the SUR18-azido ATP complex at 25 C is 7.5 min. B, Competition of 8-azido ATP labeling by unlabeled 8-azido ATP4-. Membranes were incubated with 2 µM [32P]8-azido ATP and the indicated concentrations of 8-azido ATP in 150 mM NaCl, 50 mM Tris-HCl, 2 mM EDTA, pH 7.5 for 10 min, diluted with 20 volumes of the same buffer (ice cold), pelleted in a microfuge for 15 min at 4 C, resuspended in 20 µl of the same buffer, and then irradiated at 254 nm (total energy = 1 J/cm2) on ice. C, Competition of 8-azido ATP labeling by ATP4-. The conditions were as described in panel B.

2. ATP can interact directly with K_IR6.2. Tucker et al. (51) have shown that expression of C-terminally truncated K_IR6.2 subunits generated K⁺ channels that were inhibited by ATP. Since these truncated subunits were not associated with SUR (see Fig. 6), the result indicated nucleotides could interact directly with a "primary" inhibitory ATP-binding site on K_IR6.2. Removal of 18, 26, and 36 (C18, C26, and C36, respectively) amino acids from the C terminus of K_IR6.2 produced K⁺ channels, while C41 showed no activity. The C26 and C36 channels were the most active and had conductance properties similar to the SUR/K_IR6.2 channels. The truncated channels showed rundown and refreshment as described above, but were not activated by MgADP or potassium channel openers such as diazoxide and were not inhibited by sulfonylureas. Thus, these regulatory functions were presumed to require SUR, and coexpression of K_IR6.2C subunits with SUR1 restored these properties. The IC₅₀ values for inhibition of the K_IR6.2C channels by ATP^4- were 100–200 µM with no significant Mg²⁺ dependence. These values were 20- to 40-fold higher than for the SUR1/K_IR6.2 channels. Coexpression of SUR1 with K_IR6.2C26 reduced the IC₅₀ for ATP inhibition, leading to the idea that SUR1 could "enhance" the inhibitory effect of ATP. The nature of the ATP-binding site was not clear, although the authors put forward a simple charge argument and showed that substitution of glutamine for lysine 185 markedly reduced the sensitivity to ATP (IC₅₀ > 1 mM). An extensive series of site-directed mutations did not define a binding site but did indicate that substitution at positions near the mouth of the pore and in the N terminus would also reduce the sensitivity to ATP. The interpretation of the site-directed mutation experiments has been confounded by the realization that a given substitution can alter both the apparent affinity for ATP and the open channel probability, making it difficult to determine whether a particular residue is part of a binding site or part of a link between a binding site and the channel gate. The results with C-terminally truncated channels are important and provide direct evidence that K_IR6.2 forms the K_ATP channel pore and that ATP can directly affect its activity.

Shyng et al. (238) have proposed a model in which SUR1 acts to "sensitize" the K_IR6.2 channel to inhibition by ATP, a process akin to the enhancement described by Tucker et al. (51). Shyng et al. (238) propose that SUR1 can be switched into a state, designated the "off" state, which cannot sensitize K_IR6.2. ATP hydrolysis, at one or both NBFs, switches SUR to the off state, thus blocking sensitization, while MgADP and potassium channel openers, like diazoxide, are argued to stabilize the off state and block sensitization. Mutations in the NBFs that prevent or slow hydrolysis are expected to reduce entry into the off state, thus maintaining these channels in a sensitized state. Although other interpretations are possible, in the simplest incarnation of this model, the function of SUR1 is to control the affinity of the primary inhibitory ATP-binding site on K_IR6.2, with higher affinity for ATP equating to a reduced open probability, Po, of a single channel.

C. C-terminally truncated K_IR6.2 channels show abnormal kinetics
While the single-channel conductance of the homomeric K_IR6.2C channels was equivalent to that of the SUR/K_IR6.2 channels, analysis of their single-channel kinetics revealed substantial differences in basic gating properties (240). As shown in Fig. 1, SUR1/K_IR6.2 channels exhibit bursting behavior with a burst consisting of a number of fast openings defined by brief closures. Bursting in the K_IR6.2C channels is markedly attenuated with less than two openings in an average burst (see Fig. 8). The average lifetime of these fast openings is also reduced by about 3-fold. The K_IR6.2C channels spend most of their time in long closed states analogous to the interburst intervals seen in SUR1/K_IR6.2 channels. The result is a marked decrease in the maximal open probability. Under equivalent conditions, we estimate the maximal Po of the K_IR6.2C channels to be 0.09 vs. 0.69 for SUR1/K_IR6.2 channels.

View larger version (30K): [in this window] [in a new window]   Figure 8. Comparison of wild-type and truncated KATP channels. Single-channel records from the three types of channels illustrated in Fig. 6 are shown. The (KIR6.2C35)4 channels show abnormal bursting and have a markedly reduced probability of being in an open state. The maximal open probability, Po, estimated from the all-points histogram is 0.09. The behavior of the wild-type channel is shown in the middle record. Coexpression of KIR6.2C35 with SUR1 restores normal bursting, increasing the maximal Po. The solution conditions were as follows: internal solution (millimolarM concentration) KCl, 140; MgCl2, 1; EGTA, 5; HEPES, 5; KOH, 10; pH 7.2 (adjusted with KOH); external solution: NaCl, 140; KCl, 5; MgCl2, 1; CaCl2, 1; HEPES, 10; pH 7.4 (adjusted with NaOH). The holding potential was 0 mV. The horizontal dotted lines give the zero-current level; the upward deflections correspond to outwardly directed currents.

D. Coexpression of K_IR6.2C subunits with SUR restores normal K_ATP channel activity

E. Why are K_IR6.2 channels silent?
The initial reconstitution studies failed to detect K⁺ channels when K_IR6.2 was expressed without SUR1 (26), a result that has been confirmed by others. The reasons for this silence or latency were unclear, but recent results suggest a fascinating story, the outlines of which are just becoming clear. The finding that K_IR6.2C subunits could form K⁺ channels, albeit with abnormal properties, provided a means to measure the effects of coexpression with SUR1 on trafficking. Babenko et al. (240) showed that coexpression with SUR1 increased the transit of K_IR6.2C subunits to the plasma membrane by about 8-fold. John et al. (241) showed that very strong overexpression of full-length K_IR6.2, or K_IR6.2 tagged with GFP, the green fluorescent protein, in HEK cells produced K⁺ channels with properties similar to the C-terminally truncated K_IR6.2 channels. This was the first demonstration that full-length K_IR6.2 could generate channels and effectively ruled out the possibility that the C terminus blocked channel activity. The results suggested that the C terminus affected trafficking in some unspecified fashion. Schwappach et al. (242) have shown that the C-terminal sequence of K_IR6.2 (and K_IR6.1) has an endoplasmic reticulum (ER) retention signal that keeps full-length K_IR6.2 from reaching the plasma membrane under normal conditions. Using a quantitative assay to measure surface expression of K_IR6.2, Schwappach et al. (242) could demonstrate that coexpression with SUR1 increased transit approximately 500-fold. Transfer of the K_IR6.2 C-terminal sequence to other membrane proteins caused their retention in the ER. Truncation of the C terminus of K_IR6.2 thus allows its limited expression at the plasma membrane, yielding the results seen by Tucker et al. (51). The most likely explanation for the observations of John et al. (241) is that the retention mechanism can be overcome when levels of expression are sufficiently high. How SUR interacts with K_IR to abrogate the retention signal promises to be an interesting story, but it is not at all clear why nature has gone to such lengths to control surface expression of this inward rectifier.

F. The N terminus of K_IR6.2 limits burst duration
The C terminus of K_IR6.2 is not the only interesting cytoplasmic domain. Truncation of the N terminus of K_IR6.2 has proven to be equally informative. Deletion of 30–40 amino acids does not affect surface expression but has a profound effect on bursting activity (240). SUR1/N32K_IR6.2 channels, for example, burst continuously with few interburst intervals. The maximal open probability, Po 0.95, is the value expected from a channel displaying only the fast closures observed within a normal burst. We have interpreted these results to imply that the N terminus of K_IR6.2 limits the time the channel spends in a bursting state and speculate that SUR affects bursting through its interaction with the N terminus.

G. Where do the openers bind and how do they work?
A diverse group of compounds, referred to as potassium channel openers or KCOs, are able to open K_ATP channels and thus hyperpolarize cells. Clinically, these compounds have been used for the treatment of hyperinsulinemic states and as smooth muscle relaxants for hypertension. The literature in this area is voluminous, and a complete review is beyond the scope of this article. Reviews may be found in Refs. 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253 . This area has been hampered by the lack of means to study specific K_ATP channel isoforms, but this problem should now be more accessible by reconstitution of the SUR2A and SUR2B receptors with both K_IR6.1 and K_IR6.2 (23, 24, 254), as described above.

Electrophysiological studies indicate that activation of K_ATP channels in excised patches requires hydrolysable nucleotides and Mg²⁺ (238, 255, 256, 257, 258, 259, 260, 261, 262, 263), consistent with a requirement for ATP hydrolysis. Several of these studies point out that ADP in the presence of ATP or ADP alone will potentiate the effect of KCOs. The latter observations are consistent with the presence of adenylate kinase and conversion of ADP to ATP; thus it is not clear whether ADP and KCOs can act synergistically. Several reports have argued that the stimulatory effect of KCOs is through direct competition with ATP (257, 264, 265), although this is not firmly established.

The target site for potassium channel openers is assumed to be the sulfonylurea receptor, based on observations that the response of reconstituted K_ATP channels to either diazoxide, cromakalim, or pinacidil is correlated with the SUR subtype (23, 24). A comparison of the pharmacology of the SUR2A/K_IR6.2 (23) and SUR2B/K_IR6.2 (24) channels indicates the C-terminal end of SUR is a critical determinant for KCO response and selectivity. The SUR1 and SUR2B C termini, for example, are similar, and channels formed from these receptors appear to share responsiveness to diazoxide, while the SUR2A/K_IR6.2 channel shows essentially no response to diazoxide.

Attempts to develop membrane-binding assays for channel openers from responsive tissues have been largely unsuccessful, although in some cases tissue and whole-cell binding has been detected. The most successful efforts have employed [³H]P1075, a pinacidil analog that activates smooth muscle K_ATP channels at concentrations of 20–30 nM and channels from isolated rat aortic rings and myocytes (248, 266). This line of research has recently shown that ATP is required for [³H]P1075 binding to isolated cardiac and skeletal muscle membranes (267, 268). Biochemical studies on membranes isolated from cells transfected with SUR2A and B (269, 270) suggest two plausible reasons for the earlier difficulties: low receptor concentration and a requirement for Mg²⁺ and hydrolysable nucleotides. Schwanstecher et al. (269) show that SUR2B has a K_D of approximately 25 nM for P1075, while binding of this compound to SUR1 and SUR2A is low. Binding requires MgATP; nonhydrolysable ATP analogs will not support [³H]P1075 binding. Consistent with the electrophysiological data (189, 190, 238), binding of the opener is impaired by mutations in the NBFs. The suggestion is that nucleotide binding and hydrolysis by SUR are important for the binding of P1075. The results appear to argue against direct competition between ATP and KCOs. The availability of binding assays opens the way for the experiments with different KCOs and for the structure-function studies with the SUR subtypes needed to establish the number and location of KCO-binding sites on K_ATP channels and to correlate binding with channel subtype responses.

H. Do SURs have ATPase activity?
Although there is no direct biochemical evidence that SURs are ATPases, the indirect evidence is reasonably strong. First, the similarity of the NBFs of SURs to those of other members of the ATP-binding cassette superfamily suggests they will hydrolyze ATP. Second, as discussed, several aspects of channel function require Mg²⁺ and hydrolysable nucleotides, particularly refreshment and the regulation of channel activity by MgADP and potassium channel openers. As indicated above, refreshment is not well understood and may require additional kinases or cytoskeletal elements that require ATP. MgADP and KCOs, on the other hand, interact with SUR, and their effects show a strong requirement for divalent cations such as Mg²⁺ or Mn²⁺, and for the presence of a hydrolysable nucleotide. It is not clear, in most cases, whether ATP is required or other nucleotides will substitute. For example, MgADP has been observed to substitute for ATP and support the action of diazoxide, while ,ß-methylene ADP does not (263). This could be interpreted as a requirement for ADP hydrolysis, but probably reflects the generation of ATP by adenylate kinase. This is an area that needs to be developed and should provide insight into the regulation of these channels.

I. Do SURs have transport activity?
Membership in the ATP-binding superfamily suggests SURs may have an endogenous transport function. To our knowledge, the possibility that SURs are involved in transport has not been tested in any systematic fashion. One possibility, based on suggestions from the CFTR field, is that SUR might transport nucleotides (205). While this has not been disproved rigorously, there is no direct evidence in support of nucleotide transport at this time.

J. Is there an endogenous substrate?
A frequently raised question concerns the nature of the putative "endogenous" ligand, which sulfonylureas may mimic or replace. Virsolvy-Vergine et al. (271) have addressed this question directly using competition assays in which peptide fractions isolated from brain were tested for their ability to displace [³H]glibenclamide from either brain or ß-cell membranes. This work led to the isolation of two peptides, - and ß-endosulfines (272, 273). Both molecules were reported to interact with the high-affinity receptor, and ß-endosulfine was reported to stimulate insulin release from a ß-cell line. -Endosulfine, a 77-amino acid protein (M_r = 13,196), has been cloned and is similar to two phosphoproteins, ARPP-16 (96 amino acids) and ARPP-19 (112 amino acids), M_r 16,000 and 19,000, respectively. The sequence similarity is striking, with 65 identical amino acids over the 77 residues of -endosulfine. ARPP-16 and -19 are believed to be splice variants of a single gene since their amino acid sequences are identical except for an additional 16 amino acids at the N terminus of ARPP-19 (274). ARPP-16 is present mainly in dopamine-innervated regions of the brain, while ARPP-19 is more widely distributed being present in all tissues in the adult rat (275). The phosphorylation of both ARPP-16 and -19 is regulated by cAMP and by vaso- active intestinal peptide in cultured striatal cells (276). -Endosulfine is believed to be the product of a separate gene and has a wide tissue distribution (277). Expression of recombinant -endosulfine in a bacteria produces a protein with an apparent molecular mass of 23,000 Da, which competes with [³H]glibenclamide for binding to SUR1 (K_i 1 µM), inhibits SUR1/K_IR6.2 channels expressed in Xenopus oocytes (EC₅₀ 1 µM) and stimulates insulin release from MIN6 cells in the same concentration range. Heron et al. (277) suggest that -endosulfine may act as an endogenous regulator of K_ATP channels in ß-cells.

	VIII. Human SUR1 and KIR6.2 Genes

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
The human SUR1 and K_IR6.2 genes have been mapped to the short arm of chromosome 11 (11p15.1). The SUR1 gene has 39 exons spanning approximately 100 kb of genomic DNA. The average exon size is 124 bp with a range of 33–243 bp; the known intron sizes range from 100 bp to greater than 6.5 kb. Figure 9 provides an overview and superimposes the major structural features of the protein on the human gene, as well as summarizing the known SUR1 mutations associated with familial hyperinsulinism. We have reviewed the structure of the human SUR1-K_IR6.2 gene region elsewhere along with a comparison of the human SUR1 and SUR2 genes (19). There is new sequence available that will be of interest to those screening for disorders associated with the SUR1-K_IR6.2 complex. Evans et al. have put an 86-kb sequence into the Genbank database (Accession no. U90583). The sequence starts in a large intron (>6.5 kb) between SUR1 exon 16 and 17 and spans exons 17–39. K_IR6.2 is downstream of the SUR1 gene (4900 bp between the stop codon of SUR1 and the start codon of K_IR6.2). The entire SUR1-K_IR6.2 region plus a considerable amount of flanking sequence is available with the sequence of a pac clone, pDJ239b22, in the Genbank database (Accession no. U90583). The entire coding region including both genes is contained within a segment that spans almost 90 kb of DNA. The promoter sequences in the 5'-untranslated regions of the SUR1 and K_IR6.2 genes have begun to be explored, which should lead to understanding of how this family of channel proteins is regulated (278).

View larger version (22K): [in this window] [in a new window]   Figure 9. Representation of the human SUR1 gene. An illustration of SUR1 is depicted in the top line. The shaded boxes position the 17 predicted TMDs, the open boxes, identified with A and B to mark the Walker A and B motifs, position the two NBFs. The 39 exons of the SUR1 gene are shown below the protein. The exon boundaries are projected onto the protein. The approximate positions of known mutations in SUR1 that have been identified in patients with PHHI are given. The intron mutations are identified by intron number for clarity, and intron base changes are lowercase. Exon base changes are given as uppercase and are designated by the use of "nt."

	IX. KATP Channels and Persistent Hyperinsulinemic Hypoglycemia of Infancy (PHHI)

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

The diagnosis of hyperinsulinism, considered here as an inappropriately high insulin level, low blood ketone levels, and low free fatty acid levels at the time of hypoglycemia, can result from several genetic alterations. At the suggestion of Dr. Franz Matschinsky, we will name the familial hyperinsulinemic disorders for which a responsible gene has been identified, first by designating them as hyperinsulinemic and second by designating the affected molecule. For example, hyperinsulinism associated with a mutation in the sulfonylurea receptor would be designated "HI-SUR1." As we are reviewing K_ATP channels, we will focus mainly on mutations in channel subunits that give rise to hyperinsulinism, but information on the causes of persisting hypoglycemia in newborns is widening and teaching us much about the heterogeneity of this disease. It is clear that K_ATP channel defects are not the sole cause. Additional mutations in other pathways have been identified as discussed briefly below, and focal and diffuse histopathological forms of the disease have been identified and partially related to K_ATP channel defects. It has also become clear that nesidioblastosis is not the pathognomonic histopathological lesion for PHHI.

A. HI-glucokinase (GK)
GK, or hexokinase IV, found in ß-cells is responsible for conversion of glucose to glucose-6-phosphate and is generally considered to be the ß-cell "glucose sensor" (216). Mutations in GK have been identified as one cause of maturity onset diabetes of the young (MODY 2), where increases in the K_m of GK are correlated with an increase in the concentration of glucose needed for insulin release. Glaser et al. (279) described an informative mutation in GK which lowers the K_m and results in hypoglycemia. The mutation, valmet, V408 M, causes a rare, mild dominant form of PHHI, which responds clinically to diazoxide. No homozygotes have been identified, suggesting that two copies of the allele may be lethal.

B. HI-GlnDH
Zammarchi et al. (280) and Weinzimer et al. (281) described a mild, dominant form of hyperinsulinism associated with hyperammonemia. Several patients from unrelated families, who responded to diazoxide but were poorly responsive to octreotide, have been identified. This form of hyperinsulinism appears to be the result of excessive oxidation of glutamate via glutamine dehydrogenase. Enzyme studies indicate that the glutamine dehydrogenase in two unrelated patients with this disorder was less sensitive to GTP, a negative allosteric effector, as a result of mutations in the C terminus of the enzyme (282).

C. HI-"unknown"
Kukuvitis et al. (283) reported on PHHI in a French- Canadian kindred. Mutations in K_ATP channel subunits and GK were ruled out as potential causes. These patients responded to diazoxide, confirming they have functional K_ATP channels. Familial clustering suggested that this may be another mild dominant form of PHHI, but the susceptibility locus has not been identified. In several populations studied, approximately 50% of the cases fall within this unknown category.

D. HI-K_IR6.2
Three mutations that produce persisting hypoglycemia due to hyperinsulinism have been identified in K_IR6.2 (Fig. 5). Although as described below, mutations in SUR1 were the first to be associated with hyperinsulinism and are more frequent, mutations in K_IR6.2 produce the same phenotype and provide a genetic confirmation of the biochemical and electrophysiological data that K_IR6.2 is the partner for SUR1 in ß-cells.

Thomas et al. (284) reported on a K_IR6.2 mutation, a leupro change, L147P, near the external side of M2, which demonstrated that mutations in the inward rectifier led to PHHI. This mutation was identified in a child of Iranian origin, the progeny of a marriage of first cousins. Diagnosis of PHHI was based on an insulin level of more than 30 mU/ml with a glucose level less than 30 mg/dl and a requirement of more than 15 mg glucose kg/min to maintain euglycemia. The substitution of a proline in M2 has a large effect on K_ATP channel activity; when engineered and expressed with wild-type SUR1, K_IR6.2_L147P does not produce K_ATP channels and does not co-photolabel with [¹²⁵I]iodoazidoglibenclamide (J. Bryan, unpublished data). It is not clear whether the protein is folded correctly and/or fails to associate with SUR1.

Nestorowicz et al. (285) described a nonsense mutation in K_IR6.2 that truncates the protein after 12 amino acids, Y12X. The patient was homozygous for this mutation. When Y12X was engineered into K_IR6.2, as expected for a 12-residue peptide that is missing all of the elements required to form the K⁺ channel pore, it did not form a functional channel when coexpressed with wild-type SUR1.

Sharma and Aguilar-Bryan (unpublished data) identified a third K_IR6.2 mutant, a trparg change, W91R, near the external side of M1. This mutation was identified in a newborn, the product of a marriage of first cousins of Palestinian descent. Clinical treatment with diazoxide and somatostatin was not successful; a partial pancreatectomy was performed when the patient was 2 weeks of age, and a second resection was necessary 4 weeks later. When coexpressed with wild-type SUR1, K_IR6.2_W91R failed to produce active channels.

E. HI-SUR1
A number of studies indicated that, in most instances, PHHI was an autosomal recessive disorder (summarized in Ref. 20). Two groups (286, 287) independently mapped the major susceptibility gene to the short arm of chromosome 11 at 11p14–15.1 using multiplex families. In situ hybridization mapped the SUR1 gene to the same region (288). It was apparent that loss of K_ATP channel activity in pancreatic ß-cells might be expected to give the PHHI phenotype. As described above, K_ATP channels set the ß-cell resting membrane potential below the threshold for activation of voltage-gated Ca²⁺ channels. In the absence of some compensatory mechanism, the loss of ß-cell K_ATP channel activity would lead to constitutive membrane depolarization, spontaneous voltage-gated Ca²⁺ channel activity, and a sustained increase in intracellular Ca²⁺ levels. The loss of K⁺ channel activity would therefore effectively uncouple membrane electrical activity from metabolism and would result in persistent insulin release, regardless of the blood glucose level (18, 289).

The discovery of two separate splice site mutations that segregated with the disease phenotype in affected children from nine different families provided the first evidence that SUR1 was a PHHI susceptibility gene (288). The first two mutations discovered were in intron 32 and exon 35, respectively. [Note, in the Permutt review article (20), the authors have used an early numbering of the SUR1 exons where the 3'-most exon is defined as exon 1. The human SUR1 sequence(s) in Genbank begin with exon 1 at the N terminus; we will use this numbering convention throughout.] The intron 32 allele is a ga mutation identified in 1 PHHI affected children from consanguineous mating. This mutation eliminates an NciI restriction endonuclease recognition site; homozygous loss of this site cosegregated with the disorder within the two families. The ga mutation was at a position -9 from the 3'-end of intron 32 (Fig. 9). The resulting sequence, -gcc cag ccc cagCAC- caused usage of three cryptic splice sites within exon 33 in place of the normal wild-type splicing. The "alternative" splicing was expected to produce transcripts encoding truncated receptors of three different sizes missing NBF2. The exon 35 allele is also a GA mutation in the last position of the exon (Fig. 9). This change eliminates an MspI site; homozygous loss of this site was observed to cosegregate with the phenotype in 8 families and was missing in 12 affected children from 6 families of Saudi Arabian and 1 of German origin. The GA mutation in the last position of the exon weakens the splice site and results in the skipping of exon 35. The exon deletion causes a frame shift that results in a premature stop 24 codons beyond the end of exon 34, again producing a transcript that encodes a truncated receptor missing most of NBF2. RT-PCR was used to clone a pancreatic cDNA product missing the 109-bp segment, showing that the altered transcript was expressed.

A screen for SUR1 mutations in 25 probands from Ashkenazi Jewish families affected with PHHI uncovered an additional mutation, a deletion of codon 1388 resulting in a loss of a phenylalanine residue, F1388 (290). In some patients this mutation was present in the homozygous state and in others it was present as a compound heterozygote with known SUR1 mutations or mutations yet to be identified.

Thomas et al. (291) reported on three additional mutations in the region of NBF1. Two of these mutations are expected to lead to severe truncations of SUR1 in or near NBF1, while the third mutation results in a glyval substitution at position 716, G716V, in the Walker A motif.

Nichols et al. (189) reported on a missense mutation, G1479R, in the second NBF, which is informative about the mechanism of nucleotide regulation as discussed below. This mutation was identified by Dr. Ann Nestorowicz in Dr. Alan Permutt’s laboratory in a patient of Iraqi and Moroccan Jewish extraction (20) who was diagnosed with PHHI by Drs. Heddy Landau and Benjamin Glaser (Hebrew University, Hadassah Medical Center, Jerusalem). The patient is a compound heterozygote, with the G1479R allele on one chromosome and a second, unidentified allele on the other. As described above, when the G1479R mutation was engineered into hamster SUR1 and expressed with wild-type K_IR6.2, the resulting channel displayed a novel phenotype with normal inhibition by ATP, but reduced stimulation by MgADP.

A number of new mutations have been identified in patients with PHHI from different ethnic groups (Ref. 292) and N. Sharma and L. Aguilar-Bryan, unpublished data). We have summarized as many of these mutations as are available in Fig. 9. Some of these mutations have been engineered into the hamster or human SUR1 and examined for K_ATP channel activity after coexpression with wild-type K_IR6.2 (Ref. 292 and L. Aguilar-Bryan, N. Sharma, A. Crane, G. Gonzalez, and J. Bryan, unpublished data). A number of these mutations are associated with severe forms of the disease and show no K_ATP channel activity in the reconstitution assays. This includes a failure to show increased sulfonylurea-sensitive ⁸⁶Rb⁺ efflux upon metabolic poisoning. Several of these mutations behave like the G1479R mutation and retain some sensitivity to diazoxide. With the exception of the severe truncations and the G716V mutation, the SUR1 mutants retain high-affinity sulfonylurea binding activity, suggesting their folding is not completely aberrant. Detailed examination of these mutations should provide further insight into the regulation of K_ATP channels.

	X. Linking PHHI to Defects in KATP Channel Activity

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
A. ß-Cells from newborns diagnosed with "sporadic" PHHI lack K_ATP channel activity
Kane et al. (293) studied islet cells derived from partial pancreatectomy specimens from five patients (four of Caucasian and one of Kuwaiti origin) diagnosed with sporadic PHHI, differentiated here from familial hyperinsulinism, because there is a single affected sibling in the family and no knowledge of consanguinity. K_ATP channels could not be detected in primary cultured islet cells, although they were present in normoglycemic human tissue from unaffected individuals (1 infant, 11 months of age, and 12 adults, median age 45 yr) treated similarly. The control islet cells were electrically silent, while the patient’s islet cells exhibited spontaneous openings of voltage-gated Ca²⁺ channels that resulted in an estimated increase in [Ca²⁺]_i of about 2-fold (88 nM to 177 nM). The results confirmed the hypothesis put forward earlier that loss of K_ATP channel activity should produce the PHHI phenotype (18). The missing link in this study was a direct connection between a mutation(s) in one of the K_ATP channel subunits and the loss of channel activity. These five patients had no parental consanguinity, no other affected siblings, and a preliminary, but not exhaustive, screen for SUR1 and K_IR6.2 mutations was negative (L. Aguilar-Bryan, unpublished data). Thus, although the physiology was compelling, one could argue that the genetic defect lay elsewhere in these patients.

B. PHHI ß-cells with the SUR1 exon 35 mutation lack K_ATP channel activity
A direct demonstration that a known SUR1 mutation results in the loss of K_ATP channel activity in patient ß-cells was made for the exon 35 mutation. Dunne et al. (294) analyzed islet cells obtained by pancreatectomy from one newborn diagnosed with PHHI. Analysis of patient DNA indicated she was homozygous for the exon 35 GA mutation. She was not responsive to diazoxide or to nifedipine and required continuous infusion of glucose (18 mg/kg/min) to remain euglycemic. Subtotal (95%) pancreatectomy was insufficient to reduce her insulin levels, and removal of 99% of the pancreas was necessary to control her hypoglycemia. Electrical recording from ß-cells isolated from the surgical specimens demonstrated K_ATP channel activity was absent, but voltage-gated Ca²⁺ channels were spontaneously active. As in the five infants studied previously (293), ß-cell cytosolic Ca²⁺ levels were elevated beyond control cell values (83 vs. 115 nM) as determined by fluorescence measurements. A parallel mutation engineered into hamster SUR1 did not generate K_ATP channel activity when cotransfected into COS cells with wild-type K_IR6.2. Photolabeling studies on the parallel mutation indicate that a truncated receptor is produced and retains high-affinity sulfonylurea binding activity. Preliminary results indicate that the truncated receptor is able to associate with and co-photolabel K_IR6.2. These studies have been repeated with ß-cells from a second newborn homozygous for the same allele, with essentially the same results (M. Dunne, K. Lindley, A. Aynsley-Green, and L. Aguilar-Bryan, unpublished data). In addition, four different mutations have been identified in patients previously diagnosed with sporadic PHHI whose ß-cells were shown to lack K_ATP channel activity. The overwhelming conclusion is that mutations in SUR1 result in the loss of K_ATP channel activity that leads to PHHI. This indicates that K_ATP channels are the predominant mechanism that human ß-cells use to set their resting membrane potential; there is no evidence for redundancy of SUR1, K_IR6.2, or K_ATP channel functions. This conclusion emphasizes the key link that K_ATP channels form in the closed negative feedback loop that regulates insulin release and validates our initial hypothesis that this recessive familial form of PHHI is a potassium channel disease (18).

C. Why is there a lack of dominant negative mutations?
Although analysis of PHHI mutations in SUR1 and K_IR6.2 is at an early stage, given the multimeric structure of K_ATP channels it is not clear why dominant negative mutations in either subunit have not been discovered. It is not clear, for example, whether the truncated SUR1 subunits produced by the exon 35 mutation can assemble a tetrameric channel. The exon 35 mutation, and other identified mutations, lead to severe hypoglycemia only in the homozygous condition. The heterozygous parents are apparently normoglycemic and asymptomatic. Thus, dominant negative effects are either small or the fraction of normal channels produced in the heterozygotes is sufficient to maintain the ß-cell resting membrane potential. If assembly of a single-mutant receptor into the tetramer is sufficient to block channel activity, we expect only 6.25% of the K_ATP channels to be functional in the heterozygotes if the assembly of channel subunits is random. One possible explanation for the failure to identify dominant negative mutations could lie in the spare channel hypothesis (230), if 6–7% of the K_ATP channels in a ß-cell are sufficient for adequate regulation. Additional studies on the heterozygous parents could prove informative on this issue.

D. Development of mouse models
Miki et al. (295) have shown that a dominant negative effect can be observed by overexpression of a mutant K_IR6.2 subunit. Transgenic mice expressing K_IR6.2 carrying the "weaver" mutation, G132S, have low blood glucose at birth as a result of unregulated insulin secretion. Four weeks after birth these animals develop hyperglycemia, with reduced insulin secretion as a result of ß-cell destruction. Reconstitution experiments indicate K_IR6.2_G132S can compete with wild-type K_IR6.2 and suppress K_ATP channel activity. Interestingly the weaver mutation in K_IR3.2 (GIRK2) shows altered cation selectivity and leads to neuronal cell death (296, 297), suggesting this mutation in K_IR6.2 could have similar consequences.

Knockout mice missing both K_IR6.2 and SUR1 are being generated and should prove to be interesting and informative regarding K_ATP channel function in the pancreas and other tissues.

	XI. Other Issues

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
A. Nesidioblastosis does not cause PHHI
Since the initial description of nesidioblastosis, a term coined by Laidlaw (298) to refer to a persistent, diffuse, disseminated endocrine cell budding from pancreatic ducts, many authors have suggested that this lesion is the specific histopathological finding in the pancreas of hypoglycemic neonates. Yackovac et al. (299) described nesidioblastosis in the pancreas of newborns with persisting hypoglycemia and made the initial suggestion that the increased ß-cell mass might produce the insulin increase and intractable hypoglycemia (300, 301). Heitz et al. (302) analyzed pancreatic tissue from patients with PHHI using immunocytochemistry and electron microscopy. They reported a 5-fold increase in the mean area occupied by endocrine tissue relative to unaffected control tissue. The ratio of ß-cells to endocrine area was equivalent to that of controls.

More detailed comparisons of pancreatic tissue from patients with PHHI and normal individuals has failed to confirm this initial suggestion. Gould et al. (303, 304) reported an increase in total endocrine cell volume in PHHI patients but concluded the "typical features of nesidioblastosis" were also present in normal age-matched controls. They introduced the term "nesidiodysplasia" to define the abnormal growth, distribution, and regulation of endocrine cells when present in association with endocrine abnormality. Rahier et al. (305, 306) reached a similar conclusion after comparing 15 hypoglycemic infants diagnosed with PHHI to 23 normoglycemic controls; "nesidioblastosis was not a specific feature of the pancreas in infantile hypoglycemia, being observed in age-matched controls as well." Nesidioblastosis has also been identified in other pathologies such as multiple endocrine neoplasia, cystic fibrosis, and pancreatitis, without hypoglycemia; therefore it is clear at this point, that nesidioblastosis is not the pathognomonic lesion of PHHI (307).

B. "Diffuse" vs. "focal" forms of PHHI
In addition to the heterogeneous genetic causes of PHHI, at least two histopathological forms of hyperinsulinism have been distinguished by morphology (308, 309): a focal type, which shows nodular or adenomatous hyperplasia of a particular area within the normal pancreas, and a diffuse type, which involves the entire pancreas and is characterized by irregularly sized islets and ducto-insular complexes, both with hypertrophied ß-cells with larger nuclei. The two forms of PHHI have different times of onset and degree of severity (310); the diffuse form is more severe, develops within the first 48 h after birth, and requires a large resection or total pancreatomy to control hypoglycemia, while the focal form is less severe and is identified in patients that develop hypoglycemia several weeks after birth. The focal form can be controlled by resection of the lesion (310, 311). Sempoux et al. (310) analyzed the pancreata from 25 PHHI infants with diffuse or focal forms and evaluated ß-cell hyperactivity by determining the size of the nuclei and cytoplasm. Infants diagnosed with the diffuse form of hyperinsulinemia had larger ß-cell nuclear radii than infants diagnosed with the focal form. In addition, the cytoplasmic volume was greater in the diffuse form. These two parameters allowed discrimination between the two different forms of the disease.

Infants with PHHI who have had a 95% pancreatectomy frequently develop diabetes mellitus; therefore, distinguishing between the focal and diffuse forms of the disorder is of importance (see for example Refs. 312, 313 for discussion). Toward this end, two methods have been recently employed. Dubois et al. (314) have used pancreatic venous sampling (315, 316, 317), a technique in which catheters are used to sample blood from different areas of the pancreas and "map" local insulin release, to differentiate diffuse vs. focal forms of PHHI at time of surgery. Similarly, the analysis of small specimens from the head, isthmus, body, and tail by means of frozen sections has been used to differentiate between the focal and diffuse forms. Rahier et al. (318) employed this approach and used nuclear size to discriminate the two forms in a study of 20 infants diagnosed with PHHI in the first few hours after birth who were nonresponsive to diazoxide. These methods show promise as a means for discriminating between the focal and diffuse forms of PHHI and minimizing the extent of surgery required to control hypoglycemia (319, 320).

Is there a relationship between the genetic causes of PHHI and the two morphological forms? A recent report by de Lonlay et al. (321) suggests this possibility in some instances. These authors reported on 16 cases of "sporadic" PHHI, 10 identified with the focal form, and 6 with the diffuse form. Analysis of DNA isolated from the foci of identified focal PHHI suggested genomic imprinting, with a specific loss of maternal alleles from the chromosome 11p15.1 region where the SUR1 and K_IR6.2 genes map. A similar loss was not observed in DNA taken from cells outside the foci or in samples taken from the pancreata of patients diagnosed with the diffuse form of PHHI. de Lonlay et al. (321) show that a somatic event resulting in deletion of the maternal alleles has occurred and that a clonal expansion of the cells carrying the deletion gives rise to a focus of ß-cells missing K_ATP channels as a result of a mutation in SUR1 on the paternal chromosome. The development of this area should prove interesting.

	XII. KATP and NIDDM

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
It was of interest to determine whether mutations in SUR1 were associated with other disorders of glucose homeostasis, particularly non-insulin-dependent diabetes, NIDDM. The evidence for a connection is mixed. Hani et al. (322) and Inoue et al. (323) have suggested that the SUR1 locus may contribute to the genetic susceptibility of Caucasians to NIDDM, while Iwasaki et al. (324) and Stirling et al. (325) have concluded that this locus does not make a major contribution to this susceptibility in Japanese and Mexican-American populations, respectively. Sakura et al. (326) have described sequence variations in K_IR6.2, but failed to find linkage with NIDDM.

A. ß-Cell type K_ATP channels in the brain
Several early binding studies established the presence of high-affinity glibenclamide receptors, presumably SUR1, in the brain (148, 158, 160, 327, 328, 329), and one of the earliest reports of purification of the high-affinity receptor was from porcine brain (161). Autoradiography with ³H- and ¹²⁵I- labeled glibenclamide have been used to localize receptors to regions of the brain (330, 331, 332, 333, 334). These studies localize SUR1 in various areas of the brain. Mourre et al. (330), using [³H] glibenclamide, indicate that the density of receptors is "particularly important in the substantia nigra reticulata, the septohippocampal nucleus, the globus pallidus, the neocortex, the molecular layer of the cerebellum, and the CA3 field and dentate gyrus of the hippocampus. They note that the "hypothalamic areas, medulla oblongata and spinal cord" contained lower amounts of glibenclamide receptors. Treherne and Ashford (331), also using [³H]glibenclamide, note the "highest levels of glibenclamide binding were found in the substantia nigra with high levels in the globus pallidus, cerebral cortex, hippocampus and caudate-putamen, intermediate levels in the cerebellum, and low levels in the hypothalamus and pons." These authors mention that only low levels of binding were observed in glucose-responsive regions of the brain known to respond to sulfonylureas. Gehlert et al. (334), using [¹²⁵I]iodoglibenclamide, report the "highest levels of binding were seen in the globus pallidus and ventral pallidum followed by the septohippocampal nucleus, anterior pituitary, the CA2 and CA3 region of the hippocampus, ventral pallidum, the molecular layer of the cerebellum and substantia nigra zona reticulata. The hilus and dorsal subiculum of the hippocampus, molecular layer of the dentate gyrus, cerebral cortex, lateral olfactory tract nucleus, olfactory tubercle and the zona incerta contained relatively high levels of binding. A lower level of binding (approximately 3- to 4-fold) was found throughout the remainder of the brain."

In situ hybridization has been used to localize SUR1 and K_IR6.2 mRNAs in rodent brain (335). There was extensive overlap of the two signals with each other and extensive overlap with the earlier studies using radiolabeled glibenclamide. Figure 10 gives an example of the localization of SUR1 by in situ hybridization in adult rat brain.

View larger version (134K): [in this window] [in a new window]   Figure 10. Localization of SUR1 mRNA in rat brain. The localization was done after Largent et al. (374 ) using three 45-mer oligonucleotide probes based on the rat SUR1 sequence (22 ). The highest levels of expression were seen in the CA-2 and dentate gyrus (DG) of the hippocampus, and in the granular cell layer (gr) of the cerebellum (CB). Lower levels are in the pontine nucleus and medial habenular nucleus. Light staining is evident throughout the cortex (CTX) as well as the thalamus and inferior colliculus. [This work was done by Dr. Noam Cohen in Dr. Solomon Snyder’s laboratory at the Johns Hopkins University; the authors are indebted to Dr. Cohen for his help.]

	XIII. The Leptin Connection

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
One of the more interesting and speculative areas of research relating the functions of K_ATP channels in the brain and in ß-cells involves activation of channel activity by leptin, the product of the mouse obesity or ob gene. Leptin, a 16-kDa protein, is produced by adipocytes and is believed to regulate body weight, food intake, and energy expenditure through activation of the leptin receptor in hypothalamic neurons and peripheral tissues (see for example Refs. 364, 365). Ob/ob mice develop a profound obesity with hyperglycemia and hyperinsulinemia. Leptin treatment of ob/ob mice improves their glycemia and insulinemia and causes a reduction in food intake along with increased physical activity and thermogenesis. Leptin has been shown to activate a potassium conductance sensitive to the sulfonylurea, tolbutamide, in glucose-responsive hypothalamic neurons (198). Activation of these channels hyperpolarized the neurons inhibiting the firing of action potentials. The electrophysiological properties of these channels suggest they are distinct from the ß-cell/neuronal channels described here as they are markedly less sensitive to inhibitory ATP, their slope conductance in isolated patches is about twice that observed with ß-cell channel, and tolbutamide fails to block channels in isolated patches.

The development of hyperinsulinemia as a result of a leptin deficiency or loss of the leptin receptor in db/db mice suggested leptin might suppress insulin secretion under normal conditions (366, 367), and the long form of the leptin receptor is found in islets and ß-cell lines (368). A study on the isolated, perfused rat pancreas failed to show an effect of leptin on glucose-stimulated insulin secretion (369, 370), while leptin is reported to suppress insulin release from isolated ß-cells (197, 371) and from the perfused pancreas of ob/ob mice (371). This effect appears to involve K_ATP channels, as Kieffer et al. (197) and Harvey et al. (372) have reported that leptin activates a sulfonylurea-suppressible K⁺ conductance in the insulinoma ßTC3 and CRI-G1 insulin-secreting cell lines, respectively. The time course of channel activation is slow, occurring for more than approximately a 10-min period, suggesting an indirect link between activation of the leptin receptor and the channel. In this regard, Harvey and Ashford (201) have reported that application of tyrosine kinase inhibitors to CRI-G1 cells produced an activation of a K⁺ conductance similar to that seen with leptin, while whole-cell dialysis with the tyrosine phosphatase inhibitor orthovanadate blocked the action of leptin and tyrosine kinase inhibitors. Serine/threonine-specific protein phosphatase inhibitors neither blocked nor reversed the action of leptin on K_ATP channels. The authors conclude that leptin activation appears to require inhibition of tyrosine kinases and subsequent dephosphorylation. Harvey and Ashford (200) further suggest that opening of K_ATP channels by leptin may occur through the activation of PI 3-kinase as wortmannin, a PI 3-kinase inhibitor, blocked activation of the K⁺ conductance by tyrosine kinase inhibitors. Interestingly, Harvey and Ashford (200) report that insulin can reverse the activating effect of leptin on K⁺ channels and will block the effect if applied before leptin. Unraveling how activation of the leptin receptor up-regulates K_ATP channels promises to be exciting and to give deeper insight into the metabolic alterations associated with both obesity and NIDDM.

	XIV. Summary and Conclusions

Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 
K_ATP channels are a newly defined class of potassium channels based on the physical association of an ABC protein, the sulfonylurea receptor, and a K⁺ inward rectifier subunit. The ß-cell K_ATP channel is composed of SUR1, the high-affinity sulfonylurea receptor with multiple TMDs and two NBFs, and K_IR6.2, a weak inward rectifier, in a 1:1 stoichiometry. The pore of the channel is formed by K_IR6.2 in a tetrameric arrangement; the overall stoichiometry of active channels is (SUR1/K_IR6.2)₄. The two subunits form a tightly integrated whole. K_IR6.2 can be expressed in the plasma membrane either by deletion of an ER retention signal at its C-terminal end or by high-level expression to overwhelm the retention mechanism. The single-channel conductance of the homomeric K_IR6.2 channels is equivalent to SUR/K_IR6.2 channels, but they differ in all other respects, including bursting behavior, pharmacological properties, sensitivity to ATP and ADP, and trafficking to the plasma membrane. Coexpression with SUR restores the normal channel properties.

The key role K_ATP channels play in the regulation of insulin secretion in response to changes in glucose metabolism is underscored by the finding that a recessive form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is caused by mutations in K_ATP channel subunits that result in the loss of channel activity. K_ATP channels set the resting membrane potential of ß-cells, and their loss results in a constitutive depolarization that allows voltage-gated Ca²⁺ channels to open spontaneously, increasing the cytosolic Ca²⁺ levels enough to trigger continuous release of insulin. The loss of K_ATP channels, in effect, uncouples the electrical activity of ß-cells from their metabolic activity. PHHI mutations have been informative on the function of SUR1 and regulation of K_ATP channels by adenine nucleotides. The results indicate that SUR1 is important in sensing nucleotide changes, as implied by its sequence similarity to other ABC proteins, in addition to being the drug sensor. An unexpected finding is that the inhibitory action of ATP appears to be through a site located on K_IR6.2, whose affinity for ATP is modified by SUR1. A PHHI mutation, G1479R, in the second NBF of SUR1 forms active K_ATP channels that respond normally to ATP, but fail to activate with MgADP. The result implies that ATP tonically inhibits K_ATP channels, but that the ADP level in a fasting ß-cell antagonizes this inhibition. Decreases in the ADP level as glucose is metabolized result in K_ATP channel closure.

Although K_ATP channels are the target for sulfonylureas used in the treatment of NIDDM, the available data suggest that the identified K_ATP channel mutations do not play a major role in diabetes. Understanding how K_ATP channels fit into the overall scheme of glucose homeostasis, on the other hand, promises insight into diabetes and other disorders of glucose metabolism, while understanding the structure and regulation of these channels offers potential for development of novel compounds to regulate cellular electrical activity.

	Footnotes

 
Address reprint requests to: Joseph Bryan, Ph.D., Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 USA.

¹ Supported by NIH Grants DK-44311, DK-52771, and DK-50750, the Juvenile Diabetes Foundation International, and the American Diabetes Association.

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Top Abstract I. Introduction II. How Are KATP... III. How Do KATP... IV. KATP Channel Subunits V. Reconstitution of KATP... VI. KATP Channel Structure VII. Regulation of KATP... VIII. Human SUR1 and... IX. KATP Channels and... X. Linking PHHI to... XI. Other Issues XII. KATP and NIDDM XIII. The Leptin Connection XIV. Summary and Conclusions References

 

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				Y. Shi, Z. Wu, N. Cui, W. Shi, Y. Yang, X. Zhang, A. Rojas, B. T. Ha, and C. Jiang PKA phosphorylation of SUR2B subunit underscores vascular KATP channel activation by beta-adrenergic receptors Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1205 - R1214. [Abstract] [Full Text] [PDF]

				M. Winkler, D. Stephan, S. Bieger, P. Kuhner, F. Wolff, and U. Quast Testing the Bipartite Model of the Sulfonylurea Receptor Binding Site: Binding of A-, B-, and A + B-Site Ligands J. Pharmacol. Exp. Ther., August 1, 2007; 322(2): 701 - 708. [Abstract] [Full Text] [PDF]

				N. Baroukh, M. A. Ravier, M. K. Loder, E. V. Hill, A. Bounacer, R. Scharfmann, G. A. Rutter, and E. Van Obberghen MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic {beta}-Cell Lines J. Biol. Chem., July 6, 2007; 282(27): 19575 - 19588. [Abstract] [Full Text] [PDF]

				V. Garg and K. Hu Protein kinase C isoform-dependent modulation of ATP-sensitive K+ channels in mitochondrial inner membrane Am J Physiol Heart Circ Physiol, July 1, 2007; 293(1): H322 - H332. [Abstract] [Full Text] [PDF]

				M. Vaxillaire, A. Dechaume, K. Busiah, H. Cave, S. Pereira, R. Scharfmann, G. P. de Nanclares, L. Castano, P. Froguel, M. Polak, et al. New ABCC8 Mutations in Relapsing Neonatal Diabetes and Clinical Features Diabetes, June 1, 2007; 56(6): 1737 - 1741. [Abstract] [Full Text] [PDF]

				X. Mao, Y. Chai, and Y.-F. Lin Dual regulation of the ATP-sensitive potassium channel by caffeine Am J Physiol Cell Physiol, June 1, 2007; 292(6): C2239 - C2258. [Abstract] [Full Text] [PDF]

				M. Acosta-Martinez and J. E. Levine Regulation of KATP channel subunit gene expression by hyperglycemia in the mediobasal hypothalamus of female rats Am J Physiol Endocrinol Metab, June 1, 2007; 292(6): E1801 - E1807. [Abstract] [Full Text] [PDF]

				A. Szollosi, M. Nenquin, and J.-C. Henquin Overnight Culture Unmasks Glucose-induced Insulin Secretion in Mouse Islets Lacking ATP-sensitive K+ Channels by Improving the Triggering Ca2+ Signal J. Biol. Chem., May 18, 2007; 282(20): 14768 - 14776. [Abstract] [Full Text] [PDF]

				A. Hambrock, C. B. de Oliveira Franz, S. Hiller, A. Grenz, S. Ackermann, D. U. Schulze, G. Drews, and H. Osswald Resveratrol Binds to the Sulfonylurea Receptor (SUR) and Induces Apoptosis in a SUR Subtype-specific Manner J. Biol. Chem., February 2, 2007; 282(5): 3347 - 3356. [Abstract] [Full Text] [PDF]

				A. Szollosi, M. Nenquin, L. Aguilar-Bryan, J. Bryan, and J.-C. Henquin Glucose Stimulates Ca2+ Influx and Insulin Secretion in 2-Week-old beta-Cells Lacking ATP-sensitive K+ Channels J. Biol. Chem., January 19, 2007; 282(3): 1747 - 1756. [Abstract] [Full Text] [PDF]

				F.-F. Yan, J. Casey, and S.-L. Shyng Sulfonylureas Correct Trafficking Defects of Disease-causing ATP-sensitive Potassium Channels by Binding to the Channel Complex J. Biol. Chem., November 3, 2006; 281(44): 33403 - 33413. [Abstract] [Full Text] [PDF]

				K. Heusser, H. Yuan, I. Neagoe, A. I. Tarasov, F. M. Ashcroft, and B. Schwappach Scavenging of 14-3-3 proteins reveals their involvement in the cell-surface transport of ATP-sensitive K+ channels J. Cell Sci., October 15, 2006; 119(20): 4353 - 4363. [Abstract] [Full Text] [PDF]

				K. Fang, L. Csanady, and K. W. Chan The N-terminal transmembrane domain (TMD0) and a cytosolic linker (L0) of sulphonylurea receptor define the unique intrinsic gating of KATP channels J. Physiol., October 15, 2006; 576(2): 379 - 389. [Abstract] [Full Text] [PDF]

				N. M. Doliba, W. Qin, M. Z. Vatamaniuk, C. W. Buettger, H. W. Collins, M. A Magnuson, K. H. Kaestner, D. F. Wilson, R. D. Carr, and F. M. Matschinsky Cholinergic regulation of fuel-induced hormone secretion and respiration of SUR1-/- mouse islets Am J Physiol Endocrinol Metab, September 1, 2006; 291(3): E525 - E535. [Abstract] [Full Text] [PDF]

				P. Stiernet, Y. Guiot, P. Gilon, and J.-C. Henquin Glucose Acutely Decreases pH of Secretory Granules in Mouse Pancreatic Islets: MECHANISMS AND INFLUENCE ON INSULIN SECRETION J. Biol. Chem., August 4, 2006; 281(31): 22142 - 22151. [Abstract] [Full Text] [PDF]

				N. Yokoi, M. Kanamori, Y. Horikawa, J. Takeda, T. Sanke, H. Furuta, K. Nanjo, H. Mori, M. Kasuga, K. Hara, et al. Association Studies of Variants in the Genes Involved in Pancreatic {beta}-Cell Function in Type 2 Diabetes in Japanese Subjects. Diabetes, August 1, 2006; 55(8): 2379 - 2386. [Abstract] [Full Text] [PDF]

				C.-W. Lin, Y.-W. Lin, F.-F. Yan, J. Casey, M. Kochhar, E. B. Pratt, and S.-L. Shyng Kir6.2 Mutations Associated With Neonatal Diabetes Reduce Expression of ATP-Sensitive K+ channels: Implications in Disease Mechanism and Sulfonylurea Therapy Diabetes, June 1, 2006; 55(6): 1738 - 1746. [Abstract] [Full Text] [PDF]

				Z. T. Bloomgarden Cardiovascular Disease Diabetes Care, May 1, 2006; 29(5): 1160 - 1166. [Full Text] [PDF]

				Y. M. Leung, I. Ahmed, L. Sheu, X. Gao, M. Hara, R. G. Tsushima, N. E. Diamant, and H. Y. Gaisano Insulin Regulates Islet {alpha}-Cell Function by Reducing KATP Channel Sensitivity to Adenosine 5'-Triphosphate Inhibition Endocrinology, May 1, 2006; 147(5): 2155 - 2162. [Abstract] [Full Text] [PDF]

				N. Teramoto Physiological roles of ATP-sensitive K+ channels in smooth muscle J. Physiol., May 1, 2006; 572(3): 617 - 624. [Abstract] [Full Text] [PDF]

				A. V. Gyulkhandanyan, S. C. Lee, G. Bikopoulos, F. Dai, and M. B. Wheeler The Zn2+-transporting Pathways in Pancreatic beta-Cells: A ROLE FOR THE L-TYPE VOLTAGE-GATED Ca2+ CHANNEL J. Biol. Chem., April 7, 2006; 281(14): 9361 - 9372. [Abstract] [Full Text] [PDF]

				N. Ishiyama, M. A. Ravier, and J.-C. Henquin Dual mechanism of the potentiation by glucose of insulin secretion induced by arginine and tolbutamide in mouse islets Am J Physiol Endocrinol Metab, March 1, 2006; 290(3): E540 - E549. [Abstract] [Full Text] [PDF]

				Y.-W. Lin, C. MacMullen, A. Ganguly, C. A. Stanley, and S.-L. Shyng A Novel KCNJ11 Mutation Associated with Congenital Hyperinsulinism Reduces the Intrinsic Open Probability of beta-Cell ATP-sensitive Potassium Channels J. Biol. Chem., February 3, 2006; 281(5): 3006 - 3012. [Abstract] [Full Text] [PDF]

				T. Otonkoski, K. Nanto-Salonen, M. Seppanen, R. Veijola, H. Huopio, K. Hussain, P. Tapanainen, O. Eskola, R. Parkkola, K. Ekstrom, et al. Noninvasive Diagnosis of Focal Hyperinsulinism of Infancy With [18F]-DOPA Positron Emission Tomography Diabetes, January 1, 2006; 55(1): 13 - 18. [Abstract] [Full Text] [PDF]

				P. E MacDonald, J. W Joseph, and P. Rorsman Glucose-sensing mechanisms in pancreatic {beta}-cells Phil Trans R Soc B, December 29, 2005; 360(1464): 2211 - 2225. [Abstract] [Full Text] [PDF]

				A. Munoz, M. Hu, K. Hussain, J. Bryan, L. Aguilar-Bryan, and A. S. Rajan Regulation of Glucagon Secretion at Low Glucose Concentrations: Evidence for Adenosine Triphosphate-Sensitive Potassium Channel Involvement Endocrinology, December 1, 2005; 146(12): 5514 - 5521. [Abstract] [Full Text] [PDF]

				L. R. Conti and C. A. Vandenberg ERADication of ion channels destined for the plasma membrane. Focus on "Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels" Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1072 - C1074. [Full Text] [PDF]

				J. C. Koster, M. A. Permutt, and C. G. Nichols Diabetes and Insulin Secretion: The ATP-Sensitive K+ Channel (KATP) Connection Diabetes, November 1, 2005; 54(11): 3065 - 3072. [Abstract] [Full Text] [PDF]

				F.-F. Yan, C.-W. Lin, E. A. Cartier, and S.-L. Shyng Role of ubiquitin-proteasome degradation pathway in biogenesis efficiency of {beta}-cell ATP-sensitive potassium channels Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1351 - C1359. [Abstract] [Full Text] [PDF]

				C.-W. Lin, F. Yan, S. Shimamura, S. Barg, and S.-L. Shyng Membrane Phosphoinositides Control Insulin Secretion Through Their Effects on ATP-Sensitive K+ Channel Activity Diabetes, October 1, 2005; 54(10): 2852 - 2858. [Abstract] [Full Text] [PDF]

				H. Heissig, K. A. Urban, K. Hastedt, B. J. Zunkler, and U. Panten Mechanism of the Insulin-Releasing Action of {alpha}-Ketoisocaproate and Related {alpha}-Keto Acid Anions Mol. Pharmacol., October 1, 2005; 68(4): 1097 - 1105. [Abstract] [Full Text] [PDF]

				C. Shiota, J. V. Rocheleau, M. Shiota, D. W. Piston, and M. A. Magnuson Impaired glucagon secretory responses in mice lacking the type 1 sulfonylurea receptor Am J Physiol Endocrinol Metab, October 1, 2005; 289(4): E570 - E577. [Abstract] [Full Text] [PDF]

				E. Marthinet, A. Bloc, Y. Oka, Y. Tanizawa, B. Wehrle-Haller, V. Bancila, J.-M. Dubuis, J. Philippe, and V. M. Schwitzgebel Severe Congenital Hyperinsulinism Caused by a Mutation in the Kir6.2 Subunit of the Adenosine Triphosphate-Sensitive Potassium Channel Impairing Trafficking and Function J. Clin. Endocrinol. Metab., September 1, 2005; 90(9): 5401 - 5406. [Abstract] [Full Text] [PDF]

				R. Cruz-Cruz, A. Salgado, C. Sanchez-Soto, L. Vaca, and M. Hiriart Thapsigargin-sensitive cationic current leads to membrane depolarization, calcium entry, and insulin secretion in rat pancreatic {beta}-cells Am J Physiol Endocrinol Metab, September 1, 2005; 289(3): E439 - E445. [Abstract] [Full Text] [PDF]

				L. Li, X. Geng, M. Yonkunas, A. Su, E. Densmore, P. Tang, and P. Drain Ligand-dependent Linkage of the ATP Site to Inhibition Gate Closure in the KATP Channel J. Gen. Physiol., August 29, 2005; 126(3): 285 - 299. [Abstract] [Full Text] [PDF]

				S. J. Marx and W. F. Simonds Hereditary Hormone Excess: Genes, Molecular Pathways, and Syndromes Endocr. Rev., August 1, 2005; 26(5): 615 - 661. [Abstract] [Full Text] [PDF]