physiological and pathophysiological roles of atp-sensitive k+ channels

Progress in Biophysics & Molecular Biology 81 (2003) 133–176

Review

Physiological and pathophysiological roles of ATP-sensitiveK+ channels

Susumu Seinoa,*, Takashi Mikia,b

aDepartment of Cellular and Molecular Medicine, Graduate School of Medicine, Chiba University, 1-8-1 Inohana

Chuo-ku, Chiba 260-8760, JapanbGene Research Center, Chiba University, Chiba 260-8670, Japan

Abstract

ATP-sensitive potassium (KATP) channels are present in many tissues, including pancreatic isletcells, heart, skeletal muscle, vascular smooth muscle, and brain, in which they couple the cell metabolicstate to its membrane potential, playing a crucial role in various cellular functions. The KATP channelis a hetero-octamer comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) andthe regulatory subunit sulfonylurea receptor SUR (SUR1 or SUR2). Kir6.x belongs to the inward rectifierK+ channel family; SUR belongs to the ATP-binding cassette protein superfamily. Heterologousexpression of differing combinations of Kir6.1 or Kir6.2 and SUR1 or SUR2 variant (SUR2A or SUR2B)reconstitute different types of KATP channels with distinct electrophysiological properties and nucleotideand pharmacological sensitivities corresponding to the various KATP channels in native tissues. Thephysiological and pathophysiological roles of KATP channels have been studied primarily using KATP

channel blockers and K+ channel openers, but there is no direct evidence on the role of the KATP

channels in many important cellular responses. In addition to the analyses of naturally occurring muta-tions of the genes in humans, determination of the phenotypes of mice generated by genetic manipulationhas been successful in clarifying the function of various gene products. Recently, various geneticallyengineered mice, including mice lacking KATP channels (knockout mice) and mice expressing variousmutant KATP channels (transgenic mice), have been generated. In this review, we focus on the physiologicaland pathophysiological roles of KATP channels learned from genetic manipulation of mice and naturallyoccurring mutations in humans.r 2002 Elsevier Science Ltd. All rights reserved.

*Corresponding author. Tel.: +81-43-226-2187; fax: +81-43-221-7803.

E-mail address: [email protected] (S. Seino).

0079-6107/02/$ - see front matter r 2002 Elsevier Science Ltd. All rights reserved.

PII: S 0 0 7 9 - 6 1 0 7 ( 0 2 ) 0 0 0 5 3 - 6

1. Introduction

ATP-sensitive K+ (KATP) channels, originally discovered in heart (Noma, 1983), are widelydistributed in many tissues and cell types, including pancreatic �-cells (Ashcroft et al., 1984; Cookand Hales, 1984; Rorsman and Trube, 1985), brain (Ashford et al., 1988; Amoroso et al., 1990;Bernardi et al., 1993), skeletal (Spruce et al., 1985) and smooth muscles (Standen et al., 1989), andkidney (Hunter and Giebisch, 1988). KATP channels are inhibited by intracellular ATP andactivated by MgADP (Noma, 1983; Ashcroft, 1988). They couple the metabolic state of the cell to

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

2. General features of KATP channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136

2.1. Molecular structure and functional expression of KATP channels . . . . . . . . . . . . 136

2.2. Molecular and functional diversity of KATP channels . . . . . . . . . . . . . . . . . . 138

2.3. Regulation of the KATP channel by nucleotides . . . . . . . . . . . . . . . . . . . . . 139

2.4. Sulfonylureas binding to SUR subunits . . . . . . . . . . . . . . . . . . . . . . . . . 141

2.5. Potassium channel opener binding to SUR subunit . . . . . . . . . . . . . . . . . . . 141

2.6. Regulation of KATP channels by various factors . . . . . . . . . . . . . . . . . . . . . 142

3. Pathophysiological roles of KATP channels learned from disease states in humans . . . . . . 142

3.1. Persistent hyperinsulinemic hypoglycemia of infancy . . . . . . . . . . . . . . . . . . 142

3.2. Diabetes mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

4. KATP channel genetically manipulated mice . . . . . . . . . . . . . . . . . . . . . . . . . . 144

5. Roles of KATP channels in pancreatic �-cells . . . . . . . . . . . . . . . . . . . . . . . . . . 144

5.1. Mice expressing dominant-negative Kir6.2 in pancreatic b-cells . . . . . . . . . . . . . 144

5.2. Mice expressing Kir6.2 with reduced ATP-sensitivity in pancreatic b-cells . . . . . . . 145

5.3. Kir6.2�/� (null) mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

5.4. SUR1�/� (null) mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

6. Roles of KATP channels in brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

6.1. KATP channels in the hypothalamus . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

6.2. Role of KATP channels in the substantia nigra . . . . . . . . . . . . . . . . . . . . . . 152

6.3. Role of the KATP channels in forebrain . . . . . . . . . . . . . . . . . . . . . . . . . 153

7. Roles of the KATP channels in skeletal muscle . . . . . . . . . . . . . . . . . . . . . . . . . 153

7.1. KATP channels in muscle fatigue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

7.2. KATP channels in glucose uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154

8. Roles of KATP channels in cardiovascular tissues . . . . . . . . . . . . . . . . . . . . . . . 155

8.1. Molecular structure of sarcolemmal KATP channels of cardiomyocytes . . . . . . . . . 155

8.2. Sarcolemmal KATP channels in heart . . . . . . . . . . . . . . . . . . . . . . . . . . . 156

8.3. KATP channels in vascular smooth muscle . . . . . . . . . . . . . . . . . . . . . . . . 159

8.4. Mouse model of prinzmetal (variant or vasospastic) angina . . . . . . . . . . . . . . . 160

9. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

S. Seino, T. Miki / Progress in Biophysics & Molecular Biology 81 (2003) 133–176134

membrane potential by sensing changes in intracellular adenine nucleotide concentration. KATP

channels are thought to play important roles in the cellular responses of various tissues underaltered metabolic states, including hyperglycemia, hypoglycemia, ischemia, and hypoxia(Yokoshiki et al., 1998) (Fig. 1). The physiological role of KATP channels has been bestcharacterized in insulin-secreting pancreatic �-cells (Cook et al., 1988; Boyd 3rd, 1988; Ashcroftand Rorsman, 1989). In pancreatic �-cells, the increase in ATP concentration due to increasedglucose metabolism closes the KATP channels, depolarizing the �-cell membrane, leading to theopening of the voltage-dependent calcium channels (VDCCs), which allows calcium influx. Theresultant rise in intracellular calcium concentration ([Ca2+]i) in the �-cell triggers exocytosis ofinsulin-containing granules. Accordingly, the KATP channels in �-cells are thought to be critical inthe regulation of glucose-induced insulin secretion. In addition, sulfonylureas such as tolbutamideand glibenclamide, widely used in treatment of type 2 (non-insulin dependent) diabetes mellitus(NIDDM), stimulate insulin release by closing KATP channels directly (Sturgess et al., 1985; Trubeet al., 1986; Dunne et al., 1987). On the contrary, potassium channel openers (KCOs) such asdiazoxide inhibit insulin release by opening the KATP channels (Trube et al., 1986; Dunne et al.,1987; Sturgess et al., 1988). KATP channels in the cardiovascular system play various roles,especially in metabolic stress such as ischemia and hypoxia that decreases the intracellular ATPconcentration (Fujita and Kurachi, 2000). In heart, the KATP channels are involved in increase ofK+ efflux and shortening of action potential (Gasser and Vaughan-Jones, 1990; Faivre andFindlay, 1990; Nakaya et al., 1991; Findlay, 1994; Nichols and Lederer, 1991; Nichols et al.,1991), both of which are major factors in the electrophysiological disturbance that can inducearrhythmias (Bril et al., 1992; Tosaki et al., 1993; Wilde and Janse, 1994). Activation of KATP

channels in the heart during ischemia is thought to minimize cardiac damage by ‘‘ischemicpreconditioning’’ (Grover et al., 1992; Yao et al., 1993). In the vascular system, since relaxation ofvascular smooth muscles by KCOs (Edwards and Weston, 1993) is associated with activation ofglibenclamide-sensitive K+ currents (Quayle et al., 1995; Zhang and Bolton, 1995), the KATP

channels are thought to regulate tonus of vascular smooth muscles, thereby playing a role in theregulation of blood pressure (Edwards and Weston, 1993; Nelson and Quayle, 1995; Quayle et al.,

Fig. 1. KATP channel as metabolic sensor. KATP channels, as metabolic sensor, play an important role in the cellular

responses of various tissues under altered metabolic states, including hyperglycemia, hypoglycemia, ischemia, and

hypoxia.

S. Seino, T. Miki / Progress in Biophysics & Molecular Biology 81 (2003) 133–176 135

1997). In the coronary artery, KATP channels are believed to be involved in the vasodilationresponse during ischemia, as the vasodilation response induced by ischemia and hypoxia can beblocked by glibenclamide (Daut et al., 1990; Beech et al., 1993; Dart and Standen, 1995).In brain, KATP channels with differing properties are detected in many cell types, including

hippocampal neurons (Fujimura et al., 1997), glial cells (Zawar et al., 1999), dorsal vagal neurons(Trapp and Ballanyi, 1995), hypothalamic neurons (Ashford et al., 1990a, b; Dunn-Meynell et al.,1997), and substantia nigra (SNr) (Liss et al., 1999). Opening the KATP channels in the brainunder metabolic stress has been suggested to protect against neuronal damage andneurodegeneration (Heurteaux et al., 1995; Blondeau et al., 2000).These physiological roles of KATP channels have been proposed primarily on the studies of

cellular responses using pharmacological agents such as KATP channel blockers and openers. Amore straightforward approach to clarifying the physiological roles of KATP channels currently isthe generation of genetically modified KATP channel mice and analysis of their phenotypes.Recent studies of mice lacking a KATP channel subunit and transgenic mice expressing a mutantKATP channel subunit have revealed many expected and unexpected roles of the KATP channels invarious tissues. In this review, the physiological and pathophysiological roles of KATP channelslearned from genetically manipulated mice and naturally occurring mutations in humans will bediscussed. For more detailed review of the structure–function relationships and the regulation ofKATP channels, the reader is referred to recent reviews (Ashcroft and Gribble, 1998; Aguilar-Bryan and Bryan, 1999; Seino, 1999; Fujita and Kurachi, 2000; Ashcroft, 2000).

2. General features of KATP channels

2.1. Molecular structure and functional expression of KATP channels

The KATP channel is a hetero-octameric complex comprising two subunits: a pore-formingsubunit Kir6.x (Kir6.1 or Kir6.2) and SUR, a regulatory subunit that is a receptor forsulfonylureas, widely used for treatment of type 2 diabetes mellitus (Aguilar-Bryan and Bryan,1999; Ashcroft and Gribble, 1998; Seino, 1999) (Fig. 2). Kir6.1 (Inagaki et al., 1995a) and Kir6.2(Inagaki et al., 1995c; Sakura et al., 1995) are members of the inward rectifier K+ channel familyhaving two transmembrane domains. There is about 71% amino acid identity between Kir6.1 andKir6.2 (Inagaki et al., 1995c). Although the Gly–Tyr–Gly motif in the H5 region, which is thoughtto be critical for K+ ion selectivity, is highly conserved in inwardly rectifying K+ channels, theamino acid sequence of this motif in both Kir6.2 and Kir6.1 is Gly–Phe–Gly, suggesting that themotif may be unique to Kir6.x members. Sulfonylurea receptors belong to members of the ATP-binding cassette (ABC) protein superfamily (Higgins, 1992). There are two isoforms of thesulfonylurea receptor, SUR1 and SUR2, which are derived from two different genes. SUR1 wasfirst cloned from the pancreatic �-cell cDNA libraries, based on the information of the partialamino acid sequence of [125I]-iodoglibenclamide binding purified protein (Aguilar-Bryan et al.,1995). SUR2 (now renamed SUR2A) was subsequently cloned by homology screening usingSUR1 as probe (Inagaki et al., 1996). In addition, there are several variants of SUR2A derivedfrom alternative splicing (Isomoto et al., 1996; Chutkow et al., 1996, 1999), the major one beingSUR2B. SUR2A and SUR2B differ by only 42 amino acids in the C-terminus, due to alternativesplicing (Isomoto et al., 1996). As a consequence, the C-terminus of SUR2B is similar to that of


SUR1. The sulfonylurea receptor has been proposed to have three transmembrane domains,TMD0, TMD1, and TMD2, each of which consists of five, five, and six membrane spanningregions, respectively, based on studies with membrane-impermeable biotinylating reagent, biotinmaleimide (Conti et al., 2001) as well as the multi-sequence alignments of SUR1 and themultidrug resistance-associated protein (MRP) family (Tusnady et al., 1997) (Fig. 2). Thesulfonylurea receptor has two nucleotide binding folds (NBF-1 and NBF-2) on the cytoplasmicside (Conti et al., 2001). NBF-1 and NBF-2 are located in the loop between TMD1 and TMD2and in the C-terminus, respectively. The SUR1 protein shows a high-affinity binding capacity tothe sulfonylurea glibenclamide, indicating that SUR1 confers sulfonylurea binding (Aguilar-Bryan et al., 1995). The affinity of SUR2A for sulfonylureas is much lower than that of SUR1(Inagaki et al., 1996). SUR2A cannot be photolabeled with [125I]-iodoglibenclamide under thesame conditions that photolabels SUR1 (Inagaki et al., 1996). SUR1 is expressed at high levels inpancreatic islets and various clonal �-cells and moderate to low levels in brain. While SUR2A isexpressed predominantly in heart and skeletal muscle (Inagaki et al., 1996), SUR2B isubiquitously expressed, assessed by reverse transcription-polymerase chain reaction (RT-PCR)assay (Isomoto et al., 1996). Other splice variants of SUR2 also have been identified (Chutkowet al., 1996, 1999), and also of SUR1 (Sakura et al., 1999). Such variation might account inpart for the more subtle differences in the properties of the various KATP channels in nativetissues.

Fig. 2. Molecular structure of KATP channel. (a) Assembly of KATP channel. The KATP channel is a hetero-octamer

comprising two subunits: the pore-forming subunit Kir6.x (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea

receptor SUR (SUR1 or SUR2). (b) Membrane topology of SUR and Kir6.x: The sulfonylurea receptor has been

proposed to have three transmembrane domains, TMD0, TMD1, and TMD2, each of which consists of five, five, and

six membrane spanning regions. Kir6.x has two transmembrane domains.


As do other inward rectifier K+ channels, Kir6.2 co-assembles as a tetramer. The stoichiometryof the KATP channel has been determined using cDNAs encoding tandem dimers of SUR1 andKir6.2. The tandem dimer of SUR1 and Kir6.2 revealed electrophysiological properties similar tothose of the native pancreatic �-cell KATP channel, indicating that a 1:1 stoichiometry is sufficientto form functional channels. In contrast, the triple fusion protein of SUR1 with Kir6.2 and Kir6.2did not generate functional KATP channels, while co-expression with SUR1 restored non-functional channels. Biochemical studies revealed the molecular mass of the Kir6.2/SUR1(glycosylated form) complex to be about 950 kDa (Clement et al., 1997). Thus, the KATP channelis likely to be a hetero-octameric complex of Kir6.2 and SUR1 in 4:4 stoichiometry (Clement et al.,1997; Inagaki et al., 1997; Shyng et al., 1997a).It has been shown that both Kir6.2 and SUR1 possess RKR, an endoplasmic reticulum (ER)

retention signal that prevents the trafficking of each subunit to the plasma membrane in theabsence of the other subunit (Zerangue et al., 1999). The retention signal is present in the C-terminal region in Kir6.2 and in an intracellular loop between TM11 and NBF-1 in SUR1. Co-expression of two subunits masks these retention signals, allowing them to traffic to the plasmamembrane. Truncation of the C-terminus of Ki6.2 deletes its retention signal, allowing functionalexpression of Kir6.2 in the absence of SUR subunit (Tucker et al., 1997). The C-terminus ofSUR1 also has been suggested to have an anterograde signal containing part of a dileucine motifand downstream phenylalanine, which is required for KATP channels to exit the ER/cis-Golgicompartments and transit to the cell surface (Sharma et al., 1999). It was also shown that thedeletion of as few as seven amino acids from the C-terminus in SUR1, including thephenylalanine, markedly reduces surface expression of KATP channels (Babenko et al., 1998a).In contrast, the deletion of more residues in the C-terminus of SUR1 restores functionalexpression of the channels at the cell surface (Sakura et al., 1999). Thus, the traffic of KATP

channels to the cell surface is regulated by a complex mechanism.

2.2. Molecular and functional diversity of KATP channels

Heterologous expression of Kir6.x and SUR subunits in differing combinations reconstitutesdifferent types of KATP channel with distinct electrophysiological properties and nucleotide andpharmacological sensitivities that reflect the various KATP channels in native tissues. While Kir6.2andSUR1 constitute the pancreatic �-cell type KATP channel (Inagaki et al., 1995c), Kir6.2 andSUR2A constitute the cardiac type KATP channel (Inagaki et al., 1996), and Kir6.2 and SUR2Bprobably constitute non-vascular smooth muscle type KATP channels (Yamada et al., 1997).Kir6.1 and SUR2B constitute the vascular smooth muscle type KATP channel (KNDP), which israther insensitive to ATP, activated by nucleoside diphosphates (NDPs), and inhibited byglibenclamide (Yamada et al., 1997).Co-expression of Kir6.2 and SUR1 in COS-1 cells reconstitutes weakly inwardly rectifying K+

channel currents with unitary conductance of B76 pS (Inagaki et al., 1995c; Sakura et al., 1995).ATP inhibits reconstituted channel activity with half-maximal inhibition (Ki) at B10mM in thepresence of Mg2+. The reconstituted K+ channel currents are almost completely blocked byadenyl-50-yl imidodiphosphate (AMP-PNP), a non-hydrolyzable ATP analog. Glibenclamideblocks Kir6.2/SUR1 channels, while diazoxide activates the channels. The properties of the K+

channels reconstituted from Kir6.2 and SUR1 are similar to those of the KATP channel in native


pancreatic �-cells (Ashcroft et al., 1984; Cook and Hales, 1984; Rorsman and Trube, 1985;Aguilar-Bryan et al., 1995; Inagaki et al., 1995c). Kir6.2 and SUR1 constitute the KATP channel inventromedial hypothalamus (VMH) (Miki et al., 2001) and SNr in the brain (Liss et al., 1999).Co-expression of SUR2A and Kir6.2 in COS-1 cells reconstituted K+ channels currents

(Kir6.2/SUR2A) with unitary channel conductance ofB80 pS at a membrane potential of –60mV(Inagaki et al., 1996). ATP also inhibits the Kir6.2/SUR2A channel in a dose dependent manner,but the Kir6.2/SUR2A channel is less than a tenth as sensitive to ATP as the SUR1/Kir6.2channel, when expressed in COS-1 cells. Glibenclamide, at the concentration sufficient to blockSUR1/Kir6.2 channel currents, slightly inhibited Kir6.2/SUR2A channel currents reconstituted inCOS1-cells. However, the sensitivity to both ATP and glibenclamide of Kir6.2/SUR2A channelsis increased when Kir6.2 and SUR2A were co-expressed in Xenopus oocytes (Gribble et al., 1998),compared to mammalian cells. While the KCOs cromakalim and pinacidil stimulate channelactivity, diazoxide does not activate Kir6.2/SUR2A channels, the properties similar to those ofKATP channels in native heart (Sanguinetti et al., 1988; Arena and Kass, 1989; Escande et al.,1989; Thuringer and Escande, 1989; Faivre and Findlay, 1989; Hamada et al., 1990; Findlay,1992). However, the sensitivity of SUR2A/Kir6.2 channels to diazoxide increases in the presenceof MgADP (D’hahan et al., 1999b).Co-expression of Kir6.2 and SUR2B in a mammalian cell line, HEK 293T cells, elicited KATP

channel currents with unitary conductance of B80 pS between �100 and �20mV, which wereactivated both by diazoxide and pinacidil (Isomoto et al., 1996), properties similar to those ofnative non-vascular smooth muscles such as urinary bladder, gut, and trachea (Foster et al., 1989;McPherson and Angus, 1990; Nielsen-Kudsk et al., 1990; Longmore et al., 1991). Whether or notKir6.2 and SUR2B are constituents of the non-vascular smooth muscle KATP channels in nativetissues remains to be demonstrated.Co-expression of Kir6.1 and SUR2B in HEK239 cells has been shown to produce K+ channel

currents activated by NDPs such as UDP and ADP and blocked by glibenclamide. The Kir6.1/SUR2B channel is rather insensitive to ATP. The electrophysiological and pharmacologicalproperties of Kir6.2/SUR2B channel resemble those of the NDP-dependent (KNDP) channel invascular smooth muscle cells (Yamada et al., 1997). By comparison of the electrophysiologicaland pharmacological properties with those of KATP channels in native cells, subunit compositionof different types of KATP channel is listed in Table 1.

2.3. Regulation of the KATP channel by nucleotides

Since SUR1 possesses two NBFs, it was thought initially that the SUR subunit mediates theeffects of nucleotides. However, it was found that truncation of the C-terminal region (26–36

Table 1

Types of KATP channel

Subunit composition Type

Kir6.2/SUR1 Pancreatic �-cell (Inagaki et al., 1995c; Sakura et al., 1995)

Kir6.2/SUR2A Cardiomyocyte (Inagaki et al., 1996)

Kir6.2/SUR2B Smooth muscle (Isomoto et al., 1996)

Kir6.1/SUR2B Vascular smooth muscle (Yamada et al., 1997)


amino acids) of Kir6.2 (Kir6.2DC) in the absence of SUR1 generates K+ channel currentsinhibited by ATP and not activated MgADP (Tucker et al., 1997), indicating that the Kir6.2subunit confers the ATP-sensitivity of the KATP channel. Overexpression of wild-type Kir6.2alone in mammalian cells (John et al., 1998) or in the insect cell line Sf9 (Mikhailov et al., 1998)was shown to form functional KATP channels at least at very low density. However, the ATP-sensitivity of channels reconstituted only by Kir6.2 subunits is much lower (B10 mM) than that ofthe channels reconstituted from both Kir6.2 and SUR1. Direct evidence for ATP-binding ofKir6.2 has been provided by photoaffinity labeling of Kir6.2 by [g-32P]-8-azido-ATP (Tanabeet al., 2000). Although the precise binding site of ATP remains to be determined, mutationalanalyses of Kir6.2 suggest that Arg, Lys, Ile, Arg, and Gly at residue 50 (Tucker et al., 1998), 182(Drain et al., 1998), 185 (Tucker et al., 1997; Reimann et al., 1999), and 201 (Shyng et al., 2000),respectively, are involved in ATP-sensitivity of the channel without affecting the single-channelkinetics (Tucker et al., 1997, 1998; Drain et al., 1998). Both R50G and K185Q mutations decreaseATP-binding, suggesting that the N-terminal and C-terminal regions of Kir6.2 interactcooperatively to inhibit channel activity by ATP (Tanabe et al., 2000). Since there is one ATP-binding site per Kir6.2 monomer, there are four ATP-binding sites per KATP channel, but thebinding of ATP at one site has been shown to be sufficient to induce closure of the channel(Markworth et al., 2000).It has been shown that MgADP stimulates KATP channel activity by interacting with the NBFs

of the SUR subunit (Gribble et al., 1997; Shyng et al., 1997b). Nucleotide binding to SUR1 wasanalyzed by characterizing the binding ability of 8-azido-[32P]-ATP to wild-type and variousmutant SUR1 (Ueda et al., 1997, 1999a; Matsuo et al., 1999). SUR1 was efficiently labeled with 8-azido-[a-32P]-ATP and 8-azido-[g-32P]-ATP in the presence or absence of Mg2+. Mutations ofNBF-1 impaired photolabeling, while mutations of NBF-2 did not. Preincubation with MgADPstrongly antagonized photolabeling with 8-azido-[a-32P]-ATP and mutations of NBF-2 reducedMgADP antagonism (Ueda et al., 1997). In addition, MgATP and MgADP stabilized the bindingof prebound 8-azido-[a-32P]ATP to SUR1. Mutations in the Walker A and B motifs (Walker et al.,1982) of NBF-2 of SUR1 abolished this stabilizing effect of MgADP (Ueda et al., 1999a). Theseresults suggest that SUR1 binds ATP at NBF-1, and that MgADP, either by direct binding toNBF-2 or by hydrolysis of bound MgATP at NBF-2, induces a conformational change at NBF-2that results in stabilizing ATP-binding at NBF-1. This is supported by biochemical studies inwhich tryptic fragments of SUR1, SUR2A, and SUR2B containing the respective NBF-1 andNBF-2 were immunoprecipitated by antibodies specific for NBF-1 and NBF-2 after photoaffinitylabeling with 8-azido-[a-32P] ATP and 8-azido-[g-32P]-azido ATP (Matsuo et al., 2000). The resultsindicate that NBF-2 of SUR1, SUR2A, and SUR2B all were labeled with 8-azido-[a-32P] ATP butnot with 8-azido-[g-32P]-azido ATP in the presence of Mg2+, suggesting that NBF-2 of the SURsubunit has ATPase activity and that NBF-1 has none or little. Based on these findings, thefollowing model has been proposed (Ueda et al., 1999b). When the ATP/ADP ratio is decreased,NBF-1 binds ATP and NBF-2 binds MgADP. In this conformational state, the interaction ofSUR1 with Kir6.2 reduces the affinity of Kir6.2 to ATP, opening the KATP channels. In contrast,when the ATP/ADP ratio increases, the decrease in MgADP induces dissociation of boundMgADP from NBF-2, resulting in release of ATP from NBF-1 and closure of the channels. Thecapability of ATP hydrolysis by the NBFs of the SUR subunits has been examined using fusionproteins of the NBF-1 or NBF-2 region and maltose binding protein (MBP) (Bienengraeber et al.,


2000; Zingman et al., 2001). NBF-2 has been shown to have double the ATPase activity of NBF-1,although the activity is much lower than that of NBF-1 of CFTR (Ko and Pedersen, 1995; Liet al., 1996; Bear et al., 1997).

2.4. Sulfonylureas binding to SUR subunits

Studies of chimeric constructs between SUR1 and SUR2A (Gribble et al., 1998; Gribble andAshcroft, 1999; Babenko et al., 1999) have localized sulfonylurea binding regions, but the precisebinding sites are yet to be determined. TMD2, especially the region between TM15 and TM16, iscritical for sulfonylurea binding (Babenko et al., 1999; Ashfield et al., 1999), and Ser at 1237,located in the intracellular loop between TM15 and TM16, is critical for high-affinity block oftolbutamide. Glibenclamide, which contains both sulfonylurea and benzamido moieties, isthought to bind SUR1 at two regions; in addition to binding the site for tolbutamide (sulfonylureabinding site), it also binds the site for the benzamido derivative meglitinide (bezoamido bindingsite) (Ashcroft and Gribble, 2000a). In contrast, tolbutamide, which contains only a sulfonylureamoiety, binds to SUR1 at only one site (tolbutamide binding site) (Gribble et al., 1998). SUR1and SUR2 are both thought to possess a benzamido binding site, while SUR2 lacks thesulfonylurea binding site. Since SUR2B-containing channels are also blocked by tolbutamide(Isomoto et al., 1996), the C-terminal 42 amino acids of SUR1 and SUR2B might also be involvedin sulfonylurea binding. These differences could account in part for the tissue specificity of thevarious sulfonylureas.

2.5. Potassium channel opener binding to SUR subunit

KCOs including diazoxide, pinacidil, cromakalim, and nicorandil, are a structurally diversegroup of drugs which open KATP channels in various cell types, thereby causing hyperpolarizationof the plasma membrane and reducing electrical activity (Ashcroft and Gribble, 2000b). It hasbeen shown that different SUR subunits confer varying sensitivities to KCOs. For example,Kir6.2/SUR1 channels are activated strongly by diazoxide but not by pinacidil; Kir6.2/SUR2Achannels are activated by pinacidil and cromakalim but only weakly by diazoxide, while Kir6.2/SUR2B channels are activated by diazoxide, pinacidil, and cromakalim (Inagaki et al., 1995c,1996; Isomoto et al., 1996; Gribble et al., 1998; Babenko et al., 1998b; D’hahan et al., 1999a, b).However, Kir6.2/SUR2A channels become as sensitive to diazoxide as Kir6.2/SUR1 channels inthe presence of MgADP (D’hahan et al., 1999b). In addition, recent studies have shown thatKCOs such as pinacidil stimulate ATP hydrolysis at NBF-2 and promote channel opening bystabilizing channels in the Mg-nucleotide bound state (Bienengraeber et al., 2000; Zingman et al.,2001). These findings suggest that KCOs, interacting with nucleotides, exert their effects on KATP

channels in a complex manner. Studies of chimeric proteins between SUR1 and SURA or SUR2Bsuggest that the sensitivities of SUR2-specific KOCs such as pinacidil are conferred by twodistinct regions of TMD2 of SUR2A, part of intracellular loop between TM13 and TM14 andTM16 and TM17 (D’hahan et al., 1999a; Uhde et al., 1999; Babenko et al., 2000), and that tworesidues in TM17 are especially critical: Lys at 1249 and Thr at 1253 of SUR2A (Moreau et al.,2000). However, the precise location of the binding site for diazoxide is still unknown. Diazoxidemust bind to all of the SUR1, SUR2A, and SUR2B subunits, since it activates Kir6.2/SUR1,


Kir6.2/SUR2A (in the presence of MgADP), and Kir6.2/SUR2B (in the presence of MgATP).The effect of diazoxide requires MgADP binding at NBF-2.

2.6. Regulation of KATP channels by various factors

Activity of KATP channels is regulated by various intracellular signals. Protein kinase A (PKA)simulates activity of KATP channels in native tissues (Ribalet et al., 1989; Quayle et al., 1994;Wellman et al., 1998). The effects of PKA phosphorylation on the �-cell KATP channel have beendetermined. Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. Although the phosphorylation of Kir6.2increases channel activity, the PKA site responsible for channel stimulation is controversial.Beguin et al. (1999) have shown that Ser372 is responsible for the channel stimulation while Linet al. (2000) have shown Thr224 is responsible. On the other hand, the phosphorylation of SUR1contributes to the basal channel properties by decreasing burst duration, interburst interval andopen probability, and also increasing the number of functional channels at the cell surface (Beguinet al., 1999). These findings suggest a model of heteromultimeric ion channels in which there arefunctionally distinct roles of the phosphorylation of the different subunits (Beguin et al., 1999).Membrane phospholipids such as PIP2 and PIP3 have been shown to modulate the ATP-

sensitivity of KATP channels (Hilgemann and Ball, 1996; Fan and Makielski, 1997; Baukrowitzet al., 1998; Shyng and Nichols, 1998). PIP2 dramatically decreases the ATP-sensitivity of KATP

channels. It also blocks tolbutamide sensitivity by stabilizing the open state of the channels(Koster et al., 1999). Breakdown of PIP2 by phospholipase C enhances the ATP-sensitivity ofKATP channels (Xie et al., 1999). The effects of PIP2 have been suggested be mediated by Kir6.2subunit.Trimeric GTP-binding proteins have been shown to modulate the activity of KATP channels.

While Gai1 stimulates both Kir6.2/SUR1 and Kir6.2/SUR2A channels, Gai2 does not stimulateKir6.2/SUR1 channels, but stimulates Kir6.2/SUR2A (Sanchez et al., 1998). Although Gbg

subunits have no effect on Kir6.2/SUR1 channels (Sanchez et al., 1998), they caused a reductionof ATP-induced inhibition of Kir6.2/SUR channel activity by reducing the ATP-sensitivity (Wadaet al., 2000).

3. Pathophysiological roles of KATP channels learned from disease states in humans

3.1. Persistent hyperinsulinemic hypoglycemia of infancy

Studies of mutations of KATP channels in humans have provided important insights into thephysiological role of KATP channels as well as their structure–function relationships. So far,mutations in KATP channels have been identified only in the genes for SUR1 and Kir6.2. HumanSUR1 and Kir6.2 genes locate in a cluster at chromosome 11p15.1 (Inagaki et al., 1995c), theregion to which the severe form of persistent hyperinsulinemic hypoglycemia of infancy (PHHI)has been mapped (Glaser et al., 1994).PHHI is a clinically heterogeneous disease, characterized by severe hypoglycemia with

inappropriate, excessive insulin secretion in neonates and infants. The incidence of PHHI in the


general population is about 1/30000–1/50000 live births. It occurs at relatively high incidence(approximately 1/2500 live births) in inbred populations (Aguilar-Bryan and Bryan, 1999).Thomas et al. (1995) originally identified three genetic mutations of the SUR1 gene in patientswith PHHI. Subsequently, mutations of the Kir6.2 gene also were identified in patients withPHHI (Thomas et al., 1996; Nestorowicz et al., 1997). At present, two genes other than SUR1 andKir6.2 have been shown to cause PHHI in humans: the glucokinase gene (Glaser et al., 1998) andthe glutamate dehydrogenase gene (Stanley et al., 1998). Although most PHHI caused bymutations in SUR1 generally shows an autosomal recessive form of inheritance (Aguilar-Bryanand Bryan, 1999), a dominant form of PHHI due to an SUR1 mutation recently was identified(Huopio et al., 2000). All of the mutations in the SUR1 or Kir6.2 genes are considered to causedeterioration of pancreatic �-cell KATP channel function and lead to unregulated insulin secretionregardless of the blood glucose level (Kane et al., 1996). So far, more than 50 mutations in thehuman SUR1 and Kir6.2 genes have been identified in patients with the recessive form of PHHI.The studies of these mutations have shed light on the structure–function relationship of the KATP

channels. These mutations can be classified into two categories in respect to channel function:mutations that cause loss of function and mutations that lead to defective channel regulation.Loss of function mutations prevent trafficking of the channel to the plasma membrane (Cartieret al., 2001; Partridge et al., 2001; Taschenberger et al., 2002) or cause structural defects(Nestorowicz et al., 1997). For example, the DF1388, R1394H, and L1544P mutations in theSUR1 gene have all been shown to impair normal trafficking of SUR1 to the plasma membrane,while Kir6.2Y12X, which is truncated at amino acid residue 12, fails to form functional KATP

channels (Nestorowicz et al., 1997). The other mechanism of PHHI due to SUR1 mutations isaltered interaction between nucleotides and SUR1. For example, mutations in NBF-2 of SUR1have been shown to impair channel activation by MgADP (Nichols et al., 1996; Huopio et al.,2000). Some mutations, such as SUR1-R1420C, which gives rise to a mild-form of patients withPHHI, have been shown to affect KATP channels only slightly (Matsuo et al., 2000). Thehomozygous mutation SUR1-R1420C was found in a PHHI patient with focal adenomatoushyperplasia of pancreatic islets (Verkarre et al., 1998), and was shown to cause a mild-form ofPHHI with clinical remission (Tanizawa et al., 2000) that is resistant to diazoxide treatment.A recent study has shown that glucagon secretion in response to generalized hypoglycemia in

Kir6.2 knockout mice is severely impaired due to a defect in glucose responsiveness in the VMH(Miki et al., 2001). Although data on glucagon secretion in PHHI patients are not available, thissuggests that impairment of glucagon secretion in hypoglycemia also might contribute topersistent hypoglycemia in PHHI.

3.2. Diabetes mellitus

Although the SUR1 and Kir6.2 genes in type 2 diabetes mellitus have been analyzedextensively, the association of mutations or polymorphisms of the SUR1 or Kir6.2 gene anddiabetes remains unclear. Several studies have noted an association of mutations of SUR1 (Inoueet al., 1996; Hart et al., 1999; Rissanen et al., 2000) or Kir6.2 (Hani et al., 1998) with diabetes,while the others have not (Stirling et al., 1995; Iwasaki et al., 1996; Sakura et al., 1996; Lindneret al., 1997). Recently, a mutation of SUR1 (SUR1-E1506K), which causes dominantly inheritedPHHI, also has been shown to cause diabetes mellitus in later life (Huopio et al., 2000). This


might be due to unresponsiveness of the �-cells or to increased �-cell apoptosis (Kassem et al.,2000).

4. KATP channel genetically manipulated mice

Although it is clear that certain mutations of �-cell KATP channel genes lead to unregulatedinsulin secretion and cause PHHI, the roles of KATP channels composed of Kir6.2 and SUR1occurring in native tissues other than pancreatic �-cells have not been examined in patients withPHHI. In addition, the consequences of dysfunction of other KATP channel subunits, includingSUR2A, SUR2B, and Kir6.1 have not determined. Genetic manipulation of KATP channels is themost straightforward strategy currently available to elucidate the physiological and pathophy-siological roles of KATP channels and their molecular composition in native tissues. So far, ninedifferent genetically manipulated mice either lacking a KATP channel subunit or expressing amutant KATP channel subunit with altered function have been generated (Table 2).

5. Roles of KATP channels in pancreatic �-cells

5.1. Mice expressing dominant-negative Kir6.2 in pancreatic b-cells

The putative K+ ion permeable domain (H5) is highly conserved in K+ channels (Jan and Jan,1994), and the motif Gly–Tyr (or Phe)–Gly in H5 is critical for K+ ion selectivity (Heginbothamet al., 1992; Jan and Jan, 1994; Kerr and Sansom, 1995). A substitution of the first residue of theGly–Tyr–Gly motif with Ser (residue 156) is found in the G-protein-gated inward rectifier GIRK2(Kir3.2) of weaver mice (Patil et al., 1995). This mutation is responsible for the phenotype ofweaver mice, including the abnormalities in neuronal differentiation and development (Slesinger

Table 2

KATP channel genetically modified mice

Knockout mice

Kir6.2�/� (Miki et al., 1998)

SUR1�/� (Seghers et al., 2000)

SUR2�/� (Chutkow et al., 2001)

Kir6.1�/� (Miki et al., 2002a)

Transgenic mice

(1) Dominant-negative Kir6.2 in pancreatic �-cellsKir6.2G132S (Miki et al., 1997)

Kir6.2132A133A134A (Koster et al., 2000)

(2) Overactive Kir6.2 in pancreatic �-cellsKir6.2[DN2-30] (Koster et al., 2000)

(3) Overactive Kir6.2 in heart

Kir6.2[DN2-30, K185N] (Koster et al., 2001)

(4) Overexpression of SUR1in forebrain (Hernandez-Sanchez et al., 2001)


et al., 1996; Kofuji et al., 1996; Navarro et al., 1996; Hess, 1996). By analogy with the weavermutant, the first residue of the Gly–Phe–Gly motif of Kir6.2 was substituted with serine, and themutant was called Kir6.2G132S. Kir6.2G132S was shown to act as a dominant-negative inhibitorof the Kir6.2/SUR1 channel when co-expressed with wild-type SUR1 and wild-type Kir6.2.Transgenic mice expressing Kir6.2G132S specifically in the pancreatic �-cells under regulation ofthe human insulin promoter were generated (Miki et al., 1997). These transgenic mice showed arelatively high level of insulin despite severe hypoglycemia as neonates, indicating unregulatedinsulin secretion and suggesting a phenotype resembling PHHI. However, the blood glucose levelsin transgenic mice as adults become markedly elevated with age, and the glucose-induced insulinsecretion is markedly reduced in adult transgenic mice, as assessed by intraperitoneal glucosetolerance test. The KATP conductance is significantly reduced in the �-cells, indicating that KATP

channel function in the �-cells is impaired. Resting membrane potential and basal intracellularcalcium concentration ([Ca2+]i) of the �-cells of transgenic mice were significantly higher thanthat of wild-type mice. Histological analysis revealed abnormal architecture of the pancreaticislets in transgenic mice. Glucagon-positive alpha cells, which are present normally in theperiphery of pancreatic islets, appeared in the central region. The abnormal architecture of theislets became more evident as the number of �-cells decreased with age, due probably to the highfrequency of �-cell apoptosis in the transgenic mice.In the weaver mice, the weaver mutation in Kir3.2 (GIRK2) has been shown to alter cation

selectivity and leads to membrane depolarization by excessive Ca2+ influx, resulting in neuronalcell death. By contrast, in the �-cells of the transgenic mice, the loss of KATP channel function dueto the dominant-negative Kir6.2G132S mutation causes membrane depolarization and high basal[Ca2+]i in the �-cells, resulting in hypoglycemia from unregulated insulin secretion in neonates.Thus, although the underlying mechanisms are different, the mutations in Kir3.2 (weaver mice)and Kir6.2 (Kir6.2G132S/Tg) both elicit chronic membrane depolarization and Ca2+ overload,finally inducing apoptotic cell death, which contributes to the ataxia in the weaver mice andhyperglycemia in the adult transgenic mice. Transgenic mice expressing another dominant-negative form, Kir6.2 [AAA], in which the Gly132, Phe133, and Gly134 are each substituted byAla, also have been made (Koster et al., 2000). However, unlike Kir6.2G132S transgenic mice,there are few effects on either insulin secretion or glucose homeostasis in these mice.

5.2. Mice expressing Kir6.2 with reduced ATP-sensitivity in pancreatic b-cells

The N-terminal deletion mutant Kir6.2[DN2-30], when co-expressed with SUR1 in COSm6cells, has been shown to result in a channel with about 10-fold lower ATP-sensitivity than wild-type �-cell KATP (Kir6.2/SUR1) channels (Koster et al., 2000). Transgenic mice expressingKir6.2[DN2-30] in pancreatic �-cells showed severe hyperglycemia, hypoinsulinemia, andketoacidosis within 2 days, and typically die within 5 days. Overexpression of Kir6.2[DN2-30]in �-cells resulted in a decrease in ATP-sensitivity of KATP channels in pancreatic �-cells. Thismight lead to inhibition of glucose-induced insulin secretion, which could contribute to thedevelopment of diabetes. Nevertheless, the architecture of the islets including the distribution ofthe �-cells and a-cells is normal. Therefore, the Kir6.2[DN2-30] �-cell and the Kir6.2G132S �-cellare quite different in their threshold for the induction of the membrane excitability by glucose.The pancreatic �-cells of Kir6.2[DN2-30] transgenic mice are resistant to glucose-elicited action


potential, while the �-cells of Kir6.2G132S transgenic mice are electrically active continuously,which induces constant calcium influx into the �-cells and leads to apoptotic death and decrease inthe number of �-cells. Thus, although Kir6.2G132S transgenic and Kir6.2[DN2-30] transgenicmice both develop diabetes, their pancreatic �-cell electrical phenomena are different.

5.3. Kir6.2�/� (null) mice

The limitation of studies of transgenic mice is that the phenotypes vary depending upon theexpression levels of the transgenes. To determine the roles of gene products directly, generation ofknockout mice is the more straightforward approach. Mice lacking Kir6.2-containing KATP

channels (Kir6.2 null mice) were generated (Miki et al., 1998). Kir6.2 and SUR1 both areexpressed in all the endocrine cells in the pancreatic islets (Suzuki et al., 1997, 1999). Since theKir6.2 subunit forms the K+ ion-selective pore (Inagaki et al., 1996; Clement et al., 1997; Shyngand Nichols, 1997c), mice lacking KATP channels can be generated by disruption of Kir6.2(Kir6.2�/�). Both whole-cell and single-cell recordings showed that KATP channel activity wascompletely absent in pancreatic �-cells of Kir6.2�/� mice, indicating that the Kir6.2 subunit is anessential component of the �-cell KATP channel (Fig. 3). Kir6.2�/� mice showed transienthypoglycemia as neonates, similar to transgenic mice expressing a dominant-negativeKir6.2G132S. Although the blood glucose levels of Kir6.2�/� mice in adults in the fed statewere not significantly different from those of wild-type mice, the insulin response to glucose invivo also was impaired in Kir6.2�/� mice, as evaluated by intraperitoneal glucose tolerance test.The resting membrane potential of Kir6.2�/� �-cells (at 2.8mM glucose) was significantly higherthan that of wild-type �-cells. In the presence of low glucose (2.8mM), repetitive bursts of actionpotential were frequently found in Kir6.2�/� �-cells. In contrast to control �-cells, high glucose didnot alter the membrane potential of Kir6.2�/� �-cells. The basal levels of intracellular calcium([Ca2+]i) in single �-cells were significantly elevated in Kir6.2�/� �-cells. However, neither highglucose (16.7mM) nor tolbutamide (100mM) elicited any change in [Ca2+]i in Kir6.2�/� �-cells.This indicates that the rise of [Ca2+]i in normal pancreatic �-cells which is elicited by both glucoseand tolbutamide requires closure of the KATP channels. In contrast, acetylcholine or high K+

stimulation increased [Ca2+]i in Kir6.2�/� �-cells to levels comparable to those in control �-cells,suggesting that intracellular calcium mobilization from inositol 1,4,5 triphosphate (IP3)-sensitiveCa2+ stores and Ca2+ influx through VDCCs are independent of the activity of the KATP

channel. The insulin secretary responses to glucose and tolbutamide were determined using bothbatch incubation and perifusion of pancreatic islets of adult mice. Neither 16.7mM glucose nor100 mM tolbutamide elicited significant insulin secretion in Kir6.2�/� mice (Fig. 4). Histologicalexamination showed an abnormal architecture of the pancreatic islets in Kir6.2�/� mice:glucagon-positive a-cells, which are present primarily in the periphery in islets of normal mice,appear also in the central region in islets of Kir6.2�/� mice (Fig. 5), similar to the Kir6.2G132Stransgenic mice. Despite the severe defect in glucose-induced insulin secretion, Kir6.2�/� miceshow only a very mild impairment in glucose tolerance. However, as Kir6.2�/� and wild-type micebecame obese with age, only the Kir6.2�/� mice developed fasting hyperglycemia and glucoseintolerance. Thus, the Kir6.2�/� mouse is a diabetic animal model with a genetic defect in glucose-induced insulin secretion and an acquired insulin resistance due to environmental factorscontributing to the development of diabetes (Seino et al., 2000).


5.4. SUR1�/� (null) mice

SUR1 knockout (SUR1�/�) mice also were generated (Seghers et al., 2000). Similar to Kir6.1knockout mice, the SUR1 knockout mouse �-cells lack the �-cell type KATP channel. Themembrane potential of the �-cells of SUR1�/� mice fluctuated between �45 and �25mV as aresult of spontaneous action potentials, which were not suppressed by diazoxide but wereinhibited by the Ca2+ channel blocker nifedipine. Short-term incubation of pancreatic islets ofSUR1�/� mice (perifusion or and batch incubation) revealed glucose–induced insulin secretion tobe markedly reduced and tolbutamide–induced insulin secretion to be absent. Similar to Kir6.2�/�

mice, insulin secretion in response to intraperitoneal glucose challenge is markedly reduced inSUR1�/� mice. Although recent studies have suggested the importance of the KATP channelindependent mechanism of glucose-induced insulin secretion (Aizawa et al., 1998; Henquin, 2000),studies of dominant-negative Kir6.2-expressing mice, overactive KATP channel-expressing mice,Kir6.2�/� mice, and SUR1�/� mice clearly indicate that the KATP channel in the �-cell is critical inboth glucose-induced insulin secretion and sulfonylurea-induced insulin secretion underphysiological conditions.

Fig. 3. KATP channel currents of pancreatic �-cell isolated from control and Kir6.2�/� mice. Left, representative traces

of whole-cell recordings of pancreatic �-cells in wild-type (Kir6.2+/+, control) mice and Kir6.2�/� mice. The holding

potential was �70mV and alternate voltage pulses of 7 10mV and 200ms duration every 2 s were applied. In control

�-cells (upper), dialysis of the �-cells intracellularly with the ATP-free pipette solution (breakthrough) caused a

progressive increase in K+ conductance, and addition of 500mM tolbutamide promptly inhibited this conductance. In

contrast, no increase in K+ conductance was observed in Kir6.2�/� �-cells (lower). Right, normalized peak KATP

channel conductance of pancreatic �-cells in wild-type and Kir6.2�/� mice. (Reprinted with permission from Miki et al.,

1998 copyright 1998 National Academy of Sciences, USA.)


6. Roles of KATP channels in brain

6.1. KATP channels in the hypothalamus

In the brain, KATP channels have been found in many regions, including the SNr (Roper andAshcroft, 1995; Stanford and Lacey, 1996), neocortex (Ohno-Shosaku and Yamamoto, 1992),hippocampus (Zawar et al., 1999), and hypothalamus (Ashford et al., 1990a). Among these, thehypothalamus is known to possess KATP channels in abundance. In addition, the hypothalamusplays a critical role in glucose homeostasis by regulating the secretion of counter-regulatoryhormones, including glucagon and catecholamines, via the autonomic nervous system (Taborskyet al., 1998).Recovery from insulin-induced systemic hypoglycemia is severely impaired in Kir6.2�/� mice,

suggesting that the secretion of counter-regulatory hormones such as glucagon and catechola-mines is impaired. Although epinephrine secretion in response to insulin-induced hypoglycemia issimilar in Kir6.2�/� and wild-type mice in vivo, the glucagon secretion is markedly reduced inKir6.2�/� mice. Glucagon secretion from isolated pancreatic islets in response to a change fromhigh (16.7mM) to low (1mM) glucose concentration was similar in Kir6.2�/� and wild-type mice.In addition, the glucagon response to a synthetic choline ester, carbachol (50 mM), was notimpaired but was somewhat enhanced in Kir6.2�/� mice compared to wild-type mice. These

Fig. 4. Insulin secretary responses to glucose and tolbutamide in perifused pancreatic islets of wild-type and Kir6.2�/�

mice. There was no significant difference in the basal levels of insulin secretion in the presence of 2.8mM glucose (wild-

type, 2.04 7 0.50 pg/islet/min; Kir6.2�/�, 1.92 7 0.41 pg/islet/min). Only a trace in the first phase of the insulin

secretary response to 16.7mM glucose was detected in Kir6.2�/� mice. There was no insulin response in the second

phase in Kir6.2�/� mice. 100mM tolbutamide in the presence of 16.7mM glucose did not stimulate insulin secretion in

Kir6.2�/� mice. (Reprinted with permission from Miki et al., 1998 copyright 1998 National Academy of Sciences,

USA.)


findings suggest that the primary defect in hypoglycemia-induced glucagon secretion in these miceis upstream of the a-cells. In the brain, neuroglycopenia stimulates glucagon secretion throughactivation of autonomic neurons. 2-deoxyglucose (2DG) is known to induce neuroglycopenia inthe hypothalamus (Muller et al., 1971), thereby stimulating glucagon secretion (Borg et al., 1995,1999). In contrast to control mice, there was almost no glucagon secretion in response tointracerebroventricullarly injected 2DG in Kir6.2�/� mice (Fig. 6). In the VMH, a subset ofneurons known as glucose-responsive neurons (GRNs), increase their firing rate in response toelevation of extra-cellular glucose levels (Oomura et al., 1969; Minami et al., 1986). Threedifferent neuronal populations have been distinguished based on their spontaneous firing ratesand distinct subthreshold rebound behavior (Minami et al., 1986). Type A neurons quicklyresumed spiking at the end of hyperpolarizing current injections. In contrast, type B neuronsshowed rebound calcium spikes, while type C neurons displayed a prominent delay inrepolarization from hyperpolarized membrane potentials. Although approximately a quarter ofwild-type VMH neurons, which were either type A or C neurons, showed about a 2-fold increasein spontaneous discharge rate in response to high glucose (25mM), none of the Kir6.2�/� VMHneurons displayed changes in spiking frequency in response to increased glucose (Fig. 7). Inaddition, Kir6.2�/� VMH neurons displayed higher discharge rates at low glucose concentrationsthan wild-type mice, strongly suggesting that Kir6.2-containing KATP channels are essential forglucose responsiveness in VMH neurons. In all three types of VMH neurons in wild-type mice,

Fig. 5. Histology of pancreatic islets in wild-type and Kir6.2�/� mice. Pancreatic �-cells (a and c) and a-cells (b and d)

were stained using guinea pig anti-insulin and rabbit anti-glucagon antibodies, respectively. In the islets of a Kir6.2�/�

mouse glucagon-positive a-cells (d), which are present in the periphery of the islets of the control mouse (b), are seen

also in the central region of the islets. The �-cell population in the Kir6.2�/� mouse (c) is similar to the control mouse

(a). Scale bar, 100 mm. (Reprinted with permission fromMiki et al., 1998 copyright 1998 National Academy of Sciences,

USA.)


dialysis with an ATP-free pipette solution activated outward K+ currents that were completelyblocked by tolbutamide, indicating that they flowed through KATP channels. In contrast to wild-type neurons, dialysis with an ATP-free solution did not activate K+ currents in Kir6.2�/� VMHneurons. Thus, the Kir6.2-contaning KATP channel is essential for glucose responsiveness inGRNs. Using single-cell RT-PCR analysis, it was found that the VMH KATP channel comprisesKir6.2 and SUR1, the same composition as the �-cell KATP channel.Based on findings in Kir6.2�/� mice, a model for maintenance of glucose homeostasis by the

KATP channels has been proposed (Miki et al., 2001). As the blood glucose level rises, inhibition ofthe KATP channels in pancreatic �-cells induces insulin secretion and so lowers the glucose level.As the blood glucose level falls, activation of the KATP channels in the GRNs of thehypothalamus stimulates the autonomic input to the pancreatic a-cells, triggering glucagonsecretion that raises the glucose level. Thus, the pancreatic �-cell and the hypothalamus arefunctionally interactive: the insulin secretion system and the glucagon secretion system areintegrated in the maintenance of glucose homeostasis through a common KATP channel. Inaddition to stimulating glucagon secretion, 2DG also is known to increase food intake in normalmice, presumably by inhibiting the activity of GRNs in the hypothalamus (Bergen et al., 1996).The increment in food intake by 2DG was significantly less in Kir6.2�/� mice than in wild-typemice, indicating a functional role for hypothalamic KATP channels in the control of food intake(Fig. 8a).Since feeding behavior is critical for survival and plays a pivotal role in energy homeostasis, it

should be regulated by signals from metabolic sensors that link the changes in cellular metabolicstatus to satiety (Woods et al., 1998; Schwartz et al., 2000). These signals include large numbers ofneuropeptides and hormones that may be generated either in the brain or in peripheral tissues.While Neuropeptide Y (NPY), Agouti-related protein (AGRP), orexin, melanin-concentratinghormone (MCH), and a-melanocyte-stimulating hormone (MSH) are neuropeptides generated in

Fig. 6. (a) Changes in blood glucose levels after exogenous insulin injection. Human insulin (0.5 IU/kg) was injected

intraperitoneally to conscious male mice. Results are expressed as percent of initial blood glucose concentration. Open

circles, wild-type; filled circles, Kir6.2�/� mice (n ¼7 for each). (b) Glucagon secretion in vivo by intracerebroven-

tricular administration of 2DG in wild-type and Kir6.2�/� mice. Plasma glucagon levels were measured before and

15min after the injection of 2DG (1mg/body) (� � po0.02). Open columns and filled columns represent wild-type and

Kir6.2�/� mice, respectively. (Reprinted with permission from Miki et al., 2001 copyright 2001 Nature Publishing

Group.)


the brain, several hormones associated with appetite are generated in the peripheral tissues and acton the hypothalamus to regulate food intake via the blood stream. These hormones includeinsulin, leptin, and ghrelin (Kojima et al., 1999; Nakazato et al., 2001). Leptin and insulin now arethought to be critical signaling molecules from peripheral tissues, since the central administrationof leptin (Woods et al., 1979) and insulin (Campfield et al., 1995) both reduces food intake. Inaddition, genetic mutation in leptin (Zhang et al., 1994) or the leptin receptor (Chen et al., 1996)and genetic disruption of the insulin receptor (Bruning et al., 2000) are responsible for the obesityand/or hyperphagia in the mice. Furthermore, leptin (Spanswick et al., 1997) and insulin(Spanswick et al., 2000) have been shown to inhibit hypothalamic neurons by activating KATP

channels. Accordingly, it is tempting to learn if the anorexic effects of leptin and insulin aremediated by activation of the KATP channels. However, this was not the case in Kir6.2�/� mice,as the anorexic effect of leptin was similar in Kir6.2�/� and wild-type mice (Fig. 8b). Since theserum level of leptin (Considine et al., 1996) and insulin (Bagdade et al., 1967) correlate well tobody fat content, these two hormones are so-called ‘lipostatic’ signals that reflect the body fatcontent (Woods et al., 1998; Schwartz et al., 2000). In contrast, since blood glucose levels andintracellular [ATP] in brain continuously fluctuate, the activity of KATP channels in brain couldmediate short-term ‘‘glucostatic’’ signals (Woods et al., 1998). However, further investigation will

Fig. 7. Cell firing of VMH neurons of control and Kir6.2�/� mice. VMH neurons of Kir6.2�/� are defective in glucose

sensing. Shown are cell-attached recordings of spontaneous activity in response to increase in extra-cellular glucose

concentration from 2.5 to 25mM (upper panels). After cell-attached recordings, the same neurons were repatched for

whole-cell recordings to identify the neuronal cell type (middle panels). VMH neurons of Kir6.2�/� already exhibit

spontaneous activity with a higher frequency compared to control at 2.5mM glucose, and no further increase in activity

is observed in response to increased glucose concentration (right upper panels). Cell firing rates in low and high

concentrations of glucose are summarized in the bottom panels. The values are means7s.e.m (�po0:01). (Reprintedwith permission from Miki et al., 2001 copyright 2001 Nature Publishing Group.)


be required to clarify the interaction between leptin- or insulin-receptor signaling and KATP

channel activity.

6.2. Role of KATP channels in the substantia nigra

The most intense high-affinity binding of [3H]glibenclamide in the brain is detected in the SNr,(Mourre et al., 1989; Hicks et al., 1994), suggesting high expression of the �-cell type KATP

channel in this nucleus. Because the SNr acts as a central gating system in the propagation ofseizure (Iadarola and Gale, 1982; De Sarro et al., 1984; Depaulis et al., 1994) and generalizedseizures can be evoked by metabolic stresses such as hypoxia and hypoglycemia, these KATP

channels could well be involved in the development of seizure during ATP-depleted conditions.Studies of Kir6.2�/� mice revealed that responses to brief (150 s) hypoxia caused by oxygendeprivation differed in Kir6.2�/� and wild-type mice (Yamada et al., 2001). The wild-type mice allremained sedated during the challenge and revived normally. In contrast, the Kir6.2�/� mice allresponded with a myoclonic jerk followed by severe tonic-chronic convulsion and death.Electroencephalogram (EEG) and electromyogram (EMG) revealed a sequence of seizurepatterns in conscious Kir6.2�/� mice challenged with hypoxia (5.4% O2) contrasting with wild-type mice under the same conditions, suggesting that KATP channels participate in determiningthe threshold of seizure during hypoxia. In the normal state, the firing rate of SNr neurons wasnot significantly different in wild-type and Kir6.2�/� mice. However, in brief hypoxia (90 s), wild-type neurons showed a marked decrease in firing rate to about one-third of that before hypoxia,whereas the firing rate of the Kir6.2�/� neurons increased about 1.8-fold, suggesting that the KATP

channel-mediated suppressive effect on SNr activity is sufficient to reverse facilitation duringhypoxia (Fig. 9). By inside-out patch recordings, KATP channel currents with properties similar tothose of the �-cell KATP channel were detected in the SNr GABAergic neurons of wild-type mice,while no such currents were observed in the neurons of Kir6.2�/� mice. Single-cell RT-PCRshowed the KATP channels in SNr neurons to be composed of Kir6.2 and SUR1 (Liss et al., 1999).

Fig. 8. The effects of 2DG, leptin, and NPY on food intake in wild-type and Kir6.2�/� mice. Food intake for 3 h after

saline injection (X) was measured. Three days later, food intake for 3 h after 2DG administration (Y) was measured in

the same mice. The increment in food intake in the same mice was calculated by subtracting X from Y. The increments

were: wild-type, 0.27970.039 g/3 h, n ¼27; Kir6.2�/�, 0.09570.037 g/3 h, n ¼28, �po0:002 (b). Effect of leptin on food

intake. The decrement in food intake due to leptin was calculated similarly to (a). The cumulative food intake was

measured over 24 h. The values are means7s.e.m (NS, not significant). (Reprinted with permission from Miki et al.,

2001.)


The membrane potentials of wild-type and Kir6.2�/� SNr neurons were shifted in the oppositedirection when these neurons were perfused with hypoxic solution. Wild-type SNr neurons, whenperfused with hypoxic solution, were hyperpolarized, and the hyperpolarization was reversed bytolbutamide. Diazoxide also produced hyperpolarization. In contrast, Kir6.2�/� neurons showedno such hyperpolarization but were depolarized. In addition, using acute brain slice preparations,postsynaptic KATP channels have been shown to be critical in the hypoxia-induced inactivation ofthe SNr neurons. Accordingly, it is suggested that the KATP channel in SNr neurons acts as ametabolic-sensor that prevents seizure propagation during hypoxic stress.

6.3. Role of the KATP channels in forebrain

Mice overexpressing SUR1 in forebrain also have been made (Hernandez-Sanchez et al., 2001).These mice exhibit a 9–12-fold increase in the density of [3H]glibenclamide binding to cortex,hippocampus, and striatum. The animals are resistant to kainic acid-induced seizures. Kainicacid-treated transgenic mice showed no significant loss of hippocampal pyramidal neurons,whereas wild-type mice lost pyramidal neurons significantly in the CA1-3 regions after kainateadministration. These results indicate that overexpression of SUR1 alone in the forebrain protectmice from seizures and neuronal damage without interfering with locomotor or cognitivefunction, suggesting that SUR1-containing KATP channels play a role in protecting neurons in thisarea under acute or chronic metabolic stress such as focal or global ischemia (Heurteaux et al.,1993). Overexpression of SUR1 in forebrain also prevented the development of seizure intransegenic mice (Hernandez-Sanchez et al., 2001).

7. Roles of the KATP channels in skeletal muscle

KATP channels are also present in the skeletal muscles (Spruce et al., 1985). Thepharmacological and electrophysiological properties of the channel have been examined in

Fig. 9. Effect of hypoxia on firing rate of SNr neurons in acute slice preparations. Brief (90 s) hypoxia (solid bar)

produced a marked decrease in firing rate of SNr neuron of control mouse (a) but increased it in Kir6.2�/� mouse (b).

Insets represent traces of unit activities before, during, and after hypoxia (arrows). Spike amplitude increased in wild-

type but decreased in KO mouse during hypoxia. (c) Hypoxia-induced changes in firing rate of control (open circles,

n ¼9 from nine mice) and Kir6.2�/� mice (solid triangles, n ¼8 from eight mice). Data points represent means7SE.

(Reprinted with permission from Yamada et al., 2001 copyright 2001 American Association for the Advancement of

Science.)


native skeletal muscles isolated from several vertebrates (Weik and Neumcke, 1989; Vivaudouet al., 1991; Allard and Lazdunski, 1992, 1993). While the unitary conductance of these muscles issimilar (55B75 pS), the electrophysiological properties of the KATP channels recorded in nativeskeletal muscles vary to some extent in terms of sensitivities to ATP, glibenclamide, and KCOs(Allard and Lazdunski, 1992; Davis et al., 1991; Barrett-Jolley and Davies, 1997). Thesedifferences could be in part due to differences in experimental conditions, species, or splicevariations of SUR2 (Chutkow et al., 1999). Although the electrophysiological properties ofchannels reconstituted by Kir6.2 and SUR2A (Inagaki et al., 1996; Okuyama et al., 1998) aresimilar to those of the KATP channels recorded in native skeletal muscles, the molecularconstituents of the skeletal muscle KATP channel in native tissues has not been determined.

7.1. KATP channels in muscle fatigue

One important characteristic of metabolism in skeletal muscle is that the metabolic rate ischanged dramatically by muscle contraction. As a consequence, a large change in ATPconsumption occurs and the activity of the KATP channel of skeletal muscles is altered. Indeed,KATP channels remain inactive at rest and are activated during fatigue (Light et al., 1994).Opening of the KATP channels increases outward K+ currents that contribute to shorteningaction potential duration (Gramolini and Renaud, 1997), and protecting from intracellular Ca2+

overload (Burton and Smith, 1997). Using openers and blockers of the KATP channel, severalstudies suggested that KATP channels may contribute to a decline in tetanic force (Duty and Allen,1995), although the possibility is still controversial (Comtois et al., 1995; Van Lunteren et al.,1998). In addition, it has been shown that the KCO pinacidil suppresses contractility duringfatigue in mouse soleus muscles while the KATP channel blocker glibenclamide does not affect therate of fatigue (Matar et al., 2000). The role of KATP channels in skeletal muscle during fatiguewas examined in Kir6.2�/� mice (Gong et al., 2000). During fatigue, although there is nodifference in the decrease in tetanic force in EDL and soleus muscle between wild-type andKir6.2�/� mice, Kir6.2�/� mice showed a significant increase in resting tension when compared towild-type mice. The recovery of tetanic force after fatigue was significantly less in muscle of 1 yearold Kir6.2�/� mice than age-matched wild-type mice, suggesting that the major function of theKATP channel during fatigue is to reduce development of resting tension and that the KATP

channel plays an important role in protecting muscle function in older mice.

7.2. KATP channels in glucose uptake

In addition to the role in contractile activity of skeletal muscle, KATP channels may also beinvolved in glucose uptake in skeletal muscles (Pulido et al., 1996). Some sulfonylureas have beenshown to improve glycemic control in patients with type 2 diabetes mellitus by acting on extra-pancreatic tissues to reduce insulin resistance (Wang et al., 1989). However, no direct evidence hasbeen provided for the involvement of the skeletal muscle KATP channel in the extra-pancreaticeffects of sulfonylureas. Interestingly, the glucose-lowering effect of insulin is enhanced inKir6.2�/� mice (Miki et al., 1998), suggesting that insulin sensitivity is increased in Kir6.2�/�

mice. The basal level and insulin-stimulated glucose uptake in gastrocnemius of Kir6.2�/� micewere found to be increased significantly both in vivo and in vitro, compared to wild-type mice,


although the effect of disruption of the KATP channel vary between the different types of muscle(Miki et al., 2002b). Since there is no KATP channel activity in the plasma membrane of skeletalmuscles of Kir6.2�/� mice, these findings suggest that the KATP channel is involved in glucoseuptake in skeletal muscle.The involvement of KATP channels in glucose uptake also is indicated by SUR2 deficient

(SUR2�/�) mice (Chutkow et al., 2001). SUR2�/� mice show lower fasting and fed serum glucose,improved glucose tolerance during glucose tolerance test, and a more rapid and severehypoglycemia after administration of insulin. Enhanced glucose use was observed during in vivohyperinsulinemic euglycemic clamp studies during which SUR2�/� mice required a greater glucoseinfusion rate to maintain blood glucose level. The enhanced insulin action was intrinsic to theskeletal muscle, as in vitro insulin-stimulated glucose transport was 1.5-fold greater in SUR2�/�

muscle than in wild-type. Considering these findings together, the KATP channel in skeletalmuscles clearly participates in glucose uptake, but the mechanism is unclear at present.

8. Roles of KATP channels in cardiovascular tissues

KATP channels are present both in cardiomyocytes and vascular smooth muscles, but theirelectrophysiological properties differ greatly. The plasma membrane KATP channel incardiomyocytes exhibits single-channel conductance of B70–90 pS (Ashcroft and Ashcroft,1990), while single-channel conductance of vascular smooth muscles is B30 pS, much smallerthan that of cardiomyocytes (Kajioka et al., 1991). In addition, the KATP channel incardiomyocytes opens when the ATPi is removed (Noma, 1983), while the vascular smoothmuscle KATP channel requires nucleotide diphosphates intracellularly (NDPis) as well as thedepletion of ATPi (Kajioka et al., 1991). Furthermore, diazoxide opens the vascular smoothmuscle KATP channel (Quayle et al., 1995), while it does not open the cardiomyocyte KATP

channel (Faivre and Findlay, 1989). KATP channels in native cardiomyocytes are activated byKCOs such as pinacidil, levcromakalim, and aprikalim but not by diazoxide (Sanguinetti et al.,1988; Arena and Kass., 1989; Escande et al., 1989; Thuringer and Escande, 1989). In contrast, thesmooth muscle KATP channel is blocked by glibenclamide and tolbutamide (Faivre and Findlay,1989; Hamada et al., 1990; Findlay, 1992).Reconstitution experiments have suggested that the cardiomyocyte KATP channel is composed

of Kir6.2 and SUR2A (Inagaki et al., 1996) and the vascular smooth muscle KATP channel ofKir6.1 and SUR2B (Yamada et al., 1997). Direct evidence for the molecular composition of thesechannels in native tissues has not been provided.

8.1. Molecular structure of sarcolemmal KATP channels of cardiomyocytes

As mentioned above, the KATP channel reconstituted by Kir6.2 and SUR2A exhibits a single-channel conductance of B80 pS and is closed by glibenclamide (IC50, B150 nM) and bytolbutamide very weakly (IC50, >100mM) (Gribble et al., 1998; Okuyama et al., 1998), and isopened by the KCO pinacidil (EC50, B10 nM). In contrast, diazoxide does not activate Kir6.2/SUR2A channels (Inagaki et al., 1996; Okuyama et al., 1998). All of these characteristics areconsistent with those of the KATP channels recorded in native cardiomyocytes, suggesting that


Kir6.2 and SUR2A comprise the cardiomyocyte KATP channel. However, several reports suggestthe heteromultimerization of Kir6.1 and Kir6.2 subunits within the same channel complex (Konoet al., 2000; Pountney et al., 2001) while others do not (Seharaseyon et al., 2000). Thus, themolecular structure of the pore-forming subunit of the KATP channel in native cardiomyocyteshas remained controversial. To clarify the molecular structure of the pore-subunit of thesarcolemmal KATP channel of heart, cardiomycytes from both Kir6.1�/� and Kir6.2�/� mice wereexamined (Suzuki et al., 2001; Miki et al., 2002a, b). KATP channel currents (single-channelconductance,B79 pS) were found to be sensitive to ATP and glibenclamide in membrane patchesof wild-type but not in Kir6.2�/� cardiomyocytes. In whole-cell membrane current recordings,pinacidil produced an outward current that was inhibited by glibenclamide, confirming that itflows through KATP channels in wild-type cardiomyocytes but not in Kir6.2�/� cardiomyocytes(Fig. 10). These findings demonstrate directly that Kir6.2 is essential for sarcolemmal KATP

channel function in cardiomyocytes. This was further confirmed by Kir6.2 rescue experiments. Inneonatal myocytes of Kir6.2�/� mice, adenoviral gene transfer of Kir6.2 restored the plasmamembrane KATP channel current that was activated by P-1075 and blocked by HMR1098, whichare a specific activator and a blocker, respectively, of the sarcolemmal KATP channel (Suzuki et al.,2001; Miki et al., 2002a, b). Since the KATP channel in cardiomyocytes was absent in Kir6.2�/�

mice but present in Kir6.1�/� mice, Kir6.2 is an essential constituent of the KATP channels incardiomyocytes.

8.2. Sarcolemmal KATP channels in heart

In heart, the KATP channels are activated during ischemic and/or hypoxic conditions, and areresponsible for the increase in K+ efflux and shortening of action potential duration (APD)(Faivre and Findlay, 1990; Nichols and Lederer, 1991; Nichols et al., 1991; Findlay, 1994).Shortening of APD leads to a decrease in Ca2+ influx, which may protect against cardiac damageduring ischemia or hypoxia. APD was shortened by pinacidil in cardiomyocytes of control micebut not in those of Kir6.2�/� mice (Suzuki et al., 2001). Pinacidil elicits a negative inotropic effectby opening the KATP channels in cardiomyocytes, which prevents Ca2+ influx through VDCCsand/or Na+–Ca2+ exchangers. In fact, pinacidil significantly decreased cardiac contractilefunction in wild-type hearts, as assessed by LVDP (left ventricular developed pressure) and itsmaximal derivative (dp=dt), but did not elicit any change in either LVDP or maximal dp=dt inKir6.2�/� mice, indicating that the pinacidil-induced negative inotropic effect on the heart ismediated by opening of the sarcolemmal KATP channels (Fig. 10). The action potential shorteningin hypoxic and ischemic conditions decreases Ca2+ influx, and possibly results in reduction ofmechanical contraction, amelioration of Ca2+ overload, and energy sparing.Brief intermittent periods of ischemia paradoxically protect the myocardium against more

prolonged ischemic insult, and result in a marked reduction of infarct size, a phenomenon knownas ischemic preconditioning (IPC) (Cohen et al., 2000). Sarcolemmal KATP (sarcKATP) channelswere originally proposed to play an important role in the cardioprotective effect, because KCOsmimicked cardioprotection and the KATP channel blocker glibenclamide abolished the IPC (Grossand Auchampach, 1992; Mizumura et al., 1995; Lawrence et al., 2001). However, recent studiessuggest that activation of the mitochondrial KATP (mitoKATP) channel (Paucek et al., 1992)rather than the sarcKATP channel is responsible for the cardioprotective effect of IPC and KCOs


Fig. 10. In vivo myocardial infarction studies. (a–d) Representative photographs of myocardial slices of wild-type mice

without IPC (WT-CON) (a), wild-type mice with IPC (WT-IPC) (b), Kir6.2�/� mice without IPC (KO-CON) (c), and

Kir6.2�/� mice with IPC (KO-IPC) (d) are shown. A schema of each photograph is indicated below; infarct area is

expressed as gray, viable myocardium in area at risk (AAR) as red, and non-ischemic area as blue. Scale bar = 2mm.

(e) Myocardial infarct size expressed as percentage of AAR. The infarct size of the WT-IPC group was significantly

smaller than that of the WT-CON group. There are no significant differences in the infarct size between WT-CON and

KO-CON or between KO-CON and KO-IPC. Values are expressed as mean7SE (NS, not significant, �po0:01 vs WT-

CON). (Reprinted with permission from Suzuki et al. (2002) copyright 2002 American Society for Clinical

Investigation.)


(Garlid et al., 1997; Liu et al., 1998; Ghosh et al., 2000). mitoKATP channel current was originallyidentified in the inner membrane of rat liver mitochondria (Inoue et al., 1991; Garlid et al., 1996;Szewczyk et al., 1996). However, the molecular identity of the channel has not been established.Therefore, molecular biological approaches such as reconstitution of cloned channels or genetargeting technique are not applicable for the study of mitoKATP channel function. In this regard,the direct evidence for the role of the mitoKATP channel in IPC is still lacking. In contrast, thecontribution of sarcKATP channels to IPC can be assessed in Kir6.2�/� mice, which completelylack sarcKATP channels (Suzuki et al., 2001, 2002). Since Kir6.2�/� mice retain normalflavoprotein oxidation, mitoKATP channel, if any, does not play a major role in IPC in mice(Suzuki et al., 2001, 2002). The infarct size of wild-type mice with IPC (three cycles of 3minutes ofcoronary occlusion before long term occlusion) was significantly smaller than in wild-type micewithout IPC (Fig. 11). However, there was no significant difference between the infarct size ofKir6.2�/� mice with IPC and that of Kir6.2�/� mice without IPC. In in vitro experiments usingLangendorff-perfused hearts following global ischemia/reperfusion, the increase of left ventricularend-diastolic pressure (LVEDP) during ischemia was more marked in Kir6.2�/� mice than in wild-type mice, while the recovery of contractile function was less marked in Kir6.2�/� mice. Treatmentwith HMR1098, a sarcolemmal KATP channel blocker, but not with 5-hydroxydecanoate, aputative mitochondrial KATP channel blocker, produced deterioration of contractile function inhearts of wild-type mice comparable to that in Kir6.2�/� mice. These findings suggest that thesarcolemmal KATP channels in heart are important in modulating ischemia/reperfusion injury.Under stressed conditions such as life threatening events, an elevation in catecholamines

generates a superior level of bodily performance, including an escalation of cardiac function. Theelevation in catecholamines simultaneously triggers a feedback mechanism to preserve cellularhomeostasis. It recently has been shown that cardiomyocytes of Kir6.2�/� mice have a defect in anelectrical feedback mechanism responsible for cellular calcium handling, and that the mice exhibitarrythmia and sudden death under vigorous sympathetic stimulation (Zingman et al., 2002).Thus, in addition to a cytoprotective role against ischemia, the KATP channels in cardiomyocytesare also required in adaptation to stress.Elevation of the ST segment in electrocardiogram (ECG) is characteristic of acute myocardial

ischemia. Despite its clinical importance, the molecular mechanism underlying ST elevationremains unclear. Activation of sarcolemmal KATP channels by ischemic ATP depletion has beensuggested to be involved in this mechanism (Wilde, 1996; Kleber, 2000). During cardiac ischemia,intracellular ATP decreases, which activates the KATP channels, leading to the shortening ofaction potential. These cellular events cause electrical inhomogeneity within the heart, generatinginjury currents between ischemic and normal cells and shifting the ST segment in the ECG.Whether or not ST elevation occurs during acute myocardial ischemia was evaluated in theKir6.2�/� mice by ligating the left anterior descending (LAD) artery. Interestingly, while LADligation elicited distinct ST elevation in wild-type mice, ST elevation was markedly suppressed inKir6.2�/� mice, indicating that the Kir6.2-pore-forming KATP channels play a role in thiselectrocardiographic phenomenon (Li et al., 2000).Mice expressing the double mutant Kir6.2 [DN2-30, K185Q] in heart have also been generated

(Koster et al., 2001). This mutant results in the KATP channel having about 30-fold lower ATP-sensitivity when co-expressed with SUR1 compared to the wild-type Kir6.2/SUR1 channel. As aresult, the sarcolemmal KATP channels in heart of the transgenic mice were extremely insensitive


to ATP inhibition (half-maximal inhibition K1/2, B2.7mmol/l), compared to wild-type mice(K1/2, B51mmol/l), as assessed by excised patches. Despite the severe insensitivity to ATP, thechannels are inactive in intact cells, and there is no obvious difference in cardiac electrical activity.Thus, in striking contrast to the effects of a similar transgene in pancreatic �-cells (Koster et al.,2000), there is a profound tolerance for reduced ATP-sensitivity in cardiac KATP channels. Thus,the metabolic regulation of KATP channels in the heart and pancreatic �-cells differs significantly.

8.3. KATP channels in vascular smooth muscle

KATP channel currents have been recorded in many vascular smooth muscles (Quayle et al.,1997). Most were recorded in excised or cell-attached patches and identified as KATP channels onthe criteria of inhibition by ATP or glibenclamide, requirement for the presence of NDPs at thecytoplasmic face of the patch, and activation by metabolic inhibition or KCOs (Standen et al.,1989; Kajioka et al., 1991; Beech et al., 1993). Nevertheless, they appear to be heterogeneous at

Fig. 11. Effects of the KCO pinacidil (PIN) and coapplication of glibenclamide (GLB) on whole-cell membrane

currents (a and b) and action potentials (d and e) recorded from cardiomyocytes of wild-type (control) (a and d) and

Kir6.2�/� mice (b and e). Current densities at 0mV in control and Kir6.2�/� cardiomyocytes are summarized in panel

(c) and (f). Summary of changes in action potential duration (APD90) after (in mmol/l) application of PIN 100 and PIN

100 plus GLB 1 in wild-type and Kir6.2�/� cardiomyocytes. There is no shortening of APD by pinacidil �Po0:05; � �po0:01 vs control (CON); #po0.01 vs 100mmol/l pinacidil. (Reprinted with permission from Suzuki et al., 2001

copyright 2000 American Heart Association.)


least in unitary conductance of the channels, but they can be roughly divided into two broadgroups: channels with small or medium conductance and channels with large conductance,suggesting diversity in the molecular structure of vascular smooth muscle KATP channels.Reconstituted channels from Kir6.1 and SUR2B are activated by NDPs such as UDP and are

inhibited by the sulfonylurea glibenclamide, properties similar to those of the KATP channels innative vascular smooth muscle, also called the KNDP channel (Yamada et al., 1997). The unitaryconductance of the channel reconstituted in HEK293T was 33 pS, comparable to that of small ormedium conductance KATP channels in native vascular smooth muscles (Quayle et al., 1997).Recent studies of Kir6.1�/� mice and SUR2�/� mice have shown that while smooth muscles inaorta of wild-type mice exhibit pinacidil-induced K+ channel currents that are blocked byglibenclamide, those of Kir6.1�/� mice and SUR2�/� mice both lack such currents. Accordingly,Kir6.1 and SUR2 (most likely Kir6.1/SUR2B) constitute the KATP channel in vascular smoothmuscles.KCOs are greatly divergent chemical compounds, including cromakalim, pinacidil, levcroma-

kalim, minoxidil, nichorandil, and diazoxide (Jahangir et al., 2001). They exhibit a wide variety oftherapeutic potential for treatment of bronchial asthma, myocardial ischemia, glaucoma, andmale baldness (Jahangir et al., 2001). In addition, most KCOs are known to lower arterial bloodpressure by relaxing vascular smooth muscles, presumably by opening the vascular KATP channels(Weston and Edwards, 1992). However, there has been no direct evidence that the vasodilatingeffects of KCOs are mediated by opening the KATP channels. In addition, the consequence ofdisruption of vascular smooth muscle KATP channels in vivo is unknown. Recently, a clue wasobtained from studies of two animal models; Kir6.1�/� mice (Miki et al., 2002a) and SUR2�/�

mice (Chutkow et al., 2002). Interestingly, both mouse models exhibit very similar phenotypes. Inboth knockout mice there is no KATP channel activity in aortic smooth muscles and thevasodilating effect by pinacidil is abolished. These findings indicate that the vasodilating effects ofKCOs are mediated by opening of Kir6.1/SUR2 channels in the vascular smooth muscles.

8.4. Mouse model of prinzmetal (variant or vasospastic) angina

Kir6.1 null mice are prone to premature death, so-called sudden death, the majority dyingbetween 30 and 40 days after birth (Fig. 12). Surprisingly, all of the knockout mice examinedshowed spontaneous elevation of ST segments, followed by atrioventricular (AV) blocks ofvarious degrees (Fig. 13). In contrast, there were no such abnormalities in the control mice.Apparently, myocardial ischemia is the cause of sudden death in these Kir6.1�/� mice. In situhybridization revealed that Kir6.1 signals are detected widely in heart, but most clearly in thecardiomyocytes of wild-type mice. In addition, the signals are also evident in the vascular smoothmuscles of the coronary arteries of wild-type mice. No such signals were detected incardiomyocytes and coronary arteries of Kir6.1�/� mice. In both wild-type mice and Kir6.1�/�

mice, pinacidil produced outward currents that were blocked by glibenclamide, indicating anKATP current and showing that Kir6.1 is not a component of the sarcolemmal KATP channel incardiomyocytes. KCOs are known to lower blood pressure by relaxing vascular smooth muscles,presumably by opening the KATP channel. Intravenous injection of pinacidil decreased the meanarterial pressure significantly in control mice but not in Kir6.1�/� mice, indicating a loss of thevasodilatation response to pinacidil in Kir6.1�/� mice. The vasodilation response of aorta to


pinacidil in Kir6.1�/� mice also was reduced remarkably in vitro, as assessed by changes in theisometric tension of aortic rings. Pinacidil elicited significant K+ currents that were blocked byglibenclamide in aortic smooth muscle cells in wild-type mice, but failed to evoke significant K+

currents in the knockout mice, clearly indicating that Kir6.1 is an essential component of theKATP channel in vascular smooth muscles. Vasospasms were induced in Kir6.1�/� mice both invivo and in vitro (Fig. 14) by application of methylergometrine, an ergot alkaloid that stimulatesserotonergic receptors and directly triggers vasoconstriction of vascular smooth muscles. Thesefindings together indicate that the phenotype of Kir6.1�/� mice resembles that of Prinzmetal(variant or vasospastic) angina in humans (Prinzmetal et al., 1959; Maseri, 1987). It also has beenshown that disruption of SUR2 also mimics the phenotype of Prinzmetal (variant or vasospastic)angina (Chutkow et al., 2002). Interestingly, SUR2�/� mice exhibit significantly elevated restingblood pressures, and the coronary arteries of SUR2�/� mice show focal arterial narrowings. Invivo monitoring demonstrated transient coronary artery vasospasm in SUR2�/� mice, andtreatment with a calcium channel antagonist reduced the vasospastic episodes. Studies ofKir6.1�/� and SUR2�/� mice make it clear that the Kir6.1/SUR2 channel is critical in theregulation of vascular tonus, especially in the coronary arteries.Prinzmetal angina is a relatively rare form of unstable angina (Prinzmetal et al., 1959)

characterized by the occurrence of the attack at rest and by elevation of ST segments on ECGduring the attack (Maseri, 1987). The pathophysiology of Prinzmetal angina is associated withhypercontractility of epicardial coronary arteries. Prinzmetal angina is diagnosed by elevated STsegments on ECG during the attack or by the induction of coronary spasm using ergot alkaloidsor acetylcholine. Indeed, the coronary artery pressure was increased under the resting conditions,

Fig. 12. Survival of Kir6.1+/+ (wild-type) mice (thick line, n ¼ 36), Kir6.1+/� mice (dotted line, n ¼ 67), and Kir6.1�/�

(thin line, n ¼ 27) mice after birth. (Reprinted with permission from Miki et al., 2002 copyright 2002 Nature Publishing

Group.)


and administration of the ergot alkaloid methylergometrine elicited a significant elevation of STsegments in Kir6.1�/� heart.Many signal molecules are thought to be involved in the occurrence of coronary vasospasm

(Feliciano and Henning, 1999), including adenosine (Belardinelli et al., 1989), nitric oxide (NO)

Fig. 14. Methylergometrine-induced changes in coronary perfusion pressure (CPP). Representative traces of CPP

measured in control (left) and Kir6.1�/� (right) hearts are shown. A coronary spasm in Kir6.1�/� mouse (right) is

indicated by arrow. (Reprinted with permission from Miki et al., 2002 copyright 2002 Nature Publishing Group.)

Fig. 13. Representative ECGs from control and Kir6.1�/� mice using radio telemetry. (a) Spontaneous ST elevations

and AV block in ECG recorded from Kir6.1�/� mouse. A longer period recording (16 s) including ST elevation (solid

line) and AV block (dotted line) is shown. Typical waveforms at time points indicated in the chart (1, 2, and 3) are

shown at higher magnification. ST elevation appears in all Kir6.1�/� mice examined (n ¼ 4). The elevation lasted for a

short time, and then returned to baseline. After a short latency period, the ST elevation was followed by AV blocks of

various degrees as shown in c. (b) ECGs of 1st (left), 2nd (middle), and 3rd (right) AV block in Kir6.1�/� mice. ‘‘P’’

indicates P wave. (Reprinted with permission from Miki et al., 2002 copyright 2002 Nature Publishing Group.)


(Ishibashi et al., 1998), and other endothelium-derived factors (Mombouli and Vanhoutte,1999) such as prostacyclin and endothelin-1 (Kinlay et al., 2001; Schiffrin et al., 1997). Adenosineis generated by degradation of adenine nucleotides (ADP and ATP) when the energy sourcefor ATP is depleted in cardiomyocytes by metabolic inhibition or hypoxia. As a result, adenosineis released from the cardiomyocytes into the interstitial space and reaches to the coronaryartery where it elicits the coronary dilatory effects. Nitric oxide (NO) is generated in theendothelium by endothelial nitric oxide synthase (eNOS), which activates guanylatecyclase thereby increasing intracellular cyclic guanosine monophosphate (cGMP), whichcauses dilatation of coronary arteries. However, no gene associated with coronary vasospasmhas been identified, nor have animal models of coronary vasospasm been available. Accordingly,both Kir6.1�/� and SUR2�/� mice are useful to investigate the molecular mechanism of thepathogenesis of coronary vasospasm. At present, it remains unknown if mutations in theKir6.1 gene (Inagaki et al., 1995b) or SUR2 gene (Chutkow et al., 1996; Aguilar-Bryan et al.,1998) cause Prinzmetal angina in humans. Although vasospastic angina is rare in Caucasians,many reports suggest that it is more common in Japanese (Yasue and Kugiyama, 1997;Beltrame et al., 1999; Pristipino et al., 2000). Therefore, it is important to learn if geneticalterations of Kir6.1 or SUR2 are associated with Prinzmetal angina in various ethnicpopulations.

9. Concluding remarks

KATP channels belong to the inward rectifier K+ channel family. Distinct from otherinward rectifier K+ channels, in which pore-forming subunits by themselves producechannel function, KATP channels are unique in that the pore-forming subunit Kir6.x and theregulatory subunit SUR both are required for functional expression. KATP channels areinvolved in many cellular responses by coupling cell metabolism to membrane potential. Recently,genetic manipulation of the KATP channel subunits, Kir6.2, Kir6.1, SUR1, and SUR2,has directly demonstrated many expected and unexpected physiological and pathophysiologicalroles of KATP channels. Based on studies of KATP channel knockout mice and transgenicmice as well as naturally occurring mutations in humans, it is now clear that KATP channels,as metabolic sensors, are critical in protective mechanisms against a variety of acute meta-bolic stresses, including hyperglycemia, hypoglycemia, ischemia, and hypoxia. However,many issues remain to be resolved. For example, the physiological and pathophysiologicalroles of Kir6.2-containing KATP channels in brain are largely still unknown. Although Kir6.1is expressed at high levels in cardiomyocytes, its significance is unclear. The role of Kir6.1-containing KATP channels in kidney, in which the expression of Kir6.1 is induced markedlyby ischemia at transcription level (Akao et al., 1997; Sgard et al., 2000), is also unclear. Tissuespecific disruption of each KATP channel subunit (conditional knockout) would determinethe roles of each subunit in the various tissues more specifically. KATP channel subunit-nullmice and various transgenic mice expressing a mutant KATP channel subunit should be usefulfor further elucidation of the pathophysiological roles of KATP channels in various metabolicand cardiovascular disorders and for the development of novel drugs targeting KATP

channels.


Acknowledgements

The authors especially thank the coworkers and collaborators involved in the studies fromour laboratory who are cited in this review. The studies in our laboratory are supported byGrant-in-Aid for Creative Basic Research (10NP0201) from the Ministry of Education, Science,Sports and Culture, by a Scientific Research Grant from the Ministry of Health, Labour, andWelfare, Japan; and by grants from Novo Nordisk Pharma Ltd., from Takeda ChemicalIndustries Ltd., and from the Yamanouchi Foundation for Research on Metabolic Disorders.

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physiological and pathophysiological roles of atp-sensitive k+ channels

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