Molecular aspects of ATP-sensitive K+ channels in the cardiovascular system and K+ channel openers

Pharmacology & Therapeutics 85 (2000) 39–53

Associate editor: M. Endoh

Molecular aspects of ATP-sensitive K1 channels in the cardiovascular system and K1 channel openers Akikazu Fujita, Yoshihisa Kurachi* Department of Pharmacology II, Faculty of Medicine and Graduate School of Medicine, Osaka University, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan

Abstract ATP-sensitive K1 (KATP) channels are inhibited by intracellular ATP (ATP i) and activated by intracellular nucleoside diphosphates and thus, provide a link between cellular metabolism and excitability. KATP channels are widely distributed in various tissues and may be associated with diverse cellular functions. In the heart, the K ATP channel appears to be activated during ischemic or hypoxic conditions, and may be responsible for the increase of K 1 efflux and shortening of the action potential duration. Therefore, opening of this channel may result in cardioprotective, as well as proarrhythmic, effects. These channels are clearly heterogeneous. The cardiac K ATP channel is the prototype of KATP channels possessing z80 pS of single-channel conductance in the presence of z150 mM extracellular K1 and opens spontaneously in the absence of ATPi. A vascular KATP channel called a nucleoside diphosphate-dependent K 1 (KNDP) channel exhibits properties significantly different from those of the cardiac KATP channel. The KNDP channel has the single-channel conductance of z30–40 pS in the presence of z150 mM extracellular K1, is closed in the absence of ATP i, and requires intracellular nucleoside di- or triphosphates, including ATPi to open. Nevertheless, KATP and KNDP channels are both activated by K1 channel openers, including pinacidil and nicorandil, and inhibited by sulfonylurea derivatives such as glibenclamide. It recently was found that the cardiac K ATP channel is composed of a sulfonylurea receptor (SUR)2A and a two-transmembrane-type K 1 channel subunit Kir6.2, while the vascular K NDP channel may be the complex of SUR2B and Kir6.1. By precisely comparing the functional properties of the SUR2A/Kir6.2 and the SUR2B/Kir6.1 channels, we shall show that the single-channel characteristics and pharmacological properties of SUR/Kir6.0 channels are determined by Kir and SUR subunits. respectively, while responses to intracellular nucleotides are determined by both SUR and Kir subunits. © 1999 Elsevier Science Inc. All rights reserved. Keywords: ATP-sensitive K1 channel; Sulfonylurea receptor; Inwardly rectifying K1 channel; Kir6.0; K1 channel openers; Cardiovascular system Abbreviations: ADPi, intracellular ADP; ATPi, intracellular ATP; ha, hamster; IKATP, ATP-sensitive K1 conductance; KATP, ATP-sensitive K1; KCO, K1 channel opener; KNDP, nucleoside diphosphate-dependent K1; m, mouse; NBF, nucleotide-binding fold; NDP, nucleoside diphosphate; NDPi, intracellular nucleoside diphosphate; PIP2, phosphatidylinositol bisphosphate; r, rat; RT-PCR, reverse transcription-polymerase chain reaction; SUR, sulfonylurea receptor.

Contents 1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Regulation of ATP-sensitive K1 channels by intracellular nucleotides . . . . . . . . . . . . . . . . 3. Pharmacological regulation of ATP-sensitive K1 channels . . . . . . . . . . . . . . . . . . . . . . . . . 4. Molecular structure of ATP-sensitive K1 channels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Molecular heterogeneity of sulfonylurea receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Molecular mechanism of ATP-sensitive K1 channel inhibition by intracellular ATP. . . . . 7. Molecular mechanism of response to intracellular nucleoside diphosphates . . . . . . . . . . . . 8. Molecular mechanism of rundown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. Tel.: 181-6-6879-3512; fax: 181-6-6879-3519. E-mail address: [email protected] (Y. Kurachi) 0163-7258/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S0163-7258(99)00 0 5 0 - 9

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A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53

1. Introduction 1

The ATP-sensitive K (KATP) channel is a weakly inward-rectifying K1 channel that is inhibited by intracellular ATP (ATPi) and activated by intracellular nucleoside diphosphates (NDPis). Thus, it provides a link between the cellular metabolism and excitability. The KATP channel, which was first discovered in heart muscle, has been identified also in a variety of tissues, including pancreatic b-cells, skeletal muscle, smooth muscle, and renal tubular cells, and in the CNS. These KATP channels have been associated with diverse cellular functions, such as insulin secretion from pancreatic b-cells, smooth muscle relaxation, regulation of skeletal muscle excitability, and neurotransmitter release. KATP channels in the cardiovascular system might have a physiological role in modulating cardiac function, particularly under conditions of metabolic stress, such as hypoxia, ischemia, and metabolic inhibition when ATPi is reduced. The KATP channels also exhibit characteristic pharmacological properties: they are selectively inhibited by antidiabetic sulfonylurea derivatives, such as glibenclamide and tolbutamide, and activated by a certain class of vasorelaxants, such as pinacidil, levcromakalim, and nicorandil, which are collectively termed K1 channel openers (KCOs) (Table 1). The cardiac KATP channel may be involved in the increase of K1 efflux and shortening of the action potential duration in the ischemic heart. Both are major factors contributing to the electrophysiological abnormalities that predispose the heart to the development of re-entrant arrhythmias. On the other hand, opening of the cardiac KATP channel also has been implicated as a cardioprotective mechanism underlying ischemia-related preconditioning. In the coronary arteries, the KATP channel is believed to mediate coronary vasodilation, particularly during ischemia, because the vasodilation induced by ischemia, hypoxia, and metabolic inhibition can be prevented by glibenclamide, a blocker of KATP channels, and can be mimicked by cromakalim, one of the KCOs. The KCOs also have been shown to relax vascular smooth muscle in organs other than the heart, and this is also glibenclamide-sensitive. Thus, KATP channels play important roles not only in the pathology of heart muscle, but also in vasculature, and thus, may have potential importance in treatment of heart diseases, such as ischemic heart diseases and hypertension. However, the physiological role of the KATP channels in cardiac myocytes is unclear at present. The pancreatic, cardiac, and skeletal muscle KATP channels all exhibit the single-channel conductance of z70–90 pS under the symmetrical 150 mM K1 conditions (Table 1; Ashcroft, 1988; Terzic et al., 1995; Yamada et al., 1998). This class of KATP channels has often been referred to as “classical-type” KATP channels. Their responses to intracellular nucleotides or to pharmacological agents, however, are diverse (Table 1). The pancreatic and skeletal muscle KATP channels are more sensitive to Mg21-free than Mg21-bound ATPi, while the cardiac KATP channel is equally sensitive to

both forms of ATPi. The pancreatic KATP channel is inhibited by tens of micromolar of tolbutamide, while submillimolar concentrations of this agent are needed to inhibit the cardiac KATP channel. The pancreatic KATP channel is activated by diazoxide, but not by pinacidil, while vice versa for cardiac and skeletal muscle KATP channels. Thus, KCOs exhibit clear tissue specificity. These KATP channels, therefore, can be similar, but distinct, members of the same family of K1 channels. The fact that various KCOs, such as pinacidil and diazoxide, potently induce vasorelaxation in a sulfonylurea derivative-sensitive manner indicates that vascular smooth muscle may also possess some types of KATP channels (Edwards & Weston, 1993). Indeed, many distinct types of K1 channels have been reported as KATP channels in vascular smooth muscle cells (Table 1) (Quayle et al., 1997). However, despite the similarity in pharmacology, these channels exhibit single-channel characteristics and nucleotide-regulation distinct from those of the classical KATP channels. For example, the most commonly observed vascular KATP channel, which is often called the “small-conductance” KATP channel or the “nucleoside diphosphate-dependent” K1 (KNDP) channel (Zhang & Bolton, 1995, 1996; Kamouchi & Kitamura, 1994; Beech et al., 1993a, 1993b; Kajioka et al., 1991), exhibits less than one-half of the single-channel conductance of the classical KATP channels (Table 1). The classical KATP channels open spontaneously when ATPi is removed from the internal surface of the membrane, while the KNDP channel requires NDPis to open. Furthermore, the KNDP channel is reported to be activated rather than inhibited by ATPi. Such striking differences between classical KATP channels and vascular KNDP channels have yielded some confusion as to the identity of so-called KATP channels in vascular smooth muscle cells. In this article, we will review recent progress in molecular dissection of cardiovascular KATP channels and show that the apparent differences between cardiac KATP and vascular KNDP channels can be explained in terms of different combination of subunits with a similar molecular structure.

2. Regulation of ATP-sensitive K1 channels by intracellular nucleotides The KATP channels are known to be regulated by various intracellular factors, such as ATPi (Fig. 1) and nucleoside diphosphates (NDPs). ATPi is the main regulator of classical KATP channels and has two functions: to close the channels and to maintain channel activity in the presence of Mg21 (Takano et al., 1990; Ohno-Shosaku et al., 1987; Findlay & Dunne, 1986; Trube & Heschler, 1984). The first action of ATPi is referred to as the “ligand action” because the binding of ATPi to the KATP channel is assumed to be required for the action and it persists as long as ATPi is bound to the channel. Typically, KATP channels have a very low probability of being open at physiological concentrations of

z1 z2 z2 z2 z1.5 .70% inhibition at 1 mM .90% inhibition at 3 mM

z150 nM

z2–10 nM

z25 nM

z10–200 nM

z5 nM

z5–30 nM

Glibenclamide (IC50)

.70 inhibition at 500 mM

z120 mM

z5–30 mM

z350 mMg

z50 mM

z400 mM

z5–20 mM

Tolbutamide (IC50)

Stimulatory at >1 mMk

z1 mM at 200 mM

z10 mM

No effect at 1 mM

z0.5 mMg

z100 mM

z10–30 mM

.500 mM

Pinacidil (EC50)

Stimulatory at 200 mMk

No effect at 200 mM Stimulatory

z50 mM

z40 mMg

No effect, or inhbitory at 500 mM No effect

220–100 mM

Diazoxide (EC50)

Yamada et al., 1997; Satoh et al., 1998

Inagaki et al., 1995a; Sakura et al., 1995; Nichols et al., 1996; Gribble et al., 1997a Inagaki et al., 1996; Okuyama et al., 1998 Isomoto et al., 1996b

Ashcroft and Ashcroft, 1990; Hamada et al., 1990; Findlay, 1992; Faivre and Findlay, 1989 Weik and Neumcke, 1989, 1990; Vivaudou et al., 1991; Benton and Haylett, 1992; Allard and Lazdunski, 1993; McKillen et al., 1994; Allard et al., 1995 Kajioka et al., 1991; Beech et al., 1993a, 1993b; Kamouchi and Kitamura, 1994; Zhang and Bolton, 1995, 1996; Quayle et al., 1995, 1997

Ashcroft and Ashcroft, 1990

Reference

b

a

The values under the symmetrical 150 mM K1 or similar conditions. Only applicable when ATP inhibits the channels. c In the absence of intracellular Mg21. d In the presence of intracellular Mg21, whose concentration differs among studies, but usually several hundred micromolar to several millimolar in total. e The small-conductance KAPT channel or the KNDP channel. f There is a controversy on the effect of ATP. Some groups reported only a stimulatory effect, while others showed both stimulatory and inhibitory effects. g These data were obtained from smooth muscle cells of the mesenteric artery in which the KNDP channels are known to exist. Because the experiments were performed in the whole-cell configuration (Quayle et al., 1995), however, the single-channel conductance of the channels responding to these agents was unknown. h So-called large-conductance of KATP channels in smooth muscle cells, which are so heterogeneous that no representative values are given to these channels in this table. i No stimulatory effect. Inhibitory effect could not be examined because this channel does not possess spontaneous opening and because neither NDPs nor KCOs activated the channel in the absence of Mg21. j Effect of ATP on its own in the absence of spontaneous activity, NDPs, or KCO. k These effects of KCOs are completely dependent on intracellular Mg21 and nucleoside di- or tri-phoshates.

z33 pS

z80 pS

SUR2B/Kir6.2

SUR2B/Kir6.1

Mg21 (2): z150 mM Mg21 (1): z100–150 mM Mg21 (2): z70 mM Mg21 (1): z300 mM Mg21 (2): no effecti Mg21 (1): stimulatory at 1 mM, inhibitory at 10 mMj

z80 pS

SUR2A/Kir6.2

z30 pSe z100–250 pSh

Smooth muscle cells

Mg21 (2): z10 mM Mg21 (1): z10–300 mM

Mg21 (2): z30–200 mMf Mg21 (1): stimulatory at mM or inhibitory at z1 mMf

z55–75 pS

Skeletal muscle cells

z75 pS

z1.5–2 z1.5–2

Mg21 (2): z10–20 mM Mg21 (1): z200 mM

z70–90 pS

Cardiac myocytes

SUR.Kir channels SUR1/Kir6.2

z1 z1 z2–3

Mg21 (2)c: z5 mM Mg21 (1)d: z15–45 mM Mg21 (2) and (1): z20–100 mM

Hill coefficientb

z50–90 pS

(IC50)b

Effect of ATPi

Native KATP channels Pancreatic b-cells

Single-channel conductancea

Table 1 Biophysical and pharmacological properties of the native and cloned KATP channels

A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53 41

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A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53

ATPi. Half-maximum inhibition of the KATP channel in cardiac muscle cells is achieved by 20–30 mM of ATPi, which is independent of ATPi forms because both forms of ATPi in the free-acid form (ATP42) and Mg21-bound form (MgATP) can inhibit the KATP channel activity (Terzic et al., 1995; Ashcroft & Ashcroft, 1990; Findlay, 1988). In pancreatic b-cells, on the other hand, it has been shown that Mg21 increased the half-maximum concentration for inhibition of the channel from 4 to 26 mM, suggesting that ATP42 is a more potent inhibitor of the pancreatic KATP channel than MgATP (Ashcroft & Kakei, 1989). Although information about the ATPi inhibition of KATP channels in smooth muscle is limited, MgATP is shown to be less effective than ATP42 (Nelson & Quayle, 1995; Kajioka et al., 1991). Half-maximum inhibition of the KATP channel in the rat portal vein is achieved by 29 mM of ATP42, whereas MgATP is ineffective. The second action of ATPi is referred to as “hydrolysisdependent” because it apparently requires the hydrolysis of ATP in the presence of Mg21 and can last for several tens of minutes after the removal of ATPi. The effect of ATPi on KATP channels depends on the state of the channel protein. When the channels are operative, ATPi inhibits channel opening. When the channels are not operative, treatment with MgATP restores channel opening. Recently, it has been shown that phosphatidylinositol bisphosphate (PIP2) added to the intracellular side of the membrane could restore KATP channel activity after rundown, as described in Section 8.

NDPs are also major regulators of KATP channel activity. NDPs have two distinct actions: (1) attenuating ATP-induced channel inhibition by competing with the binding of ATPi to the KATP channels and (2) permitting KATP channel opening, even after rundown (Terzic et al., 1994a; Beech et al., 1993a; Dunne & Petersen, 1991; Tung & Kurachi, 1991; Faivre & Findlay, 1989). The regulation of channel activity by nucleotides is modulated by several factors exogenous to the channel protein such as hormones, including galanine and somatostatin, which are known to inhibit insulin secretion via activation of G-proteins (Wille et al., 1989, 1988). While the acetylcholine-activated K1 channels are activated by Gbg, the KATP channels are activated by a GTP-bound form of Gia (Sánchez et al., 1998; Terzic et al., 1994b; Ito et al., 1992). Information on regulation of “KATP” channels in smooth muscle cells by intracellular nucleotides is limited. These channels have been reported to be activated by NDPs or by certain KCOs and inhibited by sulfonylureas. On the other hand, regulation of the “KATP” channels in smooth muscle cells by ATPi is divergent among various tissues, as well as species. Whole cell “KATP” currents in canine coronary arteries are inhibited by ATPi, with a half-maximal inhibition at z350 mM (Xu & Lee, 1994), whereas those in porcine coronary arteries are only partially inhibited by 1 mM ATPi (Miyoshi et al., 1992). The “KATP” channel in rabbit mesenteric arteries is completely inhibited by 1 mM ATPi in the presence of Mg21. In smooth muscle cells isolated from rat

Fig. 1. Inhibition of KATP channels by ATPi. Under cell-attached configuration of the patch-clamp technique (holding potential 275 mV), no activity of the KATP channel can be recorded due to millimolar levels of ATP inside the cardiac cell. After patch excision in an ATP-free solution, several KATP channels immediately open. Application of micromolar concentrations of ATP to the cytosolic side of the inside-out patch inhibits KATP channel openings. Note that Kir current can be recorded even in the presence of ATP. Reproduced from Terzic et al. (1995), with permission of the copyright holder, American Physiological Society, Bethesda.

A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53

portal vein, two kinds of “KATP” channels, LK and MK channels, with conductances of 50 and 22 pS in 60 mM extracellular K1, respectively, have been reported (Zhang & Bolton, 1996). The LK channel, like cardiac and pancreatic b-cell KATP channels, is inhibited by micromolar concentrations of ATPi and its spontaneous openings can be observed upon creating an inside-out patch into the nucleotide-free solution. Half-maximal inhibition of the LK channel for ATP42 or ATP in the presence of 1 mM Mg21 occurs at 11 and 23 mM, respectively. However, the LK channel is insensitive to KCOs and to glibenclamide. This channel seems to be a Ca21-activated K1 channel that had been described previously (Xiong et al., 1991). The MK channel is activated by NDPs, but is much less sensitive to ATP. While the channel is active in the cell-attached mode, its activity contrariwise declines upon creating an inside-out patch into the nucleotide-free solution. This finding is also observed in the rabbit portal vein “KATP” channel (Beech et al., 1993a, 1993b; Kajioka et al., 1991). Based on these observations, it was suggested that NDPs, instead of ATP, are the more important regulators of the MK channel. Thus, these channels were designated KNDP channels. The MK (or KNDP) channel is activated by KCOs and is inhibited by glibenclamide (Zhang & Bolton, 1996; Beech et al., 1993a, 1993b). The MK channel is activated by 1 mM ATPi in the presence of 3 mM Mg21, but inhibited when ATPi is increased to higher concentrations. However, the “KATP” channel in the rabbit portal vein is not activated by 1 mM MgATP, but rather, partially inhibited. This channel is inhibited by ATP42,with a half-maximal inhibition at 29 mM (Kajioka et al., 1991). Thus, the sensitivity of the smooth muscle “KATP” channels to ATPi seems to be divergent and is clearly different from that of classic KATP channels identified in cardiac and skeletal muscle cells or pancreatic b-cells. This confusion might be derived from the molecular heterogeneity of “KATP” channels in smooth muscle cells.

3. Pharmacological regulation of ATP-sensitive K 1 channels KATP channels in various tissues including cardiac muscle are the targets of two important classes of drugs: (1) the antidiabetic sulfonylureas, which block the channels and (2) a series of compounds called KCOs, which tend to maintain the channels in an open conformation. Sulfonylureas, including glibenclamide and tolbutamide, are hypoglycemic agents that stimulate insulin secretion by blocking the KATP channel, resulting in membrane depolarization and thus, an increase of Ca21 influx (Dunne & Petersen, 1991). As shown in Table 1, at concentrations higher than those that block the pancreatic b-cell KATP channel, these drugs can also block KATP channels in cardiac and smooth muscle cells (Nelson & Quayle, 1995; Beech et al., 1993a, 1993b; Findlay, 1992; Dunne & Petersen, 1991; Ashcroft & Ashcroft, 1990; Standen et al., 1989; Fosset et al., 1988). KATP

43

channels are also blocked by less-specific organic K1 channel blockers such as tetraethylammonium ions and certain antiarrhythmic drugs (Davies et al., 1989; Haworth et al., 1989). KCOs include divergent chemical compounds, such as cromakalim, pinacidil, levcromakalim, nicorandil, diazoxide, and minoxidil sulphate. These agents may possess high therapeutic potential in treating various clinical conditions, including hypertension, acute and chronic myocardial ischemia, or congestive heart failure, as well as in managing bronchial asthma, urinary incontinence, and certain skeletal muscle myopathies. These effects are ascribed to an increase in the open probability of KATP channels (Nichols & Lederer, 1991; Arena & Kass, 1989; Faivre & Findlay, 1989). KATP channels in different tissues exhibit considerable variations in response to KCOs (Table 1) (Nelson & Quayle, 1995; Terzic et al., 1995; Nichols & Lederer, 1991; Ashcroft & Ashcroft, 1990; Garrino et al., 1989). The pancreatic b-cell KATP channel is activated by diazoxide (EC50, 20–100 mM) (Trube et al., 1986), but only weakly by pinacidil (EC50, .100 mM) (Ashcroft & Ashcroft, 1990). The cardiac KATP channel is activated by pinacidil (EC50, 65 mM) (Escande et al., 1989), but not by diazoxide (Faivre & Findlay, 1989). Most of the smooth muscle “KATP” channels are activated by both of these agents (Quayle et al., 1995; Xu & Lee, 1994; Kovacs & Nelson, 1991; Ashcroft & Ashcroft, 1990; Standen et al., 1989). For example, the “KATP” currents in smooth muscle cells from rabbit mesenteric arteries are activated by pinacidil (EC50, 0.6 mM), as well as diazoxide (EC50, 37 mM) (Quayle et al., 1995). It should be noted that the LK channel in the rat portal vein, which is inhibited by ATPi, is not activated effectively by cardiac KATP channel openers such as pinacidil and levcromakalim, although the effect of diazoxide on this channel was not examined (Zhang & Bolton, 1996). Thus, the properties of KATP channels vary among tissues, leading to the premise that these channels may be composed of heterogeneous channel proteins. Recent molecular biological dissection of KATP channels indicates that the pharmacology of KATP channels to various KCOs is due to different subtypes of sulfonylurea receptors (SURs) expressed in these tissues (see Section 5).

4. Molecular structure of ATP-sensitive K1 channels In 1993, an ATP-dependent Kir channel, ROMK/Kir1.1 (Ho et al., 1993), and a classic Kir channel, IRK1/Kir2.1 (Kubo et al., 1993a), were cloned by the expression cloning technique from the outer medulla of rat kidney and a mouse macrophage cell lines, respectively. They have a common molecular motif in the primary structure, i.e., two putative membrane-spanning regions (M1 and M2) and one potential pore-forming loop (H5) (Fig. 2B, part a). Thus, the primary structure of these Kir channel subunits resembles that of the S5, H5, and S6 segments of Kv channels (Fig. 2A, part a).

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A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53

Recent studies have provided evidence that the IRK1 channel, as well as a functional Kv channel, can be a tetrameric channel (Yang et al., 1995; Jan & Jan, 1994; MacKinnon, 1991). This tetrameric structure has also been suggested for GIRK channels (Inanobe et al., 1995). Thus, it seems likely that as in the case of Kv channels, four Kir channel subunits are assembled to form a functional channel (Figs. 2A, part b and 2B, part a). In the Kv channels, the S4 region with the repeated positively charged amino acid residues is thought to be the voltage-sensor region of the channels. Because the Kir channel subunits do not have the segment corresponding to the S4 region in Kv channels, the channel may not be controlled by the potential difference across the membrane. Actually, it was shown that the blockade of outwardly flowing currents through Kir channels is caused by intracellular substances, such as Mg21 and polyamines, but not by intrinsic voltage-dependent gating of the channels. The cDNA encoding one of the pore-forming subunits of KATP channels (uKATP-1/Kir6.1), as well as that of acetylcholine-activated K1 channels (GIRK1/Kir3.1) has also been cloned subsequently. All of these channel subunits exhibit the same primary structure. So far, at least 11 cDNAs encoding Kir chan-

nel subunits have been isolated in mammals. These cloned Kir channel subunits can be classified into four groups (Fig. 3): (1) Kir2.0/IRK subfamily, classical Kir channels (Takahashi et al., 1994; Morishige et al., 1993, 1994; Kubo et al., 1993a); (2) Kir3.0/GIRK subfamily, G-protein-activated K1 channels (Isomoto et al., 1996a; Lesage et al., 1994; Kubo et al., 1993b; Dascal et al., 1993); (3) Kir1.0 and Kir4.0/KAB subfamily, K1-transport K1 channels (Takumi et al., 1995; Bond et al., 1994; Ho et al., 1993); and (4) Kir6.0/KATP subfamily, KATP channels (Inagaki et al., 1995a, 1995b; Sakura et al., 1995). BIR9, which is referred to as Kir5.1, does not produce functional Kir channels when expressed by itself in Xenopus oocytes and may be able to associate specifically with Kir1.2/Kir4.1/KAB-2 to form heteromultimeric Kir channels (Pessia et al., 1996; Bond et al., 1994). However, Kir5.1 does not possess a Walker type-A ATP-binding motif, which is present in the KAB subfamily, and has only 36% sequence identity with both Kir1.1 and Kir1.2/Kir4.1 and 42% identity to other Kir channel subunits. Thus, Kir5.1 may belong to another subfamily of Kir channels. Kir2.4 (Töpert et al., 1998) and Kir7.1 (Döring et al., 1998, Krapivinsky et al., 1998) were also isolated recently, but they are expressed in brain

Fig. 2. Hypothetical structures of a pore-forming subunit of an a-subunit of Kv channels (A) and Kir channels (B). Putative transmembrane segments are numbered. H5, as well as P, are putative pore-forming regions. S4 may be the voltage-sensing segment.

A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53

45

Fig. 3. The evolutionary tree of the inward rectifying K1-channel family with two membrane-spanning domains. The evolutionary relationships of the sequences were calculated by the unweighted pair-group method with arithmetic mean.

both Kir6.1 and Kir6.2 appear to require coupling with SURs to form functional KATP channels (Yamada et al., 1997; Inagaki et al., 1995a, 1996; Isomoto et al., 1996b; Sakura et al., 1995). Inagaki et al. (1995a, 1996) demonstrated that the pancreatic b-cell and cardiac KATP channels are a complex composed of Kir6.2 and a member of the SURs, SUR1 and SUR2A, respectively (Fig. 4). A stoichiometry of SURs and Kir subunits in the KATP channels was investigated by the studies on the expression of SUR1/ Kir6.2 fusion constructs (Clement et al., 1997; Inagaki et al., 1997; Shyng & Nichols, 1997). These studies indicated that the KATP channels are hetero-octamers, consisting of four SURs interacting with four Kir subunits. Although Ämmälä et al. (1996) demonstrated that Kir6.1 also couples to SUR1 and acquires sulfonylurea sensitivity, other features of KATP channels, such as regulation by ATPi and NDPs and activation by KCOs, have not been identified on the channel reconstituted from Kir6.1 and SUR1. Recently, we found that Kir6.1 could form a smooth muscle KNDP

motor neurons and in brain cerebellum and hippocampus, respectively, and not in the heart. So far, two Kir subunits belonging to the KATP subfamily, Kir6.1/uKATP-1 and Kir6.2/BIR, have been isolated (Inagaki et al., 1995a, 1995b; Sakura et al., 1995). The predicted amino acid sequences show that Kir6.1 and Kir6.2 have z70% identity with each other, and 40–50% identity with other members of the Kir channel family. The highly conserved motif of Gly-Tyr-Gly in the H5 region among various K1 channel families is Gly-Phe-Gly in both Kir6.1 and Kir6.2. Inagaki et al. (1995a, 1995b) examined the distribution of their mRNAs by Northern blot analysis and showed that Kir6.1 is ubiquitously expressed in various tissues and Kir6.2 in pancreas, heart, skeletal muscle, and brain (Table 2). While members of the Kir2.0/IRK, Kir3.0/ GIRK or Kir1.1, Kir1.2/Kir4.1/KAB-2 subfamily themselves can function as Kir channels when expressed heterologously (Krapinvinsky et al., 1995; Jan & Jan, 1992, 1994; Ho et al., 1993; Kubo et al., 1993a, 1993b; Pongs, 1992),

Table 2 Distribution of mRNA for SUR and Kir6.x Small Urinary Skeletal Brain Heart Lung Liver Pancreas Spleen Kidney Stomach intestine Colon Adrenal Testis Ovary Uterus bladder muscle Methodsa Reference SUR1 SUR2A SUR2B Kir6.1 Kir6.2 a b

6b 1 1 1 1

6 1 1 1 1

2 2 1 1 2

2 2 1 1 2

11 2 1 1 11

NE 2 1 NE NE

2 2 1 1 2

2 2 1 1 2

2 2 1 1 2

2 2 1 1 2

2 NE NE 1 2

2 NE NE 1 2

2 2 1 1 2

NE 2 1 NE NE

NE 1 1 NE NE

Northern, Northern analysis of expression of mRNA; RT-PCR, analysis of expression of mRNA with the RT-PCR. 2, undetectable; 6, moderately expressed; 1, expressed; 11, strongly expressed; NE, not examined.

2 1 1 1 1

Northern RT-PCR RT-PCR Northern Northern

Inagaki et al., 1995a Isomoto et al., 1996b Isomoto et al., 1996b Inagaki et al., 1995b Inagaki et al., 1995a

46

A. Fujita, Y. Kurachi / Pharmacology & Therapeutics 85 (2000) 39–53

Fig. 4. Molecular structure of KATP channels. Pancreatic, cardiac, and skeletal muscle KATP channels are composed of two distinct subunits: a SUR and a K1 (Kir) channel subunit, Kir6.2 (Inagaki et al., 1995a, 1996; Sakura et al., 1995; Isomoto et al., 1996b). On the other hand, the complex of SUR2B and Kir6.1 corresponds to the “small-conductance KATP” or “KNDP” channel in vascular smooth muscle (Yamada et al., 1997).

channel with another member of the SURs, SUR2B (see the next section) (Satoh et al., 1998; Yamada et al., 1997).

5. Molecular heterogeneity of sulfonylurea receptors The first SUR, SUR1, was cloned from insulinoma cells by Aguilar-Bryan et al. (1995). Its protein is assumed to possess 17 potential transmembrane regions (Fig. 4) (Tusnády et al., 1997), 2 nucleotide-binding folds (NBFs) with Walker A and B consensus motifs, 2 N-linked glycosylation sites, and several protein kinase A- and C-dependent phosphorylation sites. Co-expression of hamster (ha)-SUR1 and mouse (m)-Kir6.2 elicits KATP conductance (IKATP), which is inhibited by glibenclamide (half-maximal at 1.8 nM) and activated by diazoxide (half-maximal at 60 mM) (Inagaki et al., 1995a). The single-channel conductance is 76 pS in symmetric 140 mM K1 solution. ATPi inhibits the haSUR1/m-Kir6.2 channel activity, with a half-maximal value of 10 mM. These properties are the same as those of the pancreatic b-cell KATP channel (Dunne & Petersen, 1991; Garrino et al., 1989; Findlay et al., 1985; Cook & Hales, 1984). Northern blot analysis has revealed that ha-SUR1 mRNA is expressed at a high level in pancreatic islets and at a low level in heart and skeletal muscle (Table 2) (Inagaki et al., 1995a; Aguilar-Bryan et al., 1995). Chromosome mapping data show that both Kir6.2 and SUR1 genes are clustered on human chromosome 11 at 11p15.1 (Inagaki et al., 1995a). Thus, SUR1 seems to form the pancreatic b-cell KATP channel with Kir6.2. Mutations in the SUR1 protein have been shown to cause a nonfunctional KATP channel, resulting in

persistent hyperinsulinemic hypoglycemia of infancy, a disease associated with unregulated insulin secretion (Kane et al., 1996; Thomas et al., 1995). Two additional homologs of SUR1 have been isolated subsequently. The second member of SURs, designated SUR2, was first cloned from rat brain by Inagaki et al. (1996). We have also cloned a mouse homolog of SUR2 and a novel splicing isoform of SUR2 from a mouse heart cDNA library (Isomoto et al., 1996b). Sequence analysis indicates that this novel member of SUR2 is divergent from SUR2 only at 42 amino acid residues in the C-terminal end. Based on these results, we proposed renaming of the original SUR2 as SUR2A and designated the third member SUR2B (Isomoto et al., 1996b). The amino acid sequence of rat (r)-SUR1 has 66% and 67% identity with those of m-SUR2A and m-SUR2B, respectively. A further splice variant of m-SUR2A, which has a deletion of 35 predicted amino acid residues in the intracellular loop between the 11th and 12th membrane-spanning domains, has also been cloned, but its function is not clear (Chutkow et al., 1996). It is now designated SUR2C (Ashcroft & Gribble, 1998). Co-expression of Kir6.2 and either SUR2A or SUR2B also elicits IKATP with a single-channel conductance of z80 pS in symmetric z145 mM K1 solution. The r-SUR2A/ m-Kir6.2 channel activity is inhibited by ATPi in a concentration-dependent manner, with a half-maximal value of 100 mM (Inagaki et al., 1996). It was only partially inhibited by glibenclamide at 1 mM (Inagaki et al., 1996). In contrast to the ha-SUR1/m-Kir6.2 channel, the m-SUR2A/r-Kir6.2 channel is activated by pinacidil, but not by diazoxide. These are the features of cardiac and skeletal muscle KATP

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channels (Terzic et al., 1994c, 1995; Findlay, 1992; Nichols et al., 1991; Nichols & Lederer, 1991; Ashcroft & Ashcroft, 1990; Faivre & Findlay, 1990; Fosset et al., 1988). The m-SUR2B/r-Kir6.2 channel is activated by both pinacidil and diazoxide, suggesting that the features of this channel closely resemble the responses of smooth muscle to KCOs (Nelson & Quayle, 1995; Beech et al., 1993b; Lorenz et al., 1992; Kajioka et al., 1991; Standen et al., 1989). Thus, pharmacological properties of KATP channels to KCOs may be determined by their SURs. Because diazoxide activates KATP channels containing SUR1 or SUR2B, but not SUR2A, the alternative splicing region between SUR2A and SUR2B may be a functional domain important for diazoxide activation of KATP channels. Interestingly, the sequence of the last 42 amino acids of SUR2B exhibits 74 and 33% identity with those of the corresponding region of r-SUR1 and m-SUR2A, respectively. On the other hand, the functional domain for pinacidil may be in regions different from the C-terminal end because pinacidil activates KATP channels reconstituted from SUR2A or SUR2B, but not from SUR1 (Isomoto et al., 1996b). Northern blot analysis for rat tissues, using the probe that includes the common nucleotide sequence in both isoforms of SUR2, revealed that mRNA for either SUR2A or SUR2B in rat is expressed at high levels in heart, skeletal muscle, and ovary; at moderate levels in brain, tongue, and pancreatic islets; at low levels in lung, testis, and adrenal gland; and at very low levels in stomach, colon, thyroid, and pituitary (Inagaki et al., 1996). On the other hand, the reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that m-SUR2A mRNA is expressed in heart, skeletal muscle, cerebellum, eye, and urinary bladder, whereas m-SUR2B mRNA ubiquitously distributes not only in these tissues, but also in forebrain, lung, liver, pancreas, kidney, spleen, stomach, small intestine, colon, uterus, ovary, and fat tissue (Isomoto et al., 1996b). In situ hybridization using the probe that is common to SUR2 isoforms showed that SUR2 isoforms are expressed in the parenchyma of the heart and skeletal muscle and in the vasculatures of various tissues (Chutkow et al., 1996). Because SUR2A is believed to be the cardiac and skeletal muscle type SUR, other isoforms of SUR2 would be expressed in the vasculature. This supports the idea that SUR2B represents the SUR in the vascular smooth muscle type KATP channels. However, to clarify the distribution of each isoform of SURs, further studies using probes specific for each isoform are needed. SUR2B is thought to be one of the subunits reconstituting smooth muscle type “KATP” channels because of its pharmacological properties and tissue distribution. However, the SUR2B/Kir6.2 channel expressed IKATP, with single-channel conductance of z80 pS, which is distinct from those of physiological smooth muscle “KATP” channels. In addition, mRNA for Kir6.2 is expressed in restricted tissues, whereas that of SUR2B is in a variety of tissues. These findings suggest that SUR2B may reconstitute some types of

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“KATP” channels in smooth muscle cells by coupling with members of the Kir channel family other than Kir6.2. Recently, we demonstrated that co-expression of SUR2B and Kir6.1 forms a functional K1 channel with the features of a smooth muscle K1 channel, which is described as the KNDP channel in rat portal vein (Satoh et al., 1998; Yamada et al., 1997). The m-SUR2B/m-Kir6.1 channel possesses a single channel conductance of z33 pS in symmetric z145 mM K1 solution, which is activated by diazoxide, pinacidil, and nicorandil. Surprisingly, the channel does not spontaneously open on patch excision, even in the absence of ATPi. In excised patches, the channel activity is activated by NDPs and inhibited by sulfonylureas, but not by ATPi. More than 100 mM ATPi on its own activates the channel. Thus, the SUR2B/Kir6.1 channel closely resembles a KNDP channel in vascular smooth muscle at the following points: (1) activation by internal nucleoside di- and triphosphates, (2) activation by KCOs and inhibition by sulfonylureas, (3) little inhibition by ATPi, and (4) a single-channel conductance of 22 pS with 60 mM extracellular K1 (Zhang & Bolton, 1996). It should be also noteworthy that SUR2 and Kir6.1 genes are clustered in the distal region of mouse chromosome 6 (Isomoto et al., 1997). 6. Molecular mechanism of ATP-sensitive K1 channel inhibition by intracellular ATP One of the hallmarks of the classical KATP channels is the inhibition of channel activity by micromolar concentrations of ATPi (Terzic et al., 1995; Ashcroft, 1988; Noma, 1983). Tucker et al. (1997) recently found that the Kir6.2, whose last 26 amino acids at the C-terminus are deleted (Kir6.2DC26), can be functionally expressed in the absence of SUR. A charge-neutralization mutation on Lys185 of Kir6.2DC26 reduces the ATPi sensitivity of the Kir6.2DC26 channel by z40 times. The ATPi sensitivity of the Kir6.2DC26 channel was increased 5-8 times by co-expression of SUR1. Thus, it is likely that the primary inhibitory ATPi-binding site resides in Kir6.2, while SUR1 increases the ATPi sensitivity of Kir6.2. Koster et al. (1998) showed that the complex of SUR1 and the Kir6.2, whose N-terminal 30 amino acids were deleted (Kir6.2DN30), exhibited z10 times lower ATPi sensitivity than the SUR1/Kir6.2 channel. Interestingly, the SUR1/Kir6.2DN30 channel was also less sensitive to intracellular ADP (ADPi) and tolbutamide than the SUR1/Kir6.2 channel. Therefore, Kir6.2 might interact with SUR1 through its N-terminus, and the low ATPi sensitivity of the SUR1/Kir6.2DN30 channel might be due to impaired coupling between SUR1 and Kir6.2. It is unknown how SUR1 increases the ATPi sensitivity of Kir6.2. Gribble et al. (1997b) found that the ATPi sensitivity of the SUR1/Kir6.2 channel was not modified by mutations on either or both of the two conserved lysine residues in the Walker A motifs in the first or the second NBF of SUR1 (K719A and K1384M, respectively). On the other

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hand, Ueda et al. (1997) demonstrated that SUR1 possesses two distinct ATPi-binding sites with high and low affinities. The high-affinity binding site was saturated with 10 mM ATPi in the absence of Mg21i. Substitution of the conserved lysine residue in the Walker A motif (K719R and K719M) or the aspartate residue in the Walker B motif (D854N) in the first NBF all abolished the high-affinity ATPi-binding, while the corresponding mutations in the second NBF did not cause any significant effect. Because Ueda et al. (1997) and Gribble et al. (1997b) used different mutations (K719R, K719M, or D854N vs. K719A, respectively), it is not clear whether the ATPi binding found by Ueda et al. (1997) underlies the sensitization of Kir6.2 to ATPi by SUR1. No corresponding studies have been done on SUR2s. However, ATPi inhibits the SUR2A/Kir6.2 and the Kir6.2DC26 channels with similar potencies (z100 mM) (Okuyama et al., 1998; Tucker et al., 1997; Inagaki et al., 1996). Thus, SUR2A may not substantially enhance the ATPi sensitivity of Kir6.2. However, the native cardiac KATP channel has been reported to be z3-10 times more sensitive to ATPi than the SUR2A/Kir6.2 channel (Table 1). Thus, some unidentified factors in cardiac myocytes might sensitize the SUR2A/Kir6.2 channel to ATPi in vivo. Actually, various factors, including intracellular polyvalent cat-

ions and actin polymerization, have been suggested to affect the ATPi sensitivity of the cardiac KATP channel (Hiraoka et al., 1996; Terzic & Kurachi, 1996; Deutsch et al., 1994). The SUR2A/Kir6.2 channel was equally sensitive to Mg21-free and Mg21-bound ATPi (Fig. 5A) while the SUR2B/Kir6.2 channel was more sensitive to Mg21-free than Mg21-bound ATPi (Fig. 5B). This difference should be ascribed to the difference in amino acid sequence of the C-terminus between SUR2A and SUR2B (Isomoto et al., 1996b). As stated previously, the sequence of the last 42 amino acids in the C-terminus of SUR2B is more similar to that of the corresponding part of SUR1 than SUR2A, and the SUR1/Kir6.2 channel is more sensitive to Mg21-free than Mg21-bound ATPi (Nichols et al., 1996). Therefore, the last 42 amino acids in the C-terminus of SURs may be involved in discrimination of Mg21-bound and Mg21-free ATPi by KATP channels. This part is very close to the second NBF in the primary structure, and the second NBF is known to play a crucial role in NDPi-induced activation of KATP channels (Gribble et al., 1997b; Nichols et al., 1996). Ueda et al. (1997) found in SUR1 that the binding of ADPi to the second NBF potently antagonized the ATPi binding to the first NBF of the same protein in the presence of Mg21i. Therefore, the C-terminal tail of SURs might serve to regu-

Fig. 5. Inhibition of the SUR2A/Kir6.2 and SUR2B/Kir6.2 channels by ATPi. (A) The SUR2A/Kir6.2 channel. (a) Effect of ATPi in inside-out patch membranes in the presence (upper trace) or the absence (lower trace) of z1.4 mM intracellular free Mg21. (b and c) The relationship between total ATP concentrations and channel activity in the presence (b) and the absence (c) of Mg21. The data are expressed as a percentage of the value obtained in the absence of ATP. The different symbols indicate the data obtained from different patches. Lines are the fit of each set of the data with the Hill equation. Modified from Okuyama et al. (1998). (B) The SUR2B/Kir6.2 channel. (a) Effect of ATP in inside-out patch membranes in the presence (upper trace) or the absence (lower trace) of z1.4 mM free Mg21. (b) The relationship between ATP concentrations and channel activity in the presence (open circles) and the absence (filled circles) of Mg21. Symbols and bars indicate the mean 6 SEM (n 5 3 for each point). Open and filled circles are the plot against the total ATP concentrations, whereas open diamonds are the estimated concentrations of ATP not complexed with Mg21 in this solution. Lines are the best fit of each set of data by the Hill equation. Modified from Okuyama et al. (1998) and Isomoto et al. (1996b).

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late either ATPi hydrolysis on or ADPi binding to the second NBF in the presence of Mg21i. ATPi inhibited both SUR2A/Kir6.2 and SUR2B/Kir6.2 channels, with a Hill coefficient significantly larger than unity (z1.8). No cooperativity, however, was detected with the SUR1/Kir6.2 channel and the Kir6.2DC26 channel (Gribble et al., 1997a, 1997b; Tucker et al., 1997). Therefore, SUR2s, but not SUR1, may function to create the cooperative interaction between ATPi and KATP channels through an unknown molecular mechanism.

7. Molecular mechanism of response to intracellular nucleoside diphosphates NDPis such as UDP exhibit distinct effects on the cardiac KATP channel before and after rundown (Terzic et al., 1994a, 1995). UDP antagonizes the inhibitory effect of ATPi before rundown. After rundown, UDP restores the channel activity without attenuating the ATPi sensitivity of the channels (Tung & Kurachi, 1991). The SUR2A/Kir6.2 channel well-

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mimicked such a dualistic response of the cardiac KATP channel to NDPis (Fig. 6) (Okuyama et al., 1998). As shown in Fig. 6A (part a), ATPi exhibited a weaker inhibitory effect on spontaneous channel activity of the SUR2A/Kir6.2 channel in the presence than in the absence of UDP. Removal of UDP almost completely restored the channel’s ATPi sensitivity. Thus, UDP antagonized the ATPimediated inhibition of channel activity before rundown. After rundown, UDP activated the channel in a concentration-dependent manner (Fig. 6A, part b), with an EC50 of 240 mM (Fig. 6A, part c). This effect was completely dependent on Mg21i (not shown). ATPi inhibited the channel activity induced by UDP (Fig. 6A, part d) in a concentrationdependent manner (symbols in Fig. 6A, part e) as potently as inhibiting spontaneous activity in the absence of UDP (a line in Fig. 6A, part e, which is the average of the data shown in Fig. 5A, part b). Thus, the SUR2A/Kir6.2 and native cardiac KATP channels respond to NDPi in a very similar way. UDP activated the post-rundown SUR2B/Kir6.2 channel in a concentration-dependent manner with an EC50 of 71.7 mM and a Hill coefficient of 1.74 (Fig. 6B). This response

Fig. 6. Effect of NDPs on the SUR2A/Kir6.2 and SUR2B/Kir6.2 channels. (A) The SUR2A/Kir6.2 channel. (a, b, and d) Inside-out patch recordings. (c) The relationship between UDP concentrations and channel activity after rundown. Channel activity is expressed as a percentage of the maximum activity induced by 3 mM UDP. Symbols and bars indicate the mean 6 SEM (n 5 3–12 for each point). The line is the best fit of the data with the Hill equation. (e) The concentration-dependent inhibitory effect of ATP on channel activity induced by 3 mM UDP after rundown in the presence of Mg21 (symbols). Different symbols indicate data from different patches. The line indicates the average inhibitory effect of ATP on the spontaneous channel activity in the absence of UDP (Fig. 5A, part b). Modified from Okuyama et al. (1998). (B) The SUR2B/Kir6.2 channel. (a) An inside-out patch recording. (b) The relationship between UDP concentrations and channel activity after rundown. Channel activity is expressed as a percentage of the maximum activity induced by 1 mM UDP. Symbols and bars indicate the mean 6 SD (n 5 3 for each point). The line is the best fit of the data with the Hill equation. Reproduced from Okuyama et al. (1998), with permission of the copyright holder, Springer-Verlag, Heidelberg.

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was also dependent on Mg21i (not shown). UDP also antagonized the inhibitory effect of ATPi on SUR2B/Kir6.2 channel before, but not after, rundown, as is the case for the SUR2A/Kir6.2 channel (unpublished observation). Overall, the responses to NDPi were very similar between SUR2A/ Kir6.2 and SUR2B/Kir6.2 channels. Nichols et al. (1996) found that a human persistent hyperinsulinemic hypoglycemia of infancy mutation (G1479R) in the second NBF of SUR1 abolished the antagonizing effect of ADPi on the ATPi-induced inhibition of the SUR1/ Kir6.2 channel. Tucker et al. (1997) demonstrated that MgADPi inhibited the Kir6.2DC26 channel, but activated the SUR1/Kir6.2DC26 channel. Gribble et al. (1997b) showed that either the K719A or K1384M mutation of r-SUR1 abolished the stimulatory effects of ADPi on the partial rundown SUR1/Kir6.2 channel, both in the presence and in the absence of ATPi. Furthermore, hydrolysis-resistant b-methylene ADP failed to activate the SUR1/Kir6.2 channel. Thus, it is likely that hydrolysis of MgADPi (and probably also the other NDPis) at the NBFs of SUR1 may be critically involved in the activating effect of the nucleotides. Although no corresponding studies have been done in SUR2s, a similar mechanism would underlie the activating effect of NDPi on the SUR2/Kir6.2 channel. The molecular mechanism responsible for the dualistic responses of the SUR2/Kir6.2 channels to NDPi, however, is unknown.

8. Molecular mechanism of rundown Rundown of channel activity in inside-out patch membranes is not necessarily a phenomenon specifically associated with KATP channels, but can be seen in other Kir channels as well. Nevertheless, many investigators have been interested in the mechanism underlying the rundown of KATP channels because KATP channels run down more prominently than other Kir channels (Terzic et al., 1995; Ashcroft, 1988). The KATP channels can be reactivated after rundown with ATPi in the presence, but not in the absence, of Mg21i (Takano et al., 1990; Ohno-Shosaku et al., 1987; Findlay & Dunne, 1986). Nonhydrolyzable ATP analogues cannot mimic this effect of ATPi, even in the presence of Mg21i (Takano et al., 1990; Ohno-Shosaku et al., 1987). Therefore, rundown/refreshment of KATP channel activity might be crucially related to hydrolysis of ATPi or phosphorylation/dephosphorylation of KATP channels (Terzic et al., 1995; Ashcroft, 1988). Kir6.2DC26 channels also exhibit rundown and can be reactivated with MgATPi (Tucker et al., 1997), indicating that the rundown/refreshment is primarily associated with a certain functional alteration of Kir6.2. Hilgeman and Ball (1996) showed that PIP2 added to the intracellular side of the membrane could restore KATP channel activity after rundown. Recently, Huang et al. (1998) showed that various Kir channels, including Kir1.1, Kir2.1, Kir3.2 homomeric, and Kir3.1/Kir3.4 heteromeric channels, could be reactivated by PIP2 after rundown. Furthermore, the ATPi-medi-

ated restoration of activity was inhibited by antibodies against PIP2. Thus, PIP2 and its generation by ATP-dependent lipid kinases appear to be critically involved in spontaneous Kir channel activity. The dualistic behavior of KATP channels to NDPis (Terzic et al., 1994a, 1995) may have to be re-examined from this aspect. More recently, it has been shown that PIP2 shifts the dose-response curves for ATP inhibition of KATP channels toward higher concentrations. This result suggests that PIP2 metabolism may physiologically regulate the ATP sensitivity of KATP channels (Shyng & Nichols, 1998; Baukrowitz et al., 1998). 9. Conclusions Recent molecular dissection of Kir channels and SURs has identified molecular structures of KATP channels in the cardiovascular system. Further understandings at the molecular level of the KATP channels in the cardiovascular system may enable us to clarify the roles of these channels in cardiovascular physiology and pathophysiology, which may allow further development of strategy and pharmacological agents to treat various cardiovascular diseases. References Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E. I., González, G., Herrera-Sosa, H., Nguy, K., Bryan, J., & Nelson, D. A. (1995). Cloning of the b cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. Science 268, 423–426. Allard, B., & Lazdunski, M. (1993). Pharmacological properties of ATPsensitive K1 channels in mammalian skeletal muscle cells. Eur J Pharmacol 236, 419–426. Allard, B., Lazdunski, M., & Rougier, O. (1995). Activation of ATP-dependent K1 channels by metabolic poisoning in adult mouse skeletal muscle: role of intracellular Mg21 and pH. J Physiol (Lond) 485, 283–296. Ämmälä, C., Moorhouse, A., Gribble, F., Ashfield, R., Proks, P., Smith, P. A., Sakura, H., Coles, B., Ashcroft, S. J., & Ashcroft, F. M. (1996). Promiscuous coupling between the sulfonylurea receptor & inwardly rectifying potassium channels. Nature 379, 545–548. Arena, J. P., & Kass, R. S. (1989). Enhancement of potassium-sensitive current in heart cells by pinacidil: evidence of modulation of the ATPsensitive potassium channel. Circ Res 65, 436–445. Ashcroft, F. M. (1988). Adenosine 59-triphosphate-sensitive potassium channels. Annu Rev Neurosci 11, 97–118. Ashcroft, F. M., & Gribble, F. M. (1998). Correlating structure and function in ATP-sensitive K1 channels. Trends Neurosci 21, 288–294. Ashcroft, F. M., & Kakei, M. (1989). ATP-sensitive K1 channels in rat pancreatic b-cells: modulation by ATP and Mg21 ions. J Physiol (Lond) 416, 349–367. Ashcroft, S. J., & Ashcroft, F. M. (1990). Properties and functions of ATPsensitive K-channels. Cell Signal 2, 197–214. Baukrowitz, T., Schulte, U., Oliver, D., Herlitze, S., Krauter, T., Tucker, S. J., Ruppersberg, J. P., & Fakler, B. (1998). PIP2 and PIP as determinants for ATP inhibition of KATP channels. Science 282, 1141–1144. Beech, D. J., Zhang, H., Nakao, K., & Bolton, T. B. (1993a). K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells. Br J Pharmacol 110, 573–582. Beech, D. L., Zhang, H., Nakao, K., & Bolton, T. B. (1993b). Single channel and whole-cell K-currents evoked by levcromakalim in smooth muscle cells from the rabbit portal vein. Br J Pharmacol 110, 583–590.

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