Cellular Signalling Vol. 2, No. 3, pp. 197-214, 1990.

0898-6568/90 $3.00 + .00 © 1990PergamonPress plc

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MINI REVIEW PROPERTIES A N D FUNCTIONS OF ATP-SENSITIVE K-CHANNELS STEPHEN J. H . ASHCROFT*'~ a n d FRANCES M . ASHCROFT3~

*Nutiield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford OX3 9DU, U.K. and :~UniversityLaboratory of Physiology, Parks Road, Oxford OXl 3PT, U.K.

(Received 23 December 1989; and accepted 19 February 1990) Key words: Ion channel, ATP-sensitive K + channel, sulphonylurea. INTRODUCTION

classes of channel are highly selective for K ÷ ions. By contrast, in certain fl-cell lines [16] and pancreatic acinar cells [17] a non-selective cation channel (Type 4) blocked by micromolar ATP has been described. This channel is also inhibited by micromolar AMP and activated by millimolar Ca 2+. Non-selective ion channels (Type 5), permeable to both cations and anions have been found in cortical neurones l13]; intracellular ATP produces a flickery block of these channels. Brief reports of a number of other channels permeable to K + and blocked by ATP have also appeared [139, 122]: currently, information available about these channels is too little to allow classification or discussion. In this review we attempt to provide an overview of the biophysics, pharmacology and regulation of these different types of K-ATP channels. We shall, however, focus principally on the Type 1 K-ATP channel of the pancreatic flcell, as less is known about the other types of K-ATP channel.

A K-CHANNEL inhibited by intracellular ATP was first described in cardiac muscle [1]. It has since become clear that such channels (K-ATP channels) occur also in other tissues as a heterogeneous family and provide a means of linking cellular metabolism to electrical activity in the plasma membrane. Table 1 summarizes the main classes of KATP channel that have been described to date. The best characterized of these (Type 1) is found in cardiac muscle [1], skeletal muscle [2, 3], smooth muscle [4], pancreatic fl-cells [5-7] and vertebrate axons [149]. Activity of Type 1 K-ATP channels is blocked by micromolar concentrations of ATP applied to the intracellular membrane surface and by sulphonylureas, a class of hypoglycaemic drugs used to treat noninsulin dependent diabetes mellitus (NIDDM) [8-11]. Channel activity is relatively insensitive to calcium and to voltage. In cortical and hypothalamic neurones [12, 13] related channels (Type 2) have been described which are much less sensitive to ATP, requiring millimolar concentrations to produce significant inhibition. A third type of K-ATP channel (Type 3) found in epithelial cells (nasal polyps, Amphiuma distal tubule) is similar to the neuronal channel in its sensitivity to ATP but differs in being activated by micromolar Ca `'+ [14, 15, 32]. These three

BIOPHYSICAL PROPERTIES OF K-ATP CHANNELS The biophysical properties of Type 1 K-ATP channels have been extensively covered in a number of recent reviews [18-21]. Here we summarize the main findings and compare these to data obtained for other types of K-ATP channels.

t A u t h o r to w h o m correspondence should be addressed. liLtS ~:3.-A

] 9"7

198

S. J. H. ASHCROFT and F. M. ASHCROFT TABLE 1. TYPES OF K - A T P CHANNELS

Type

Location

1 Cardiac muscle Hypertrophied cardiac muscle Skeletal muscle Smooth muscle Vertebrate axon Pancreatic #-cell

2

Human #-cell Foetal rat//-cell HIT T15 #-cell RINm5F #-cell CRI-G1 #-cell VMH neurone

3

Cortical neurone Respiratory epithelia

Amphiuma tubule t t 4

CRI-G1 #-cell Human insulinoma Pancreatic acinar cell 5 Cortical neurone

Conductance (pS)

K~ for ATP (/~M)

80 25* 77 42t 74 135t 445 50-65

17-100

20-30§ 50-65 57 52 50-90 55 150 6511 100" 300 23011 120-300 25 19 25 585:~

250 135 53 31 10-20 42 10 14 56 78 13 3000 2000 1000 5000 8

2000

References 1, 49, 51, 137, 140, 147 25 130 2, 30 3 4 149 5, 7, 40, 42 138 26, 27, 100 I1, 123, 142 138 113, 145 41, 122 139 12 12 13 15 15 32 16 142 17 13

Single channel conductances and the concentration of ATP required for half-maximal inhibition of channel activity (K~) were measured in the inside-out patch with about 140 mM [K]o and 140 mM [K]~, except where stated otherwise. * in 5 mM [K]o; "~in 60 mM [K]o; $ with 105 mM [K]o, 105 mM [K]i; § in 5 mM [K]o and the absence of internal blocking ions; II in 5 mM [K]o, calculated chord conductance at 0 mV; t t early distal tubule; $:~ between 0 and + 50 mV.

Single channel conductance The single channel conductances of K - A T P channels, measured in the inside-out patch, with approximately 140 m M K + on each side of the membrane, are summarized in Table i. With normal ionic gradients, current flow is outward at potentials positive to the K-equilibrium potential (about - 7 0 m V ) and single channel conductances are lower (see Table 1). Type 1 K - A T P channels show saturation of the single channel conductance at high K-concentrations (K~ > 200 mM [22, 23]). In cell-attached patches, Type l K - A T P

channels show a pronounced inward rectification, with the current amplitude increasing little at potentials above + 2 0 i n V . It is now clear that this rectification results from a voltagedependent block by internal ions, primarily Na + and Mg 2+ [22, 24-26]. High concentrations of other substances such as Tris, MOPS, N-methyiglucamine, sucrose, mannitol and choline are also able to block the channel from the inside, suggesting that it has a fairly wide internal mouth [14, 15, 27]. There is apparently also some intrinsic rectification of the single channel current, since rectification of the current-voltage (I-V) relation occurs even in the absence of

ATP-sensitive K-channels internal blocking ions [27, 28]. External Na + and Mg 2+ do not block the channel, but a voltage-dependent block of the K-ATP channel by external Cs + and Ba ~+ has been described [29]. The properties of saturation and block found for Type 1 K-ATP channels are not consistent with the Goldman-Hodgkin-Katz equation and suggest that the channel is a multi-ion pore [29]. Eyring rate theory models which assume two binding sites and three energy wells provide a reasonable description of the steady-state conductance properties of the Type 1 K-ATP channel [22, 30]. No studies of other types of K-ATP channels have directly addressed the question of whether intracellular cations can produce a voltagedependent block of outward currents. However, Type 2 K-ATP channels do not show inward rectification when 1 mM Mg 2+ is present in the internal solution [12]: since this concentration of Mg 2+ produces a marked rectification of the I-V relation of the Type 1 K-ATP channel [22, 24, 26], Type 2 K-ATP channels may not be as sensitive to intracellular blocking ions. Similar arguments can be made for Type 3 [14] and Type 4 [17] K-ATP channels.

Selectivity The permeability ratio (PNJPK) for Type l K-ATP channels ranges between 0.007 in/~-cells [27] and 0.015 in frog [30] or rabbit [31] skeletal muscle. Type3 K-ATP channels are equally selective (PNJPK > 0.025 in respiratory epithelial cells [14], 0.033 in Amphiuma tubule [32]). In both these channels, the Rb + permeability is comparable to that of K +, but the Rb ÷ conductance is very low [14, 27, 30]. Type2 K-ATP channels in brain neurones are also strongly Kselective [12]. By contrast, Type 4 K-ATP channels do not discriminate between Na + and K + [16, 17] and the Type 5 K-ATP channel found in brain neurones is permeable both to monovalent cations and to anions [13].

Voltage-dependence and kinetics Detailed discussions of the voltage-dependence and kinetic properties of Type 1 K-ATP

199

channels can be found in earlier reviews [1821]. Here we briefly describe the main findings for the purpose of comparison. Both whole-cell and single channel current recordings indicate that the Type 1 K-ATP channel shows little voltage-dependence [7, 163]. The characteristic kinetics of the Type 1 K-ATP channel consists of bursts of openings separated by longer interburst intervals. The mean closed and open times within the bursts (0.3-0.8 and 2-3ms, respectively, at - 7 0 mV) are not very different between/~-cells [7, 23], cardiac [28] and skeletal muscle [30]. The voltage-dependence of the open and closed times within the bursts has been studied most fully in cardiac myocytes [28]. In this tissue, channel gating depends on both the K-gradient and the membrane potential with open times being maximal, and closed times minimal, at the potassium equilibrium potential [28]. The long closed times separating the bursts are more difficult to measure under steady state conditions as long recording periods are needed to obtain sufficient data and channel activity often decreases with time in excised patches (rundown, see below), or is subject to modulation by metabolism in intact cells. Indeed, the mean duration of the long closed intervals shows considerable variability both between patches and between tissues. One possible reason for this variability is that it results from metabolic regulation of the channel, since the main effect of ATP on the channel kinetics also appears to be to increase the frequency and duration of this long closed state (or states) [2, 30, 33]. A similar effect is seen when glucose is applied to cell-attached patches on pancreatic//-cells [23, 81]. ATP also has the additional effect of increasing the frequency of a very short-lived open state [30, 33]. K-ATP channel kinetics have also been investigated by analysis of the time-course of the macroscopic currents in cell-attached patches in response to instantaneous changes in [ATP]~. This study [163] showed that the current decreased exponentially on increasing ATP, and following a variable latency, increased exponentially when ATP was decreased, suggesting that the gating results from the binding and unbinding of one

200

s.J.H. ASHCROFTand F. M. ASHCROFr

ATP molecule. The channel was able to exist in two states which had different sensitivities to ATP [163]. Much less is known about the other types of K-ATP channel, but in contrast to Type 1 channels the open state probability of Type 3 [14, 32] and Type4 [16] K-ATP channels is voltagedependent and increases with membrane depolarization.

REGULATION OF K-ATP CHANNELS A number of previous reviews have dealt with the regulation of K-ATP channels [18, 21, 35-37].

Regulation by adenine nucleotides It is now clear that ATP has more than one action on Type 1 K-ATP channels for, in addition to closing the channel, the presence of ATP is necessary for maintenance of channel activity. A dual effect of ATP has also been reported for Type5 K-ATP channels [13]. Whether this is the case for other types of KATP channel has not been investigated, but the lack of significant rundown (see below) of these channels in isolated patches would suggest that activation by ATP plays only a minor role (if any) in channel regulation.

Channel activation. In heart and fl-cell membranes, channel activity decreases rapidly (rundown) following patch excision if MgATP is not present in the intracellular solution; channel activity can be recovered following brief exposure of the patch to MgATP [38-40]. A role for protein phosphorylation in this refreshment of activity by ATP is suggested by the requirement for the presence of Mg 2÷ and by the fact that non-hydrolysable analogues of ATP are ineffective. Since ATP alone is sufficient to activate channels in excised patches the kinase responsible for this phosphorylation must be closely associated with the membrane. It seems possible that lower ATP concentrations are needed to activate K-ATP channels than to produce

significant inhibition, since channel activity is often greater at low ATP levels (0.1-1/~M) than in the absence of ATP [41, 42]. Direct evidence for the involvement of phosphorylation in channel activation in RINm5F /3-cells has been provided by studies showing activation of K-ATP channels in inside-out patches by the catalytic subunit of protein kinase A (PKA) [43]. The ability of PKA inhibitor ('Walsh inhibitor' or PKI) to decrease channel activity in the isolated patch is also consistent with a role for PKA as the endogenous kinase mediating channel refreshment [43]. Whether these observations are applicable to other cell types, however, has not been tested. It seems unlikely that phosphorylation by protein kinase C (PKC) is involved in the refreshment of channel activity by ATP, since in normal/3cells PKC activation has no effect on K-ATP channel activity [44]. Curiously, in RINm5F/3cells, PKC activation has been reported both to inhibit [45] and to activate [46] K-ATP channels. Rundown proceeds much more slowly in patches excised from skeletal muscle [2, 30, 47] or smooth muscle [4]. Since phosphorylation increases K-ATP channel activity it seems possible that rundown might be a consequence of dephosphorylation by endogenous phosphatases present in the excised patch. This possibility has been explored in some detail in cardiac myocytes using Ca2÷-free solutions (to prevent activation of Ca2+-dependent phosphatases) or fluoride (a non-specific phosphatase inhibitor, which also chelates Ca2+). Although the presence of F in the intracellular solution prevented rundown of K-ATP channels in myocytes [28] it is not clear whether this effect of fluoride was mediated by inhibition of phosphatases, by activation of a G-protein (see below) or by a non-specific effect of the anion. Rundown was enhanced by 5-100/zM intracellular Ca 2+, but channel activity did not return on Ca 2÷ removal and only recovered on exposure of the patch to MgATP [80]. Since neither F- nor leupeptin (a protease inhibitor) were able to prevent the Ca2+-evoked rundown the role of Ca2÷-dependent proteases

ATP-sensitive K-channels or phosphatases in rundown remains speculative [80]. Indeed, trypsin (2mg/ml) actually appears to increase channel activity in insideout patches from mouse pancreatic fl-cells [20]. Interestingly, this protease not only increased the open probability and/or the number of active channels, but also decreased the sensitivity to the inhibitory action of ATP [20]. Channel inhibition. Half-maximal inhibition for Type l K-ATP channels and for the nonselective cation (Type 4) channels is produced by about 10/~M ATP applied to the intracellular (but not extracellular) surface of isolated membrane patches (Table 1). Millimolar ATP concentrations produce complete channel inhibition. Type 2 and 3 K-ATP channels, however, require as much as 1-5 mM ATP to produce half maximal inhibition in the inside-out patch (Table 1). It is unclear whether inhibition of Type 1 K-ATP channels requires the binding of more than one molecule of ATP since Hill coefficients between 1 and 2 have been reported for the inside-out patch [5, 7, 30]. The stoichiometry of the other types of K-ATP channels has been investigated only for the Type 4 channel of a fl-cell line (Hill coefficient 1.65 [16]). A number of studies have indicated that phosphorylation is not involved in the inhibition of Type 1, 2 or 3 K-ATP channels by ATP, since Mg 2+ is not required and non-hydrolysable analogues are also effective [12, 13, 15, 30, 32, 42, 51]. Despite this, Ribalet et aL [43] have suggested that phosphorylation could mediate the inhibitory effect of ATP on Type 1 K-ATP channels via binding of the nucleotide to PKI, the protein kinase A inhibitor. This favours the interaction between PKI and the catalytic subunit of protein kinaseA, promoting dephosphorylation and channel closure. Unfortunately, this novel hypothesis cannot explain the fact that free ATP (in the absence of Mg 2+) can inhibit the channel. The mechanism of block by ATP may be different in Type 5 K-ATP channels; millimolar ATP induces a flickery block of outward currents which is not mimicked by AMP-PNP [13]. The lack of effect of the non-hydrolysable ana-

20!

logue suggests that in this case inhibition may involve a phosphorylation. In the Type l K-ATP channel of the//-cell it appears that the active inhibitory species are the free ionized forms, ATP 4 and ATPH 3 [42, 48]. By contrast, MgATP 2 is more effective than free ATP in cardiac muscle [49], and in skeletal muscle [47, 50] there appears to be little difference in the efficacy of the Mg-bound and free ATP forms. Other adenine nucleotides are considerably less potent than ATP in blocking Type 1 K-ATP channel activity, the order of potency being ATP > ADP > AMP > adenine; other nucleotide triphosphates are also less effective [5, 30, 51]. In the case of Type4 KATP channels, however, AMP is as effective as ATP [52]. No data are yet available for the nucleotide sensitivity of Types 2 and 3 K-ATP channels. Sulphydryl-modifying reagents such as Nethylmaleimide have been reported to block irreversibly Type 1 K-ATP channels in skeletal muscle [47]. The ability of ATP to prevent this inhibition suggests that a functionally important sulphydryl group may be located at, or close to, the ATP-binding site. Modulation of inhibition. In both the fl-cell [40, 53-55] and cardiac muscle [51, 56], the inhibitory action of ATP on Type 1 K-ATP channels is reduced in the presence of ADP, suggesting that the ATP/ADP ratio may be more important than [ATP] itself in regulating channel activity in the intact cell. The effect of ADP can be mimicked by GDP, GTP and the non-hydrolysable analogues ADPflS, GDPflS and GTP),S [26]. ADP is also able to relieve ATP inhibition of Type 3 K-ATP channels [15]. The mechanism by which ADP relieves channel inhibition is complex and not fully understood. It has been suggested that ADP competes with ATP for the ATP-binding site, since ADP alone is inhibitory and 2 m M ADP increases the K~ for ATP [53]. At high ATP concentrations such a mechanism may well be operative. However, three observati'~)ns suggest that the main modulatory action of ADP may take place at a site distinct from that associated

202

s.J.H. ASHCROF'rand F. M. ASHCROFT

with inhibition. First, concentrations of ADP (10-100/~M) capable of relieving ATP-inhibition do not themselves inhibit channel activity. Secondly, Mg ~+ must be present for ADP to reduce the ATP-sensitivity of the channel [51, 56] but is not required for the inhibitory action of ADP [42, 51]. Thirdly, the modulatory effect of ADP decreases with time following patch excision, whereas the ATP-sensitivity remains unchanged [57].

Effects of pyridine nucleotides Although in inside-out patches on RINm5F fl-celis pyridine nucleotides produce activation of Type 1 K-ATP channels at low concentration and inhibition at higher concentrations [58], these effects are probably unlikely to have a regulatory role, since both the oxidized and reduced forms of the pyridine nucleotide are effective. It has been suggested, however, that NAD contributes to the tonic inhibition of the channel in the intact cell [58].

Regulation by cellular metabolism Recordings from cell-attached patches on a variety of tissues have demonstrated that KATP channel activity in the intact cell may be modulated by changes in cellular metabolism, for example in response to elevated glucose (//cells, brain neurones) or anoxia (muscle and brain). It is currently believed that the ATP/ ADP ratio is the best candidate for the physiological second messenger linking metabolism and channel inhibition. The arguments in favour of this hypothesis mostly derive from studies of Type 1 K-ATP channels in pancreatic //-cells and include [18]: (1) ATP blocks the channel in inside-out patches; (2) in cellattached patches, those substances which raise [ATP]~ reduce channel activity and those which lower [ATP]~ result in channel activation; (3) there is a good correlation between both the glucose concentration dependence and timecourse of channel inhibition [59, 60] and that of the increase in the ATP/ADP ratio. Although in RINm5F//-cells de novo synthesis of diacyl-

glycerol may be of importance for Type l K-ATP channel regulation [45], this mechanism does not appear to operate in normal fl-cells [44]. There is evidence that glycolytic metabolism may be more effective than oxidative metabolism in suppressing Type 1 K-ATP channel activity in cardiac myocytes [61, 164], an effect attributed to the localization of key glycolytic enzymes near the channel. This concept is unlikely to apply to the//-cell, however, in view of the marked effects on K-ATP channel activity of 2-ketoisocaproate whose metabolism is entirely intramitochondrial [62]. Measured concentrations of total cytosolic ATP range betwen 3.5 mM in the absence of glucose and 7.5 mM in 20 mM glucose in//-cells [63]. It has therefore been argued that the high ATP-sensitivity of the K-ATP channel in the isolated patch is inconsistent with the proposed regulatory role for intracellular ATP. Indeed, the channel should be more than 99% closed in the absence of glucose. However, when assessed in intact cells and correlated with measured cytosolic ATP concentrations, it is apparent that K-ATP channel activity varies with ATP concentrations in the millimolar range [64, 65]. A lower ATP sensitivity is also found in outside-out patches and in the whole-cell configuration [66, 67]. The ability of ADP to reduce the effect of ATP discussed above may at least partially account for this altered sensitivity. Additionally, submembrane concentrations of ATP may be considerably lower than the measured whole-cell concentrations. This possibility is supported by studies on intact HIT T15//-cells in which the activity of K-ATP channels and of Na, K-ATPase was assessed by measurement of Rb-fluxes [65]. When intracellular ATP was varied in the millimolar range, by exposing the cell to graded concentrations of oligomycin, marked changes in the activity of both membrane proteins were observed. Perforated patch recordings suggest that around 10% of K-ATP channels are active in the resting fl-cell [146]. This is in reasonable agreement with estimates from whole-cell recordings [7, 11] and flux studies on intact cells

ATP-sensitive K-channels

203

The effects of pH and ATP on Type I K-ATP channels appear to be interdependent. In insideout patches from normal//-cells, changes of pH~ in ATP-free solution had little effect on ATP channel activity until pH~ was reduced below 6.3, when the channels abruptly closed [69]. In the presence of ATP, however, channel activity decreased when pH~ was reduced to 6.3 and increased when pHi was elevated to 7.9. Evidence was also obtained from cell-attached patches for modulatory effects of pHi on channel activity. Possible explanations for these findings include effects of pH on the ATPbinding site and pH-dependent changes in the relative concentrations of the ionized forms of free ATP. The situation is quite different in skeletal muscle where, in the presence of 0.5 mM ATP, channel activity actually increases as pH~ is reduced. The single channel current amplitude is also reduced at acid pH [50]. A decrease in pH produced a small increase in K-ATP channel activity in cardiac muscle, apparently as a result of a shift in the ATP-dependence since the K i increased from 25 to 50#M [51]. No effect of pH (6.9-7.9) was found on Type 3 K-ATP channels in respiratory epithelial cells [15].

activity which requires the presence of Mg 2+ ions. The requirement for Mg 2+ suggests that a GTP-binding protein (G-protein) may be involved in this activation. This idea is supported by finding that both the non-hydrolysable analogue, GTP?S, and A1F4 (which mimics the action of GTPTS) produce K-ATP channel activation in muscle [31] and RINm5F //-cells [71]. G-protein modulation of K-ATP channel activity may be of physiological importance in mediating the effects of certain hormones and neurotransmitters. For example, in the //-cell, insulin secretion can be inhibited by galanin, by somatostatin and by ~2-adrenergic agonists [72, 73]. All three agents have been shown to produce membrane hyperpolarization, a decrease in action potential frequency, and a reduction in cytosolic calcium concentration; these effects can be abolished by preincubation with pertussis toxin [72], suggesting the involvement of a G-protein(s). In patch clamp studies both galanin [71, 74] and somatostatin [46, 75] have been shown to activate K-ATP channels in RINm5F //-cells. However, there are a number of findings which suggest that this simple picture may be incomplete. First, conclusive patch clamp evidence that these hormones activate K-ATP channels in normal //-cells is lacking [76]. Secondly, pertussis toxin was without effect on galanin activation of K-ATP channels in RINm5F //-cells [74], whereas in the same cell line pertussis toxin abolished the galaninevoked hyperpolarization and inhibition of insulin release [71]. Finally, studies from islets [72, 77] and from permeabilized cells [78, 79] indicate that all these agents can have effects on insulin secretion at a site distal to events at the plasma membrane.

Regulation by G-proteins

Regulation by divalent cations

Activation of K-ATP channels by guanine nucleotides has been reported in insulin-secreting RINm5F/~-cells [70] and in T-tubular membranes isolated from skeletal muscle [31]. In RINm5F //-cells, GTP and GDP evoke a reversible, dose-dependent increase in channel

In addition to the voltage-dependent block of outward currents described above, divalent cations can influence the open probability of KATP channels. In cardiac muscle and in fl-cells, millimolar concentrations of divalent cations (Mg 2+, Ca 2÷, Sr 2+) produce a decrease in the

[64, 65]. Thus, it is the closure of only the last few channels that appears to be responsible for the depolarization produced by glucose. It has been suggested that the reason only a small percentage of channels are active in the intact cell is that this ensures the maximum sensitivity to metabolism with the minimal expenditure of energy (the spare channel hypothesis [68]).

Regulation by intracellular pH

204

S.J.H. ASHCROFTand F. M. ASHCROFT

open probability of Type 1 K-ATP channels [25, 42, 80]. About 5 mM free Mg 2+ is needed to produce 50% reduction in channel activity in the//-cell [42]. As described above, the inhibitory effect of Ca 2+ can only be reversed by application of MgATP [80, 81]. The activity of both Type3 and 4 K-ATP channels is markedly potentiated by intracellular Ca 2+. In Type 3 K-ATP channels the K~ for channel activation is about 1/~M Ca 2+ and the channel is almost completely closed at 10-SM Ca 2+ [15, 32]. The possibility that the Type 3 K-ATP channel is identical to the maxi (BK) CaZ+-activated K-channel [82] cannot be excluded. No activity of Type4 channels in acinar membranes is found at 10 -7 M Ca 2+, but significant activation is found at 10 -6 M [17]. In //-cells higher Ca 2+ concentrations are required for channel activation: the channel is not active at 10-6M Ca 2+, and around l mM Ca 2+ is needed for 50% activation [16], suggesting that the channel is not open under physiological conditions. PHARMACOLOGY OF K-ATP CHANNELS The pharmacology of K-ATP channels has been recently reviewed [83-85], as have the actions of sulphonylureas and K-channel openers [86]. Channel blockers

Sulphonylureas (Table2) are a class of hypoglycaemic drugs that have been used for many years in the treatment of NIDDM. It has recently become evident that the mechanism by which these drugs increase insulin release is by inhibition of the Type 1 K-ATP channel [8, 9, 64, 81, 87, 88, 90-93]. Sulphonylureas appear to be highly specific for the K-ATP channel in//cells and cardiac muscle [9, 81, 93], although this point has not been investigated in other tissues. They have also been shown to block K-ATP channels in skeletal muscle [3] and smooth muscle [4] at the single channel level. The relative potency of sulphonylureas on K-ATP channel activity has only been studied

in detail in the//-cell [64, 92, 93] where the order of potency for inhibition of whole-cell currents (Ki) is glibenclamide (4 nM) > glipizide (6 nM) > meglitinide (HB699; 2 #M) > tolbutamide (4#M) [92]. This correlates well with inhibition of insulin secretion [90]. The structures of these drugs are somewhat different: glibenclamide contains both sulphonylurea and non-sulphonylurea moieties; HB699 consists only of the non-sulphonylurea moiety; tolbutamide consists of the suiphonylurea part of glibenclamide. It has therefore been suggested that the high potency of the second generation sulphonylureas (such as glibenclamide) results from their interaction with the receptor at more than one site. Tolbutamide and glibenclamide have been most widely studied and are effective when applied to either the intracellular or extracellular membrane surface [94]. Unlike tolbutamide, the inhibitory action of glibenclamide is usually slow and irreversible [81, 96]. The mechanism by which sulphonylureas produce channel inhibition is unclear. Doseresponse curves for tolbutamide are well fitted by the Hill equation using a Hill coefficient of l, suggesting that there is a one-to-one binding between the drug and the channel [92, 94]. This may not be the case for glibenclamide since Hill coefficients of 1.8 [92] and 1.5 [139] have been reported. Tolbutamide affects K-ATP channel kinetics in a manner similar to glucose, decreasing the burst duration and increasing the burst interval [81]. It has been suggested that the drugs reach their target site by dissolving in the lipid phase of the membrane [95]. Consistent with this idea, the rate at which tolbutamide blocks K-ATP channels is slowed when extracellular pH is increased, suggesting that the undissociated form is the effective species [96]. Tolbutamide is more potent in the presence of intracellular ADP which may explain the observation that the sulphonylurea is more effective in whole-cell recordings [97]. Other nucleotides (ATP, AMP, GTP, GDP) have no effect on the efficacy of tolbutamide inhibition [94, 97]. The interaction of sulphonylureas with other types of K-ATP channel has received less atten-

ATP-sensitive K-channels

205

TABLE 2. K-ATP CHANNELBLOCKERS Type

Blocker

1

Tolbutamide

2 3 4 1

Glibenclamide

2 3 4 1 I 1 l

Meglitinide Glipizide Charybdotoxin TEA

3 4 1 4 3 4 1 1 1 1

Sparteine Amantadine Ligustrazine 4-Aminopyridine

3 I 1 1 1

Lidocaine Pentobarbitone Thiopentane Secobarbitone Phenobarbitone

Quinine Quinidine

Location CRI-G1 fl-cell Human fl-cell Rodent #-cell Skeletal muscle Cardiac muscle VMH neurone Epithelia CRI-G1 fl-cell Mouse fl-cell Human #-cell CRI-G1 fl-cell Smooth muscle VMH neurone Epithelia CRI-G1 fl-cell Mouse fl-cell Mouse fl-cell Smooth muscle Mouse fl-cell Cardiac muscle Skeletal muscle Axon Epithelia CRI-G1 fl-cell Mouse fl-cell CRI-G1 fl-cell Epithelia CRI-G1 fl-cell Rat fl-cell Rat fl-cell Mouse fl-cell Cardiac muscle CRI-GI fl-cell Epithelia CRI-GI fl-cell CGR-G1 fl-cell CRI-GI fl-cell CRI-G1 fl-cell

Efficacy (Ki) 17/~M 5-18 pM 3-7 #M 60 pM 380 pM Inhibits at 100/tM* Ineffective t Ineffective at ! mM 4 nM 50 nM 27 nM Inhibits at 20 pM Ineffective at 100 pM$ Ineffective t Ineffective at 10/~M 2 #M 6 nM Ineffective at 100 nM > 20 mM Inhibits at 0.5 mM 26 mM[[ 3 mM~ < 5 mM Ineffective at 10 mM Inhibits at 10 pM Inhibits at 10 pM** Inhibits at 1 mM Inhibits at 10/tM 200 #M 150 pM < 1 mM Inhibits at 500 #M Inhibits at 2 m M t t Inhibits at 5 mM 360 pM 60 #M 250 pM 17% decrease by 1 mM

Reference 94 11, 123 9, 81, 92 3 93 98 94 92 123 94 4 98 94 92 92 4 100 147 101 149 15 16 100 16 15 16 $$ ++~ 143 147 16 15 151 151 151 151

The whole-cell configuration was used to determine the concentration of the sulphonylureas tolbutamide, glibenclamide (glyburide), meglitinide (HB699) and glipizide required to produce half-maximal inhibition (K~). Other drugs were tested on single channel currents in inside-out patches except where stated otherwise. * In cell-attached patch; no effect in inside-out patch; t Greger R, personal communication; :~ in inside-out patch; 1[ 7 mM when applied from inside; ¶ applied internally; ** both external and internal. Block increases with depolarization; t t no block by 10 mM external 4-aminopyridine; $:1:Ashcroft F. M., Gibson J. and Kerr A. J. (unpublished observation).

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tion. In the ventromedial hypothalamic neurones the effects of sulphonylureas on Type 2 KATP channels are anomalous [98]. Whereas tolbutamide inhibits channel activity in cellattached patches, glibenclamide is without effect, although it is capable of preventing the action of tolbutamide. Furthermore, tolbutamide is not effective in excised membrane patches. Sulphonylureas do not appear to inhibit the K-ATP channels (Type 3) in epithelial cells (Greger R., personal communication) and are without effect on Type 4 K-ATP channels [94]. The high sensitivity of the K-ATP channel to glibenclamide (Ki = 2 nM) raises the interesting possibility that an endogenous ligand for the sulphonylurea receptor may exist. An endogenous peptide capable of displacing glibenclamide binding from brain membranes has been extracted from rat brain [99]. A number of other K-channel blockers (TEA, quinine, sparteine, amantadine) are known to interact with the Type 1 K-ATP channel (Table 2). Unlike the sulphonylureas, many of these agents also have effects on other types of K-channel. For example, in fl-cells TEA blocks K-ATP channels, but less effectively than Ca-activated and delayed rectifier K-channels; conversely, quinine is more potent on K-ATP channels (K~ < 10 pM versus K~ > 100/tM for the Ca-activated K-channel [100102]. Ligustrazine is an interesting exception since this drug is a selective blocker of Type 1 K-ATP channels in fl-cells, with a K i of < l mM [143]. Type l K-ATP channels are also inhibited by the barbiturates thiopentane and pentobarbitone [141, 151], which means that these general anaesthetics should be avoided in whole-animal experiments on the role of KATP channels. Less information is available regarding the inhibition of other types of K-ATP channels by K-channel blockers. Channel openers

A number of drugs are known to increase Type 1 K-ATP channel activity and lead to membrane hyperpolarization (Table3). Patch

clamp studies have shown that the efficacy of these agents varies greatly between tissues. For example, in the mouse fl-cell, diazoxide has a very potent effect, pinacidil produces some activation, nicorandii is without detectable effect and (at high concentration), minoxidil and cromakalim are inhibitory [103, 104] (but see [!527 153] for different effects on insulinsecreting RINm5F fl-cells). By contrast, in smooth muscle cromakalim produces a pronounced activation of K-ATP channels [4]. Cromakalim, RP4356 and pinacidil are activators of K-ATP channel activity in cardiac muscle [106-108] and have also been shown to activate a tolbutamide-sensitive K-conductance in human skeletal muscle [110]. Surprisingly, diazoxide inhibits K-ATP channels in cardiac muscle [109]. Like the sulphonylureas, K-channel openers have been reported to have effects on a wide variety of other tissues, such as the pulmonary vasculature, aorta, intestine, trachea and bladder [86]. In many of these tissues the effects of K-channel openers can be reversed by sulphonylureas, suggesting the involvement of K-ATP channels. Direct evidence for the presence of KATP channels in these tissues is lacking, however, and the possibility that other mechanisms may be involved in the action of these drugs must be kept in mind. Indeed, the selectivity of K-channel openers has not been investigated in detail (although in heart they are without effect on the inward rectifier [106]). The mechanism of action of K-ATP channel openers is unclear, although it is known that they have no effect on the single channel current amplitude but rather increase the time the channel spends in the open state. In the heart, activation of K-ATP channels by cromakalim could only be demonstrated at 37°C and not at room temperature [150]. It is possible this may simply result from differences in the rate of access of the drug to the target site since, in the E-cell, diazoxide is more effective when applied to the inside (rather than to the outside) of the membrane in excised patches [103]. The action of diazoxide requires the presence of MgATP at the intracellular membrane surface [9, 81, 88,

ATP-sensitive K-channels

207

TABLE 3. K - A T P CHANNEL ACTIVATORS

Type

Activator

Location

Efficacy

Reference

1

Diazoxide

1

Pinacidil

!

Nicorandil

1 1

Minoxidil sulphate Cromakalim

1

RP 49356

1

SR44866

Mouse fl-cell CRI-GI fl-cell RINm5F fl-cell Cardiac muscle Mouse fl-cell RINm5F fl-cell Cardiac muscle Mouse fl-cell RINm5F fl-cell Mouse fl-cell Mouse fl-cell CRI-G1 #-cell RINm5F fl-cell Cardiac muscle Smooth muscle Cardiac muscle RINm5F fl-cell Cardiac muscle

Increases (Ki; 20-102/~M)~:t Increasest Increases at 100 #M* Inhibits at 500/~M* 35% increase by 500/~M~" Increases at 200 pM*§ Increases at 300 #M No effect at 500 #Mr Increases at 200 ~Mtll 29% decrease at 500 #Mr 49% decrease at 500/~Mt No effect at 100/~M* Increases at 1-200/~MII Increases at 300 #M* Increases at 1 /IM* Increases at 30 #M* Increases at 200 #M*§ Increases at 50/~Mt*

92 94, 103 88, 155 109 104 153 106 104 153 104 104 103 152 107, 106 4 144, 106 153 162

t Indicates the whole-cell configuration was used; * indicates drugs were tested on single channel currents; :~20/~M with 0.3/~M ATP i and 102/~M with 1 /~M ATPi; § whole-cell currents in presence of cytosolic ATP; I[ single channel currents tested in presence of cytosolic ATP. 92, 94, 103]; diazoxide has little effect in the absence of MgATP [103, 88] and is actually inhibitory in the presence of ATP 4" [103]. Activation of K-ATP channels in RINm5F flcells by pinacidil, RP 49356, nicorandil and cromakalim also requires cytosolic ATP [152, 153]. Since non-hydrolysable ATP-analogues cannot substitute for ATP it has been suggested that the actions of these drugs involves phosphorylation of the channel or an associated control protein [103, 155]. The K-channel opener RP 49356 has been reported to reduce the sensitivity of the channel to inhibition by ATP in cardiac cells [144]. Diazoxide is unable to reverse inhibition produced by quinine [100] or glibenclamide [94], but can reverse inhibition by tolbutamide or ligustrazine [143]. THE S U L P H O N Y L U R E A RECEPTOR The high potency and specificity of the effects of sulphonylureas on K-ATP channels has

prompted the use of these drugs as ligands for channel isolation. High affinity sulphonylurea binding sites have been described in #-cells [64, 90, 111-114], heart [89] and brain [112, 114116]. In the brain they appear to be localized to those regions such as the substantia nigra and the globus pallidus, which are associated with movement control [117]. The relative ability of various sulphonylureas to displace [3H]glibenclamide from #-cell [90, 111] and heart membranes [89] correlates with their efficacy as K-ATP channel blockers. It is not yet clear, however, whether the sulphonylurea receptor is itself the K-ATP channel or whether it is a separate protein. The Kd (nM) for binding of glibenclamide to membranes has been variously reported as 0.4 in mouse #-cells [90]; 0.7 [11 !] or 1.12 [113] in HIT T15 #-cell membranes; 2 in cardiac muscle [89]; 0.3 in RINm5F #-cells [64]; 0.8 in pig brain microsomes [116]. A second class of low-affinity sulphonylurea-binding sites has been found in HITT15 #-cell membranes [113] and in rat cerebral cortex membranes

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S.J.H. AS.CROFTand F. M. AsncaoFx

[115]. The number of high affinity sulphonylurea-binding sites (pmol/mg protein) in isolated membrane preparations has been reported as 0.02 in cardiac muscle [89]; 0.11 in cerebral cortex [115]; 0.93 or 1.38 in mouse fl-cells [90]; 0.15 in RINm5F /~-cells [64]; 0.4 in pig brain [116]; 0.55 [113] or 1.09 [lll] in HIT T15 fl-cell membranes. In intact cells, however, the density of binding sites is much greater, probably due to protein degradation or inactivation during membrane preparation; estimates of up to approximately 18,500 glibenclamide-binding sites per cell have been obtained in HIT T15 flcells (Niki I., personal communication). Diazoxide does not compete with glibenclamide for binding to HIT T15 fl-cells, suggesting that these two classes of drugs bind to different sites [113]. This is in agreement with electrophysiological data [103]. ATP also fails to displace bound [3H]glibenclamide. However, ADP (0.5-1 mM) specifically reduces [3H]glibenclamide binding to isolated HIT T15 fl-cell membranes [113]. A similar effect is found in intact HIT cells both with ADP and ADP-agarose (Niki I., personal communication), suggesting that the binding site for sulphonylureas is at the external side of the membrane. A sulphonylurea-binding protein has been solubilized from pig brain membranes and purified ( ~ 2000-fold) tO apparent homogeneity on polyacrylamide gels; the molecular weight was 154,000 on gel electrophoresis under both reducing and non-reducing conditions [116]. This molecular weight is similar to that reported for a protein species from rat fl-cell tumour membranes photoaffinity labelled with glibenclamide [118]. No sequence data have yet been reported, nor has the protein been functionally reconstituted and shown to possess ionophoretic activity. Since, at this stage, it is unclear whether the sulphonylurea receptor constitutes the K-ATP channel there remains the possibility that the receptor may couple to different channels (or other membrane proteins) in tissues in which K-ATP channels have not yet been identified. Other receptors for sulphonylureas also cannot be excluded: for example, suiphonylurea binding sites have recently been

described in adipocytes, but the affinity for glibenclamide is about three-fold lower [154]. PHYSIOLOGICAL ROLES OF K-ATP CHANNELS The physiological role of the K-ATP channel is to couple cellular metabolism to electrical activity and ionic fluxes. This coupling may have different functional consequences in different tissues. Insulin secretion

In the pancreatic /3-cell it is clear that the Type 1 K-ATP channel plays a key role in the insulin secretory response both to the major physiological regulator, glucose, and to the main pharmacological stimulant, the sulphonylureas (for reviews see [18-21, 35, 37, 76, 85, 119). The K-ATP channel is the channel principally responsible for maintenance of the resting potential in the unstimulated/~-cell [23]. In cellattached patches, channel activity is inhibited by a rise in the extracellular concentration of glucose or other nutrient secretagogues [23, 40, 121]. There is good evidence that this effect requires intracellular metabolism of the stimulant [121-123], as previously documented for the effect of these agents on insulin secretion [120]. It appears possible that a product of mitochondrial metabolism mediates the effect of the sugar on channel activity since 2-ketoisocaproate has similar effects to glucose on KATP channel activity [62], and inhibitors of mitochondrial ATP synthesis reverse the effects of glucose [40, 161]. The closure of the channel by glucose metabolism has two main consequences. First, it depolarizes the/3-cell to the threshold potential at which electrical activity is initiated [18]. Second, at higher concentrations further closure of these channels increases the frequency of fl-cell action potentials [87, 124, 146]. The Ca 2+ influx associated with the action potential stimulates insulin secretion and there is a close correlation between the glucose dependence of action potential frequency and that of insulin

ATP-sensitive K-channels release. Inhibition of K-ATP channels has been shown to precede the depolarization-evoked increase in intracellular Ca 2÷ [59]. As discussed above there is also some evidence that the modulation of K-ATP channels by hormones and transmitters may be the mechanism by which these substances influence insulin secretion. The identification of the K-ATP channel as the site of action of the sulphonylureas is a landmark in our understanding of the action of antidiabetic drugs. In non-insulin dependent diabetic patients insulin secretion in response to glucose is reduced, but sulphonylureas are still effective [125]. This suggests that events distal to closure of K-ATP channels may be unimpaired and that the secretory defect resides in the coupling of glucose metabolism to K-ATP channel activity. Studies on E-cells from noninsulin dependent diabetic patients are required to test this possibility. In this context it is relevant that the failure of glucose to stimulate secretion from foetal E-cells results from defective metabolism of the sugar rather than altered K-ATP channel properties [138].

Cardiac ischaemia It appears likely that the K-ATP channels in cardiac muscle are almost completely closed under normal conditions, since sulphonylureas have little effect on the cardiac action potential [89]. In ischaemia, however, the action potential duration shortens and K+-efflux increases, as previously reviewed [126, 127]; both of these effects can be blocked by sulphonylureas suggesting that they result from activation of K-ATP channels [128]. Similar results are found during metabolic blockade [129]. This mechanism may serve two functions. First, increased extracellular K ÷ will lead to vasodilation and increased blood supply to the tissue. Secondly, the decreased contractile force resulting from the reduced action potential duration will help preserve cellular ATP levels. There is currently controversy as to whether cellular ATP can serve as the coupling factor in ischaemia because it is difficult to demonstrate significant

209

changes in ATP levels in the anoxic heart [34, 129]. The ATP concentration producing half-maximal inhibition of channel activity is increased in myocytes from hypertrophied hearts (from 75 to 250/~M) [130]. This reduced ATP-sensitivity has been suggested to have a protective function. Silent ischaemia (ischaemia without pain) has been reported in diabetic patients treated with sulphonylureas. Although this may result from diabetic neuropathy, an alternative explanation could be that sulphonylurea inhibition of KATP channels prevents the K+-efflux that normally leads to the sensation of pain.

Skeletal muscle function The physiological role of the K-ATP channels in skeletal muscle is not well understood, since cell-attached recordings of channel activity in this tissue have not been reported. During exercise K + is lost from working muscle resulting in a rise in plasma potassium which may have both a local vasodilatory effect [! 56] and also serve to stimulate the carotid body to increase ventilation [131]. Activation of K-ATP channels may contribute to the K-efflux from muscle. Indeed, Rb + efflux from metabolically exhausted muscle shows a pharmacological sensitivity characteristic of Type 1 K-ATP channels [132]. Again, clear evidence that changes in cellular ATP are sufficient to modulate channel activity is lacking. Recent evidence suggests that a more likely coupling factor may be the decrease in intracellular pH that occurs in skeletal muscle during exercise [50]. In this tissue a decrease in intracellular pH markedly reduces the inhibitory effect of ATP in excised patches ([50] and see above). However, the cytosolic ATP/ADP ratio may link metabolism to K-ATP channel inhibition in subjects with McArdle's syndrome, who cannot catabolize glycogen [159]. In these subjects a marked rise in K-efflux during exercise is also found [157, 159], which (unlike normal subjects) is associated with a reduced muscle phosphorylation potential [158]

210

S.J.H. ASnCROFTand F. M. ASHCROFT

and an increase in blood pH [157, 159] (and presumably, therefore, intracellular pH). Smooth muscle tone Recent evidence suggests that K-ATP channels may play a major role in the regulation of smooth muscle tone and thus in the local regulation of blood flow. These channels have been described [4] in mesenteric artery smooth muscle where they can be activated by K-channel openers such as cromakalim (1 #M) and inhibited by glibenclamide (20/~M). They may thus be a major target for antihypertensive drugs. Activation of K-ATP channels may also be of physiological importance in the mechanism of action of endogenous vasorelaxants, such as endothelium-derived hyperpolarizing factor, vasoactive intestinal peptide and calcitonin-gene-related peptide [4, 164]. Neuronal functions The glucose-sensing neurones of the ventromedial hypothalamus (VMH) play an important part in appetite control. Recent studies have implicated the Type 2 K°ATP channel in the glucose-sensing mechanism of these neurones [12, 133]. In response to glucose-free solution, or to metabolic inhibition by mannoheptulose, VMH neurones hyperpolarize and cease to fire action potentials, due to activation of KATP channels [12]. In addition, the activation of the K-ATP channel may underlie the rise in extracellular K + observed in response to cerebral anoxia. Support for this idea comes from the finding that anoxia elicits a neuronal hyperpolarization that can be blocked by sulphonylureas [117, 134, 135]. As in the heart, this may be a mechanism for short-term conservation of cellular ATP levels. It may also serve to increase local blood flow by a mechanism involving glial cells. In response to an increase in extracellular K +, glial cells translocate K + to endfeet located on the capillary endothelium, where release of K + produces a vasodilation which increases blood flow to anoxic regions [148].

The recent finding that Type i K-ATP channels are present in the nodal regions of vertebrate axons suggests that they may play a role in regulating nerve conduction: activation of KATP channels would be expected to lead to a failure of transmission. It also seems possible that K-ATP channels may serve to communicate the metabolic state of the axon to the adjacent glia. It is believed that a rise in extracellular K + stimulates glia cells to release nutrients to the adjacent nerve [148]: this K + might be the result of electrical activity in the nerve or result from activation of K-ATP channels under conditions of metabolic stress. F U T U R E PROSPECTS The compelling evidence to implicate K-ATP channels in a wide variety of normal and pathological cellular functions indicates the urgent need for detailed structural information on this family of channels. A first step towards the cloning of the Type 1 K-ATP channel has been the expression of K-ATP channel activity in Xenopus laevis oocytes after microinjection of m R N A from H I T T i 5 fl-cells [136]. Cloning of the different types of K-ATP channel will allow molecular characterization of their function and regulation and will open up the possibility of the rational design of new and more selective channel openers and blockers. The possible roles of K-ATP channels in disease is an important subject for future studies. Acknowledgements--Work in our own laboratories has been supported by the British Diabetic Association, the Medical Research Council, the Wellcome Trust, the Parkinson's Disease Society, the Royal Society, the E.P. Abraham Fund, Nordisk U.K., the Smith Kline and French Foundation. F. M. AsncRorr is a Royal Society 1983 University Research Fellow. REFERENCES 1. Noma A. (1983) Nature 305, 147-148. 2. Spruce A. E., Standen N. B. and Stanfield P. R. (1985) Nature 316, 736--738. 3. Woll K. H., Lonnendonker U. and Neumcke B. (1989) Pfliigers Arch. 414, 622-628.

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