Pfliigers Arch (1992) 422:185-192

Joumal of Physiology 9 Springer-Verlag1992

Nucleotide diphosphates activate the ATP-sensitive potassium channel in mouse skeletal muscle Bruno Allard and Michel Lazflunski

Institut de Pharmacologie Mol6culaire et Cellulaire, 660 route des Lucioles, Sophia Antipolis, F-06560 Valbonne, France Received June 22, 1992/Received after revision July 28, 1992/Accepted July 31, 1992 Abstract. Patch-clamp techniques were used to study the

effects of internal nucleotide diphosphates on the KATe channel in mouse skeletal muscle. In inside-out patches, application of G D P (100 gM) and A D P (100 gM) reversibly increased the channel activity. In the presence of internal Mg 2+ (1 mM), low concentrations of A D P ( < 300 gM) enhanced channel activity and high concentrations of A D P ( > 300 gM) limited channel opening while G D P activated the channel at all concentrations tested. In the absence of internal Mg 2+, A D P decreased channel activity at all concentrations tested while G D P had no noticeable effect at submillimolar concentrations and inhibited channel activity at millimolar concentrations. G D P [flS] (100 ~tM), which behaved as a weak G D P agonist in the presence of Mg 2+ , stimulated ADPevoked activation whereas it inhibited GDP-evoked activation. The K + channel opener pinacidil was found to activate the KATP channel but only in the presence of internal GDP, A D P and G D P [flS]. The results are discussed in terms of the existence of multiple nucleotide binding sites, in charge of the regulation of the KATe channel. Key words: Patch-clamp - K~Tp channel muscle - Nucleotide diphosphates

Skeletal

Introduction

shortening of the cardiac action potential during ischaemia in cardiac myocytes [7, 24, 39, 43]. The KATP channel from skeletal muscle has not been as extensively studied as in cardiac or pancreatic cells and the role and the mechanisms underlying activation of these channels under physiological conditions remain to be completely elucidated. This channel has been described in sarcolemmal vesicles [371 and sarcolemmal blebs of frog muscle [42] and in sarcolemmal vesicles and sarcolemmal blebs of h u m a n muscles [5, 32]. It has also been observed in transverse tubules and in the surface membrane of adult mouse skeletal muscle [30, 46]. Effects of nucleotide diphosphates (NDP) on cardiac and pancreatic fl cells have been well studied but their effects on the KATe channel of skeletal muscle have not been analysed in detail. In the absence of Mg 2§ , Spruce et al. [37] found that A D P induced an inhibitory action on the channel activity that was three times weaker than that of ATP on sarcolemmal vesicles of frog muscle. A D P did not appear to reduce the effectiveness of A T e in closing channels. On the other hand, Vivaudou et al. [42] reported that in sarcolemmal blebs of frog muscle the sensitivity to ATP was reduced by A D P in a manner consistent with a competition between A D P and ATP for the same inhibitory binding site. We have investigated in this paper the effect of N D P on the activity of the KATPchannel in isolated membrane patches of mouse skeletal muscle. It was found that A D P and G D P have large modulatory effects on the NATP channel activity, which are dependent on whether Mg 2+ is present or absent in the cytosol. The results are discussed in terms of a model of a multi-nucleotide-binding site for the muscle KATP channel.

ATe-sensitive K + channels (KATe) are present in cardiac and skeletal muscle cells [29, 36], as well as in insulin-secreting pancreatic islets cells [9] and in neurons [3]. This K + channel plays a crucial physiological role in pancreatic fl cells and in cardiac muscle. KATp channels are strongly implicated in the regulation of insulin secretion in pancreatic fl cells [1, 17] and are responsible for the

Isolation o f skeletal muscle fibres. Mouse flexor digitorum brevis mus-

Correspondence to: M. Lazdunski

cles were incubated at 37 ~ in Tyrode solution containing collagenase (Sigma type I) (2 mg/ml) for 1.5 h. After enzyme treatment, muscles were rinsed with Tyrode and stored in Tyrode at 5 ~ until use. Before each experiment, whole muscles were transferred into disposable 35-ram

Materials and m e t h o d s

186 tissue-culture dishes (Falcon) and intact skeletal muscle fibres were separated from the muscle mass by gently triturating the muscle with a plastic pasteur pipette.

Table 1. Calculated concentration of free ADP and Mg-ADP for various added ATP concentrations in mouse skeletal muscle Concentration (gM) after addition of total ADP at

Electrophysiology. Single-channel currents were recorded at 0 mV from

inside-out or outside-out membrane patches at room temperature using a voltage patch-clamp amplifier RK 300 (Bio-Logic, Grenoble, France). Pipettes were coated with Sylgard resin to reduce current noise. Currents were stored in digitized format on digital audio tapes using a Bio-Logic DTR 1201 recorder for further analysis. Currents flowing respectively into the pipette in the inside-out configuration and out of the pipette in the outside-out configuration were represented as positive. The cutoff frequency of the chart recorder (Kipp & Zonen BD 100, Delft, Holland) was 5 Hz. Short segments of channel activity were filtered at 500 Hz and sampled at 1 kHz. Channel activity was found from the average current (1) as N P o = I / i in each patch were i is the single-channel current, N the number of open channels and Po, the open-state probability. For each solution, N P o was measured over 60-s records after filtering at 500 Hz and sampling at 1 kHz. Average changes in channel activity (NPo) are expressed as means_+SEM, with the number of experiments in parentheses. Records generally began 2 rain after the patch was formed. External Tyrode solution contained (in mM) 135NaC1, 4 KC1, 2.5 CaC12, 1 MgC12, 10 HEPES/NaOH, pH 7.4. The internal solution contained (in mM) 150 KC1, 1 MgC12 (or zero when mentioned), 0.5 EGTA, 10 HEPES/KOH, pH 7.4. After excision, membrane patches were exposed to different solutions by placing them in the mouth of a perfusion tube from which flowed the rapidly exchanged solutions. GDP (Sigma) and ATP (Sigma) were used as sodium salts and ADP (Sigma) and GDP [flS] (Boehringer) as lithium salts, all buffered at pH 7.4 with KOH. Glibenclamide and pinacidil were first dissolved in dimethylsulphoxide at 0.1 M and then diluted in adequate solutions at the desired concentrations. Table 1 indicates the resulting calculated concentrations of free ADP and MG-ADP for various concentrations of total ADP added in an internal solution containing 0.5 mM EGTA and 1 mM MgC12. The condiSolutions.

A

Free ADP Mg-ADP

I0 ~tM

30 ~tM

100 ~tM

300 pM

1000 ~tM

4.9 5.1

15.7 14.3

52.5 47.5

162 138

623 377

tional stability constants at t = 20~ and pH 7.4, obtained from Fabiato [19] and used to calculate these concentrations, are 107 M -1 for Ca-EGTA, 101'S2M- t for Mg-EGTA, 102'7M -1 for Ca-ADP and 103 M 1 for Mg-ADP. The contaminating Ca 2+ was supposed to be no more than 10 gM. Resulting concentrations of free GDP and Mg-GDP were assumed to be the same as those of free ADP and Mg-ADP.

Results In the presence of physiological external K § concentrations and internal EGTA, excised membrane patches of mouse skeletal muscle contained one type of ionic channel with an amplitude of 1 . 8 - 2 . 5 pA at 0 mV. Although basal activity was highly variable from one patch to another, frequent and sustained openings of this K + channel were generally observed in isolated patches with the inside-out or outside-out configurations. During an experiment, generally of 30 min duration, the activity of the channel did not significantly run down. The single-channel activity was almost completely abolished by application of 0.1 mM ATP to the cytoplasmic side of the membrane (Fig. 1A). In this patch, N P o was decreased from

B

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Fig. 10 A Effects of intracellular ATP on channel activity in an inside-out patch (top trace: chart recording; b o t t o m trace: segments of the main trace on expanded scales). B Concentration-dependent effects of glibenclamide on channel activity in an outside-out patch (top trace: chart recordings; b o t t o m trace: segments of the main trace on expanded scales). C Conductance properties of the channel current in an inside-out patch (left: example of channel currents at various membrane potentials; right." current/voltage relationship)

187

In this patch, N P o was increased from 0.35 to 1.03 by ADP and from 0.1 to 1.9 by GDR These effects were fully reversible. Effects of ADP were then further examined over a range of concentrations. The response to ADP depended on the concentration used (Fig. 2 B). At all concentrations below 0.3 mM, ADP stimulated channel activity. At concentrations greater than 0.3 mM, ADP reduced channel activity. In this patch, N P o was increased from 0.15 in control conditions to 0.54, 2.6 by 1 0 p M A D P and 100 gM ADP respectively. In the presence of 1 mM ADP, N P o was further reduced to 0.2. It was also found that N P o was multiplied by a factor of 4.6+_1.7 (n = 5), 8.6+_2.3 (n = 10) and 3.6+_1.7 (n = 4) in the presence of respectively 10 pM ADP, 100 gM ADP and 1 mM ADP. Wash-out of ADP then caused a very significant potentiation of channel activity ( N P o = 2.4), which lasted 40 s before returning to the control value. This potentiation of channel activity can be interpreted as a "refreshment" phenomenon as observed after wash-out of Mg-ATP (Fig. 1A) and already described in insulin-secreting cells [23]. Table 1 indicates that at 300 gM total ADP, the resulting concentrations of free ADP and Mg-ADP are 162 ~tM and 138 gM respectively. In order to determine whether the limited activation observed in the presence of high concentrations of total ADP may be due to inhibition by free ADP in this concentration range, similar experiments were conducted in the absence of Mg 2+ . Upon removal of Mg 2+ from the internal solution, the unitary conductance of the outward current was increased as already reported for frog skeletal muscle [42] and for insulin-secreting cells [21]. In this patch, the amplitude of the unitary current was increased from J.8 pA to 2.4 pA (Fig. 3 A). No other significant modification of channel activity was observed. Figure 3B shows that 100 ~tM ADP decreased channel activity in the absence of internal Mg 2+ . A subsequent addition of 1 mM internal

0.42 to 0.02 in the presence of ATP. Upon wash-out of ATP, channel activity was restored and even potentiated as shown in Fig. 1A, where N P o was estimated to be 1.24. Figure 1B shows that, in outside-out membrane patches, channel activity was inhibited by the sulphonylurea glibenclamide externally applied in submicromolar concentrations (100-300 nM). Complete blockade was achieved in the presence of micromolar concentrations ( 3 - 1 0 gM). In this patch, N P o was reduced from 2.38 to 0.05 by 3 gM glibenclamide. Inhibition was partially reversible ( N P o = 0.3) after wash-out of the drug with a delay of 20 s. A similar pattern of inhibition was seen in eight patches. A comparable glibenclamide sensitivity was found by Vivaudou et al. [42] on sarcolemmal blebs of frog muscle. Skeletal muscle appears to be much less sensitive to sulphonylureas than cardiac muscle or pancreatic B cells where block has been observed with glibenclamide concentrations in the nanomolar range [24, 34]. A typical current/voltage relationship is illustrated in Fig. 1 C. The single-channel current/voltage relationship was linear over the potential range from - 60 mV to + 40 mV in physiological external K + concentrations. At potentials positive to +40mV, saturation of unitary current amplitude was apparent. The current/voltage relationship has a slope of 24 pS. All these results taken together indicate that this channel is the KATP channel, which has also been described by Woll et al. [46] and Weik and Neumcke [44] in elevated external K + concentrations on the same preparation. Effects of NDP were tested on channel activity of inside-out membrane patches. In the presence of internal Mg 2+ , single-channel activity was enhanced. Figure 2A shows that, in the same inside-out membrane patch, channel activity was successively enhanced by 0.1 mM ADP and 0.1 mM GDP applied to the cytoplasmic side.

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Fig. 2. A Effects of intracellular ADP and GDP on channel activity in an inside-out patch (top trace: chart recording; bottom trace: segments of the main trace on expanded scales). B Concentration-dependent effects of intracellular ADP on channel activity in an inside-out patch in the presence of 1 mM internal Mg2+ (top trace: chart recording; bottom trace." segments of the main trace on expanded scales)

188

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Mg 2+ then markedly increased channel activity above the control. This activatory effect of Mg 2+ was reversible. When the concentration of ADP was further enhanced to 1 mM in the absence of internal Mg 2+, channel activity was entirely suppressed. Upon wash-out of ADP the channel activity returned to its control value. Clearly ADP behaved as an activator or an inhibitor of the KAyP channel depending on the internal Mg 2+ concentration. The effect of different concentrations of ADP in the absence of Mg 2+ is shown in Fig. 3 C. A significant inhibition was observed at 100 ~tM, where N P o was decreased from 0.28 to 0.08 and an almost complete suppression of openings was seen at 1raM ADP ( N P o = 0.04). This inhibition was reversible upon washout of ADP (Fig. 3 C). We found, in three patches, that N P o was decreased to 37~ _+8 and 11 ~ _+7 of control by 100 ~tM and l mM ADP respectively. Average changes in channel activity at 10 ~tM were not significant.

A

Fig. 3. A Effects of intracellular Mg 2+ on the unitary current in an inside-out patch. B Effects of internal Mg 2+ upon the response of the channel to the application of intracellular A D P in an inside-out patch (chart recording). C Concentratio,n-dependent effects o f intracellular A D P in the absence of internal Mg 2+ in an inside-out patch (top trace." chart recording; bottom trace: segments of the main trace on expanded scales)

The effects of GDP on channel opening were also examined. In the presence of internal Mg 2+, GDP up to 1 raM, applied to the internal side of the membrane patch, increased channel activity in a concentration-dependent fashion (Fig. 4A). In this patch, N P o (0.01 in the control) increased to 0.1, 1.2 and J.2 after addition of 10 gM, 100 gM and 1 mM GDP respectively. The high resulting concentration of free GDP (around 600 ~tM, see Table 1) in the presence of 1 mM total GDP, which could be just slightly inhibitory (see below), can account for the limited activation observed during 1 mM GDP. It was also found that N P o was multiplied by a factor of 27_+ 11 (n = 9) and 38+15 (n = 9) by 100 gM and 1 m M G D P respectively. Conversely, in the absence of internal Mg 2+ , channel activity was not significantly affected by GDP up to 1 mM. At 3 mM, free GDP induced a large and reversible decrease of channel opening (Fig. 4B). In this patch, N P o was reduced from 0.6 in the control to 0.34 and 0.01

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Fig. 4. A Concentration-dependent effects of intracellular G D P on channel activity in an inside-out patch in the presence of 1 m M internal Mg a+ (top trace." chart recording; bottom trace: segments of the main trace on expanded scales). B Concentration-dependent effects of intracellular G D P on channel activity in an inside-out patch in the absence of internal Mg 2+ (top trace: chart recording; bottom trace." segments of the main trace on expanded scales)

189

0.1mMGDP[BS] in the continued presence of GDP dramatically reduced channel opening. In this patch, N P o was increased from 0.53 to 4.6 by 100 BM GDR Addition of 100 gM GDP[flS] further decreased N P o to 1.46. This effect was also reversible upon return to the GDP-containing solution. N P o was reincreased to 3.2 in the sole presence of GDP. Average changes in channel activity from several patches indicated that N P o potentiated by 100pMGDP was decreased to 49%+11 by 10 BM GDP[flS] (n = 6). It has been reported that K + channel openers such as pinacidil, or cromakalim, activate the KATp channel of mouse skeletal muscle [45]. This channel activation by openers was found to require the presence of ATP (0.1 raM) at the cytoplasmic side. It was therefore of interest to determine whether cytoplasmic NDP could have an effect on pinacidil activation as already described in cardiac cells [35, 41] and in the rabbit portal vein where, in the cell-free patch configuration, the simultaneous presence of external pinacidil and internal GDP was required to activate a 15-pS KATp channel [26]. As previously reported [45], we found that in the absence of nucleotides, pinacidil up to 1 mM failed to induce channel stimulation (not shown). On the other hand, when channel activity was initially augmented by the presence of 0.1 mM internal GDP, a subsequent addition of 0.3 mM pinacidil further abruptly enhanced channel activity (Fig. 6A). In this patch, after GDP-evoked stimulation, N P o was augmented from 1.25 to 5.1 by 300 gM pinacidil. This pinacidil-evoked stimulation was reversed

by 1 mM and 3 mM GDP respectively. In addition, we found in four patches that 100 gM and 300 gM GDP did not affect significantly channel activity whereas N P o was reduced to 35%+5 and 16%+8 of control by 1 mM and 3 mM GDP respectively. These results indicate that MgGDP behaves as a potent activator of the KATP channel whereas free GDP can be considered as a weak inhibitor of the channel. It was observed that GTP (1 mM) had no stimulating action on channel activity and that it even produced a slight inhibition (not shown). Effects of the GDP analog, GDP[flS], were also investigated in the presence of Mg2+. When exposed to the internal side of inside-out membrane patches, GDP[flS] (0.1 raM) had either no noticeable effect on channel opening or it slightly increased it (Fig. 5A). Average changes in channel activity from several patches indicated that N P o was weakly stimulated by a factor of 1.35+0.2 (n = 11) in the presence of 100 ~tM GDP[flS]. However, in combination with other NDP, GDP[flS] did influence channel activity. Figure 5B shows that GDP[flS] potentiates the activation by 0.1 mM ADP. In this patch, N P o was increased from 0.14 to 0.51 by 100 BM ADP. Addition of GDP[flS] further increased NPo to 2.8. GDP[flS]-induced stimulation was entirely reversible ( N P o returned to 0.75 in the sole presence of ADP). Average changes in channel activity from several patches indicated that N P o potentiated by 50 gM or 100 ~tM ADP was increased to 240% +50 by 100 BM GDP[flS] (n = 8). Conversely, Fig. 5 C shows that if channel activity is first potentiated by 0.1 mMGDP, subsequent addition of

A

50 s

50 s

GDPBS100;M GDPBS100pM

GDPBS 100 pM

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~_ ~ m ~ ' ~ ' ~ b r ~

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Fig. 5. A Effects of intracellular GDP[flS] on channel activity in inside-out patches. B Effects of GDP[BS] on the ADP-evoked stimulation of channel activity. C Effects of GDP[BS] on the GDP-evoked stimulation of channel activity (top trace: chart recording; bottom trace: segments of the main trace on expanded scales)

190

A i

I, 31

,

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pinacidil 300 pM

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upon return to the GDP activating solution. Similar results were obtained in three patches. ADP was also capable of revealing pinacidil action (Fig. 6B). In this patch, after ADP-evoked stimulation, N P o was augmented from 0.15 to 0.75 by 300 gM pinacidil. A similar potentiation has been observed in another patch. Finally GDP[flS], although it had no or a very small activatory effect on the KATP channel by itself, also permitted pinacidil activation of the channel (Fig. 6C). In this patch, after application of 100 [aM GDP[flS], N P o was augmented from 0.05 to 0.44 by 300 ~tM pinacidil. A similar activation by pinacidil was seen in another patch.

Discussion

Mechanisms of regulation of KATP channels have been extensively studied in pancreatic fl cells and in cardiac cells (reviewed in [2, 13, 17, 28]) particularly in relation to modulation by intracellular nucleotides. KATP channels probably play an important function in muscle physiology [11, 36]. However, they seem to belong to a subtype that is distinct from subtypes found in the pancreatic fl cells or in the cardiac cell. The muscle

_

_ I

control GDPBS100 pM pinaeidil 300 ~tM

Fig. 6. Increase of channel activity by pinacidil in inside-out patches in the presence of intracellular GDP (A), intracellular ADP (B) and intracellular GDP[flS] (C)

KATP channel is 100-1000 times more resistant to blockade by antidiabetic sulphonylureas than KATp channels

in cardiac and pancreatic fl cells [24, 34]. It is differently affected by the K + channel openers. For instance, the K + channel opener diazoxide has no effect on skeletal muscle KATP channels [45] and inhibits cardiac muscle KATP channels [20], whereas it strongly stimulates the KATP channel in insulin-secreting cells [18, 40]. This emphasizes the differences in channel population between different tissues. Finally, the skeletal muscle KATP channel seems to be much more sensitive to activation by intracellular acidification [10, 12] than any other KAyP channel in other cell types. One of the most important properties of KATP channels is that they can be regulated not only by ATP but also by intracellular concentrations of NDP. In insulinoma cells, ADP (100-500 gM) activates NATP channels in the presence of internal Mg 2+, while it inhibits channel activity in the absence of Mg 2+ [15, 21]. The same type of situation was found in cardiac cells [22, 27, 41]. The easiest interpretation [27] is of course that the Mg-ADP species is an activator whereas free ADP is an inhibitor. Results presented in this paper for mouse skeletal muscle lead us to the same conclusion. Mg-ADP activates the muscle KATP channel, free ADP inhibits it.

191 Not only ADP but also GDP has a modulatory activity on muscle KATP channels. In the presence of internal Mg 2§ , GDP increased channel activity at all tested concentrations up to 1 mM. In the absence of internal Mg 2§ , GDP did not noticeably alter channel activity up to 1 mM and it took a concentration of 3 mM GDP to inhibit channel opening. GDP-evoked KATp channel stimulation in the presence of Mg 2+ has also been observed in insulin-secreting cells [16] as well as in cardiac myocytes [27, 41]. In the absence of Mg 2+, GDP fails to activate KATP channels in cardiac myocytes [411; under those conditions inhibition of channel activity has even been observed [271. Mg-GDP clearly behaves as a potent activator of the skeletal muscle KATp channel whereas free GDP can be viewed as a weak inhibitor of the channel. All these results taken together suggest that basic mechanisms for the modulation by NDP in skeletal muscle cells are similar to those described in pancreatic fl cells and in cardiac cells. How many intracellular nucleotide binding sites are required for this modulation? The simplest interpretation of the results is that there are two main types of nucleotide binding sites (there may be several sites per type), a first one for free ATP, free ADP and free GDP, which would be in charge of inhibition, and another one for Mg-ADP and Mg-GDP, which would be in charge of activation. Such a view would be consistent with a recent model proposed for ventricular cells [22, 27], and for insulinoma cells [141 for which affinity labelling of the activating site was even found to be possible, to provide permanent irreversible activation of a KATP channel. Although the interpretation involving two types of site (activatory and inhibitory) has the merit of being simple, one should also consider the possibility that both Mg-ADP and Mg-GDP serve as sensors of the intracellular bioenergetic situation and thay they regulate channel activity at distinct NDP sites. Such a model would then imply three types of modulatory sites, one inhibitory site where the main ligand is ATP and two distinct activatory sites for Mg-ADP and Mg-GDP respectively. Observations made in this work with GDP[flS] are intriguing. It has been previously reported that in insulinsecreting cells GDP[flS] had the same stimulatory effect as GDP [16, 21]. The situation is different for skeletal muscle where GDP[flS] has a stimulatory activity, which is very small, when it is present, as compared to the activating effect of GDP. In fact GDP[flS] can inhibit the activation of the KAyp channel by GDP in the presence of Mg 2+ . The simplest explanation for such a finding is that GDP[flS] behaves as an antagonist or a very partial agonist of GDP (with a similar or even higher affinity) at the Mg-GDP activating site. If GDP[flS] behaves as a partial agonist of the action of GDP, what is then its effect on ADP action in the presence of Mg2§ It was found that Mg-ADP activation of muscle NATP channels is substantially increased by GDP[flS]. Thus GDP[flS] has two opposite actions on the activatory effects of NDP, it inhibits GDP action and stimulates the effects of ADP. A possible interpretation of all these results is again that sites for Mg-GDP and Mg-ADP on the KATP channel are distinct. GDP[flS] would behave, as previously in-

dicated, as a partial GDP agonist (i.e. as an antagonist of GDP action) at the GDP site but would still allosterically improve the efficacy of Mg-ADP at the ADP site. It would actually make sense to have two different types of site to sense independently intracellular variations of ADP (which reports on ATP variations) and G D P (which reports on GTP variations) if skeletal muscle KATp channels really play an important role during metabolic exhaustion of skeletal muscle as previously suggested [8]. Analysis of the mutual interactions between MgGDP, or Mg-ADP, or GDP[flS] in the presence of Mg 2+ and the K § channel opener pinacidil offered another way to gain information about skeletal muscle NATP channel NDP binding sites. Pinacidil was found to be inactive in the absence of GDP but pinacidil activation of KATP channels was large in the presence of G D R There is clearly, as in vascular smooth muscle cells [261, a synergy between GDP and pinacidil, which strongly suggests an allosteric interaction between the internal Mg-GDP site and the pinacidil binding site, which is expected to be situated on the external surface of the channel. Interestingly GDP[flS], which by itself acts as an antagonist of direct GDP activation, also potentiates pinacidil effects. Finally ADP in the presence of Mg 2§ not only increases KATP channel activity by itself but it also potentiates the action of pinacidil. Clearly the channel opened by Mg-ADP or Mg-GDP is a much better target for pinacidil action than the channel with its NDP binding sites not occupied by the dinucleotides. These observations do not indicate whether the intracellular sites for Mg-ADP and Mg-GDP are independent but they clearly indicate a strong allosteric interaction between these sites and the binding site [4, 31] for the K + channel opener. These results may be important if treatment of some muscle diseases by openers could be envisaged as previously suggested [25, 33, 38]. The relevance of these mechanistic results in terms of physiological responsiveness of the muscle in vivo is fairly clear. Active exercise will increase ADP (from about 10 gM to about 120 gM) [6] and probably also GDP concentrations. These increases of ADP and GDP in the presence of Mg 2§ concomitant with acidification and other unknown factors, will greatly contribute to the activation of the KATp channel, produce hyperpolarization, decrease contraction and thereby save the remaining intracellular ATP [11]. Acknowledgements. Thanks are due to Dr. G. Romeyand Dr. J.R. De

Weille for fruitful discussions. The authors thank F. Aguila and C. Roulinat for skilful technical assistance. This work was supported by the Centre National de la RechercheScientifique.B.A. is a recipient of a fellowship from the Association Fran~aise contre les Myopathies. References 1. Ashcroft FM (1988)Adenosine 5'-triphosphate-sensitivepotassium channels. Annu Rev Neurosci 11:97-118 2. Ashcroft SJH, Ashcroft FM (1990) Properties and functions of ATP-sensitiveK channels. Cell Signalling 2:197-214 3. Ashford MLJ, Sturgess NC, Trout NJ, Gardner NJ, Hales CN (1988) Adenosine5'-triphosphate-sensitiveion channels in neonatal rat cultured central neurones. Pfl~igersArch 412:297-304

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