Naunyn-Schmiedeberg's Arch Pharmacol (1990) 342: 258 - 263

Naunyn-Schmiedeberg's

Archivesof

Pharmacology

© Springer-Verlag1990

Effects of potassium channel openers on single potassium channels in mouse skeletal muscle R. Weik* and B. Neumcke I. Physiologisches Institut der Universitfit des Saarlandes D-6650 Homburg/Saar, Federal Republic of Germany Received January 31, 1990/Accepted April 18, 1990

Summary. The patch-clamp technique was used to study the effects of the potassium channel openers cromakalim, pinacidil, RP 49356 and diazoxide on single potassium channels in mouse skeletal muscle. In excised patches in the inside-out configuration, one type of potassium channel, the ATP-sensitive potassium channel, could be activated by internally applied RP 49356 even in the absence of internal ATP. At a concentration of 0.4 and 0.8 retool/l, RP 49356 increased the open-probability of the channels by a factor of 2.7 and 17.4 respectively. The stimulating effect of cromakalim (0.2-0.8 retool/l) and pinacidil (0.4 retool/l) depended on the presence of ATP (0.1 mmol/1) at the cytoplasmic side of the patch membrane. The two drugs were able to restore the open-probability of the channels blocked by internal ATP (0.1 retool/l) to 5 0 - 9 0 % of its value in ATP-free solution. No channel reactivation could be observed at a higher ATP concentration (1 mmol/1). Diazoxide (0.4 retool/l) had almost no effect. None of these channel openers could stimulate the other prominent type of potassium channel in skeletal muscle, the large-conductance Ca 2 +-activated potassium channel. The results show that cromakalim, pinacidil and RP 49356 are specific openers of ATP-sensitive potassium channels in skeletal muscle. It is suggested that the drugs displace the channel blocker ATP and that RP 49356 in addition recruits inactive channels. Key words: Patch clamp - Adenosine triphosphate-sensitive potassium channel - Calcium-activated potassium channel - Potassium channel opener - Skeletal muscle

Introduction The recently discovered K + channel openers, e.g. cromakalim, pinacidil, RP 49356 and diazoxide, increase * Present address. Max-Plank-Institut ffir experimentelle Medizin,

Hermann-Rein-Str. 3, D-3400 G6ttingen, Federal Republic of Germany Send offprint requests to B. Neumcke at the above address

the permeability of smooth muscle cells to K + ions (for a review see Hamilton and Weston 1989). ATP-sensitive K + channels are probably the target sites for these drugs (Standen et al. 1989, Quast and Cook 1989a). Cromakalim, pinacidil and RP 49356 also activate ATP-sensitive K + channels in cardiac muscle (Escande et al. 1988, 1989) and diazoxide opens the channels in pancreatic islet cells (Trube et al. 1986). In addition, activating effects of the channel opener cromakalim on the macroscopic K + conductance of human skeletal muscle have been demonstrated by Spuler et al. (1989). Therefore, it was of interest to study the effects of K + channel openers on single K + channels in skeletal muscle and to compare the results with those obtained in other tissues.

Materials and methods The preparation of single muscle fibres has already been described in detail elsewhere (Woll et al. 1989, Weik and Neumcke 1989). In short, muscles from the hindfeet of adult female mice were isolated and dissociated into single fibres with collagenase (Sigma Type I, Sigma, Deisenhofen, FRG, 3 mg/ml Na+-rich solution) at 35°C for 1.5 h. Single fibres were stored refrigerated in Na+-rich solution and could be used for experiments within 10 h. Experiments were done at room temperature (19-24°C) except studies made with internally applied cromakalim, which were performed both at 30 31°C and at room temperature. Pipettes (borosilicate glass capillaries, GC 150-15, Clark Electromedical Instruments, Reading, UK) were pulled in two stages and filled with the following solution (in retool/l): 155 KC1, 3 MgC12, 0.5 EGTA (ethylene glycol bis(fl-aminoethyl ether)N,N,N',N'-tetraacetic acid), 10 HEPES (4-(2-hydroxyethyl)-l-piperazine ethanesulphonic acid), pH 7.4. After forming GQ seals, currents through single K + channels were recorded in the insideout configuration of the patch-clamp technique (Hamill et al. 1981) using an L/M-EPC-7 amplifier (List, Darmstadt, FRG). Solutions. The solution used for storage of the muscle cells was a Na +-rich solution composed of(mmol/1) 150 NaC1, 5 KC1, 2 CaC12, 1 MgCI2, 10 HEPES, pH 7.4. After forming the seal and excising the patch, this solution was subsequently exchanged in a small chamber beside the main pool against K +-rich solutions. A complete solution exchange was achieved within about 5 s. To increase the open-probabilityof ATP-sensitive K + channels in the determination of the concentration-response curve of cromakalim (Fig. 5), the

259 patches were excised in a nominally Ca z +-free solution composed of(mmol/1) 160 NaC1, 1 MgC12, 0.5 EGTA, 10 HEPES, pH 7.4 and then transferred to a K+-rich solution. The K+-rich solution used to study ATP-sensitive K + channels contained (mmol/1) 160 KC1, I MgC12, 10 HEPES, pH 7.4. For experiments with Ca2+-activated K + channels it was composed of (mmol/1) 160 KC1, 1 MgC12, 5 CaClz, 5 EGTA, 10 HEPES, pH 7.4, which results in a free Ca 2+ concentration of about 20 gmol/1 calculated according to Trube (i 979) and with stability constants for EGTA as taken from Martell and Smith (1974). The K+-rich solutions were titrated to pH 7.4 by addition of 1 N KOH. For adjusting the pH value of the bathing solution, 1 N NaOH was used. When the effects of channel openers on Ca ~+-activated K + channels were studied, ATP-sensitive K + channels were irreversibly blocked by internally applied chloramineT (0.5 mmol/1), (Fluka, Neu-Ulm, FRG), see Weik and Neumcke (1989). ATP was added to the internal bath solution as NazATP (Boehringer, Mannheim, FRG), if necessary the pH was readjusted to 7.4 by addition of I N KOH. Drugs used in this study. Diazoxide was a kind gift from Professor P. Grate (University of Munich, Munich, FRG), the other drugs were generously provided by the respective manufacturers: Cromakalim (BRL 349•5 [(+)-6-cyano-3,4-dihydro-2,2-dimethyltrans-4-(2-oxo-l-pyrrolidyl)-2H-benzo[b]pyran-3-ol] by Beecham Pharmaceuticals (Betchworth, UK), pinacidil (LY 164021) [(_+)-(N"cyano-N-4-pyridyl-N'-l,2,2-trimethyl-propylguanidine] by Lilly Research Laboratories (Indianapolis, USA) and RP 49356 [Nmethyl - 2 - (3 - pyridyl) - tetra - hydrothiopyran - 2- carbothi oamide - 1oxide] by Rhfne Poulenc Sant6 (Vitry sur Seine, France). Stock solutions of diazoxide and pinacidil (each 40 mmol/1) were prepared using 0.1 N KOH (diazoxide) or 0.15 N KOH (pinacidil). The pH of the final solutions was readjusted to 7.4 with i N HC1. Dimethylsulphoxide (DMSO) was used to dissolve cromakalim (200 and 440 mmol/1) and RP 49356 (250 and 500 mmol/1). DMSO alone (l.6 gI/ml) was without effect (see Figs. 2, 3 E and 4). Analysis. Membrane currents were recorded on pen-recorder

(0.1 kHz filtered, linearcorder Mark VII, WR 3310, Type MK7-102, Graphtec Corporation, March, FRG), see Fig. 3, and stored on video tape. Stored currents were replayed, filtered at 0.4 kHz corner fiequency using a four-pole low-pass Bessel filter (Datel FLJ-DC, Munich, FRG) and digitized at 0.3 ms intervals (DEC LSI 11/73, Digital Equipment Corporation, MA, USA). Current recordings of 10 to 30 s duration were analyzed using the half-amplitude criterion (Colquhoun and Sigworth 1983) to detect channel openings and closures. Since the precise number of channels within a patch is difficult to determine, we normalized the open-probabilities Po (defined as the sum of open times of all channels divided by the total time period of analysed records) with respect to control po values calculated before and after application of the differenf drugs (see Weik and Neumcke 1989). The resulting relativepo values are given in percent-units.

O. 4-retool / I

Results ATP-sensitive K + channels. The first set o f experiments

was d o n e to test direct effects o f the channel openers on ATP-sensitive K + channels w i t h o u t internal ATP. Figure 1 illustrates that ATP-sensitive K + channels have a low o p e n - p r o b a b i l i t y p o at a m e m b r a n e potential o f - 50 m V in a drug-free K + - r i c h internal solution (Po ~ 0.15, c o m pare Fig. 4 o f Woll et al. 1989). Internally applied R P 49356 (0.4 retool/l) induced an increase in the o p e n - p r o b ability after a delay o f a b o u t 30 to 60 s (Fig. 1), which is m u c h longer than the time required for the solution exchange. The other drugs tested, for instance diazoxide (0.4 mmol/1, see Fig. 1), had no effect on u n b l o c k e d ATPsensitive K ÷ channels. The results o f several experiments are summarized in Fig. 2. Dimethylsulphoxide ( D M S O ) alone at a c o n c e n t r a t i o n o f 1.6 ~tl/ml induced no channel activation (Fig. 2, last c o l u m n ) whereas R P 49356 0.4 mmol/1) p r o d u c e d a 2.7-fold increse in the o p e n - p r o b ability po (n = 9). In additonal experiments with a higher R P 49356 c o n c e n t r a t i o n o f 0.8 mmol/1 the open-probability was increased by a factor o f 17.4 _+ 9.3 (mean + S E M , n -- 5) at r o o m temperature. Such a large increase o f Po c a n n o t be achieved by a p r o l o n g a t i o n o f the channel open time but m u s t be due to the recruitment o f new channels which were inactive before drug application (Thuringer and Escande, 1989). All other internally applied drugs including diazoxide (0.4 mmol/1), pinacidil (0.4 mmol/1) and c r o m a k a l i m (0.1 mmol/1) p r o d u c e d no significant increase in Po (Fig. 2). In six other patches, c r o m a k a l i m (0.4 mmol/1) was dissolved in the pipette solution a n d applied to the outside o f the p a t c h membrane. N o channel activation, c o m p a r e d to seals w i t h o u t c r o m a k a l i m in the pipette, could be detected at r o o m temperature (not shown). The effects o f several channel openers, such as diazoxide in R I N m 5 F cells (Dunne et al. 1987) and in mouse pancreatic islet cells (Trube et al. 1986) as well as c r o m a k a l i m in s m o o t h muscle (Standen et al. 1989) depend on the presence o f A T P as internal blocker. We, therefore, tested the effects o f the drugs on ATP-sensitive K ÷ channels, whose activity had been decreased by ATP. Figure 3 shows the p r o t o c o l used and Fig. 4 presents quantitative results. A t r o o m temperature, internally applied A T P (0.1 retool/l) reduced the open-probability po

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Fig. 1. Different effects of internally applied RP 49356 or diazoxide (each 0.4 mmol/l) on ATP-sensitive K + channels in the absence of internal ATP. Bars above current traces indicate application periods of the different drugs. Arrows denote the closed state of all channels, channel openings are plotted downwards. Two different patches. Membrane potential - 50 mV. Room temperature

260

of ATP-sensitive K + channels to 2.1 + 1.0% (mean + SEM, n = 36) of the controls (second column in Fig. 4). Subsequent treatment with diazoxide (0.4 mmol/1) hardly had an effect (Fig. 3 A, third column in Fig. 4), whereas pinacidil (0.4 mmol/1), cromakalim (0.2 mmol/1) and RP 49356 (0.4mmol/1) strongly reactivated ATP-blocked channels (Fig. 3 B - D, fourth to sixth columns in Fig. 4). Note again the delay after application of RP 49356 before

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Fig.& Effects of internally applied diazoxide, pinacidil (each 0.4 mmol/1), cromakalim (0.2 retool/l), RP 49356 (0.4 mmol/1) and DMSO (1.6 pl/ml) on ATP-sensitive K + channels in the presence of internal ATP (0.1 mmol/1). Application time of substances is indicated by bars above the current traces. Records A to E from different patches. Membrane potential - 50 mV, channel openings are plotted downwards. Abscissa and ordinate units as indicated in part A. Room temperature except in part C, where temperature was 30°C

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Fig. 2. Relative open-probabilities (relpo) ofATP-sensitive K + channels during treatment with different drugs in the absence of internal ATP. The probabilities are normalized with respect to measurements on the same patches in drug-free K+-rich internal solution. The drugs used were diazoxide, pinacidil (each 0.4 mmol/1), cromakalim (0.1 mmol/l) and RP 49356 (0.4 mmol/1). Additional control experiments with DMSO (1.6 pJ/ml) in drug-flee K+-rich internal solution. Bars and numbers above the columns indicate the SEM values and the corresponding number of measurements. Membrane potential - 50 inV. Room temperature

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Fig. 4. Relative open-probabilities (rel Po) of ATP-sensitive K + channels during treatment with several drugs in the presence of internal ATP (0.1 mmol/1). The probabilities are normalized with respect to measurements on the same patches in drug-free K +-rich internal solution without ATP (first column on the left). The drugs used were diazoxide, pinacidil, RP 49356 (each 0.4 mmol/1) and cromakalim (0,2 mmol/1). DMSO (1.6 gl/ml) without added drugs showed no effects. Bars and numbers above the columns indicate SEM values with corresponding number of measurements. Membrane potential - 50 inV. Temperatures 1 9 - 2 4 ° C (ATP without drug, diazoxide, pinacidil, RP 49356 and DMSO) and 30°C (cromakalim)

261

the channels reopen (Fig. 3 D). DMSO (1.6 gl/ml) alone induced no prominent activation of ATP-blocked channels (Fig. 3E, last column in Fig. 4). The effect of cromakalim was usually tested at 30°C. In additional experiments at 20°C, application of cromakalim (0.2 retool/l) in the presence of ATP (0.1 retool/l) also reopened ATP-sensitive K + channels, but not as strongly as at 30°C (relative open-probability = 20.3_+ 7.1%, mean + SEM, n = 8). The large standard error obtained in experiments performed with pinacidil (0.4 retool/l), see bar above fourth column in Fig. 4, results from the fact

rel

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that in 3 out of 9 patches no stimulating effect could be detected during an application period of between I and 1.5 rain. If the concentration of internal ATP was increased to 1 retool/l, RP 49356 (0.4 retool/l) was not able to reactivate the blocked ATP-sensitive K + channels at room temperature when applied for a period of longer than 1 rain. The other drugs were ineffective when applied for 20 to 30 s (n = 4 to 10 measurements, not shown). Figure 5 shows the concentration-response curve of the effects of cromakalim on the open-probability of ATP-sensitive K + channels. The measurements were performed on patches excised in a solution of low C a 2 + concentration (see Methods) and at 30-31°C. Under these conditions internal ATP (0.1 retool/l) reduced the open-probabilitypo to 20.6 4- 5.9% (mean _+ SEM, n = 21), while the relative open-probability at the same ATP concentration but at room temperature declined to 2.1% (second column in Fig. 4). A significant increase of the Po value in the presence of internal ATP (0.1 retool/l) to 35.9% was observed at a cromakalim concentration of 0.1 retool/1 (second point in Fig. 5), at the highest cromakalim concentration tested (0.8 mmol/1) the relative po value was 88.7%. Some evidence exists (K16ckner et al. 1989, Gelband et al. 1989) that largeconductance CaZ+-activated K + channels in smooth muscle are modified by cromakalim and diazoxide. We, therefore, tested the effects of the drugs RP 49356, diazoxide, pinacidil and cromakalim o n C a 2 +-activated K + channels in skeletal muscle at a free C a 2 + concentration of about 20 gmol/1. As can be seen in Fig. 6, internally applied RP 49356 (0.4 mmol/1) did not change the channel current and open-probabilities of C a 2 + - a c t i v a t e d K + channels at different voltages. The voltage-dependence of the open-probability of Ca 2 +-activated K + channels under control conditions and in the presence of the four channel openers is shown in Fig. 7. Obviously, different CaZ+-activated K + channels exhibit different control Po curves, probably owing to varying affinities for C a 2 + (see also Moczydlowski and Latorre 1983). We, therefore,

Ca2+-activated K + channels.

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Fig. 5. Concentration-response curve of the effects of internally applied cromakalim on the relative open-probability (rel po) of ATPsensitive K + channels in the presence of internal ATP (0.1 mmol/1). The probabilities are normalized with respect to measurements on the same patches in drug-free K +-rich internal solution. The relative open-probability in the presence of ATP (0.1 mmol/1) without cromakalim is indicated by the interrupted line (rel po = 20.6%). The curve through the mean values (open circles) is drawn by eye. Bars and numbers denote the SEM values and the corresponding number of measurements, M e m b r a n e potential - 4 0 to - 50 mV. Temperature 30 - 31 ° C

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Fig. 6. Segments of current recordings from a Ca z +-activated K + channel at various membrane potentials as indicated. Addition of RP 49356 (0.4 mmol/1) to the internal solution (retool/l) 160 KC1, 1 MgC12, 5 CaC12, 5 EGTA, 10 HEPES, pH 7.4 has no effect. Arrows denote channel closure, channel openings are plotted upwards. All records from one path. R o o m temperature

262

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Fig. 7. Open-probabilities of Ca 2+-activated K + channels at different membrane potentials (E). Filled circles denote control values obtained with (mmol/1) 160 KC1, 1 MgClz, 5 CaC12, 5 EGTA, 10 HEPES, pH 7.4 as internal solution. Curves through circles are drawn by eye. Open squares show values obtained after addition of A) RP 49356 (0.4 retool/l), B) diazoxide (0.4 mmol/1), C) pinacidil (0.4 retool/l) or D) cromakalim (0.1 mmol/1). Records from different patches. Room temperature

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present results of typical experiments, which were confirmed on a total number of 3 to 9 patches. None of the applied drugs was able to induce a significant change of the open-probability curve.

Discussion

The findings of this study are in good agreement with the experiments of Spuler et al. (1989) performed on human skeletal muscle. These authors described pronounced effects of cromakalim on the macroscopic K + conductance of muscle fibres using the two-microelectrode technique. They found that externally applied cromakalim was able to hyperpolarize muscle cells; and that this could be antagonized by tolbutamide, a blocker of ATP-sensitive K + channels (Trube et al. 1986). Similar effects of cromakalim were described for smooth muscle by Standen et al. (1989), whereas in cardiac myocytes this drug is able to activate ATP-sensitive K + channels even in the absence ofATP (Escande et al. 1988). As described in the Results, all stimulating effects of the drugs in skeletal muscle could already be elicited at room temperature. In contrast, in intact papillary muscle fibres of guineapig hearts, externally applied cromakalim activated the K + conductance only at a temperature of 36°C (Sanguinetti et al. 1988), at which the cell membrane might have become drug permeable. These findings favour the idea that the channel openers act primarily from the inside of the cell membrane, as was already suggested for cardiac myocytes by Escande et al. (1989). The strong temperature dependence of the activating effect of cromakalim (see Results) further strengthens the view that the membrane fluidity plays an essential role in the

40

E(mV)

interaction between the channel openers and ATP-sensirive K + channels. As measure of the channel activation by various drugs we have used the increase of the open-probability of all ATP-sensitive K + channels in the patch (Figs. 2, 4, 5). The increase could be caused by an increase of the channel open time, a decrease of the channel closed time, a shorter gap between bursts of channel activity or a recruitment of inactive channels. The contribution of these various factors is difficult to estimate from our experiments which Were usually performed on membrane patches containing several active ATP-sensitive K + channels. Hence, we cannot give an analysis of the kinetic properties of ATPsensitive K + channels as in the study of Woll et al. (1989) in which single-channel patches were investigated. An unresolved question is why no drug-induced activation of ATP-sensitive K + channels could be observed in excised patches in the presence of I mmol/1 internal ATP. The mean concentration of ATP in intact frog skeletal muscle was measured to be 4 retool/1 (Fink et al. 1983). Hence, no effects of cromakalim in intact muscle fibres would be expected according to our experiments, in contrast to the positive findings of Spuler et al. (1989). One possible explanation could be that an ATP-concentration gradient exists within the cell due to ATP-consuming reaction centres beneath the cell membrane. In addition, the intracellular ATP/ADP ratio modulates the activity ofATP-sensitive K + channels, and in vivo activating stimuli on ATP-sensitive K + channels may exist which are lost during patch excision (for a review on the regulation of ATP-sensitive K + channels see De Weille and Lazdunski, 1990). There exists a remarkable difference between the actions of RP #9356 and the other two effective channel

263 openers, c r o m a k a l i m a n d pinacidil. R P 49356 is the o n l y s u b s t a n c e tested, w h i c h activates A T P - s e n s i t i v e K + c h a n nels even in the a b s e n c e o f i n t e r n a l A T P (Figs. 1, 2). Thus, R P 49356 acts p r i m a r i l y n o t b y d i s p l a c i n g A T P f r o m its b i n d i n g sites, b u t seems to r e c r u i t inactive c h a n n e l s ( T h u r i n g e r a n d E s c a n d e , 1989). T h e d e l a y b e f o r e c h a n n e l a c t i v a t i o n (Figs. l , 3 D ) is a f u r t h e r h i n t t h a t R P 49356 acts b y a different m e c h a n i s m to c r o m a k a l i m a n d p i n a c i d i l w h i c h s h o w n o d e l a y (Fig. 3 B, C). A c t i v a t i n g effects o f R P 49356 w i t h o u t i n t e r n a l A T P were also des c r i b e d for p u l m o n a r y a r t e r y (Eltze 1989) a n d c a r d i a c m y o c y t e s ( E s c a n d e et al. 1989). P i n a c i d i l a n d c r o m a k a l i m were o n l y effective in skeletal muscle in the presence o f i n t e r n a l A T P (Figs. 3 B, C a n d 4), w h e r e a s these d r u g s are a b l e to a c t i v a t e A T P - s e n s i t i v e K + c h a n n e l s in c a r d i a c m y o c y t e s w i t h o u t A T P ( E s c a n d e et al. 1988, 1989). D i a z o xide, the f o u r t h d r u g tested, was h a r d l y effective in skeletal muscle at a c o n c e n t r a t i o n o f 0.4 mmol/1 (Figs. 3 A, 4) a n d even i n h i b i t e d A T P - s e n s i t i v e K + c h a n n e l s in vent r i c u l a r m u s c l e cells ( F a i v r e a n d F i n d l a y 1989), b u t s h o w e d r e m a r k a b l e a c t i v a t i n g effects at c o n c e n t r a t i o n s o f 50 - 100 gmol/1 in s m o o t h m u s c l e ( S t a n d e n et al. 1989, Q u a s t a n d C o o k 1989 b) o r p a n c r e a t i c islet cells ( G a r r i n o et al. 1989). T h e different effects o f d i a z o x i d e i n d i c a t e t h a t A T P - s e n s i t i v e K + c h a n n e l s in the surface o f m o u s e skeletal m u s c l e r e s e m b l e m o r e t h o s e in c a r d i a c cells t h a n in s m o o t h m u s c l e a n d p a n c r e a t i c islet cells. O u r results (Figs. 6, 7) exclude a significant role o f the l a r g e - c o n d u c t a n c e C a 2 + - a c t i v a t e d K + c h a n n e l as a t a r g e t site for the d r u g s tested. In c o n t r a s t , in s m o o t h muscle K16ckner et al. (1989) a n d G e l b a n d et al. (1989) d e m o n s t r a t e d a n a c t i v a t i o n o f this t y p e o f i o n c h a n n e l b y diazoxide and cromakalim. I n c o n c l u s i o n , c r o m a k a l i m , p i n a c i d i l a n d R P 49356 are specific o p e n e r s o f A T P - s e n s i t i v e K + c h a n n e l s in excised m e m b r a n e p a t c h e s o f m a m m a l i a n skeletal muscle. C h a n n e l a c t i v a t i o n b y c r o m a k a l i m a n d p i n a c i d i l is o n l y o b s e r v e d in the p r e s e n c e o f i n t e r n a l A T P a n d is likely to be d u e to a d i s p l a c e m e n t o f the c h a n n e l b l o c k e r ATP. O n the o t h e r side, R P 49356 a c t i v a t e s A T P - s e n s i t i v e K + c h a n n e l s even in the a b s e n c e o f i n t e r n a l A T P v i a a n additional mechanism which may involve recruitment of inactive c h a n n e l s ( T h u r i n g e r a n d E s c a n d e , 1989).

Acknowledgements. We thank Professor H. Meres and Dr. T. Plant for helpful comments on the manuscript and Dr. U. L6nnendonker for writing computer programs. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246).

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