Neuroscience Letters, 119 (1990) 191-194

191

Elsevier Scientific Publishers Ireland Ltd. NSL 07285

Two different types of potassium channels in human skeletal muscle activated by potassium channel openers S. Q u a s t h o f f 2, C. F r a n k e l, H . H a t t I a n d M. R i c h t e r - T u r t u r 3 tPhysiologisches Institut der Technischen Universitiit Miinehen, Munich ( F.R.G. ) , 2Physiologisches Institut der Universitdt Miinchen, Munich ( F.R.G ) and 3Chirurgische Klinik, Munich (F.R.G.)

(Received 14 May 1990; Revisedversion received 13 July 1990; Accepted 23 July 1990) Key words:

Potassium channel opener; ATP-sensitiveK ÷ channel; Inside-out patch; Membrane bleb; Cromakalim; RP 49356; EMD 52692

The inside-out patch clamp technique was used to record the effectsof K ÷ channel openers (EMD 52692, RP 49356 and Cromakalim) on single channel currents in membrane blebs of human skeletalmuscle. Two types of K ÷ channels were activated by these drugs: an ATP-sensitiveK ÷ channel which was inhibited by 3 mM ATP and 5 pM Glibenclamideand an ATP insensitive K ÷ channel. The open probability of both types was strongly increased by K ÷ channel openers. Glibenclamideantagonized the action of the K ÷ channel openers.

Potassium channel openers act on a wide variety of tissues [12]. They enhance the membrane conductance of human skeletal muscle by activating a K + conductance [13, 14]. The resulting membrane hyperpolarization may be of therapeutic benefit in muscle diseases in which membrane depolarization causes inexcitability [8], or in muscle diseases where an abnormal ratio of the ion conductances of the muscle membrane leads to uncontrolled firing of action potentials. U p to now, only pharmaceutical evidence indicated which type of K + channels might be involved [13, 14]. However, in other tissues some evidence has emerged suggesting that K + channel openers act specifically on ATP-sensitive K + channels [12]. In the present study we have attempted to demonstrate that K + channel openers act on ATP-sensitive K + channels in membrane blebs of human skeletal muscle, using the patch-clamp technique. Skeletal muscle fibers obtained from orthopedic surgery and skeletal muscle fibers from specimens of routinely performed open muscle biopsies [9] from patients susceptible to malignant hyperthermia but with negative test results were used for the present study. All procedures were in accordance with the Helsinki convention and were approved by the Ethics Commission of the Technical University of Munich. The thin, about 4 cm long, muscle bundle was fixed at both ends with a string in a preparation chamber, and was superfused at 21 °C with a solution of the following composition (in mM): E G T A Correspondence: S. Quasthoff, Physiologisches lnstitut der Universit~it M/inchen, Pettenkoferstr. 12, 8000 M~nchen 2, F.R.G.

0304-3940/90/$ 03.50 © 1990 ElsevierScientific Publishers Ireland Ltd.

1, potassium K+-methylsulfate 120, K O H 7.5, MgCI 3, HEPES 5, with a total osmolarity of 283.5 mosmol, at p H 7.2. In this solution, the muscle membrane depolarized and the fibers relaxed. After a few minutes it was possible to remove single fibers from the bundle without damaging or stretching it. Fibers contained in this manner were transferred to a recording chamber containing the same solution, and were fixed at one end to the chamber bottom, the other being held with a fine forceps. By rapid pulling on the free end of the fiber (as described by Stein and Pelade [15]) and by shortening to near rest length and then fixing the free end at the bottom, membrane blebs were elicited at the surface of the fiber [10]. These membrane blebs are very similar to those described by Fink et al. [6] in adult mammalian muscle, Vivaudou and Villaz [17] and Stein and Palade [15] in frog skeletal muscle, and to the sarcolemmal vesicles from human and other mammalian muscles as reported by Burton et al. [2]. Contrary to [2] we did not need enzyme treatment, and obtained rapid access to the untreated sarcolemma of the human skeletal muscle. The standard patch-clamp technique was employed to obtain inside-out patches [9] from membrane blebs with a high rate of success. The patch pipette usually contained the same solution as that in the recording chamber, causing the same ion concentration on the in- and outside of the patch. Drugs and A T P solution were applied to the patch in a liquid filament as described by Franke et al. [7]. Stock solution (100 mmol/l) of Gtibenclamide (H6chst Frankfurt), BRL 34915 = Cromakalim (Beecham Pharmaceuticals), E M D 52962 (Merck D a r m -

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stadt) [3] and RP 49356 (Rhrne Poulenc) were prepared in dimethylsulfoxide (DMSO). In the concentration range considered, DMSO did not mimic the effects of these drugs when applied alone. Patch-clamp current was amplified and recorded on video tape [7]. Evaluation of the data was performed as described by Franke et al. [7]. A computer program constructed 'idealized traces'. These traces contain the information whether the channel is open or closed and the open probability was calculated normally during 1 min. The 12 best patches were evaluated. Fig. 1A shows current traces from inside-out patches. A high K + solution was applied to both sides of the patch (in mM: EGTA 1, KMS 120, KOH 7.5, MgCI 3, HEPES 5). In the absence of ATP at the cytoplasmic side of the patch, openings of K + channels were regularly 0 ATP

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observed. In the experiment shown in Fig. 1A (first trace), the open probability (o.p.) of this channel was 0.38. The single channel activity could be completely inhibited by application of 3 mM ATP (Fig. 1A, second trace) or by 5/aM Glibenclamide (Fig. IA, third trace) to the intracellular side of the membrane. EMD 52962 100/aM applied to the intracellular side of the membrane appeared to act as a potent activator of ATP-sensitive K + channels in 0-ATP solution (Fig. 1A, 4th trace, o.p. 0.74). Similar effects were observed using Cromakalim and RP 49356 (data not shown). Channel block caused by 3 mM ATP could be antagonized by 1 /aM EMD 52962 (Fig. 1A, 5th trace, o.p. 0.12). In most experiments, the patch contained only 1, 2, or maximally 3 ATP-sensitive channels. The I/V curve of the ATP-sensitive channel showed a 0

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Fig. 3. A: current-voltage relationship for the channel currents shown in Fig. 2 under the same conditions. Note the pronounced outward rectification of this channel at positive potentials. B: current-voltage relationship for the channel which is not influenced by ATP, K + channel openers or,Glibenclamide.It was present in most of the patches with similar activity.

conductance of about 120 pS at negative potentials, and of 85 pS at positive potentials (Fig. 1B). Occasionally, single channel currents typical for 0ATP solutions were not observed, but a small conductance channel was active (see below). In these patches, however, K + channel openers such as RP 49356 (Fig. 2) activated a channel type with rapid flickering between open and closed states. E M D (10/zM) and BRL 34915 (Cromakalim, 100 /zM) activated this second channel (Fig. 2) even in the presence of 3 m M ATP. When 5/zM Glibenclamide was added in the presence of K + channel openers, the open probability dropped to zero (Fig. 2). In Fig. 3A, the I/V curve o f this ATP independent channel is shown, the conductance is 30 pS at negative and 51 pS at positive potentials. The opening behavior and the conductance of this channel type were obviously different from that of the ATP-sensitive K + channels (Fig. 1). A third type of channel with a low elementary conductance of about 21 pS in 130 m M K + symmetrical solution and an almost ohmic I/V curve between - 4 0 mV and + 40 mV could be recorded additionally in most of the patches (Fig. 3B). This channel type was insensitive to ATP, K + channel openers and Glibenclamide. ATP-sensitive potassium channels which can be inhibited by ATP at the intracellular side of the membrane have been shown to be present in a variety of cell types [12]. We found ATP-sensitive K + channels on membrane blebs of human skeletal muscle with similar characteristics to those recently described [2, 8, 18]. This channel type was almost always present in our preparation although membrane blebs probably contain less

ATP-sensitive channels than the sarcolemma of native muscle. We found on average only 1 or 2 channels in one patch, in contrast to Escande et al. [4] in cardiac myocytes and Vivaudou et al. [17] in blebs from frog skeletal muscle. Up to now, it was unclear whether K + channel openers act exclusively on the ATP-sensitive K + channel type or also on other K + channels such as the large conductance Ca2+-dependent K + channel (BKca) (in vascular smooth muscle cells [12, 16]) or the delayed rectifier K + channel [1]. We have demonstrated that in human skeletal muscle an ATP independent K + channel is also involved in the increase in K + conductance caused by K + channel openers. The I/V curve and the opening behavior of this channel type was different from the ATPsensitive channel. Up to now, Glibenclamide has been known and used as a specific blocker of the ATP-sensitive K + channel. We have shown that Glibenclamide also inhibits ATPindependent K + channels activated by the K + channel openers. Possibly only the binding site for K + channel openers is blocked by Glibenclamide in a competitive manner, as has been proposed by Beech et al. [1]. In our experiments, the K + channel openers activated the ATP-insensitive channel time independently, and the effects were reversible. A decrease in the ATP-sensitive K + channel activity with time (run down) as described recently [4] was never observed. We therefore have no indication for reactivation of ATP-sensitive K + channels by K + channel activators. The small K + channel with a unitary conductance of 21 pS could be identical to the inward rectifying K +

194 c h a n n e l described recently ([4], [5]). I n w a r d rectification over the range of + 40 m V to - 4 0 m V could have been m a s k e d by the use of symmetrically high K + solutions used in o u r experiments. I n conclusion, we f o u n d that in h u m a n skeletal muscle K + c h a n n e l openers activate two different K + channels a n d do n o t act specifically o n ATP-sensitive K + c h a n nels. F u r t h e r m o r e , o u r d a t a indicate that a n t a g o n i s m by G l i b e n c l a m i d e c a n n o t be used as the only a r g u m e n t for the i n v o l v e m e n t of ATP-sensitive K + channels. This work was s u p p o r t e d by the W i l h e m Sander Stift u n g (S.Q.) a n d by the H e r r m a n - u n d - L i l l y - S c h i l l i n gStift u n g (C.F. a n d H.H.). The a u t h o r s would like to t h a n k Prof. Dr. P. Grafe for helpful discussion, Drs. H. Vivaud o u a n d M. Villaz (Grenoble, F r a n c e ) for teaching us the s k i n n e d single muscle fiber p r e p a r a t i o n , Ms. B. Weich for technical assistance, a n d Prof. Dr. J. D u d e l a n d Dr. K. Feasey for reading the m a n u s c r i p t . 1 Beech, D.J. and Bolton, T.B., Properties of the cromakaliminduced potassium conductance in smooth muscle cells isolated from the rabbit portal vein, Br. J. Pharmacol., 98 (1989) 851-864. 2 Burton, F., D6rstelmann, U. and Hutter, O.F., Single-channelactivity in human and other mammalian muscle, Muscle Nerve, 11 (1988) 1029-1038. 3 De Peyer, D.E., Lues, I., Gericke, R. and H~iusler,G., Characterization of a novel K ÷ channel activator, EMD 52962, in electrophysical and pharmacological experiments, PfliigersArch., 414 (1989) S191. 4 Escande, D., Thuringer, D., Le Guern, S., Laville, M. and Cavero, I., Potassium channel openers act through an activation of ATPsensitive K ÷ channels in guinea-pig cardiac myocytes, Pfliigers Arch., 414 (1989) 669-675. 5 Fan, Z., Nakayama, K. and Hiraoka, M., Pinacidil activates the ATP-sensitive K ÷ channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes, PfliigersArch., 415 (1990) 387-394. 6 Fink, R.H.A., Breatg, A.H., Feutrill, C.W. and Kyriacou, K., Single K + and CI- channels from native sarcolemma vesicles of adult mammalian muscle, Proc. Int. Union Physiol. Sci., 1989, P2422.

7 Franke, Ch., Hatt, H. and Dudel, J., Liquid filament switch for ultra-fast exchanges of solutions at excised patches of synaptic membrane of crayfish muscle, Neurosci. Lett., 77 (1987) 199-204. 8 Grafe, P., Quasthoff, S., Strupp, M. and Lehmann-Horn, F., Enhancement of K + conductance improves in vitro the contraction force of skeletal muscle in hypokalemic periodic paralysis, Muscle Nerve, in press. 9 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. and Sigworth, F.J., Improved patch-clamp technique for high-resolution current recording from cells and cell-free membrane patches, Pfliigers Arch., 391 (1981) 85-100. 10 Hart, H., Franke, Ch. and Quasthoff, S., Activation of ATP-sensitive K ÷ channels from human skeletal muscle: a new method allows recording without enzyme treatment, Pfliigers Arch., 415 (Suppl 1) (1990) R 17. 11 Lehmann-Horn, F. and laizzo, P.A., Resealed fiber segments for the study of the pathophysiology in human skeletal muscle, Muscle Nerve, 13 (1990) 222-231. 12 Quast, U. and Cook, N.S., Moving together: K ÷ channel openers and ATP-sensitiveK ÷ channels, TIPS, 10 (1989) 431435. 13 Quasthoff, S., Spuler, A., Lehmann-Horn, F. and Grafe, P., Cromakalim, pinacidil and RP 49356 activate a tolbutamide-sensitive K + conductance in human" skeletal muscle, Pfliigers Arch., 414 (Suppl 1) (1989) S179-Sl80. 14 Spuler, A., Lehmann-Horn, F. and Grafe, P., Cromakalim (BRL 34915) restores in vitro the membrane potential of depolarized human skeletal muscle, Naunyn-Schmiedeberg's Arch. Pharmacol., 339 (1989) 327-331. 15 Stein, P. and Palade, P., Patch clamp of sarcolemmal spheres from stretched skeletal muscle fibers, Am. Physiol. Soc. (Spec. Commun.), 0363-6143 (1989) C434-C440. 16 Trieschmann, U., Pichelmaier, M., K1fckner, U. and Isenberg, G., Vasorelaxation due to K-agonists. Single channel recordings from isolated human vascular myocytes, Pfliigers Arch., 411 (1988) R199. 17 Vivaudou, M.B., Arnoult, C. and Villaz, M., Skeletal muscle ATPsensitive K ÷ channel recorded from sarcolemmal vesicles obtained by a new non-enzymatic method, Biophys. J., 57 (part 2) (1990) W 382. 18 Weik, R. and Neumcke, B., ATP-sensitive potassium channels in adult mouse skeletal muscle: characterization of the ATP-binding site, J. Membrane Biol., 110 (1989) 217-226.