INTRACELLULAR CA2-ACTIVATED K+ CHANNEL ACTIVATION-INDUCED BY ACETYLCHOLINE AT THE END-PLATE OF MOUSE SKELETAL-MUSCLE FIBERS( CHANGES AND CA2+)

1. Enzymatically isolated skeletal muscle fibres were used to investig ate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+](i)) measured usin g the indicator indo-1 and single channel activity using the patch cla mp technique. 2. Using a Tyrode solution containing 5 mu M tetrodotoxi n (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) o nto the endplate induced intracellular free calcium transients the mea n maximal amplitude of which was 360 +/- 30 nM from a mean resting val ue of 72 +/- 7 nM (n = 13). In cells bathed with a K+-rich solution (1 45 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca 2+](i) from a mean resting value of 53 +/- 7 nM to a maximum of 222 +/ - 24 nM (n = 33). 3. In cell-attached membrane patches at the endplate membrane of muscle fibres bathed in a K+-rich external solution, usin g a pipette filled with Tyrode solution, external application of 0.1 m M ACh could induce a transient burst opening of channels carrying an o utward current of an average amplitude of 4.6 +/- 0.2 pA at 0 mV (n = 8). 4. These channels were characterized as Ca2+-activated K+ channels . At 0 mV, in inside-out patches excised from the endplate membrane ar ea, they displayed a conductance of 60 and 224 pS in the presence of T yrode and K+-rich solution in the pipette, respectively. Half-maximum activation was found for a [Ca2+](i) close to 4 mu M. The channels sho wed a typical voltage dependence. In outside-out patches these channel s were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTS (5 mu M), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the applica tion of ACh.6. Although the physiological relevance of this ACh-induce d K+ channel activation remains unclear, results suggest that, in the presence or a physiological extracellular [Ca2+], Ca2+ entry through t he endplate nicotinic receptors can produce a local increase in [Ca2+] (i), sufficient to trigger the opening of Ca2+-activated K+ channels i n the adjacent surface membrane.