title: Intracellular Ca2+ changes and Ca2+-activated K+ channel activation induced by acetylcholine at the endplate of mouse skeletal muscle fibres.
abstract: 1. Enzymatically isolated skeletal muscle fibres were used to investigate the effects of applying acetylcholine (ACh) onto the endplate area on intracellular free calcium concentration ([Ca2+]i) measured using the indicator indo-1 and single channel activity using the patch clamp technique. 2. Using a Tyrode solution containing 5 microM tetrodotoxin (TTX) as extracellular solution, ACh applications (at 0.1 or 1 mM) onto the endplate induced intracellular free calcium transients the mean maximal amplitude of which was 360 +/- 30 nM from a mean resting value of 72 +/- 7 nM (n = 13). In cells bathed with a K(+)-rich solution (145 mM K+), applications of ACh (0.1 mM) induced transient rises in [Ca2+]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, using a pipette filled with Tyrode solution, external application of 0.1 mM ACh could induce a transient burst opening of channels carrying an outward 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 area, they displayed a conductance of 60 and 224 pS in the presence of Tyrode and K(+)-rich solution in the pipette, respectively. Half-maximum activation was found for a [Ca2+]i close to 4 microM. The channels showed a typical voltage dependence. In outside-out patches these channels were shown to be blocked by 100 nM charybdotoxin (CTX). 5. In fibres bathed in a Tyrode solution containing TTX (5 microM), CTX had no clear effect on the change in membrane voltage, recorded near the endplate with a single intracellular microelectrode, in response to the application of ACh. 6. Although the physiological relevance of this ACh-induced K+ channel activation remains unclear, results suggest that, in the presence of a physiological extracellular [Ca2+], Ca2+ entry through the endplate nicotinic receptors can produce a local increase in [Ca2+]i, sufficient to trigger the opening of Ca2+-activated K+ channels in the adjacent surface membrane.
authors.affiliation: Laboratoire de Physiologie des Eléments Excitables, CNRS UMR 5578, Université C. Bernard, Villeurbanne, France.
authors.forename: B
authors.initials: B
authors.lastname: Allard
authors.forename: J C
authors.initials: JC
authors.lastname: Bernengo
authors.forename: O
authors.initials: O
authors.lastname: Rougier
authors.forename: V
authors.initials: V
authors.lastname: Jacquemond
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology; D018999:Charybdotoxin / Q000494:pharmacology; D066298:In Vitro Techniques; D051379:Mice; D009045:Motor Endplate / Q000187:drug effects / Q000502:physiology*; D018485:Muscle Fibers, Skeletal / Q000502:physiology*; D018482:Muscle, Skeletal / Q000294:innervation*; D018408:Patch-Clamp Techniques; D011188:Potassium / Q000494:pharmacology; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D013779:Tetrodotoxin / Q000494:pharmacology
chemical_list: D015221:Potassium Channels; D018999:Charybdotoxin; D013779:Tetrodotoxin; D000109:Acetylcholine; D011188:Potassium; D002118:Calcium