Acetylcholine-activated inward current induces cytosolic Ca2+ mobilization in mouse C2C12 myotubes

We examined the spatiotemporal pattern of intracellular Ca2+ liberation in mouse myotubes by means of fluorescence imaging of cytosolic free Ca2+ together with the simultaneous recording of membrane whole-cell currents. Acetylcholine (ACh) applications to C2C12 myotubes equilibrated in Ca(2+)-free medium and voltage clamped at -50 mV evoked localized fluorescence transients of variable amplitude with less than 0.5 s delay. Under the same experimental conditions, fluorescence transients were elicited by ACh also in mouse primary myotubes. Ca2+ transients were inhibited in myotubes clamped at depolarized potentials (-10 mV to +50 mV), or equilibrated in a Na+,Ca(2+)-free medium as well as in cells loaded with heparin, or with inositol (1,4,5) trisphosphate (InsP3). To investigate whether InsP3 could induce Ca2+ mobilization, [Ca2+]i determinations were carried out in myotubes loaded with InsP3 through the whole-cell patch-clamp recording pipette or by extracellular application in permeabilized cells. InsP3 diffusion into the myoplasm caused Ca2+ spikes with 5 +/- 1 s (mean +/- SEM) delay from the rupture of the membrane patch. Spikes were followed by sustained increases in fluorescence or by damped oscillations. In permeabilized myotubes, InsP3 induced the release of sequestered 45Ca2+ with a half-maximally effective concentration (EC50) of 0.28 +/- 0.05 microM, and Hill coefficient of 0.79 +/- 0.09. It is concluded that the ACh-activated inward current in mouse myotubes is coupled to cytosolic Ca2+ mobilization from internal InsP3-sensitive pools.