Calmodulin modulates initiation but not termination of spontaneous Ca2+ sparks in frog skeletal muscle

Calmodulin is a ubiquitous Ca2+ sensing protein that binds to and modulates the sarcoplasmic reticulum Ca2+ release channel, ryanodine receptor (RYR). Here we assessed the effects of calmodulin on the local Ca2+ release properties of RYR in permeabilized frog skeletal muscle fibers. Fluorescently labeled recombinant calmodulin in the internal solution localized at the Z-line/triad region. Calmodulin (0.05-5.0 muM) in the internal solution (free [Ca2+](i) similar to50-100 nM) initiated a highly cooperative dose-dependent increase in Ca2+ spark frequency, with a half-maximal activation (K) of 1.1 muM, a Hill coefficient (n) of 4.2 and a fractional maximal increase in frequency (R) of 17-fold. A non-Ca2+ binding mutant of calmodulin elicited a similar highly cooperative dose-dependent increase in spark frequency (K=1.0 muM; n=3.7; R=12-fold). Spatiotemporal properties of Ca2+ sparks were essentially unaffected by either wild-type or mutant calmodulin. An N-terminal extension of calmodulin, (N+3) calmodulin, that binds to but does not activate RYR at nM [Ca2+] in sarcoplasmic reticulum vesicles, prevented the calmodulin-induced increase in spark frequency. These data suggest that exogenous Ca2+-free calmodulin cooperatively sensitizes the Ca2+ release channel to open, but that Ca2+ binding to the added calmodulin does not play a significant role in the termination of Ca2+ sparks.