Calmodulin Modulates Initiation but Not Termination of Spontaneous Ca 2+ Sparks in Frog Skeletal Muscle

Abstract

Calmodulin is a ubiquitous Ca 2+ sensing protein that binds to and modulates the sarcoplasmic reticulum Ca 2+ release channel, ryanodine receptor (RYR). Here we assessed the effects of calmodulin on the local Ca 2+ 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 μM) in the internal solution (free [Ca 2+] i ∼50–100 nM) initiated a highly cooperative dose-dependent increase in Ca 2+ spark frequency, with a half-maximal activation ( K) of 1.1 μM, a Hill coefficient ( n) of 4.2 and a fractional maximal increase in frequency ( R) of 17-fold. A non-Ca 2+ binding mutant of calmodulin elicited a similar highly cooperative dose-dependent increase in spark frequency ( K = 1.0 μM; n = 3.7; R = 12-fold). Spatiotemporal properties of Ca 2+ 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 [Ca 2+] in sarcoplasmic reticulum vesicles, prevented the calmodulin-induced increase in spark frequency. These data suggest that exogenous Ca 2+-free calmodulin cooperatively sensitizes the Ca 2+ release channel to open, but that Ca 2+ binding to the added calmodulin does not play a significant role in the termination of Ca 2+ sparks.