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Calmodulin Modulates Initiation but Not Termination of Spontaneous Ca²⁺ Sparks in Frog Skeletal Muscle

Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201

Correspondence: Address correspondence to M. F. Schneider, Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 N. Greene St., Baltimore, MD 21201. Tel.: 410-706-7812; Fax: 410-706-8297; E-mail: mschneid{at}umaryland.edu.

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 µM) in the internal solution (free [Ca²⁺]_i 50–100 nM) initiated a highly cooperative dose-dependent increase in Ca²⁺ 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-Ca²⁺ 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 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.

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