Calmodulin modulates initiation but not termination of spontaneous Ca2+ sparks in frog skeletal muscle.
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 micro M) in the internal solution (free [Ca(2+)](i) approximately 50-100 nM) initiated a highly cooperative dose-dependent increase in Ca(2+) spark frequency, with a half-maximal activation (K) of 1.1 micro M, 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 micro M; 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.
Rodney
George G
GG
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Schneider
Martin F
MF
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects* / Q000502:physiology*; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018482:Muscle, Skeletal / Q000187:drug effects* / Q000502:physiology*; D011894:Rana pipiens; D012503:Saponins / Q000494:pharmacology*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects* / Q000502:physiology*
D002147:Calmodulin; D012503:Saponins; D002118:Calcium