Evidence of a role for calmodulin in the regulation of calcium release from skeletal muscle sarcoplasmic reticulum

Abstract

The effect of calmodulin and calmodulin inhibitors on the "Ca2+ release channel" of "heavy" skeletal muscle sarcoplasmic reticulum (SR) vesicles was investigated. SR vesicles were passively loaded with 45Ca2+ in the presence of calmodulin and its inhibitors, followed by measurement of 45Ca2+ release rates by means of a rapid-quench-Millipore filtration method. Calmodulin at a concentration of 2-10 microM reduced 45Ca2+ efflux rates from passively loaded vesicles by a factor of 2-3 in media containing 10(-6)-10(-3) M Ca2+. At 10(-9) M Ca2+, calmodulin was without effect. 45Ca2+ release rates were varied 1000-fold (k1 approximately equal to 0.1-100 s-1) by using 10(-5) M Ca2+ with either Mg2+ or the ATP analogue adenosine 5'-(beta,gamma-methylenetriphosphate) in the release medium. In all instances, a similar 2-3-fold reduction in release rates was observed. At 10(-5) M Ca2+, 45Ca2+ release was half-maximally inhibited by about 2 X 10(-7) M calmodulin, and this inhibition was reversible. Heavy SR vesicle fractions contained 0.1-02 micrograms of endogenous calmodulin/mg of vesicle protein. However, the calmodulin inhibitors trifluoperazine, calmidazolium, and compound 48/80 were without significant effect on 45Ca2+ release at concentrations which inhibit calmodulin-mediated reactions in other systems. Studies with actively loaded vesicles also suggested that heavy SR vesicles contain a Ca2+ permeation system that is inhibited by calmodulin.

Apocalmodulin and Ca2+-Calmodulin Bind to Neighboring Locations on the Ryanodine Receptor

“…Most likely, additional regions of the RyR1, particularly within domain 3 and the clamps (domains 5-10), are involved in the interaction with DHPR (31,52,55,56). The existence of two closely spaced but distinct sites for apoCaM and Ca 2ϩ -CaM on RyR1, together with the proximity of these sites to a region of RyR1 that potentially interacts with DHPR, leads us to speculate that CaM might play a more direct role in the molecular mechanism of E-C coupling than previously considered (6,13,15,57).…”

“…This is apparently necessary and sufficient for RyR1 activation, which results in a global intracellular Ca 2ϩ increase from above pCa 7 to pCa 6 -4, a level of Ca 2ϩ that triggers contraction of the muscle fiber (5). RyR1 itself is regulated by Ca 2ϩ in a biphasic fashion with an activation peak at pCa ϳ5.5 and inhibition below pCa 3 or above 7 (6). In addition to its vital function in skeletal muscle, RyR1 is an abundant RyR isoform in cerebellar neurons (7) where it also appears to communicate with the plasma membrane DHPR (8).…”

“…At pCa 4, Ca 2ϩ -saturated CaM (Ca 2ϩ -CaM) binds directly to the RyR1 with a stoichiometry of one mole of apoCaM per RyR1 subunit (i.e. four CaMs per tetrameric RyR1 complex) (11,12) and partially inhibits channel activity (6,13), but at pCa 7, Ca 2ϩ -depleted CaM (apoCaM) becomes an activator (14 -17). In both cases CaM reduces the direct effect produced by Ca 2ϩ alone acting on the RyR1.…”

Ion channel regulation by calmodulin binding

“…The RyR/CaCh of skeletal muscle, the best studied, passes all cations and is strongly regulated by many factors, one of which is calmodulin. Although calmodulin dependent kinases can phosphorylate the channel protein, Ca"-calmodulin interacts directly with the channel in the absence of ATP and inhibits 45Ca uptake into SR vesicles by > 1 ,OOO-fold [12] and reduces reconstituted channel activity by 2-fold [13]. Based on the deduced amino-acid sequence of RyR/CaCh, 2-3 putative calmodulin binding sites have been proposed [14,15].…”

A Calmodulin Binding Domain of RyR Increases Activation of Spontaneous Ca2+ Sparks in Frog Skeletal Muscle

“…Each RyR1 subunit binds one molecule of CaM or four CaM molecules per RyR1 homotetramer, regardless of the cytosolic [Ca 2ϩ ] (2). In SR vesicle preparations CaM displays Ca 2ϩ dependence in its functional effects on RyR1; at nanomolar [Ca 2ϩ ] Ca 2ϩ -free CaM activates RyR1, whereas at millimolar [Ca 2ϩ ] Ca 2ϩ -CaM inhibits RyR1 (3,4). This Ca 2ϩ -dependent bi-functional regulation of RyR1 function was found to require Ca 2ϩ binding to CaM (3), in particular at the two C-terminal Ca 2ϩ binding sites (3,5).…”