SUMMARY1. Experiments were done to test the hypothesis that parvalbumin (PA) promotes relaxation in frog skeletal muscle. Single fibres and purified PA from Rana temporaria skeletal muscle were used to determine the relationship between PA concentration ([PA]), Ca2" and Mg2" dissociation rates from PA and changes in rate of relaxation as a function of tetanus duration at 0°C. 3. Relaxation rate from an isometric tetanus slows progressively as a function of tetanus duration with an exponential time course and a rate constant of 1P18 + 0 35 s-' (n = 17). Time course of recovery of relaxation rate after a prolonged tetanus is exponential with a rate constant of 0O12 + 0-02 s-1 (n = 14).4. The extent of recovery of relaxation rate after a prolonged tetanus was correlated with total [PA] in fibres (correlation coefficient (r) = 0-80, n = 7; P < 005).5. Dissociation rate constants for Mg2+ and Ca2+ from purified PA are 0-93+0-02 s-' (n = 5) and 0 19+0-01 s-' (n = 5), respectively. Dissociation rate constants were not significantly different for PA isoforms IVa and IVb. These rate constants are similar to the rate constants determined for the time courses of slowing and recovery of relaxation rate, respectively. 6. Results suggest that the time courses of slowing and recovery of relaxation rate may be controlled, to a large extent, by Mg2+ and Ca2+ dissociation from PA, respectively. This evidence supports a role for PA in facilitating relaxation during a tetanus in frog skeletal muscle at 0 'C.
Calmodulin (CaM) may function as a regulatory subunit of ryanodine receptor (RYR) channels, modulating both channel activation and inhibition by Ca2+; however, mechanisms underlying differences in CaM regulation of the RYR isoforms expressed in skeletal muscle (RYR1) and cardiac muscle (RYR2) are poorly understood. Here we use a series of CaM mutants deficient in Ca2+ binding to compare determinants of CaM regulation of the RYR1 and RYR2 isoforms. In submicromolar Ca2+, activation of the RYR1 isoform by each of the single-point CaM mutants was similar to that by wild-type apoCaM, whereas in micromolar Ca2+, RYR1 inhibition by Ca2+CaM was abolished by mutations targeting CaM's C-terminal Ca2+ sites. In contrast to the RYR1, no activation of the cardiac RYR2 isoform by wild-type CaM was observed, but rather CaM inhibited the RYR2 at all Ca2+ concentrations (100 nM to 1 mM). Consequently, whereas the apparent Ca2+ sensitivity of the RYR1 isoform was enhanced in the presence of CaM, the RYR2 displayed the opposite response (RYR2 Ca2+ EC50 increased 7-10-fold in the presence of 5 microM wild-type CaM). CaM inhibition of the RYR2 was nonetheless abolished by each of four mutations targeting individual CaM Ca2+ sites. Furthermore, a mutant CaM deficient in Ca2+ binding at all four Ca2+ sites significantly activated the RYR2 and acted as a competitive inhibitor of RYR2 regulation by wild-type Ca2+CaM. We conclude that Ca2+ binding to CaM determines the effect of CaM on both RYR1 and RYR2 channels and that isoform differences in CaM regulation reflect the differential tuning of Ca2+ binding sites on CaM when bound to the different RYRs. These results thus suggest a novel mechanism by which CaM may contribute to functional diversity among the RYR isoforms.
The influences of sarcomere uniformity and Ca2+ concentration on the kinetics of relaxation were examined in skinned frog skeletal muscle fibers induced to relax by rapid sequestration of Ca2+ by the photolysis of the Ca2+ chelator, diazo-2, at 10°C. Compared with an intact fiber, diazo-2-induced relaxation exhibited a faster and shorter initial slow phase and a fast phase with a longer tail. Stabilization of the sarcomeres by repeated releases and restretches during force development increased the duration of the slow phase and slowed its kinetics. When force of contraction was decreased by lowering the Ca2+concentration, the overall kinetics of relaxation was accelerated, with the slow phase being the most sensitive to Ca2+ concentration. Twitchlike contractions were induced by photorelease of Ca2+ from a caged Ca2+ (DM-Nitrophen), with subsequent Ca2+ sequestration by intact sarcoplasmic reticulum or Ca2+ rebinding to caged Ca2+. These twitchlike responses exhibited relaxation kinetics that were about twofold slower than those observed in intact fibers. Results suggest that the slow phase of relaxation is influenced by the degree of sarcomere homogeneity and rate of Ca2+ dissociation from thin filaments. The fast phase of relaxation is in part determined by the level of Ca2+ activation.
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