There is a considerable controversy in the literature concerning the effects of higher concentrations of calcium chelators (e.g. BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid) or fura-2) on the intracellular Ca transients in muscle. We induced calcium release from sarcoplasmic reticulum (SR) in the triad preparation by chemical depolarization of the T-tubule in the presence of various concentrations of BAPTA-calcium buffer ([Ca] = 0.1 µM) and investigated the effects of the BAPTA concentration on the time courses of conformational changes in the junctional foot protein (JFP) and calcium release from SR. Upon stimulation, the JFP underwent biphasic conformational changes, as determined by stopped-flow fluorometry of the JFP-bound conformational probe. The first phase of protein conformational change, which preceded calcium release from SR, was virtually unaffected by the BAPTA concentration. However, the magnitude of the second phase increased in an inversely proportional fashion to the BAPTA concentration. An abrupt increase in [Ca] from 0.1 µM up to 1.0 µM (Ca), concurrently with T-tubule depolarization, produced biphasic protein conformational changes: a Ca-independent first phase and a Cadependent second phase. Similar Ca jump experiments under non-depolarizing conditions produced a slow monophasic conformational change equivalent to the second phase described above. These results suggest that the first phase of protein conformational change represents the activation of JFP by T-tubule depolarization to induce calcium release, and the second phase the secondary activation by the released Ca. Activation of the JFP by the released Ca resulted in an acceleration of both (i) the rate of initial calcium release, and (ii) the subsequent attenuation of calcium release. The acceleration of both was suppressed by higher concentrations of BAPTA. These results provide a reasonable explanation for both of the apparently contradictory views in the literature; high concentrations of calcium buffer (a) suppress the initial activation and (b) prevent the subsequent attenuation of calcium release.

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