Ca2+-dependent inactivation of Ca2+-induced Ca2+ release in bullfrog sympathetic neurons

摘要

We studied inactivation of Ca²⁺-induced Ca²⁺ release (CICR) via ryanodine receptors (RyRs) in bullfrog sympathetic neurons. The rate of rise in [Ca²⁺]i due to CICR evoked by a depolarizing pulse decreased markedly within 10–20 ms to a much slower rate despite persistent Ca²⁺ entry and little depletion of Ca²⁺ stores. The Ca²⁺ entry elicited by the subsequent pulse within 50 ms, during which the [Ca²⁺]i level remained unchanged, did not generate a distinct [Ca²⁺]i rise. This mode of [Ca²⁺]i rise was unaffected by a mitochondrial uncoupler, carbonyl cyanide p-trifluromethoxy-phenylhydrazone (FCCP, 1 μm). Paired pulses of varying interval and duration revealed that recovery from inactivation became distinct ≥ 50 ms after depolarization and depended on [Ca²⁺]i. The inactivation was prevented by BAPTA (≥ 100 μm) but not by EGTA (≤ 10 mm), whereas the activation was less affected by BAPTA. When CICR was partially activated, some of the non-activated RyRs were also inactivated directly. Thus, the inactivation in these neurons is induced by Ca²⁺ binding to the high-affinity regulatory sites residing very close to Ca²⁺ channels and/or RyRs, although the sites for activation are located much closer to those Ca²⁺ sources. The rate of [Ca²⁺]i decay after the pulse decreased with increasing pulse duration longer than 10 ms, and this was abolished by BAPTA. Thus, some mechanism counteracting Ca²⁺ clearance is induced after full inactivation and potentiated during the pulse. Possible models for RyR inactivation were proposed and the roles of inactivation in Ca²⁺ signalling were discussed.