Modulation of Ca2+ signalling in rat atrial myocytes: possible role of the α1c carboxyl terminal

摘要

Ca²⁺ influx through L-type Cav1.2 (α1c) Ca²⁺ channels is a critical step in the activation of cardiac ryanodine receptors (RyRs) and release of Ca²⁺ via Ca²⁺-induced Ca²⁺ release(CICR). The released Ca²⁺, in turn, is the dominant determinant of inactivation of the Ca²⁺ current (ICa) and termination of release. Although Ca²⁺ cross-signalling is mediated by high Ca²⁺ fluxes in the microdomains of α1c-RyR complexes, ICa-gated Ca²⁺ cross-signalling is surprisingly resistant to intracellular Ca²⁺ buffering and has steeply voltage-dependent gain, inconsistent with a strict CICR mechanism, suggesting the existence of additional regulatory step(s). To explore the possible regulatory role of the carboxyl (C)-terminal tail of α1c in modulating Ca²⁺ signalling, we tested the effects of introducing two α1c C-terminal peptides, LA (1571–1599) and K (1617–1636) on the central α1c-unassociated Ca²⁺-release sites of atrial myocytes, using rapid (240 Hz) two-dimensional confocal Ca²⁺ imaging. The frequency of spontaneously activating central sparks increased by approximately fourfold on dialysing LA- but not K-peptide into myocytes voltage-clamped at -80 mV. The rate but not the magnitude of caffeine (10 mM)-triggered central Ca²⁺ release was significantly accelerated by LA- but not K-peptide. Individual Ca²⁺ spark size and flux were larger in LA- but not in K-peptide-dialysed myocytes. Although LA-peptide did not change the amplitude or inactivation kinetics of ICa, LA-peptide did strongly enhance the central Ca²⁺ transients triggered by ICa at -30 mV (small ICa) but not at +20 mV (large ICa). In contrast, K-peptide had no effect on either ICa or the local Ca²⁺ transients. LA-peptide with a deleted calmodulin-binding region (LM1-peptide) had no significant effects on the central spark frequency but suppressed spontaneous spark frequency in the periphery. Our results indicate that the calmodulin-binding LA motif of the α1c C-terminal tail may sensitize the RyRs, thereby increasing their open probability and providing for both the voltage-dependence of CICR and the higher frequency of spark occurrence in the periphery of atrial myocytes where the native α1c-RyR complexes are intact.