Endogenous and Exogenous Ca²⁺ Buffers Differentially Modulate Ca²⁺-dependent Inactivation of Ca_V2.1 Ca²⁺ Channels^*

Abstract

Voltage-gated Ca²⁺ channels undergo a negative feedback regulation by Ca²⁺ ions, Ca²⁺-dependent inactivation, which is important for restricting Ca²⁺ signals in nerve and muscle. Although the molecular details underlying Ca²⁺-dependent inactivation have been characterized, little is known about how this process might be modulated in excitable cells. Based on previous findings that Ca²⁺-dependent inactivation of Ca_v2.1 (P/Q-type) Ca²⁺ channels is suppressed by strong cytoplasmic Ca²⁺ buffering, we investigated how factors that regulate cellular Ca²⁺ levels affect inactivation of Ca_v2.1 Ca²⁺ currents in transfected 293T cells. We found that inactivation of Ca_v2.1 Ca²⁺ currents increased exponentially with current amplitude with low intracellular concentrations of the slow buffer EGTA (0.5 mm), but not with high concentrations of the fast Ca²⁺ buffer BAPTA (10 mm). However, when the concentration of BAPTA was reduced to 0.5 mm, inactivation of Ca²⁺ currents was significantly greater than with an equivalent concentration of EGTA, indicating the importance of buffer kinetics in modulating Ca²⁺-dependent inactivation of Ca_v2.1. Cotransfection of Ca_v2.1 with the EF-hand Ca²⁺-binding proteins, parvalbumin and calbindin, significantly altered the relationship between Ca²⁺ current amplitude and inactivation in ways that were unexpected from behavior as passive Ca²⁺ buffers. We conclude that Ca²⁺-dependent inactivation of Ca_v2.1 depends on a subplasmalemmal Ca²⁺ microdomain that is affected by the amplitude of the Ca²⁺ current and differentially modulated by distinct Ca²⁺ buffers.