Measuring Ca²⁺-Dependent Modulation of Voltage-Gated Ca²⁺ Channels in HEK-293T Cells

Abstract

Voltage-gated Ca²⁺ (Ca_v) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Ca_v channels are subject to diverse forms of regulation, including those involving the Ca²⁺ ions that permeate the pore. High voltage-activated Ca_v channels undergo Ca²⁺-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Ca_v1 (L-type) and Ca_v2 (P/Q-, N-, and R-type) channels. Human embryonic kidney cells transformed with SV40 large T-antigen (HEK-293T) are advantageous for studying CDI and CDF of a particular type of Ca_v channel. HEK-293T cells do not express endogenous Ca_v channels, but Ca_v channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca²⁺-dependent modulation of recombinant Ca_v channels in HEK-293T cells.