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

Abstract

Voltage-gated Ca(2+) (Cav) channels regulate a variety of biological processes, such as muscle contraction, gene expression, and neurotransmitter release. Cav channels are subject to diverse forms of regulation, including those involving the Ca(2+) ions that permeate the pore. High voltage-activated Cav channels undergo Ca(2+)-dependent inactivation (CDI) and facilitation (CDF), which can regulate processes such as cardiac rhythm and synaptic plasticity. CDI and CDF differ slightly between Cav1 (L-type) and Cav2 (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 Cav channel. HEK-293T cells do not express endogenous Cav channels, but Cav channels can be expressed exogenously at high levels in these cells by transient transfection. This protocol describes how to characterize and analyze Ca(2+)-dependent modulation of recombinant Cav channels in HEK-293T cells.

Figure 1. CDI of Ca_v2.2 channels

A,B) Top, Voltage protocols and representative current traces. Currents were leak-subtracted using the P/4 method. Bottom, I_res/I_peak represents residual current amplitude at the end of the pulse normalized to the peak current amplitude, and is plotted against test voltage. In A, internal recording solution contained 0.5 mM or 10 mM EGTA. In B, internal recording solution contained 0.5 mM and extracellular solution contained 10 mM Ca²⁺ (I_Ca ) or Ba²⁺ (I_Ba ). CDI = I_res/I_peak for I_Ba - I_res/I_peak for I_Ca, where I_res/I_peak for I_Ba = 0.69 ± 0.02 and I_Ca = 0.19 ± 0.07 for test pulse to 0 mV. Currents and averaged data for I_Ca (n=10) and I_Ba (n=8) are from different cells.

Figure 2. CDF of Ca_v2.1 channels

A) Top, voltage protocol and representative I_Ca and I_Ba evoked by 10-ms test pulses to 0 mV and -10 mV, respectively, before (grey) and after (red) a 50-ms prepulse to +30 mV. Currents were leak-subtracted using the P/4 method. B) The ratio of P2/P1 current amplitudes is plotted against prepulse voltage. CDF = the difference in P2/P1 for I_Ca and I_Ba using a +30-mV prepulse, where P2/P1 for I_Ca =1.4 ± 0.03 (n=4) and for I_Ba =1.2 ± 0.05 (n=4) for a +30-mV prepulse. Currents were recorded in 10 mM extracellular Ca²⁺ or Ba²⁺ and 10 mM intracellular EGTA. Currents and averaged data for I_Ca and I_Ba are from different cells.