CaBP1 Regulates Both Ca and Ba currents through Ca(v)1.2 (L-type) Calcium Channels

Abstract

The main goal of this work was to study the mechanism of inactivation and gating of the L-type voltage-dependent calcium channel (L-VDCC) - Ca(v)1.2 - by calcium-binding protein 1 (CaBP1).Previously it was shown that Ca2+ dependent inactivation (CDI) is calmodulin (CaM)-dependent, while CaBP1 totally prevents the process. It has been suggested that the amino terminal of the pore forming subunit of the channel - Ca(v)1.2-NT plays a crucial role in mediating the effects of CaBP1 on inactivation.Electrophysiological assay was done in Xenopus oocyte expression system, using two-electrode voltage clamp (TEVC) that monitors whole cell currents. Interactions between different radiolabeled and GST- fused proteins was studied in vitro by pull down assays.We mapped the interaction sites of both CaM and CaBP1 on the Ca(v)1.2-NT, and discovered that these are separated sites. The functional study showed an opposite effect of CaBP1 on Ca(v)1.2 inactivation: it abolished CDI but enhanced the voltage-dependent inactivation (VDI). CaBP1 shifted the current-voltage (IV) curve of Ca2+ and Ba2+ currents to positive values. Surprisingly, removing CaBP1 binding site on the Ca(v)1.2-NT, reduced but did not fully eliminate the changes caused by CaBP1. However, we found an essential contribution of the β subunit in both inactivation and CaBP1 effect. These findings suggest that multiple determinants influence the regulation of Cav1.2 by Ca2+ binding proteins.