Analysis of Competition between CaBP1 and Calmodulin on the Voltage-Gated Calcium Channel Cav1.2

Abstract

Voltage-gated calcium channel (CaV) activity is regulated by calcium sensors including calmodulin (CaM) and calcium-binding protein 1 (CaBP1). CaBP1 inhibits CaM-mediated calcium-dependent inactivation (CDI). We investigated the origins of functionally important differences between CaM and CaBP1 by creating a number of CaM/CaBP1 chimeras. The exchange of C-terminal lobes between CaM and CaBP1 is accompanied by few functional changes, suggesting CaBP1 and CaM C-lobe can substitute for each other. In contrast, we identified the linker and the N-terminal lobe of CaBP1 as elements that set it functionally apart from CaM. CaBP1 and CaM are thought to modulate CaV function by competing for binding to the CaV C-terminal IQ-domain, but this assumption has never been tested directly. By determining CaV1.2 CDI under conditions with different ratios of CaM and CaBP1, we demonstrate direct competition between CaBP1 and CaM for its CaV1.2 binding site. In order to extend our analysis of CaBP1/CaM competition we used isothermal titration calorimetry to determine the affinity of both CaM and CaBP1 in both calcium-bound and apo-states for the IQ domain, suggesting that competition occurs mainly in the apo-state. Analysis of an IQ domain mutant shows unexpected differences between CaM and CaBP1 in IQ domain binding, suggesting a possible mechanism for their different functional effects on CaV1.2 inactivation. Overall, the data reveal those parts of CaBP1 that set it functionally apart from CaM and provide a framework for understanding how CaBP1 and CaM differentially regulate CDI on CaV1.2.