Competition between Calcium Sensors 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 which suggest that both calcium sensors use their C-lobes for high affinity binding to the pore-forming CaV alpha-subunit, while their N-terminal lobe are responsible for the stark functional differences of the calcium sensors. 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 in Xenopus oocytes under conditions with different ratios of CaM and CaBP1, we demonstrate direct competition between both calcium sensors for their 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. Overall, our data provide a framework for understanding how CaBP1 and CaM differentially regulate CDI on CaV1.2.