Half-calcified Calmodulin Promotes Basal Activity and Inactivation of the Calcium Channel Ca _V 1.2

The L-type Ca ²⁺ channel Ca _V 1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs Ca _V 1.2 open probability (Po) and Ca ²⁺ -dependent inactivation (CDI) but the mechanisms remain unclear. We identified a half Ca ²⁺ -saturated CaM species (Ca ₂ /CaM) with Ca ²⁺ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca ²⁺ concentrations (50-100 nM) that constitutively pre-associates with Ca _V 1.2 to promote Po and CDI. We present an NMR structure of a complex between the Ca _V 1.2 IQ motif (residues 1644-1665) and Ca ₂ /CaM _12’ , a calmodulin mutant in which Ca ²⁺ binding to EF1 and EF2 is completely disabled. The CaM _12’ N-lobe does not interact with the IQ motif. The CaM _12’ C-lobe bound two Ca ²⁺ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca ²⁺ binding to EF3 and EF4 in the CaM ₃₄ mutant when compared to wildtype CaM. Accordingly, a previously unappreciated Ca ₂ /CaM species promotes Ca _V 1.2 Po and CDI identifying Ca ₂ /CaM as an important mediator of Ca signaling.