The voltage-gated L-type Ca²⁺ channel Ca_V1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of Ca_V1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of Ca_V1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal Ca_V1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous Ca_V1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to Ca_V1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α₁1.2 subunit of Ca_V1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that Ca_V1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant Ca_V1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α₁1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of Ca_V1.2 but also augments its ion conducting activity.

Ca_V1.2 channels are critical players in cardiac excitation-contraction coupling, yet we do not understand how they are affected by an important therapeutic target of heart failure drugs and regulator of blood pressure, angiotensin II. Signaling through G_q-coupled AT1 receptors, angiotensin II triggers a decrease in PIP₂, a phosphoinositide component of the plasma membrane (PM) and known regulator of many ion channels. PIP₂ depletion suppresses Ca_V1.2 currents in heterologous expression systems but the mechanism of this regulation and whether a similar phenomenon occurs in cardiomyocytes is unknown. Previous studies have shown that Ca_V1.2 currents are also suppressed by angiotensin II. We hypothesized that these two observations are linked and that PIP₂ stabilizes Ca_V1.2 expression at the PM and angiotensin II depresses cardiac excitability by stimulating PIP₂ depletion and destabilization of Ca_V1.2 expression. We tested this hypothesis and report that Ca_V1.2 channels in tsA201 cells are destabilized after AT1 receptor-triggered PIP₂ depletion, leading to their dynamin-dependent endocytosis. Likewise, in cardiomyocytes, angiotensin II decreased t-tubular Ca_V1.2 expression and cluster size by inducing their dynamic removal from the sarcolemma. These effects were abrogated by PIP₂ supplementation. Functional data revealed acute angiotensin II reduced Ca_V1.2 currents and Ca²⁺ transient amplitudes thus diminishing excitation-contraction coupling. Finally, mass spectrometry results indicated whole-heart levels of PIP₂ are decreased by acute angiotensin II treatment. Based on these observations, we propose a model wherein PIP₂ stabilizes Ca_V1.2 membrane lifetimes, and angiotensin II-induced PIP₂ depletion destabilizes sarcolemmal Ca_V1.2, triggering their removal, and the acute reduction of Ca_V1.2 currents and contractility.

The L-type Ca²⁺ channel Ca_V1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs Ca_V1.2 open probability (Po) and Ca²⁺-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca²⁺-saturated CaM species (Ca₂/CaM) with Ca²⁺ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca²⁺ concentrations (50-100 nM) that constitutively preassociates with Ca_V1.2 to promote Po and CDI. We also present an NMR structure of a complex between the Ca_V1.2 IQ motif (residues 1644-1665) and Ca₂/CaM_12', a calmodulin mutant in which Ca²⁺ binding to EF1 and EF2 is completely disabled. We found that the CaM_12' N-lobe does not interact with the IQ motif. The CaM_12' C-lobe bound two Ca²⁺ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca²⁺ binding to EF3 and EF4 in the CaM₃₄ mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca₂/CaM species promotes Ca_V1.2 Po and CDI, identifying Ca₂/CaM as an important mediator of Ca signaling.

The L-type Ca²⁺ channel Ca_V 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of Ca_V 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts Ca_V 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The Ca_V 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits Ca_V 1.2 to defined surface regions and simultaneously boosts its open probability so that Ca_V 1.2 is mostly active when appropriately localized.

The L-type Ca²⁺ channel Ca_V 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of Ca_V 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts Ca_V 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The Ca_V 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits Ca_V 1.2 to defined surface regions and simultaneously boosts its open probability so that Ca_V 1.2 is mostly active when appropriately localized.