α-Actinin Promotes Surface Localization and Current Density of the Ca 2+ Channel Ca V 1.2 by Binding to the IQ Region of the α1 Subunit.

The voltage-gated L-type Ca2+ channel CaV 1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of CaV 1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of CaV 1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal CaV 1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous CaV 1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to CaV 1.2 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 α1 1.2 subunit of CaV 1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV 1.2 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 CaV 1.2 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 α1 1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of CaV 1.2 but also augments its ion conducting activity.