α-Actinin Promotes Surface Localization and Current Density of the Ca Channel Ca1.2 by Binding to the IQ Region of the α1 Subunit.

The voltage-gated L-type Ca channel Ca1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of Ca1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of Ca1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal Ca1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous Ca1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to Ca1.2 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 α1.2 subunit of Ca1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that Ca1.2 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 Ca1.2 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 α1.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of Ca1.2 but also augments its ion conducting activity.