Alpha-Actinin Promotes Surface Localization and Ion Conducting Activity of the L-Type Ca2+ Channel by Binding to the IQ Region of CaV1.2

Abstract

The voltage-gated L-type Ca2+ channel CaV1.2 is crucial for initiating heart beat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of CaV1.2 subunits result in serious health problems including arrhythmia, autism, immunodeficiency, and hypoglycemia. Thus precise control of CaV1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal CaV1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduced localization of endogenous CaV1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that α-actinin interacts with CaV1.2 to support 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 subunit α11.2, which individually impaired α-actinin binding. CaV1.2 channels composed of corresponding α-actinin-binding-deficient mutants exhibited a reduction of 60-75% in current density. To determine whether these mutations also influenced the deposition of the channel to the plasma membrane, we employed surface biotinylation and flow cytometry. Whereas all three mutations did reduce surface expression, this reduction was much smaller (∼40%) than the reduction in current density. Taken together our work indicates that α-actinin not only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity.