3 years ago

α-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the α1 Subunit

α-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the α1 Subunit
Tommaso Patriarchi, Carlota Montagut-Bordas, Mark W. Lillya, Johannes W. Hell, Boram Lee, Andrea M. Coleman, Mary C. Horne, Peter B. Henderson, Anne C. Hergarden, Pang-Yen Tseng
The voltage-gated L-type Ca2+ channel CaV1.2 is crucial for initiating heartbeat 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 spectrum disorders, 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 reduces localization of endogenous CaV1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to CaV1.2 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 CaV1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV1.2 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 CaV1.2 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 CaV1.2 but also augments its ion conducting activity.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00359

DOI: 10.1021/acs.biochem.7b00359

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