Bourdin Benoîte, Shakeri Behzad, Tétreault Marie-Philippe, Sauvé Rémy, Lesage Sylvie, Parent Lucie
From the Département de Physiologie, Montreal Heart Institute Research Centre, and.
Département de Microbiologie, Infectiologie et Immunologie, Centre de Recherche de l'Hôpital Maisonneuve-Rosemont, Université de Montréal, Montréal, Québec H3C 3J7, Canada.
J Biol Chem. 2015 Jan 30;290(5):2854-69. doi: 10.1074/jbc.M114.597930. Epub 2014 Dec 19.
L-type Ca(2+) channels play a critical role in cardiac rhythmicity. These ion channels are oligomeric complexes formed by the pore-forming CaVα1 with the auxiliary CaVβ and CaVα2δ subunits. CaVα2δ increases the peak current density and improves the voltage-dependent activation gating of CaV1.2 channels without increasing the surface expression of the CaVα1 subunit. The functional impact of genetic variants of CACNA2D1 (the gene encoding for CaVα2δ), associated with shorter repolarization QT intervals (the time interval between the Q and the T waves on the cardiac electrocardiogram), was investigated after recombinant expression of the full complement of L-type CaV1.2 subunits in human embryonic kidney 293 cells. By performing side-by-side high resolution flow cytometry assays and whole-cell patch clamp recordings, we revealed that the surface density of the CaVα2δ wild-type protein correlates with the peak current density. Furthermore, the cell surface density of CaVα2δ mutants S755T, Q917H, and S956T was not significantly different from the cell surface density of the CaVα2δ wild-type protein expressed under the same conditions. In contrast, the cell surface expression of CaVα2δ D550Y, CaVα2δ S709N, and the double mutant D550Y/Q917H was reduced, respectively, by ≈30-33% for the single mutants and by 60% for the latter. The cell surface density of D550Y/Q917H was more significantly impaired than protein stability, suggesting that surface trafficking of CaVα2δ was disrupted by the double mutation. Co-expression with D550Y/Q917H significantly decreased CaV1.2 currents as compared with results obtained with CaVα2δ wild type. It is concluded that D550Y/Q917H reduced inward Ca(2+) currents through a defect in the cell surface trafficking of CaVα2δ. Altogether, our results provide novel insight in the molecular mechanism underlying the modulation of CaV1.2 currents by CaVα2δ.
L型Ca(2+)通道在心脏节律中起关键作用。这些离子通道是由形成孔道的CaVα1与辅助亚基CaVβ和CaVα2δ组成的寡聚复合物。CaVα2δ可增加峰值电流密度并改善CaV1.2通道的电压依赖性激活门控，而不增加CaVα1亚基的表面表达。在人胚肾293细胞中重组表达完整的L型CaV1.2亚基后，研究了与较短复极QT间期（心电图上Q波与T波之间的时间间隔）相关的CACNA2D1（编码CaVα2δ的基因）基因变异的功能影响。通过并行进行高分辨率流式细胞术分析和全细胞膜片钳记录，我们发现CaVα2δ野生型蛋白的表面密度与峰值电流密度相关。此外，在相同条件下表达的CaVα2δ突变体S755T、Q917H和S956T的细胞表面密度与CaVα2δ野生型蛋白的细胞表面密度无显著差异。相比之下，CaVα2δ D550Y、CaVα2δ S709N和双突变体D550Y/Q917H的细胞表面表达分别降低，单突变体降低约30 - 33%，双突变体降低60%。D550Y/Q917H的细胞表面密度比蛋白质稳定性更显著受损，表明CaVα2δ的表面转运被双突变破坏。与CaVα2δ野生型相比，与D550Y/Q917H共表达显著降低了CaV1.2电流。结论是，D550Y/Q917H通过CaVα2δ细胞表面转运缺陷减少内向Ca(2+)电流。总之，我们的结果为CaVα2δ调节CaV1.2电流的分子机制提供了新的见解。
