Negatively charged residues in the first extracellular loop of the L-type CaV1.2 channel anchor the interaction with the CaVα2δ1 auxiliary subunit
Negatively charged residues in the first extracellular loop of the L-type CaV1.2 channel anchor the interaction with the CaVα2δ1 auxiliary subunit
复制标题
DOI：
10.1074/jbc.m117.806893
复制
发表时间：
2017-10-20
影响因子：
4.8
通讯作者：
Parent, Lucie
中科院分区：
文献类型：
--
作者：
Bourdin, Benoite;Briot, Julie;Parent, Lucie
心肌细胞中电压门控L型Ca（V）1.2通道以异聚体复合物的形式存在。Ca-V α 2 δ 1与Ca-V β/Ca-V α 1蛋白的共表达重建了天然L型电流的功能特性，但Ca（V）1.2/ Ca-V α 2 δ 1界面处的相互作用结构域尚不清楚。在此，Ca（V）1.2的基于同源性的模型鉴定了在重复序列I（IS 1 S2和IS 5S 6）、II（IS 5S 6）和III（IIIS 5S 6）中Ca-V α 2 δ 1的细胞外结构域与Ca-V α 1蛋白的细胞外环之间的蛋白质界面。在IS 1 S2中插入9个残基的血凝素表位，但在IS 5S 6或IS 5S 6中不插入，阻止了CaV1.2与Ca-V α 2 δ 1的免疫共沉淀。IS 1 S2含有一簇三个保守的带负电荷的残基Glu-179、Asp-180和Asp-181，这些残基可能有助于与Ca-V α 2 δ 1的非键合相互作用。CaV1.2 Asp-181的取代损害了Ca-V β/Ca（V）1.2与Ca-V α 2 δ 1的免疫共沉淀以及电压依赖性激活门控中的Ca-V α 2 δ 1依赖性移位。相比之下，在同一环中相邻位置的Ca（V）1.2中的单个取代（179、180和182-184）没有显著改变Ca（V）1.2全细胞电流的功能性上调。然而，在位置180处的带负电荷的残基对于在激活门控中传递Ca-V α 2 δ 1介导的移位是必需的。我们还发现了一个更温和的贡献，从带正电荷的Arg-1119在细胞外孔区的重复III的Ca（V）1.2。我们的结论是，Ca（V）1.2 Asp-181锚定的物理相互作用，促进Ca-V α 2 δ 1介导的Ca（V）1.2电流的功能调节。通过稳定Ca（V）1.2的第一细胞外环，Ca-V α 2 δ 1可以通过促进与通道的开放状态相关的电压传感器的构象来上调电流。
Voltage-gated L-type Ca(V)1.2 channels in cardiomyocytes exist as heteromeric complexes. Co-expression of Ca-V alpha 2 delta 1 with Ca-V beta/Ca-V alpha 1 proteins reconstitutes the functional properties of native L-type currents, but the interacting domains at the Ca(V)1.2/ Ca-V alpha 2 delta 1 interface are unknown. Here, a homology- based model of Ca(V)1.2 identified protein interfaces between the extracellular domain of Ca-V alpha 2 delta 1 and the extracellular loops of the Ca-V alpha 1 protein in repeats I (IS1S2 and IS5S6), II (IIS5S6), and III (IIIS5S6). Insertion of a 9-residue hemagglutinin epitope in IS1S2, but not in IS5S6 or in IIS5S6, prevented the co-immunoprecipitation of CaV1.2 with Ca-V alpha 2 delta 1. IS1S2 contains a cluster of three conserved negatively charged residues Glu-179, Asp-180, and Asp-181 that could contribute to non-bonded interactions with Ca-V alpha 2 delta 1. Substitutions of CaV1.2 Asp-181 impaired the co-immunoprecipitation of Ca-V beta/Ca(V)1.2 with Ca-V alpha 2 delta 1 and the Ca-V alpha 2 delta 1-dependent shift in voltage-dependent activation gating. In contrast, single substitutions in Ca(V)1.2 in neighboring positions in the same loop (179, 180, and 182-184) did not significantly alter the functional up-regulation of Ca(V)1.2 whole-cell currents. However, a negatively charged residue at position 180 was necessary to convey the Ca-V alpha 2 delta 1-mediated shift in the activation gating. We also found a more modest contribution from the positively charged Arg-1119 in the extracellular pore region in repeat III of Ca(V)1.2. We conclude that Ca(V)1.2 Asp-181 anchors the physical interaction that facilitates the Ca-V alpha 2 delta 1-mediated functional modulation of Ca(V)1.2 currents. By stabilizing the first extracellular loop of Ca(V)1.2, Ca-V alpha 2 delta 1 may up-regulate currents by promoting conformations of the voltage sensor that are associated with the channel's open state.