The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels
The metal-ion-dependent adhesion site in the Von Willebrand factor-A domain of α2δ subunits is key to trafficking voltage-gated Ca2+ channels
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DOI：
10.1073/pnas.0504183102
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发表时间：
2005-08-09
影响因子：
11.1
通讯作者：
Dolphin, AC
中科院分区：
文献类型：
--
作者：
Cantí, C;Nieto-Rostro, M;Dolphin, AC
电压门控钙通道的所有辅助性α2β亚基都含有一个胞外Von Willebrand因子-A(VWA)结构域，在α(2)β-1和-2中有一个完美的金属离子依赖的黏附位点(MIDAS)。对a(2)Delta-2VWA结构域的模拟表明，它极有可能结合二价阳离子。突变负责二价阳离子结合的三个关键MIDAS残基导致MIDAS突变体α(2)Delta-2亚基在单独表达时仍能正常加工和运输。然而，与WTα(2)Delta-2不同的是，Midas突变体a(2)Delta-2亚基没有增强，在某些情况下，通过使用Ba2+或Na+作为离子，进一步减弱了与β1b共表达的Cav1.2、-2.1和-2.2电流。此外，MIDAS突变体α(2)Delta-2的表达降低了细胞表面的表达，并在Cos-7和NG108-15细胞中观察到表达的最早时间显著增加了Caval亚基的核周滞留。尽管存在内源性α(2)Delta亚基，但在分化的NG108-15细胞中异源表达的α(2)Delta-2进一步增强了内源性高阈值钙电流，而这种增强被MIDAS突变所阻止。我们的结果表明，在功能质膜通道出现之前，α(2)Delta亚基通常在成熟早期与Caval亚基相互作用，并且α(2)Delta亚基中完整的MIDAS基序需要通过类整合素开关促进α1亚单位向质膜的运输。这一发现为VWA结构域在多聚体复合体的细胞内运输中的主要作用提供了证据，而不是在其他表面VWA结构域中结合细胞外配体的更常见的作用。
All auxiliary alpha 2 delta subunits of voltage-gated Ca2+ (Cav) channels contain an extracellular Von Willebrand factor-A (VWA) domain that, in alpha(2)delta-1 and -2, has a perfect metal-ion-dependent adhesion site (MIDAS). Modeling of the a(2)delta-2 VWA domain shows it to be highly likely to bind a divalent cation. Mutating the three key MIDAS residues responsible for divalent cation binding resulted in a MIDAS mutant alpha(2)delta-2 subunit that was still processed and trafficked normally when it was expressed alone. However, unlike WT alpha(2)delta-2, the MIDAS mutant a(2)delta-2 subunit did not enhance and, in some cases, further diminished Cav1.2, -2.1, and -2.2 currents coexpressed with beta 1b by using either Ba2+ or Na+ as a permeant ion. Furthermore, expression of the MIDAS mutant alpha(2)delta-2 reduced surface expression and strongly increased the perinuclear retention of Caval subunits at the earliest time at which expression was observed in both Cos-7 and NG108-15 cells. Despite the presence of endogenous alpha(2)delta subunits, heterologous expression of alpha(2)delta-2 in differentiated NG108-15 cells further enhanced the endogenous high-threshold Ca2+ currents, whereas this enhancement was prevented by the MIDAS mutations. Our results indicate that alpha(2)delta subunits normally interact with the Caval subunit early in their maturation, before the appearance of functional plasma membrane channels, and an intact MIDAS motif in the alpha(2)delta subunit is required to promote trafficking of the alpha 1 subunit to the plasma membrane by an integrin-like switch. This finding provides evidence for a primary role of a VWA domain in intracellular trafficking of a multimeric complex, in contrast to the more usual roles in binding extracellular ligands in other exofacial VWA domains.