Gonzalez-Gutierrez Giovanni, Miranda-Laferte Erick, Neely Alan, Hidalgo Patricia
Centro de Neurociencia de Valparaíso, Universidad de Valparaíso 2349400 Chile.
J Biol Chem. 2007 Jan 26;282(4):2156-62. doi: 10.1074/jbc.M609071200. Epub 2006 Nov 16.
High voltage-gated calcium channels enable calcium entry into cells in response to membrane depolarization. Association of the auxiliary beta-subunit to the alpha-interaction-domain in the pore-forming alpha1-subunit is required to form functional channels. The beta-subunit belongs to the membrane-associated guanylate kinase class of scaffolding proteins containing a Src homology 3 and a guanylate kinase domain. Although the latter is responsible for the high affinity binding to the alpha-interaction domain, the functional significance of the Src homology 3 domain remains elusive. Here, we show that injection of isolated beta-subunit Src homology 3 domain into Xenopus laevis oocytes expressing the alpha1-subunit reduces the number of channels in the plasma membrane. This effect is reverted by coexpressing alpha1 with a dominant-negative mutant of dynamin, a GTPase involved in receptor-mediated endocytosis. Full-length beta-subunit also down-regulates voltage-gated calcium channels but only when lacking the alpha-interaction domain. Moreover, isolated Src homology 3 domain and the full-length beta-subunit were found to interact in vitro with dynamin and to internalize the distantly related Shaker potassium channel. These results demonstrate that the beta-subunit regulates the turnover of voltage-gated calcium channels and other proteins in the cell membrane. This effect is mediated by dynamin and depends on the association state of the beta-subunit to the alpha1-pore-forming subunit. Our findings define a novel function for the beta-subunit through its Src homology 3 domain and establish a link between voltage-gated calcium channel activity and the cell endocytic machinery.
高压门控钙通道可使钙离子在细胞膜去极化时进入细胞。功能性通道的形成需要辅助β亚基与形成孔道的α1亚基中的α相互作用结构域相结合。β亚基属于膜相关鸟苷酸激酶类支架蛋白，包含一个Src同源3结构域和一个鸟苷酸激酶结构域。尽管后者负责与α相互作用结构域的高亲和力结合，但Src同源3结构域的功能意义仍不清楚。在此，我们表明，将分离的β亚基Src同源3结构域注射到表达α1亚基的非洲爪蟾卵母细胞中，会减少质膜中通道的数量。共表达α1与发动蛋白的显性负性突变体（一种参与受体介导内吞作用的GTP酶）可逆转这种效应。全长β亚基也会下调电压门控钙通道，但仅在缺乏α相互作用结构域时才会如此。此外，发现分离的Src同源3结构域和全长β亚基在体外与发动蛋白相互作用，并使远亲的Shaker钾通道内化。这些结果表明，β亚基调节细胞膜中电压门控钙通道和其他蛋白质的周转。这种效应由发动蛋白介导，并取决于β亚基与α1孔道形成亚基的结合状态。我们的发现通过其Src同源3结构域为β亚基定义了一种新功能，并在电压门控钙通道活性与细胞内吞机制之间建立了联系。
