The α1-β-subunit interaction that modulates calcium channel activity is reversible and requires a competent α-interaction domain
The α1-β-subunit interaction that modulates calcium channel activity is reversible and requires a competent α-interaction domain
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DOI：
10.1074/jbc.m605930200
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发表时间：
2006-08-25
影响因子：
4.8
通讯作者：
Neely, Alan
中科院分区：
文献类型：
--
作者：
Hidalgo, Patricia;Gonzalez-Gutierrez, Giovanni;Neely, Alan
高压门控钙通道由一个成孔亚基（α(1)）和三个非同源亚基（α (2)/ δ、β和γ）组成。虽然β -亚基促进了通往质膜的通道的交通并改变了它们的活性，但与α(1)-亚基相互作用的可逆性仍然存在争议。在这里，我们通过检测纯化β (2)a蛋白对爪蟾卵母细胞中表达的Ca(V)1.2和Ca(V)2.3通道的影响来解决这个问题。β (2a)-亚基在体外结合α(1)-相互作用域（AID），当注射到卵母细胞中时，它改变了激活的电压依赖性，并增加了电荷运动到CaV1.2通道的离子电流偶联。这种增加依赖于AID的完整性，但并没有被巴非霉素消除，这表明通过AID位点的α (1)- β相互作用可以在质膜上发生。此外，注射β (2a)蛋白可抑制CaV2.3通道的失活，并将快速失活的Ca(V)2.3/ β (1b)通道转化为缓慢失活的通道。在表达Ca(V)2.3/beta（1b）通道的卵母细胞中，抑制失活需要比单独表达CaV2.3更高浓度的β (2a)，但与通过单一结合位点竞争相互作用时达到的最大水平相同。总之，我们的数据表明，α (1)- β相互作用在完整细胞中是可逆的，并将钙通道β亚基定义为调节蛋白，而不是化学计量亚基。
High voltage-gated calcium channels consist of a pore-forming subunit (alpha(1)) and three nonhomologous subunits (alpha(2)/delta,beta, and gamma). Although it is well established that the beta-subunit promotes traffic of channels to the plasma membrane and modifies their activity, the reversible nature of the interaction with the alpha(1)-subunit remains controversial. Here, we address this issue by examining the effect of purified beta(2)a protein on Ca(V)1.2 and Ca(V)2.3 channels expressed in Xenopus oocytes. The beta(2a)-subunit binds to the alpha(1)-interaction domain ( AID) in vitro, and when injected into oocytes, it shifts the voltage dependence of activation and increases charge movement to ionic current coupling of CaV1.2 channels. This increase depended on the integrity of AID but was not abolished by bafilomycin, demonstrating that the alpha(1)-beta interaction through the AID site can take place at the plasma membrane. Furthermore, injection of beta(2a) protein inhibited inactivation of CaV2.3 channels and converted fast inactivating Ca(V)2.3/beta(1b) channels to slow inactivating channels. Inhibition of inactivation required larger concentration of beta(2a) in oocytes expressing Ca(V)2.3/beta(1b) channels than expressing CaV2.3 alone but reached the same maximal level as expected for a competitive interaction through a single binding site. Together, our data show that the alpha(1)-beta interaction is reversible in intact cells and defines calcium channels beta-subunits as regulatory proteins rather than stoichiometric subunits.