Characterization of Cav1.4 Complexes (α1 1.4, β2, and α2δ4) in HEK293T Cells and in the Retina
Characterization of Cav1.4 Complexes (α1 1.4, β2, and α2δ4) in HEK293T Cells and in the Retina
复制标题
DOI：
10.1074/jbc.m114.607465
复制
发表时间：
2015-01-16
影响因子：
4.8
通讯作者：
Haeseleer, Francoise
中科院分区：
文献类型：
--
作者：
Lee, Amy;Wang, Shiyi;Haeseleer, Francoise
在光感受器突触终末，电压门控钙(V)1.4通道介导视觉刺激传递所需的钙信号。与其他高压激活的Ca-v通道一样，Ca(V)1.4通道由一个主要的成孔Ca(V)1.4α(1)亚基和辅助的β和α(2)β亚基组成。在四种不同类型的β和α(2)增量中，β(2)和α(2)增量(4)被认为与光感受器中的Ca(V)1.4α(1)亚单位共同组装。然而，对这种钙-V亚基组合的功能性质缺乏了解。在这里，我们提供了证据，Ca(V)1.4α(1)，β(2)和α(2)Delta(4)有助于视网膜中的Ca(V)1.4通道复合体，并描述了它们在电生理记录中的特性。此外，我们发现了一种β(2)的变体，这里称为beta(2x13)，它与beta(2a)一起存在于光感受器终端中。从HEK293细胞和小鼠视网膜的裂解物中免疫共沉淀出CA(V)1.4α(1)、β(2)和α(2)Delta(4)，并通过邻近连接实验发现它们相互作用于含有光感受器突触终末的视网膜外丛状层。在HEK293T细胞的全细胞膜片钳记录中，含有α(2)β(4)的通道(Ca(V)1.4α(1)+β(2x13))表现出比含有α(2)β(1)的通道更弱的电压依赖激活。此外，与含β(2a)的通道(Ca(V)1.4α(1)+α(2)β(4))相比，含β(2x13)的通道表现出更强的电压依赖性失活。后者是Ca(V)1.4所特有的，因为它在Ca(V)1.2通道上看不到。我们的结果首次提供了形成天然光感受器Ca(V)1.4通道的Ca(V)1.4亚基的详细功能分析，并表明这些通道中潜在的异质性是由beta(2a)和beta(2x13)变体所赋予的。
In photoreceptor synaptic terminals, voltage-gated Ca(v)1.4 channels mediate Ca2+ signals required for transmission of visual stimuli. Like other high voltage-activated Ca-v channels, Ca(v)1.4 channels are composed of a main pore-forming Ca(v)1.4 alpha(1) subunit and auxiliary beta and alpha(2)delta subunits. Of the four distinct classes of beta and alpha(2)delta, beta(2) and alpha(2)delta(4) are thought to co-assemble with Ca(v)1.4 alpha(1) subunits in photoreceptors. However, an understanding of the functional properties of this combination of Ca-v subunits is lacking. Here, we provide evidence that Ca(v)1.4 alpha(1), beta(2), and alpha(2)delta(4) contribute to Ca(v)1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of beta(2), named here beta(2X13), which, along with beta(2a), is present in photoreceptor terminals. Ca(v)1.4 alpha(1), beta(2), and alpha(2)delta(4) were coimmunoprecipitated from lysates of transfected HEK293 cells and mouse retina and were found to interact in the outer plexiform layer of the retina containing the photoreceptor synaptic terminals, by proximity ligation assays. In whole-cell patch clamp recordings of transfected HEK293T cells, channels (Ca(v)1.4 alpha(1) + beta(2X13)) containing alpha(2)delta(4) exhibited weaker voltage-dependent activation than those with alpha(2)delta(1). Moreover, compared with channels (Ca(v)1.4 alpha(1) + alpha(2)delta(4)) with beta(2a), beta(2X13)-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Ca(v)1.4 because it was not seen for Ca(v)1.2 channels. Our results provide the first detailed functional analysis of the Ca(v)1.4 subunits that form native photoreceptor Ca(v)1.4 channels and indicate potential heterogeneity in these channels conferred by beta(2a) and beta(2X13) variants.