Lee Amy, Wang Shiyi, Williams Brittany, Hagen Jussara, Scheetz Todd E, Haeseleer Françoise
From the Departments of Molecular Physiology and Biophysics, Otolaryngology Head-Neck Surgery, and Neurology, University of Iowa, Iowa City, Iowa 52242.
the Departments of Ophthalmology and Visual Sciences and Biomedical Engineering, University of Iowa, Iowa City, Iowa 52242, and.
J Biol Chem. 2015 Jan 16;290(3):1505-21. doi: 10.1074/jbc.M114.607465. Epub 2014 Dec 2.
In photoreceptor synaptic terminals, voltage-gated Cav1.4 channels mediate Ca(2+) signals required for transmission of visual stimuli. Like other high voltage-activated Cav channels, Cav1.4 channels are composed of a main pore-forming Cav1.4 α1 subunit and auxiliary β and α2δ subunits. Of the four distinct classes of β and α2δ, β2 and α2δ4 are thought to co-assemble with Cav1.4 α1 subunits in photoreceptors. However, an understanding of the functional properties of this combination of Cav subunits is lacking. Here, we provide evidence that Cav1.4 α1, β2, and α2δ4 contribute to Cav1.4 channel complexes in the retina and describe their properties in electrophysiological recordings. In addition, we identified a variant of β2, named here β2X13, which, along with β2a, is present in photoreceptor terminals. Cav1.4 α1, β2, and α2δ4 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 (Cav1.4 α1 + β2X13) containing α2δ4 exhibited weaker voltage-dependent activation than those with α2δ1. Moreover, compared with channels (Cav1.4 α1 + α2δ4) with β2a, β2X13-containing channels exhibited greater voltage-dependent inactivation. The latter effect was specific to Cav1.4 because it was not seen for Cav1.2 channels. Our results provide the first detailed functional analysis of the Cav1.4 subunits that form native photoreceptor Cav1.4 channels and indicate potential heterogeneity in these channels conferred by β2a and β2X13 variants.
在光感受器突触终末，电压门控性Cav1.4通道介导视觉刺激传递所需的Ca(2+)信号。与其他高电压激活的Cav通道一样，Cav1.4通道由一个主要的孔形成Cav1.4 α1亚基以及辅助性β和α2δ亚基组成。在四种不同类型的β和α2δ中，β2和α2δ4被认为在光感受器中与Cav1.4 α1亚基共同组装。然而，目前尚缺乏对这种Cav亚基组合功能特性的了解。在此，我们提供证据表明Cav1.4 α1、β2和α2δ4对视网膜中的Cav1.4通道复合物有贡献，并在电生理记录中描述了它们的特性。此外，我们鉴定出一种β2的变体，在此命名为β2X13，它与β2a一起存在于光感受器终末。通过邻近连接分析发现，Cav1.4 α1、β2和α2δ4从转染的HEK293细胞和小鼠视网膜的裂解物中共同免疫沉淀，并在含有光感受器突触终末的视网膜外网状层中相互作用。在转染的HEK293T细胞的全细胞膜片钳记录中，含有α2δ4的通道（Cav1.4 α1 + β2X13）表现出比含有α2δ1的通道更弱的电压依赖性激活。此外，与含有β2a的通道（Cav1.4 α1 + α2δ4）相比，含有β2X13的通道表现出更大的电压依赖性失活。后一种效应是Cav1.4特有的，因为在Cav1.2通道中未观察到。我们的结果首次对构成天然光感受器Cav1.4通道的Cav1.4亚基进行了详细的功能分析，并表明β2a和β2Xl3变体赋予了这些通道潜在的异质性。
