Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
Proc Natl Acad Sci U S A. 2010 Aug 17;107(33):14887-92. doi: 10.1073/pnas.1007543107. Epub 2010 Aug 2.
The Rem, Rem2, Rad, and Gem/Kir (RGK) family of small GTP-binding proteins potently inhibits high voltage-activated (HVA) Ca(2+) channels, providing a powerful means of modulating neural, endocrine, and muscle functions. The molecular mechanisms of this inhibition are controversial and remain largely unclear. RGK proteins associate directly with Ca(2+) channel beta subunits (Ca(v)beta), and this interaction is widely thought to be essential for their inhibitory action. In this study, we investigate the molecular underpinnings of Gem inhibition of P/Q-type Ca(2+) channels. We find that a purified Gem protein markedly and acutely suppresses P/Q channel activity in inside-out membrane patches, that this action requires Ca(v)beta but not the Gem/Ca(v)beta interaction, and that Gem coimmunoprecipitates with the P/Q channel alpha(1) subunit (Ca(v)alpha(1)) in a Ca(v)beta-independent manner. By constructing chimeras between P/Q channels and Gem-insensitive low voltage-activated T-type channels, we identify a region encompassing transmembrane segments S1, S2, and S3 in the second homologous repeat of Ca(v)alpha(1) critical for Gem inhibition. Exchanging this region between P/Q and T channel Ca(v)alpha(1) abolishes Gem inhibition of P/Q channels and confers Ca(v)beta-dependent Gem inhibition to a chimeric T channel that also carries the P/Q I-II loop (a cytoplasmic region of Ca(v)alpha(1) that binds Ca(v)beta). Our results challenge the prevailing view regarding the role of Ca(v)beta in RGK inhibition of high voltage-activated Ca(2+) channels and prompt a paradigm in which Gem directly binds and inhibits Ca(v)beta-primed Ca(v)alpha(1) on the plasma membrane.
Rem、Rem2、Rad 和 Gem/Kir（RGK）家族的小分子 GTP 结合蛋白强烈抑制高电压激活（HVA）Ca(2+)通道，为调节神经、内分泌和肌肉功能提供了有力手段。这种抑制的分子机制存在争议，在很大程度上仍不清楚。RGK 蛋白直接与 Ca(2+)通道β亚基（Ca(v)β）结合，这种相互作用被广泛认为是其抑制作用所必需的。在这项研究中，我们研究了 Gem 抑制 P/Q 型 Ca(2+)通道的分子基础。我们发现，纯化的 Gem 蛋白在膜内向外膜片中显著且急性地抑制 P/Q 通道活性，这种作用需要 Ca(v)β，但不需要 Gem/Ca(v)β相互作用，并且 Gem 以 Ca(v)β独立的方式与 P/Q 通道α(1)亚基（Ca(v)α(1)）共免疫沉淀。通过构建 P/Q 通道和 Gem 不敏感的低电压激活 T 型通道之间的嵌合体，我们确定了一个包含 Ca(v)α(1)第二同源重复中的跨膜片段 S1、S2 和 S3 的区域，对于 Gem 抑制至关重要。在 P/Q 和 T 通道 Ca(v)α(1)之间交换该区域，可消除 Gem 对 P/Q 通道的抑制，并使携带 P/Q I-II 环（Ca(v)α(1)的细胞质区域，与 Ca(v)β结合）的嵌合 T 通道获得 Ca(v)β依赖性的 Gem 抑制。我们的结果挑战了关于 Ca(v)β在 RGK 抑制高电压激活 Ca(2+)通道中的作用的主流观点，并提示 Gem 直接结合并抑制质膜上 Ca(v)β-引发的 Ca(v)α(1)的范例。
