异聚体嗅觉环核苷酸门控离子通道的化学计量和排列
Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels.
作者信息
Shapiro M S, Zagotta W N
机构信息
Department of Physiology and Biophysics, Box 357290, University of Washington, Seattle, WA 98195-7290, USA.
出版信息
Proc Natl Acad Sci U S A. 1998 Nov 24;95(24):14546-51. doi: 10.1073/pnas.95.24.14546.
Native cylic nucleotide-gated (CNG) channels are composed of alpha and beta subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the alpha and beta subunits. Dimers contained three subunit types: alphawt, betawt, and alpham. The alpham subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of alphawtalphawt dimers and from alphawt monomers were indistinguishable. Channels from injection of alphawtalpham dimers had apparent affinities 3-fold lower than alphawt homomultimers, suggesting a channel with two alphawt and two alpham subunits. Channels from coinjection of alphawtalphawt and betabeta dimers were indistinguishable from those composed of alpha and beta monomers and shared all of the characteristics of the alpha+beta phenotype of heteromeric channels. Coinjection of alphawtalpham and beta beta dimers yielded channels also of the alpha+beta phenotype but with an apparent affinity 3-fold lower, indicating the presence of alpham in the tetramer and that alpha+beta channels have adjacent alpha-subunits. To distinguish between an alpha-alpha-alpha-beta and an alpha-alpha-beta-beta arrangement, we compared apparent affinities for channels from coinjection of alphawtalphawt and betaalphawt or alphawtalphawt and betaalpham dimers. These channels were indistinguishable. To further argue against an alpha-alpha-alpha-beta arrangement, we quantitatively compared dose-response data for channels from coinjection of alphawtalpham and beta beta dimers to those from alpha and beta monomers. Taken together, our results are most consistent with an alpha-alpha-beta-beta arrangement for the heteromeric olfactory CNG channel.
天然环核苷酸门控（CNG）通道由α和β亚基组成。嗅觉CNG通道由大鼠cDNA克隆在非洲爪蟾卵母细胞中表达，并在内外翻膜片上进行研究。我们使用由连接亚基组成的串联二聚体，研究了α和β亚基的化学计量和排列。二聚体包含三种亚基类型：αwt、βwt和αm。αm亚基缺乏对门控有很大影响的氨基末端结构域，使通道对配体的表观亲和力降低9倍，使其成为包含在四聚体中的报告分子。注射αwtαwt二聚体和αwt单体产生的同源通道无法区分。注射αwtαm二聚体产生的通道表观亲和力比αwt同源多聚体低3倍，表明通道有两个αwt和两个αm亚基。共注射αwtαwt和ββ二聚体产生的通道与由α和β单体组成的通道无法区分，并具有异源通道α+β表型的所有特征。共注射αwtαm和ββ二聚体产生的通道也是α+β表型，但表观亲和力低3倍，表明四聚体中存在αm，且α+β通道有相邻的α亚基。为了区分α-α-α-β和α-α-β-β排列，我们比较了共注射αwtαwt和βαwt或αwtαwt和βαm二聚体产生的通道的表观亲和力。这些通道无法区分。为了进一步反对α-α-α-β排列，我们定量比较了共注射αwtαm和ββ二聚体产生的通道与α和β单体产生的通道的剂量反应数据。综合来看，我们的结果与异源嗅觉CNG通道的α-α-β-β排列最为一致。
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