Toloue M M, Woolwine Y, Karcz J A, Kasperek E M, Nicholson B J, Skerrett I M
Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA.
Cell Commun Adhes. 2008 May;15(1):95-105. doi: 10.1080/15419060802013463.
Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the alpha-class (Cx50) and one from the beta-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.
通过半胱氨酸扫描诱变，作者比较了两种连接蛋白跨膜结构域内的位点，一种来自α类（Cx50），另一种来自β类（Cx32），氨基酸替代会破坏间隙连接通道的功能。在Cx32中，11个位点导致通道无功能，或出现一种异常的电压门控表型，称为“反向门控”，而在Cx50中，鉴定出7个这样的位点。在这两种连接蛋白中，这些位点都位于跨膜螺旋的特定面上，表明这些可能是跨膜结构域相互作用的位点。在Cx32中，鉴定出M1跨膜结构域的一个宽面和M3的一个较窄的极性面，包括一个在关闭状态下显示与M4紧密相邻的位点。在Cx50中，鉴定出M3的同一面，但M1中的位点与Cx32不同。在Cx50中，M1中破坏通道功能的位点要少得多，而且那些位点位于与Cx32中敏感位点不同的螺旋面上。对Cx32的M1和M2中的两个位点进行的更深入研究表明，侧链长度或分支对维持正常通道行为很重要，这与该位点是跨膜结构域相互作用的位点一致。
