Tang Qingxiu, Dowd Terry L, Verselis Vytas K, Bargiello Thaddeus A
Dominic P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
J Gen Physiol. 2009 Jun;133(6):555-70. doi: 10.1085/jgp.200910207.
The structure of the pore is critical to understanding the molecular mechanisms underlying selective permeation and voltage-dependent gating of channels formed by the connexin gene family. Here, we describe a portion of the pore structure of unapposed hemichannels formed by a Cx32 chimera, Cx32Cx43E1, in which the first extracellular loop (E1) of Cx32 is replaced with the E1 of Cx43. Cysteine substitutions of two residues, V38 and G45, located in the vicinity of the border of the first transmembrane (TM) domain (TM1) and E1 are shown to react with the thiol modification reagent, MTSEA-biotin-X, when the channel resides in the open state. Cysteine substitutions of flanking residues A40 and A43 do not react with MTSEA-biotin-X when the channel resides in the open state, but they react with dibromobimane when the unapposed hemichannels are closed by the voltage-dependent "loop-gating" mechanism. Cysteine substitutions of residues V37 and A39 do not appear to be modified in either state. Furthermore, we demonstrate that A43C channels form a high affinity Cd2+ site that locks the channel in the loop-gated closed state. Biochemical assays demonstrate that A43C can also form disulfide bonds when oocytes are cultured under conditions that favor channel closure. A40C channels are also sensitive to micromolar Cd2+ concentrations when closed by loop gating, but with substantially lower affinity than A43C. We propose that the voltage-dependent loop-gating mechanism for Cx32Cx43E1 unapposed hemichannels involves a conformational change in the TM1/E1 region that involves a rotation of TM1 and an inward tilt of either each of the six connexin subunits or TM1 domains.
孔道结构对于理解连接蛋白基因家族形成的通道选择性通透和电压依赖性门控背后的分子机制至关重要。在此，我们描述了由Cx32嵌合体Cx32Cx43E1形成的未对接半通道的部分孔道结构，其中Cx32的第一个细胞外环（E1）被Cx43的E1所取代。当通道处于开放状态时，位于第一个跨膜（TM）结构域（TM1）和E1边界附近的两个残基V38和G45的半胱氨酸取代显示与硫醇修饰试剂MTSEA-生物素-X发生反应。当通道处于开放状态时，侧翼残基A40和A43的半胱氨酸取代不与MTSEA-生物素-X反应，但当未对接的半通道通过电压依赖性“环门控”机制关闭时，它们与二溴双马来酰亚胺反应。残基V37和A39的半胱氨酸取代在两种状态下似乎都未被修饰。此外，我们证明A43C通道形成一个高亲和力的Cd2+位点，该位点将通道锁定在环门控关闭状态。生化分析表明，当卵母细胞在有利于通道关闭的条件下培养时，A43C也能形成二硫键。当通过环门控关闭时，A40C通道对微摩尔浓度的Cd2+也敏感，但亲和力远低于A43C。我们提出，Cx32Cx43E1未对接半通道的电压依赖性环门控机制涉及TM1/E1区域的构象变化，该变化涉及TM1的旋转以及六个连接蛋白亚基或TM1结构域各自的向内倾斜。
