Pfahnl A, Dahl G
Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101, USA.
Biophys J. 1998 Nov;75(5):2323-31. doi: 10.1016/S0006-3495(98)77676-3.
Cysteine replacement mutagenesis has identified positions in the first transmembrane domain of connexins as contributors to the pore lining of gap junction hemichannels (Zhou et al. 1997. Biophys. J. 72:1946-1953). Oocytes expressing a mutant cx46 with a cysteine in position 35 exhibited a membrane conductance sensitive to the thiol reagent maleimidobutyryl biocytin (MBB). MBB irreversibly reduced the single-channel conductance by 80%. This reactive cysteine was used to probe the localization of a voltage gate that closes cx46 gap junction hemichannels at negative potentials. MBB was applied to the closed channel either from outside (whole cell) or from inside (excised membrane patches). After washout of the thiol reagent the channels were tested at potentials at which the channels open. After extracellular application of MBB to intact oocytes, the membrane conductance was unaffected. In contrast, channels treated with intracellular MBB were blocked. Thus the cysteine in position 35 of cx46 is accessible from inside but not from the outside while the channel is closed. These results suggest that the voltage gate, which may be identical to the "loop gate" (Trexler et al. 1996. Proc. Natl. Acad. Sci. USA. 93:5836-5841), is located extracellular to the 35 position. The voltage gate results in regional closure of the pore rather than closure along the entire pore length.
半胱氨酸置换诱变已确定连接蛋白第一个跨膜结构域中的一些位置是间隙连接半通道孔衬里的组成部分（Zhou等人，1997年。《生物物理学杂志》72:1946 - 1953）。表达在第35位带有半胱氨酸的突变型cx46的卵母细胞表现出对硫醇试剂马来酰亚胺基丁酰生物胞素（MBB）敏感的膜电导。MBB使单通道电导不可逆地降低了80%。这个具有反应性的半胱氨酸被用于探测在负电位下关闭cx46间隙连接半通道的电压门的定位。MBB从外部（全细胞）或内部（切除的膜片）施加到关闭的通道上。在洗去硫醇试剂后，在通道开放的电位下对通道进行测试。将MBB细胞外施加到完整的卵母细胞后，膜电导未受影响。相反，用细胞内MBB处理的通道被阻断。因此，在通道关闭时，cx46第35位的半胱氨酸可从内部而非外部接触到。这些结果表明，电压门可能与“环门”相同（Trexler等人，1996年。《美国国家科学院院刊》93:5836 - 5841），位于第35位的细胞外。电压门导致孔的局部关闭而非沿整个孔长度的关闭。
