LOCALIZATION OF A VOLTAGE GATE IN CONNEXIN46 GAP JUNCTION HEMICHANNELS

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. 72:1946-19 53). Oocytes expressing a mutant cx46 with a cysteine in position 35 e xhibited a membrane conductance sensitive to the thiol reagent maleimi dobutyryl biocytin (MBB). MBB irreversibly reduced the single-channel conductance by 80%. This reactive cysteine was used to probe the local ization of a voltage gate that closes cx46 gap junction hemichannels a t negative potentials. MBB was applied to the closed channel either fr om outside (whole cell) or from inside (excised membrane patches). Aft er 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 cystei ne in position 35 of cx46 is accessible from inside but not from the o utside while the channel is closed. These results suggest that the vol tage gate, which may be identical to the ''loop gate'' (Trexler et al. 1996. Proc. Natl. Acad. Sci USA. 93:5836-5841), is located extracellu lar to the 35 position. The voltage gate results in regional closure o f the pore rather than closure along the entire pore length.