IDENTIFICATION OF A PORE LINING SEGMENT IN GAP JUNCTION HEMICHANNELS

The ability of certain connexins to form open hemichannels has been ex ploited to study the pore structure of gap junction (hemi)channels. Cy steine scanning mutagenesis was applied to cx46 and to a chimeric conn exin, cx32E(1)43, which both form patent hemichannels when expressed i n Xenopus oocytes. The thiol reagent maleimido-butyryl-biocytin was us ed to probe 12 cysteine replacement mutants in the first transmembrane segment and two in the amino-terminal segment. Maleimido-butyryl-bioc ytin was found to inhibit channel activity with cysteines in two equiv alent positions in both connexins: I33C and M34C in cx32E(1)43 and I34 C and L35C in cx46. These two positions in the first transmembrane seg ment are thus accessible from the extracellular space and consequently appear to contribute to the pore lining. The data also suggest that t he pore structure is complex and may involve more than one transmembra ne segment.