Stoichiometry of transjunctional voltage-gating polarity reversal by a negative charge substitution in the amino terminus of a Connexin32 chimera

Gap junctions are intercellular channels formed by the serial, head to head arrangement of two hemichannels. Each hemichannel is an oligomer of six pr otein subunits, which in vertebrates are encoded by the connexin gene famil y. All intercellular channels formed by connexins are sensitive to the rela tive difference in the membrane potential between coupled cells, the transj unctional voltage (V-j), and gate by the separate action of their component hemichannels (Harris, A.L,, D.C. Spray, and M.V. Bennett. 1981. J. Gen. Ph ysiol. 77:95-117). We reported previously that the polarity of V-j dependen ce is opposite for hemichannels formed by two closely related connexins, Cx 32 and Cx26, when they are paired to form intercellular channels (Verselis, V.K., C.S. Ginter, and T.A. Bargiello. 1994. Nature. 368:348-351). The opp osite gating polarity is due to a difference in the charge of the second am ino acid. Negative charge substitutions of the neutral asparagine residue p resent in wild-type Cx32 (Cx32N2E or Cx32N2D) reverse the gating polarity o f Cx32 hemichannels from closure at negative V-j to closure at positive V-j . In this paper; we further examine the mechanism of polarity reversal by d etermining the gating polarity of a chimeric connexin, in which the first e xtracellular loop (E1) of Cx32 is replaced with that of Cx43 (Cx43E1). The resulting chimera, Cx32*Cx43E1, forms conductive hemichannels when expresse d in single Xenopus oocytes and intercellular channels in pairs of oocytes (Pfahnl, A., X.W. Zhou, R. Werner, and G. Dahl. 1997. Pflugers Arch. 433:73 3-779). We demonstrate that the polarity of V-j dependence of Cx32*Cx43E1 h emichannels in intercellular pairings is the same as that of wild-type Cx32 hemichannels and is reversed by the N2E substitution. In records of single intercellular channels, V-j dependence is characterized by gating transiti ons between fully open and subconductance levels. Comparable transitions ar e observed in Cx32*Cx43E1 conductive hemichannels at negative membrane pote ntials and the polarity of these transitions is reversed by the N2E substit ution. We conclude that the mechanism of V-j dependence of intercellular ch annels is conserved in conductive hemichannels and term the process V-j gat ing. Heteromeric conductive hemichannels comprised of Cx32*Cx43E1 and Cx32N 2E*Cx43E1 subunits display bipolar V-j gating, closing to substates at both positive and negative membrane potentials. The number of bipolar hemichann els observed in cells expressing mixtures of the two connexin subunits coin cides with the number of hemichannels that are expected to contain a single oppositely charged subunit. We conclude that the movement of the voltage s ensor in a single connexin subunit is sufficient to initiate V-j gating. We further suggest that V-j gating results from conformational changes in ind ividual connexin subunits rather than by a concerted change in the conforma tion of all six subunits.