Effects of In Vivo expression of gap junction mutant Hve- innexin1PL on neural coupling & coexpression of other innexins

Gap junctions (GJs) are membrane channels that form between apposed cells and mediate electrical communication as well as the transfer of small molecules. GJ hemichannels are hexamers comprised of subunits called innexins (Inxs) in invertebrates and pannexins (Pnxs) or connexins (Cxs) in vertebrates. In addition to some of their shared structural motifs, Inxs, Pnxs, and Cxs also possess a highly conserved Proline residue in their second transmembrane domain (TM2). Studies with Cxs describe this Proline as introducing a kink into the protein's three- dimensional structure, which has been implicated in mediating conformational changes in the hemichannel in response to voltage. To study the role of the Proline residue in TM2 of Inxs in the developing CNS of the leech Hirudo verbana, it was mutated to a Leucine in pan- neuronally expressed Hve-inx1 (Hve-inx1PL). Through a series of experiments involving the expression of this mutant inx1PL¹ construct in developing neurons of the intact embryo, it became evident that mutant expression conferred a dominant negative effect on INX1² GJs as witnessed by the loss of GJ plaques in neuronal arbors and the decoupling of expressing neurons from their normal cellular networks. Furthermore, coexpression studies using this mutant inx1PL along with wild-type (WT) inx1 and other leech Inxs has revealed that normal punctal expression of WT Inx transgenes could also be abolished for some pairs of transgenes (inx1PL with WT inx1 or inx14), but not for others (inx1PL with inx6). Such results suggest that the dominant negative effects of INX1PL expression can be useful for not only studying INX1 function in the CNS, but also for revealing heteromeric composition of hemichannels in individual neurons