A 17MER PEPTIDE INTERFERES WITH ACIDIFICATION-INDUCED UNCOUPLING OF CONNEXIN43

Structure/function analysis shows that the carboxyl terminal (CT) doma in of connexin43 (Cx43) is essential for the chemical regulation of ce ll-cell communication. Of particular interest is the region between am ino acids 260 and 300. Structural preservation of this region is essen tial for acidification-induced uncoupling (ie, pH gating). In this stu dy, we report data showing that a 17mer peptide of the same sequence a s amino acids 271 to 287 of Cx43 (CSSPTAPLSPMSPPGYK) can prevent pH ga ting of Cx43-expressing oocytes. Experiments were carried out in pairs of Xenopus oocytes previously injected with connexin38 antisense and expressing wild-type Cx43, Junctional conductance was measured electro physiologically. pH(i) was determined from the light emission of the p roton-sensitive dye dextran-seminaphtho-rhodafluor. Intracellular acid ification was induced by superfusion with a bicarbonate-buffered solut ion gassed with a progressively increasing concentration of CO2. Injec tion of water alone into both oocytes of a Cx43-expressing pair or inj ection of a peptide from region 321 to 337 of Cx43 did not modify pH s ensitivity, However, injection of a polypeptide corresponding to amino acids 241 to 382 of Cx43 interfered with the ability of gap junctions to close on acidification. Similar results were obtained when a 17mer peptide (region 271 to 287) was injected into both oocytes of the pai r. Normal Cx43 pH gating was observed if (1) the amino acid sequence o f the 17mer peptide was scrambled or (2) the N and the C ends of the 1 7mer peptide were not included in the sequence. This is the first demo nstration of a molecule that can interfere with the chemical regulatio n of connexin channels in a cell pair. The data may lead to the develo pment of small molecules that can be used in Cx43-expressing multicell ular preparations to study the role of gap junction regulation in norm al as well as diseased states.