Chick connexin-56, a novel lens gap junction protein. Molecular cloning and functional expression

Abstract

We used primers corresponding to the amino-terminal sequence shared by rat connexin-46 and ovine MP70 and a consensus sequence of the second extracellular loop conserved in all connexins to amplify and subsequently clone from chick genomic DNA a new member of the connexin family of gap junction proteins, chick connexin-56. The derived chick connexin-56 polypeptide contains 510 amino acids with a predicted molecular mass of 55,857 daltons. Although identical in the first 70 amino acids to rat connexin-46, chick connexin-56 diverges significantly in length and composition in predicted cytoplasmic regions, which have previously been inferred to determine functional and regulatory specificity. We were able to detect hybridization of connexin-56 probes only to RNA derived from lens. Connexin-56 was functionally expressed by the stable transfection of communication-deficient Neuro2A cells. The connexin-56-transfected cells demonstrated intercellular coupling by transfer of microinjected 6-carboxyfluorescein. Double whole-cell patch clamp recordings demonstrated electrical coupling. The induced intercellular conductances were insensitive to uncoupling by heptanol, octanol, or acidification. This behavior of chick connexin-56 may explain previous observations of the unusual physiology of lens fiber gap junctions.