No abstract

Gap junctions allow direct intercellular coupling between many cells including those in the blood vessel wall. They are formed by a group of related proteins called connexins, containing conserved transmembrane and extracellular domains, but unique cytoplasmic regions that may confer connexin-specific physiological properties. We used polymerase chain reaction amplification and cDNA library screening to clone DNA encoding a human gap junction protein, connexin37 (Cx37). The derived human Cx37 polypeptide contains 333 amino acids, with a predicted molecular mass of 37,238 D. RNA blots demonstrate that Cx37 is expressed in multiple organs and tissues (including heart, uterus, ovary, and blood vessel endothelium) and in primary cultures of vascular endothelial cells. Cx37 mRNA is coexpressed with connexin43 at similar levels in some endothelial cells, but at much lower levels in others. To demonstrate that Cx37 could form functional channels, we stably transfected communication-deficient Neuro2A cells with the Cx37 cDNA. The induced intercellular channels were studied by the double whole cell patch clamp technique. These channels were reversibly inhibited by the uncoupling agent, heptanol (2 mM). The expressed Cx37 channels exhibited multiple conductance levels and showed a pronounced voltage dependence. These electrophysiological characteristics are similar to, but distinct from, those of previously characterized connexins. (J. Clin. Invest. 1993. 91:997-1004

NS5A derived from a hepatitis C virus (HCV) genotype 1b isolate has previously been shown to undergo phosphorylation on serine residues (T.

Phosphorylation of the expressed NS5A protein of hepatitis C virus (HCV), a member of the Hepacivirus genus of the familyFlaviviridae, has been demonstrated in mammalian cells and in a cell-free assay by an associated kinase activity. In this report, phosphorylation is also shown for the NS5A and NS5 proteins, respectively, of bovine viral diarrhea virus (BVDV) and yellow fever virus (YF), members of the other two established genera in this family. Phosphorylation of BVDV NS5A and YF NS5 was observed in infected cells, transient expression experiments, and a cell-free assay similar to the one developed for HCV NS5A. Phosphoamino acid analyses indicated that all three proteins were phosphorylated by serine/threonine kinases. Similarities in the properties of BVDV NS5A, YF NS5, and HCV NS5A phosphorylation in vitro further suggested that closely related kinases or the same kinase may phosphorylate these viral proteins. Conservation of this trait among three quite distantly related viruses representing three separate genera suggests that phosphorylation of the NS5A/NS5 proteins or their association with cellular kinases may play an important role in the flavivirus life cycle.

Gap junctions contain intercellular channels which are formed by members of a group of related proteins called connexins. Connexins contain conserved transmembrane and extracellular domains, but unique cytoplasmic regions which may provide connexin-specific physiologic properties. We used polymerase chain reaction (PCR) amplification and cDNA library screening to clone DNA encoding a novel member of this gene family, rat connexin40 (Cx40). The derived rat Cx40 polypeptide contains 356 amino acids, with a predicted molecular mass of 40,233 Da. Sequence comparisons suggest that Cx40 is the mammalian homologue of chick connexin42, but it has predicted cytoplasmic regions that differ from previously described mammalian connexins. Southern blots of rat genomic DNA suggest that Cx40 is encoded by a single copy gene containing no introns within its coding region. Northern blots demonstrate that Cx40 is expressed in multiple tissues (including lung, heart, uterus, ovary, and blood vessels) and in primary cultures and established lines of vascular smooth muscle cells. Cx40 is coexpressed with connexin43 in several cell types, including A7r5 cells, which contain two physiologically distinct gap junctional channels. To demonstrate that Cx40 could form functional channels, we stably transfected communication-deficient Neuro2A cells with Cx40 DNA. These Cx40-transfected cells showed intercellular passage of microinjected Lucifer yellow CH. The expression of multiple connexins (such as Cx40 and Cx43) by a single cell may provide a mechanism by which cells regulate intercellular coupling through the formation of multiple channels.