Molecular cloning and functional expression of human connexin37, an endothelial cell gap junction protein.

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

Differential Expression of Connexin43 and Desmin Defines Two Subpopulations of Medial Smooth Muscle Cells in the Human Internal Mammary Artery

“…PCR product and recombinant plasmid were characterized and identified by restriction mapping. Northern blot analysis of RNA extracted from various tissues (all containing blood vessels) show a single band at Ϸ1.8 kb as described 26 (data not shown). After linearization of the plasmids for Cx37, Cx40, Cx43, and Cx45 with appropriate restriction enzymes, the digoxigeninlabeled sense and antisense RNA probes were generated by in vitro transcription, using a kit supplied by Promega.…”

“…M96789) cDNA was obtained by PCR amplification of human genomic DNA, using base 89 to 108 as upstream primer and base 1552 to 1571 as downstream primer. 26 The PCR product was simultaneously cut with PstI and StuI endonuclease (Boehringer Mannheim), ligated into the vector pT7/T3 ␣18 (GibcoBRL) and the ligation mixture used to transform Escherichia coli. PCR product and recombinant plasmid were characterized and identified by restriction mapping.…”

Modulation of intercellular communication by differential regulation and heteromeric mixing of co-expressed connexins

“…Individually, these proteins form channels with different physiological properties (1)(2)(3). However, these connexins have all been found within the same cell type (the endothelial cell), and in a number of cases they can be identified within the same individual cell (4)(5)(6). The relative abundance of these connexins varies in different endothelia.…”

Chemistry, Physiology, and Pharmacology of β.-Adrenergic Mechanisms in the Heart. Why are .β-Blocker Antiarrhythmics Superior?

“…Cx37 is detected in the endocardial endothelium [42], while Cx50 was described in rat atrioventricular valves [43]. Cx40, Cx43 and Cx45 are the most abundant connexin isoforms of the working mammalian myocardial cells and the conduction system.…”

“…The single channel conductance is about 200 pS, 80 pS and 20 pS, for Cx40, Cx43 and Cx45 channel, respectively [42,62,63]. The conductance values can be influenced by many factors such as intracellular calcium, magnesium, sodium ion concentration, pH, hypoxia and the phosphorylation state of connexins [34,36].…”