Molecular cloning and expression of rat connexin40, a gap junction protein expressed in vascular smooth muscle

Abstract

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.

Connexin43 is overexpressed in ApcMin/+‐mice adenomas and colocalises with COX‐2 in myofibroblasts

“…We found Cx40 localised to a few epithelial cells in the top of the villi in the normal intestinal tissue of Min-mice and in endothelial tissue as previously reported. 34,35 The position of the Cx40-positive cells could suggest a function in apoptosis, but only partial colocalisation with active-caspase 3 was found, indicating that Cx40 is not generally involved in apoptosis. However, altered gap junction function seems to be related to induction of apoptosis.…”

Gap Junction Localization and Connexin Expression in Cytochemically Identified Endothelial Cells of Arterial Tissue

“…Portions of endothelium are frequently tangentially sectioned, even in transversely cut arterial rings, so that the boundary between endothelium and the underlying medial layer in sectional views varies considerably in depth. Furthermore, because two of the connexins reported to be expressed in endothelial cells, connexin43 and connexin40, have also been reported in vascular smooth muscle cells (Beyer et al 1992;Pepper et al 1992;Blackburn et al 1995;Little et al 1995a;Burt 1994,1995), the two cell types cannot be reliably distinguished on the basis of connexin immunolabeling patterns alone. All the interpretative uncertainties arising from these sources are overcome by our dual cell marking/connexin labeling technique, in which use of either a specific lectin or labeling of the characteristic endothelial product VWF (Wharton et al 1990;Sehested and Hou-Jensen 1981), permits unequivocal correlation of connexin labeling with the endothelial layer.…”

Connexins: a connection with the skin

“…Cx37 is found throughout the epidermal layers, and Cx43 and Cx26 are co-expressed in the periderm. Rodent hair follicles and sebaceous glands express Cx43, Cx40, and Cx31, while Cx37, Cx40, and Cx43 predominate in the cutaneous microvascular and smooth muscle system, and Cx43 in dermal fibroblasts (62)(63)(64)(65)(66)(67). In addition to the Cxs mentioned above, Northern blot analyses suggest that Cx30, a close homologue of Cx26, and the recently cloned Cx57 are expressed in rodent skin, although their tissue distribution remains to be elucidated (47,68).…”