Molecular cloning and functional expression of mouse connexin40, a second gap junction gene preferentially expressed in lung

Abstract

Abstract.From a mouse genomic library, a clone has been isolated that codes for a connexin-homologous sequence of 358 amino acids. Because of its theoretical molecular mass of 40.418 kD it is named connexin40 (Cx40). Based on both protein and nucleotide sequence, mouse Cx40 is more closely related to mouse Cx43 (or subgroup of connexins) than to mouse Cx32 (B subgroup). The highest overall homology detected, however, was to chick Cx42 (67% amino acid and 86% nucleotide identity), raising the possibility that Cx40 may be the mouse analogue. The coding region of Cx40 is uninterrupted by introns and is detected as a single copy gene in the mouse genome. High stringency hybridization of Northern blots with the coding sequence of Cx40 identified a single transcript of 3.5 kb that is at least 16-fold more abundant in lungsimilar to mouse Cx37-than in other adult tissues (kidney, heart, and skin). In embryonic kidney, skin, and liver the level of the Cx40 transcript is two-to fourfold higher than in the corresponding adult tissues.Microinjection of Cx40 cRNA into Xenopus oocytes induced functional cell-to-cell channels between pairs. These channels show a symmetrical and markedly cooperative closure in response to transjunctional voltage (Boltzmann parameters of Vo = +35 mV; A = 0.32) which is also fast relative to other connexin channels recorded similarly (r = 580 ms at Vj of +50 mY). Although Cx40-expressing oocytes did not couple efficiently with oocytes expressing endogenous connexins, they did couple well to Cx37-expressing oocytes. The heterotypic channels which formed had voltage-gating properties modified from those of the original homotypic forms. Transfection of mouse Cx40 DNA, under control of the SV-40 early promoter, into couplingdeficient human I-IeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.

Connexin 48.5 Is Required for Normal Cardiovascular Function and Lens Development in Zebrafish Embryos

“…the slower channels like Cx26 and Cx32 and the more rapidly gating channels such as Cx37 or Cx40 (41,42). Thus, the voltage dependence of Cx48.5 channels was quite similar to that of rodent Cx46 (31,33,43).…”

A role for heterologous gap junctions between melanoma and endothelial cells in metastasis

“…Five micrograms of total RNA extracted from cultured cells, mouse tissues, or in vivo growing tumors were fractionated on 1% agaroseformaldehyde gels, transferred to a nylon membrane, and hybridized to DNA probes labeled with [α- 32 (27), Cx40 (nt 154-826) (28), and Cx43 (nt 458-1075) (GenBank accession number X61576) were obtained using PCR. The β-actin probe was prepared in the same fashion (3).…”

Developmental regulation of connexin 40 gene expression in mouse heart correlates with the differentiation of the conduction system

“…1). As previously described (Beyer et al, 1987;Kanter et al, 1992Kanter et al, ,1993aHennemann et al, 1992a;Haefliger et al, 1992;Reed et al, 1993) a single transcript was detected for each connexin studied: a 3.5 kb mRNA encoding for CX40, a 1.7 kb mRNA encoding for CX37, a 2.3 kb mRNA encoding for CX45, and a 3.0 kb mRNA encoding for CX43. Northern blots showed that the connexin transcripts are present from 11 days post-coitum (dpc) to the adult stage, although the mRNAs are only faintly detectable at 11 dpc (Fig.…”

“…Two of these probes were cDNAs of 1.2 and 1.1 kb containing the complete coding regions of the mouse CX40 and CX37 genes, respectively (Hennemann et al, 1992a;Willecke et al, 1991). A third probe was a 0.5 kb C-terminal PstI fragment of the CX45 cDNA derived from F9 embryonic carcinoma cells (Hennemann et al, 1992b).…”

“…Thus, conduction of the electrical impulse ends by a coordinated contraction of the atria and ventricles. In mammalian heart, five connexin transcripts have been identified: CX37, CX40, CX43, CX45, and CX46 (Paul et al, 1991;Hennemann et al, 1992a;Haefliger et al, 1992;Beyer et al, 1987;Kanter et al, 1992Kanter et al, , 1993aReed et al, 1993). The first connexin identified in the heart, and thus the most extensively investigated, is (2x43.…”