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

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 (alpha subgroup of connexins) than to mouse Cx32 (beta 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 lung-similar 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 (tau = 580 ms at Vj of +/- 50 mV). 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 coupling-deficient human HeLa or SK-Hep-1 cells resulted in expression of the expected transcript and restoration of fluorescent dye transfer in transfected clones.