Molecular Cloning, Functional Expression, and Tissue Distribution of a Novel Human Gap Junction-forming Protein, Connexin-31.9

Abstract

A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or ␣11 connexin. A clone in GenBank TM containing the Cx31.9 gene localized to chromosome 17q21.2. Northern analysis of Cx31.9 showed a major 4.4-kilobase transcript, which was expressed at varying levels in all tissues analyzed. Two monoclonal antibodies generated against different domains of Cx31.9 recognized a 30 -33-kDa protein from cells overexpressing Cx31.9. Immunofluorescence of overexpressing cells indicated the presence of Cx31.9 between adjacent cells, consistent with its localization to gap junctions. Double voltage clamp analyses of Cx31.9-overexpressing cells, and of paired Xenopus oocytes injected with Cx31.9 cRNA, demonstrated junctional currents indicative of gap junction channel formation. In contrast to previously characterized connexins, Cx31.9 showed no voltage-dependent gating within a physiologically relevant range. Cx31.9 was detected in human tissues by immunoblot analysis, and immunofluorescence localized Cx31.9 expression to vascular smooth muscle cells. Furthermore, it was demonstrated that Cx31.9 interacted with ZO-1. Thus, Cx31.9 represents a novel connexin gene that in vivo generates a protein with unique voltage gating properties.Most cells in vertebrate tissues are coupled by gap junction channels, which allow the transfer of low molecular mass substances (Ͻ1 kDa) between cells. The ability of cells to communicate in this manner is thought to be important for coordinating tissue growth, development, and physiological activities. Connexins are the sole proteins required for the formation of gap junctions (1). Six connexin monomers form a hemichannel (connexon) on the cell surface, which can interact with a connexon from a neighboring cell, thus forming a channel linking the cytoplasm of the two cells.A number of different connexin isoforms are found in vertebrates. The connexins characterized to date have distinct channel permeabilities and gating properties (2, 3), as well as distinct cell and tissue expression patterns, which is presumed to orchestrate a spatially and temporally regulated diffusion of small molecules between cells. This has recently been demonstrated by studies of genetically altered mice, in which the expression of Cx43 1 was replaced by expression of Cx40 or Cx32. These mice differed functionally and morphologically from wild type mice, demonstrating that one connexin isoform cannot fully substitute for another (4). Regulation of the diffusion of small molecules can be further modulated by combining different connexin isoforms in each hemichannel (heteromeric gap junctions) and by combining different hemichannels (heterotypic gap junctions), thus creating channels with highly specific permeability and gating properties.All connexin isoforms are presumed to have a similar topology, which has been deduced from limited proteolys...

Connexin30.2 containing gap junction channels decelerate impulse propagation through the atrioventricular node

“…Gap junction channels of the human orthologue Cx31.9 also exhibit low unitary conductance (38,39). Thus, its effect on conduction characteristics in human cardiac electrophysiology and AV-nodal physiology should be investigated.…”

Protein kinase A mediates regulation of gap junctions containing connexin35 through a complex pathway

“…Such a mutation may disrupt a binding interaction that involves the tip of the C-terminus. Several connexins have been shown to interact with proteins via the C-terminal domains [13,21,32,33,45], with the scaffolding protein ZO1 being the most prevalent partner. Indeed Cx36 has recently been found to interact with ZO1 at this site [24].…”

Cx30.2 can form heteromeric gap junction channels with other cardiac connexins

“…1). Previously, investigators had shown that Cx30.2 gap junction channels exhibit little gating sensitivity to transjunctional voltage [9][10][11]. The insensitivity or activation of Cx30.2 G j at high V j may be important for the preservation of slow impulse propagation through the atriventricular node.…”

“…Recently, another cardiac connexin, mouse Cx30.2 (human Cx31.9) was discovered [9][10][11]. Cx30.2 expression was detected within the sinoatrial and atrioventricular nodes [11,12].…”