David L. Paul

BEYOND THE GAP: FUNCTIONS OF UNPAIRED CONNEXON CHANNELS

Gap junctions consist of intercellular channels that connect the cytoplasm of adjacent cells directly and allow the exchange of small molecules. These channels are unique in that they span two plasma membranes — the more orthodox ion or ligand-gated channels span only one. Each cell contributes half of the intercellular channel, and each half is known as a connexon or hemichannel. Recent studies indicate that connexons are also active in single plasma membranes and that they might be essential in intercellular signalling beyond their incorporation into gap junctions.

Gap junctions in the rat cochlea: immunohistochemical and ultrastructural analysis

Gap junctions in the rat cochlea were investigated using immunostaining for connexin26 and transmission electron microscopy. Electron microscopy of normal and pre-embedded immunostained material showed that there were gap junctions between and among all cells that light microscopy showed to have immunostained appositions. Light microscopy showed immunostaining between and among all cell types that electron microscopy showed to be joined by gap junctions. Immunostaining for connexin26 was therefore taken as providing a reasonable approximation of the locations of gap junctions throughout the cochlea and was used to provide an overview of the extent of those locations. Cells interconnected via gap junctions fell into one of two groups. The first group consists of nonsensory epithelial cells and includes interdental cells of the spiral limbus, inner sulcus cells, organ of Corti supporting cells, outer sulcus cells, and cells within the root processes of the spiral ligament. The second group consists of connective tissue cells and includes various fibrocyte types of the spiral limbus and spiral ligament, basal and intermediate cells of the stria vascularis, and mesenchymal cells which line the scala vestibuli. The present work represents a first attempt towards a description of how serial gap junctions among cochlear cells reflect a level of organization of the tissue. The organization described here, together with a great deal of information from previous investigators, suggest that serially arranged gap junctions of both epithelial and connective tissue cells serve as the strucural basis for recycling endolymphatic potassium ions that pass through the sensory cells during the transduction process.

Connexin family of gap junction proteins.

Gap junctions are composed of aggregations of membrane channels, called connexons, joined with similar connexons in adjacent cells to form intercellular pathways for the diffusion of ions and small molecules (Caspar et al., 1977; Makowski et al., 1977). The resulting intercellular communication is unique in that adjacent cells exchange cytoplasmic molecules directly, with no secretion into the extracellular space (Bennett, 1966; Loewenstein, 1966). Due to the large size of the intercellular channels formed by connexon pairs, the exchange of molecules between cells is nonspecific, and includes the entire pool of ions and small metabolites in each cell (Gilula, Reeves & Steinbach, 1972; Pitts & Simms, 1977; Simpson, Rose & Loewenstein, 1977; Goodenough, Dick & Lyons, 1980). This form of intercellular communication is ideally suited for the role of intercellular buffering of cytoplasmic ions (Corsaro & Migeon, 1977; Ledbetter & Lubin, 1979), synchronization of cellular behavior, such as the coordinated contraction of myocardial cells (Barr, Dewey & Berger, 1965) and the cell-to-cell coordination of metachronal waves (Moss & Tamm, 1987; Sanderson, Chow & Dirksen, 1988). Involvement of gap junction-mediated intercellular communication has also been suggested for growth control and embryonic differentiation (Loewenstein, 1966; Furshpan & Potter, 1968; Warner, Guthrie & Gilula, 1984; Loewenstein & Azarnia, 1988). Due to the sharing of low molecular weight substrate pools, gap junctions will also function to suppress the deleterious effects of somatic cell mutation in a variety of enzymes (Subak-Sharpe, Burk & Pitts, 1969; Cox et al., 1970).

Synchronous Activity of Inhibitory Networks in Neocortex Requires Electrical Synapses Containing Connexin36

Inhibitory interneurons often generate synchronous activity as an emergent property of their interconnections. To determine the role of electrical synapses in such activity, we constructed mice expressing histochemical reporters in place of the gap junction protein Cx36. Localization of the reporter with somatostatin and parvalbumin suggested that Cx36 was expressed largely by interneurons. Electrical synapses were common among cortical interneurons in controls but were nearly absent in knockouts. A metabotropic glutamate receptor agonist excited LTS interneurons, generating rhythmic inhibitory potentials in surrounding neurons of both wild-type and knockout animals. However, the synchrony of these rhythms was weaker and more spatially restricted in the knockout. We conclude that electrical synapses containing Cx36 are critical for the generation of widespread, synchronous inhibitory activity.

Mice lacking connexin40 have cardiac conduction abnormalities characteristic of atrioventricular block and bundle branch block

Activation of cardiac muscle is mediated by the His-Purkinje system, a discrete pathway containing fast-conducting cells (Purkinje fibers) which coordinate the spread of excitation from the atrioventricular node (AV node) to ventricular myocardium [1]. Although pathologies of this specialized conduction system are common in humans, especially among the elderly [2], their molecular bases have not been defined. Gap junctions are present at appositions between Purkinje fibers and could provide a mechanism for propagating impulses between these cells [3]. Studies of the expression of connexins - the family of proteins from which gap junctions are formed - reveal that connexin40 (Cx40) is prominent in the conduction system [4]. In order to study the role of gap junction communication in cardiac conduction, we generated mice that lack Cx40. Using electrocardiographic analysis, we show that Cx40 null mice have cardiac conduction abnormalities characteristic of first-degree atrioventricular block with associated bundle branch block. Thus, gap junctions are essential for the rapid conduction of impulses in the His-Purkinje system.

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

Connexin36 Is Essential for Transmission of Rod-Mediated Visual Signals in the Mammalian Retina

To examine the functions of electrical synapses in the transmission of signals from rod photoreceptors to ganglion cells, we generated connexin36 knockout mice. Reporter expression indicated that connexin36 was present in multiple retinal neurons including rod photoreceptors, cone bipolar cells, and AII amacrine cells. Disruption of electrical synapses between adjacent AIIs and between AIIs and ON cone bipolars was demonstrated by intracellular injection of Neurobiotin. In addition, extracellular recording in the knockout revealed the complete elimination of rod-mediated, on-center responses at the ganglion cell level. These data represent direct proof that electrical synapses are critical for the propagation of rod signals across the mammalian retina, and they demonstrate the existence of multiple rod pathways, each of which is dependent on electrical synapses.

Formation of gap junctions by expression of connexins in Xenopus oocyte pairs

RNAs coding for connexins 32, 43, and the putative lens gap junction protein MP26 were tested for their ability to induce cell-cell coupling in Xenopus oocyte pairs. Large, voltage-insensitive conductances developed when connexin32 and 43 RNA-injected oocytes were paired both with themselves and with each other. Oocyte pairs injected with water manifested small conductances, which were symmetrically voltage-dependent. MP26 RNA-injected pairs displayed no conductances above control values. Unexpectedly, connexin43/water oocyte pairs developed high, asymmetrically voltage-dependent conductances, a property not displayed by the connexin32/water pairs. In single oocytes, these proteins remained intracellular until pairing, at which time the connexins, but not MP26, concentrated at the appositional areas.

Functional defects of Cx26 resulting from a heterozygous missense mutation in a family with dominant deaf-mutism and palmoplantar keratoderma

Mutations in GJB2 encoding the gap junction protein connexin-26 (Cx26) have been established as the basis of autosomal recessive non-syndromic hearing loss. The involvement of GJB2 in autosomal dominant deafness has also been proposed, although the putative mutation identified in one family with both deafness and palmoplantar keratoderma has recently been suggested to be merely a non-disease associated polymorphism. We have observed a similar phenotype in an Egyptian family that segregated with a heterozygous missense mutation of GJB2, leading to a non-conservative amino acid substitution (R75W). The deleterious dominant-negative effect of R75W on gap channel function was subsequently demonstrated in the paired oocyte expression system. Not only was R75W alone incapable of inducing electrical conductance between adjacent cells, but it almost completely suppressed the activity of co-expressed wildtype protein. The Cx26 mutant W77R, which has been implicated in autosomal recessive deafness, also failed to form functional gap channels by itself but did not significantly interfere with the function of wildtype Cx26. These data provide compelling evidence for the serious functional consequences of Cx26 mutations in dominant and recessive deafness.

Connexin43 Is Highly Localized to Sites of Disturbed Flow in Rat Aortic Endothelium but Connexin37 and Connexin40 Are More Uniformly Distributed

Vascular endothelial cells are linked by gap junctions, which facilitate the propagation of electrical and chemical signals along the vessel wall. The aim of this study was to determine the distribution and identity of the gap junction structural proteins (connexins) expressed by endothelial cells in situ. Connexin expression in different regions of the rat aortic endothelium was analyzed with the use of indirect immunofluorescence microscopy and Western blotting. Connexin40 and connexin37 were present in most, if not all, of the thoracic and abdominal aortic endothelia in the form of maculae at cell-cell appositions. In contrast, connexin43 was undetectable in most endothelia but extremely abundant in small numbers of cells localized at the downstream edge of the ostia of branching vessels and at flow dividers, regions that experience turbulent shear stress from disturbed blood flow. To examine the relationship of shear stress and connexin43 expression, localized stress was induced by surgical coarctation of the aorta, which was sufficient to cause striking local upregulation of connexin43 within 8 days. Thus, increases in connexin43 levels are an endothelial response to mechanical stress.