Bruce J. Nicholson

Selective transfer of endogenous metabolites through gap junctions composed of different connexins

Selective transfer of endogenous metabolites through gap junctions composed of different connexins

Molecular cloning and functional expression of mouse connexin-30,a gap junction gene highly expressed in adult brain and skin.

A new gap junction gene isolated from the mouse genome codes for a connexin protein of 261 amino acids. Because of its theoretical molecular mass of 30.366 kDa, it is named connexin-30. Within the connexin gene family, this protein is most closely related to connexin-26 (77% amino acid sequence identity). The coding region of mouse connexin-30 is uninterrupted by introns and is detected in the mouse genome as a single copy gene that is assigned to mouse chromosome 14 by analysis of mouse × hamster somatic cell hybrids. Abundant amounts of connexin-30 mRNA (two transcripts of 2.0 and 2.3 kilobase pairs) were found after 4 weeks of postnatal development in mouse brain and skin. Microinjection of connexin-30 cRNA into Xenopus oocytes induced formation of functional gap junction channels that gated somewhat asymmetrically in response to transjunctional voltage and at significantly lower voltage (Vo = +38 and −46 mV) than the closely homologous connexin-26 channels (Vo = 89 mV). Heterotypic pairings of connexin-30 with connexin-26 and connexin-32 produced channels with highly asymmetric and rectifying voltage gating, respectively. This suggests that the polarity of voltage gating and the cationic selectivity of connexin-30 are similar to those of its closest homologue, connexin-26.

Paradigm of Genetic Mosaicism and Lone Atrial Fibrillation Physiological Characterization of a Connexin 43–Deletion Mutant Identified From Atrial Tissue

Background— Atrial fibrillation (AF) is the most common sustained arrhythmia observed in otherwise healthy individuals. Most lone AF cases are nonfamilial, leading to the assumption that a primary genetic origin is unlikely. In this study, we provide data supporting a novel paradigm that atrial tissue–specific genetic defects may be associated with sporadic cases of lone AF. Methods and Results— We sequenced the entire coding region of the connexin 43 (Cx43) gene (GJA1) from atrial tissue and lymphocytes of 10 unrelated subjects with nonfamilial, lone AF who had undergone surgical pulmonary vein isolation. In the atrial tissue of 1 patient, we identified a novel frameshift mutation caused by a single nucleotide deletion (c.932delC) that predicted 36 aberrant amino acids followed by a premature stop codon, leading to truncation of the C-terminal domain of Cx43. The mutation was absent from the lymphocyte DNA of the patient, indicating genetic mosaicism. Protein trafficking studies demonstrated intracellular retention of the mutant protein and a dominant-negative effect on gap junction formation of both wild-type Cx43 and Cx40. Electrophysiological studies revealed no electrical coupling of cells expressing the mutant protein alone and significant reductions in coupling when coexpressed with wild-type connexins. Conclusions— This study reports atrial tissue genetic mosaicism of a novel loss-of-function Cx43 mutation associated with lone AF. These findings implicate somatic genetic defects of Cx43 as a potential cause of AF and support the paradigm that sporadic, nonfamilial cases of lone AF may arise from genetic mosaicism that creates heterogeneous coupling patterns, predisposing the tissue to reentrant arrhythmias.

Tissue-specific distribution of differentially phosphorylated forms of Cx43.

Variants of the Cx43 gap junction protein have been detected on Western immunoblots by using an antipeptide antibody to the N-terminus of the protein. In heart ventricle, atrium, brain, retina, and uterus, different yet characteristic ratios of a broad 43-kDa band and a 39- to 40-kDa doublet were observed. These proteins (in lens epithelium, testes, and spleen) or their messages (in stomach, duodenum, kidney, and lung) were also detected in several nonexcitable systems but at consistently lower levels than found in electrically excitable tissues. The reproducible heterogeneity in electrophoretic mobility of Cx43 seen in different tissues does not appear to be due to proteolysis, since both the 43-kDa band and the 39- to 40-kDa doublet were recognized by an N-terminal as well as a C-terminal antibody. Furthermore, Northern (RNA) blots from different tissues show that both polypeptide profiles arise from indistinguishable transcripts. The conversion by alkaline phosphatase treatment of a predominantly 43-kDa profile (in heart) to a 39- to 40-kDa profile (characteristic of brain and protein translated in vitro from the RNA) suggests that the observed electrophoretic heterogeneity arises from tissue-wide differences in the phosphorylation state of Cx43.

The molecular basis of selective permeability of connexins is complex and includes both size and charge

Although gap junction channels are still widely viewed as large, non-specific pores connecting cells, the diversity in the connexin family has led more attention to be focused on their permeability characteristics. We summarize here the current status of these investigations, both published and on-going, that reveal both charge and size selectivity between gap junction channels composed of different connexins. In particular, this review will focus on quantitative approaches that monitor the expression level of the connexins, so that it is clear that differences that are seen can be attributed to channel properties. The degree of selectivity that is observed is modest compared to other channels, but is likely to be significant for biological molecules that are labile within the cell. Of particular relevance to the in vivo function of gap junctions, recent studies are summarized that demonstrate that the connexin phenotype can control the nature of the endogenous traffic between cells, with consequent effects on biological effects of gap junctions such as tumor suppression.

Gap-junction channels dysfunction in deafness and hearing loss.

Gap-junction channels connect the cytoplasm of adjacent cells, allowing the diffusion of ions and small metabolites. They are formed at the appositional plasma membranes by a family of related proteins named connexins. Mutations in connexins 26, 31, 30, 32, and 43 have been associated with nonsyndromic or syndromic deafness. The majority of these mutations are inherited in an autosomal recessive manner, but a few of them have been associated with dominantly inherited hearing loss. Mutations in the connexin26 gene (GJB2) are the most common cause of genetic deafness. This review summarizes the most relevant and recent information about different mutations in connexin genes found in human patients, with emphasis on GJB2. The possible effects of the mutations on channel expression and function are discussed, in addition to their possible physiologic consequences for inner ear physiology. Finally, we propose that connexin channels (gap junctions and hemichannels) may be targets for age-related hearing loss induced by oxidative damage.

Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.

The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.

Differences between liver gap junction protein and lens MIP 26 from rat: Implications for tissue specificity of gap junctions

Liver gap junctions and gap-junction-like structures from eye lenses are each comprised of a single major protein (Mr 28,000 and 26,000, respectively). These proteins display different two-dimensional peptide fingerprints, distinct amino acid compositions, nonhomologous N-terminal amino acid sequences and different sensitivities to proteases when part of the intact junction. However, the junctional protein of each tissue is well conserved between species, as demonstrated previously for lens and now for liver in several mammalian species. The possiblity of tissue-specific gap junction proteins is discussed in the light of data suggesting that rat heart gap junctions are comprised of yet a third protein.

Identification of amino acid residues lining the pore of a gap junction channel

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176–1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.

The hepatocyte-specific phenotype of murine liver cells correlates with high expression of connexin32 and connexin26 but very low expression of connexin43.

This investigation was initiated in order to find out whether expression of the hepatocyte-specific phenotype is accompanied by expression of certain connexin genes coding for gap junctional protein subunits. Several clones of mouse embryonic hepatocytes immortalized in serum-free MX83 medium by infection with recombinant retrovirus-expressed transcripts for connexin32, connexin26, albumin, alpha-fetoprotein, tyrosine aminotransferase, as well as aldolase A and B, at more than half of the levels found in primary mouse hepatocytes. In addition the immortalized hepatocyte clones contained low levels of connexin43 mRNA of which only trace amounts were detected in primary embryonic mouse hepatocytes and in rat liver. Two of the immortalized hepatocyte clones were shifted from serum-free MX83 medium to Dulbeccos modified Eagle medium (DMEM) containing 10% fetal calf serum and, after 2, 14, or 180 days, back to MX83 medium. We found that expression of connexin32 and connexin26 mRNAs as well as transcripts of other liver-specific proteins was reversibly decreased in serum-containing medium, whereas the expression level of connexin43 transcripts was increased in serum-containing DMEM compared to serum-free MX83 medium. The expression levels of connexin26, connexin32, or connexin43 mRNAs were altered by the addition of fetal calf serum or arginine or by the absence of hydrocortisone in MX83 medium, all of which contributed to the shift in phenotype. Furthermore several dedifferentiated cell lines derived from rat or mouse liver and cultivated in serum-containing medium were found to express little connexin32 or connexin26 mRNA but relatively high levels of connexin43 mRNA.