The canonical WNT2 pathway and FSH interact to regulate gap junction assembly in mouse granulosa cells.

WNTs are extracellular signaling molecules that exert their actions through receptors of the frizzled (FZD) family. Previous work indicated that WNT2 regulates cell proliferation in mouse granulosa cells acting through CTNNB1 (beta-catenin), a key component in canonical WNT signaling. In other cells, WNT signaling has been shown to regulate expression of connexin43 (CX43), a gap junction protein, as well as gap junction assembly.

A mechanism of gap junction docking revealed by functional rescue of a human-disease-linked connexin mutant.

Gap junctions are unique intercellular channels formed by the proper docking of two hemichannels from adjacent cells. Each hemichannel is a hexamer of connexins (Cxs) - the gap junction subunits, which are encoded by 21 homologous genes in the human genome. The docking of two hemichannels to form a functional gap junction channel is only possible between compatible Cxs, but the underlying molecular mechanism is unclear.

Novel germline GJA5/connexin40 mutations associated with lone atrial fibrillation impair gap junctional intercellular communication.

Atrial fibrillation (AF) is the most common form of sustained cardiac arrhythmia worldwide. Here, we investigate the molecular and cellular mechanisms of lone AF-linked germline mutations in the connexin40 (Cx40) gene, GJA5. The entire coding region of GJA5 was sequenced in 68 unrelated patients with lone AF.

The severity of mammary gland developmental defects is linked to the overall functional status of Cx43 as revealed by genetically modified mice.

Genetically modified mice mimicking ODDD (oculodentodigital dysplasia), a disease characterized by reduced Cx43 (connexin 43)-mediated gap junctional intercellular communication, represent an in vivo model to assess the role of Cx43 in mammary gland development and function. We previously reported that severely compromised Cx43 function delayed mammary gland development and impaired milk ejection in mice that harboured a G60S Cx43 mutant, yet there are no reports of lactation defects in ODDD patients. To address this further, we obtained a second mouse model of ODDD expressing an I130T Cx43 mutant to assess whether a mutant with partial gap junction channel activity would be sufficient to retain mammary gland development and function.

Structure and functional studies of N-terminal Cx43 mutants linked to oculodentodigital dysplasia.

Mutations in the gene encoding connexin-43 (Cx43) cause the human development disorder known as oculodentodigital dysplasia (ODDD). In this study, ODDD-linked Cx43 N-terminal mutants formed nonfunctional gap junction-like plaques and exhibited dominant-negative effects on the coupling conductance of coexpressed endogenous Cx43 in reference cell models. Nuclear magnetic resonance (NMR) protein structure determination of an N-terminal 23-amino acid polypeptide of wild-type Cx43 revealed that it folded in to a kinked α-helical structure.

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26.

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility.

Human dermal fibroblasts derived from oculodentodigital dysplasia patients suggest that patients may have wound-healing defects.

Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant human disease caused by any one of over 60 mutations in the GJA1 gene encoding the gap junction protein Cx43. In the present study, wound healing was investigated in a G60S ODDD mutant mouse model and by using dermal fibroblasts isolated from two ODDD patients harboring the p.D3N and p.

The role of amino terminus of mouse Cx50 in determining transjunctional voltage-dependent gating and unitary conductance.

Amino-terminus and carboxyl-terminus of connexins have been proposed to be responsible for the transjunctional voltage-dependent gating (V(j)-gating) and the unitary gap junction channel conductance (γ(j)). To better understand the molecular structure(s) determining the V(j)-gating properties and the γ(j) of Cx50, we have replaced part of the amino-terminus of mCx50 by the corresponding domain of mCx36 to engineer a chimera Cx50-Cx36N, and attached GFP at the carboxyl-terminus of mCx50 to construct Cx50-GFP. The dual whole-cell patch-clamp technique was used to test the resulting gap junction channel properties in N2A cells.

Sodium-hydrogen exchange inhibition attenuates glycoside-induced hypertrophy in rat ventricular myocytes.

Aims: Cardiac glycosides induce cardiomyocyte hypertrophy via yet to be defined mechanisms. These hypertrophic effects are likely related to changes in intracellular signalling secondary to Na(+)-K(+) ATPase (NKA) inhibition which would produce elevations in intracellular sodium concentrations. Sodium-hydrogen exchanger isoform 1 (NHE-1) also contributes to intracellular sodium regulation.

Fate of connexin43 in cardiac tissue harbouring a disease-linked connexin43 mutant.

Aims: More than 40 mutations in the GJA1 gene encoding connexin43 (Cx43) have been linked to oculodentodigital dysplasia (ODDD), a pleiotropic, autosomal dominant disorder. We hypothesized that even with a significant reduction in the levels of Cx43 in a mutant mouse model of ODDD (Gja1(Jrt/+)) harbouring a G60S mutation (Cx43(G60S)), cardiomyocyte function may only be moderately compromised given that a majority of mutant mice typically survive.

Methods And Results: Western blotting and quantitative reverse transcriptase-polymerase chain reaction in conjunction with immunofluorescence were used to assess the expression and localization of Cx43 in hearts and cultured cardiomyocytes from wild-type and Gja1(Jrt/+) mice.

Pannexin 1 and pannexin 3 are glycoproteins that exhibit many distinct characteristics from the connexin family of gap junction proteins.

Pannexins are mammalian orthologs of the invertebrate gap junction proteins innexins and thus have been proposed to play a role in gap junctional intercellular communication. Localization of exogenously expressed pannexin 1 (Panx1) and pannexin 3 (Panx3), together with pharmacological studies, revealed a cell surface distribution profile and life cycle dynamics that were distinct from connexin 43 (Cx43, encoded by Gja1). Furthermore, N-glycosidase treatment showed that both Panx1 (approximately 41-48 kD species) and Panx3 (approximately 43 kD) were glycosylated, whereas N-linked glycosylation-defective mutants exhibited a decreased ability to be transported to the cell surface.

D-Serine inhibits AMPA receptor-mediated current in rat hippocampal neurons.

D-Serine, a recently identified gliotransmitter, serves as an endogenous coagonist binding to the glycine site of N-methyl-D-aspartate (NMDA) receptors. However, it is not clear whether this native ligand is able to bind to and modulate alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) receptors. In the present study, we showed that D-serine was able to concentration-dependently inhibit kainate-induced AMPA receptor-mediated current in acutely isolated hippocampal neurons.

Differential potency of dominant negative connexin43 mutants in oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is a congenital autosomal dominant disorder with phenotypic variability, which has been associated with mutations in the GJA1 gene encoding connexin43 (Cx43). Given that Cx43 mutants are thought to be equally co-expressed with wild-type Cx43 in ODDD patients, it is imperative to examine the consequence of these mutants in model systems that reflect this molar ratio. To that end, we used differential fluorescent protein tagging of mutant and wild-type Cx43 to quantitatively monitor the ratio of mutant/wild-type within the same putative gap junction plaques and co-immunoprecipitation to determine if the mutants interact with wild-type Cx43.

Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma.

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments.

Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation.

Background: Atrial fibrillation is the most common type of cardiac arrhythmia and a leading cause of cardiovascular morbidity, particularly stroke. The cardiac gap-junction protein connexin 40 is expressed selectively in atrial myocytes and mediates the coordinated electrical activation of the atria. We hypothesized that idiopathic atrial fibrillation has a genetic basis and that tissue-specific mutations in GJA5, the gene encoding connexin 40, may predispose the atria to fibrillation.

Functional characterization of oculodentodigital dysplasia-associated Cx43 mutants.

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells.

A Gja1 missense mutation in a mouse model of oculodentodigital dysplasia.

Oculodentodigital dysplasia (ODDD) is an autosomal dominant disorder characterized by pleiotropic developmental anomalies of the limbs, teeth, face and eyes that was shown recently to be caused by mutations in the gap junction protein alpha 1 gene (GJA1), encoding connexin 43 (Cx43). In the course of performing an N-ethyl-N-nitrosourea mutagenesis screen, we identified a dominant mouse mutation that exhibits many classic symptoms of ODDD, including syndactyly, enamel hypoplasia, craniofacial anomalies and cardiac dysfunction. Positional cloning revealed that these mice carry a point mutation in Gja1 leading to the substitution of a highly conserved amino acid (G60S) in Cx43.

D-aspartate and NMDA, but not L-aspartate, block AMPA receptors in rat hippocampal neurons.

1 The amino acid, D-aspartate, exists in the mammalian brain and is an agonist at the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors. Here, for the first time, we studied the actions of D-aspartate on alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs) in acutely isolated rat hippocampal neurons. 2 In the presence of the NMDA receptor channel blocker, MK801, D-aspartate inhibited kainate-induced AMPAR current in hippocampal neurons.

Oculodentodigital dysplasia-causing connexin43 mutants are non-functional and exhibit dominant effects on wild-type connexin43.

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells.