Innexin-3 forms connexin-like intercellular channels.
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Innexins comprise a large family of genes that are believed to encode invertebrate gap junction channel-forming proteins. However, only two Drosophila innexins have been directly tested for the ability to form intercellular channels and only one of those was active. Here we tested the ability of Caenorhabditis elegans family members INX-3 and EAT-5 to form intercellular channels between paired Xenopus oocytes. We show that expression of INX-3 but not EAT-5, induces electrical coupling between the oocyte pairs. In addition, analysis of INX-3 voltage and pH gating reveals a striking degree of conservation in the functional properties of connexin and innnexin channels. These data strongly support the idea that innexin genes encode intercellular channels. (+info)
Downregulation of connexin 45 gene products during mouse heart development.
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The electrical activity in heart is generated in the sinoatrial node and then propagates to the atrial and ventricular tissues. The gap junction channels that couple the myocytes are responsible for this propagation process. The gap junction channels are dodecamers of transmembrane proteins of the connexin (Cx) family. Three members of this family have been demonstrated to be synthesized in the cardiomyocytes: Cx40, Cx43, and Cx45. In addition, each of them has been shown to form channels with unique and specific electrophysiological properties. Understanding the conduction phenomenon requires detailed knowledge of the spatiotemporal expression pattern of these Cxs in heart. The expression patterns of Cx40 and Cx43 have been previously described in the adult heart and during its development. Here we report the expression of Cx45 gene products in mouse heart from the stage of the first contractions (8.5 days postcoitum [dpc]) to the adult stage. The Cx45 gene transcript was demonstrated by reverse transcriptase-polymerase chain reaction experiments to be present in heart at all stages investigated. Between 8.5 and 10.5 dpc it was shown by in situ hybridization to be expressed in low amounts in all cardiac compartments (including the inflow and outflow tracts and the atrioventricular canal) and then to be downregulated from 11 to 12 dpc onward. At subsequent fetal stages, the transcript was weakly detected in the ventricles, with the most distinct expression in the outflow tract. Cx45 protein was demonstrated by immunofluorescence microscopy to be expressed in the myocytes of young embryonic hearts (8.5 to 9.5 dpc). However, beyond 10.5 dpc the protein was no longer detected with this technique in the embryonic, fetal, or neonatal working myocardium, although it could be shown by immunoblotting that the protein was still synthesized in neonatal heart. In the major part of adult heart, Cx45 was undetectable. It was, however, clearly seen in the anterior regions of the interventricular septum and in trace amounts in some small foci dispersed in the ventricular free walls. Cx45 gene is the first Cx gene so far demonstrated to be activated in heart at the stage of the first contractions. The coordination of myocytes during the slow peristaltic contractions that occur at this stage would thus appear to be controlled by the Cx45 channels. (+info)
The effect of connexin32 null mutation on hepatocarcinogenesis in different mouse strains.
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Connexin32 (Cx32) is the major gap junctional protein in mouse liver. We have shown recently that the formation of liver tumours in Cx32-deficient mice is strongly increased in comparison with control wild-type mice, demonstrating that the deficiency in gap junctional communication has an enhancing effect on hepatocarcinogenesis. We have now compared the effect of Cx32 deficiency on liver carcinogenesis in two strains of mice with differing susceptibility to hepatocarcinogenesis. Heterozygous Cx32(+/-) females were crossed with male Cx32 wild-type C57BL/6J (low susceptibility) or C3H/He (high susceptibility) mice. Since the Cx32 gene is located on the X-chromosome, the resulting F1 males segregated to the genotypes Cx32(Y/+) and Cx32(Y/-). Genotyping was performed by PCR-analysis using tail-tip DNA. Weanling male mice were i.p. injected with a single dose of N-nitrosodiethylamine and were killed 16, 21 or 26 weeks later. The number, volume fraction and size distribution of precancerous liver lesions characterized by a deficiency in the marker enzyme glucose-6-phosphatase were quantitated. The results demonstrate that Cx32 deficiency only slightly affects the number of enzyme-altered lesions, but strongly enhances their growth, both in the resistant and the susceptible mouse strain, suggesting that decreased intercellular communication results in tumour promoting activity irrespective of the genetic background of the mouse strain used. Since Cx32-deficient C3H/He hybrids were approximately 5-10 times more sensitive than C3H/He hybrids with an intact Cx32 gene, this mouse strain may prove very useful for toxicological screening purposes. (+info)
Characterization of a mouse Cx50 mutation associated with the No2 mouse cataract.
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PURPOSE: Recently, a missense mutation in the mouse connexin 50 (Cx50) gene has been associated with the nuclear opacity 2 (No2) mouse cataract. This missense mutation (D47A) resulted in an aspartate-to-alanine substitution at amino acid position 47 in the first extracellular domain of Cx50. To better understand the role of Cx50 in the pathogenesis of congenital cataract, the functional consequences of the D47A mutation in the Xenopus oocyte expression system were studied. METHODS: D47A was constructed using polymerase chain reaction (PCR) mutagenesis. Xenopus oocytes were injected with in vitro transcribed cRNA encoding wild-type mouse Cx50 (Cx50wt), wild-type rat Cx46 (Cx46wt), D47A, or combinations of wild-type and mutant connexins. The oocytes were then devitellinized and paired. Gap junctional conductance (Gj) was measured using a dual two-microelectrode voltage-clamp technique. RESULTS: Homotypic oocyte pairs expressing wild-type Cx50 or Cx46 were well coupled. In contrast, oocytes injected with D47A cRNA did not form gap junctional channels when paired homotypically. To test whether the D47A mutation could interact with wild-type connexins in a dominant negative manner, oocytes were injected with equal amounts of mutant and wild-type connexin cRNA, mimicking the heterozygous condition. Expression of D47A did not inhibit the development of junctional conductance in paired oocytes induced by wild-type Cx50 or Cx46. CONCLUSIONS: These results indicate that the D47A mutation acts as a loss-of-function mutation without strong dominant inhibition. In No2 mice, the mutation would be predicted to result in a reduction in intercellular communication, leading to cataractogenesis. It may also cause other qualitative changes such as a change in permeability for small molecules. (+info)
Differential expression of gap-junction gene connexin 31 in seminiferous epithelium of rat testes.
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Spermatogenesis, a tightly regulated developmental process of male germ cells in testis, is associated with temporal and spatial expression of certain gap-junction connexins. Our findings by RT-PCR indicate that the Cx31 gene is expressed in testis tissue of adult and postnatal rats. During the postnatal spermatogenic process, the Cx31-specific signal became detectable at 15 dpp and onward by in situ hybridization, and apparently localized in the basal compartment of seminiferous epithelium where active spermatogonia and early primary spermatocytes reside. No signal was found in the luminal region. In adult testes, spermatids of elongation phase were also Cx31 positive. Immunohistochemical analysis with mouse anti-Cx31 antibody gave a similar staining pattern, providing further evidence that the gap-junction protein is abundant in the basal seminiferous epithelium, in accordance with the cellular distribution of Cx31 mRNA. These results represent the first demonstration of Cx31 expression at both transcriptional and protein levels in the seminiferous epithelium of rat testes. Thus, Cx31 may play a role in cell-cell communication during spermatogenesis. (+info)
Endogenous casein kinase I catalyzes the phosphorylation of the lens fiber cell connexin49.
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The lens fiber cell-specific gap junction protein connexin49 is a substrate for a membrane-associated Ser/Thr protein kinase that can be extracted from lens cell membranes by 0.6 M KCl. However, the identity of this protein kinase has not been defined. In this report, evidence is presented indicating that it is casein kinase I. Thus, connexin49 was shown to be a substrate for purified casein kinase I but not for casein kinase II; the endogenous connexin49 protein kinase activity extracted from lens membranes with KCl was inhibited by the casein kinase I-specific inhibitor, N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide (CKI-7); the connexin49 protein kinase activity in the lens membrane KCl extract, which could be partially purified by gel filtration and affinity purification with a casein-Sepharose 4B column, copurified with casein kinase activity; phosphopeptide analysis showed that casein kinase I and the connexin49 protein kinase activity in the lens membrane KCl extract probably share the same phosphorylation sites in connexin49. Reverse transcription-PCR using total ovine lens RNA and casein kinase I isoform-specific oligonucleotide primers resulted in the amplification of cDNAs encoding casein kinase I-alpha and -gamma, while an in-gel casein kinase assay indicated casein kinase activity in the lens membrane KCl extract was associated with a major 39.2-kDa species, which is consistent with the 36 to 40-kDa size of casein kinase I-alpha in other animal species. These results demonstrate that the protein kinase activity present in the lens membrane 0.6 M KCl extract that catalyzes the phosphorylation of connexin49 is casein kinase I, probably the alpha isoform. (+info)
Connexin-occludin chimeras containing the ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell contacts.
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Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family. (+info)
A simple RT-PCR-based strategy for screening connexin identity.
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Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins. (+info)