Transcriptional and Translational Regulation of Zebrafish Connexin 55.5 (zf.Cx.55.5) and Connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

Transcriptional and translational regulation of zebrafish connexin 55.5 (zf.Cx.55.5) and connexin 52.6 (zf.Cx52.6)

Zebrafish connexin 55.5 (zf.Cx55.5) and connexin 52.6 (zf.Cx52.6) show highly restricted expression patterns in the nervous system. Both connexins are confined to subsets of neurons in the fish retina. In order to get initial answers to the questions of pattern definition in neuronal subsets, we elucidated molecular mechanisms responsible for their expression. Different upstream DNA fragments were subcloned into a pGL3-basic vector and transiently transfected in HeLa and N2A cells. Luciferase activity showed the presence of two putative promoter elements in zfCx55.5 and a promoter element in zfCx52.6 that showed different promoter activities in HeLa and N2A cells. Moreover, fusion constructs of zfCx55.5 with EGFP revealed the presence of a new isoform with an additional short exon I.     

Molecular cloning and functional expression of zfCx52.6: a novel connexin with hemichannel-forming properties expressed in horizontal cells of the zebrafish retina

Gap junction-mediated electrical coupling contributes to synchronous oscillatory activities of neurons, and considerable progress has been made in defining the molecular composition of gap junction channels. In particular, cloning and functional expression of gap junction proteins (connexins (Cx)) from zebrafish retina have shown that this part of the brain possesses a high degree of connexin diversity that may account for differential functional properties of electrical synapses. Here, we report the cloning and functional characterization of a new connexin, designated zebrafish Cx52.6 (zfCx52.6). This connexin shows little similarity to known connexins from fish and higher vertebrates. By combining in situ hybridization with Laser Capture Microdissection and RT-PCR, we found that this novel fish connexin is expressed in horizontal cells in the inner nuclear layer of the retina. Functional expression of zfCx52.6 in neuroblastoma cells and Xenopus oocytes led to functional gap junctional channels and, in single oocytes, induced large non-junctional membrane currents indicative of the formation of hemichannels, which were inhibited in reversible fashion by raising extracellular Ca(2+) concentrations.     

IRES-mediated translation of the carboxy-terminal domain of the horizontal cell specific connexin Cx55.5 in vivo and in vitro

Background

Changes of the interneuronal coupling mediated by electrical synapse proteins in response to light adaptation and receptive field shaping are a paramount feature in the photoreceptor/horizontal cell/bipolar cell (PRC/HC/BPC) complex of the outer retina. The regulation of these processes is not fully understood at the molecular level but they may require information transfer to the nucleus by locally generated messengers. Electrical synapse proteins may comprise a feasible molecular determinant in such an information-laden signalling pathway.

Results

Connexin55.5 (Cx55.5) is a connexin with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the PRC/HC/BPC complex in form of hemichannels where light-dependent plasticity occurs. Here we provide evidence for the generation of a carboxy-terminal domain of Cx55.5. The protein product is translated from the Cx55.5 mRNA by internal translation initiation from an in-frame ATG codon involving a putative internal ribosome entry site (IRES) element localized in the coding region of Cx55.5. This protein product resembling an 11 kDa domain of Cx55.5 is partially located in the nucleus in vivo and in vitro.

Conclusion

Our results demonstrate the generation of a second protein from the coding region of Cx55.5 by an IRES mediated process. The nuclear occurrence of a fraction of this protein provides first evidence that this electrical synapse protein may participate in a putative cytoplasmic to nuclear signal transfer. This suggests that Cx55.5 could be involved in gene regulation making structural plasticity at the PRC/HC/BPC complex feasible.     

Digenic inheritance of non-syndromic deafness caused by mutations at the gap junction proteins Cx26 and Cx31

Mutations in the genes coding for connexin 26 (Cx26) and connexin 31 (Cx31) cause non-syndromic deafness. Here, we provide evidence that mutations at these two connexin genes can interact to cause hearing loss in digenic heterozygotes in humans. We have screened 108 GJB2 heterozygous Chinese patients for mutations in GJB3 by sequencing. We have excluded the possibility that mutations in exon 1 of GJB2 and the deletion of GJB6 are the second mutant allele in these Chinese heterozygous probands. Two different GJB3 mutations (N166S and A194T) occurring in compound heterozygosity with the 235delC and 299delAT of GJB2 were identified in three unrelated families (235delC/N166S, 235delC/A194T and 299delAT/A194T). Neither of these mutations in Cx31 was detected in DNA from 200 unrelated Chinese controls. Direct physical interaction of Cx26 with Cx31 is supported by data showing that Cx26 and Cx31 have overlapping expression patterns in the cochlea. In addition, by coimmunoprecipitation of mouse cochlear membrane proteins, we identified the presence of heteromeric Cx26/Cx31 connexons. Furthermore, by cotransfection of mCherry-tagged Cx26 and GFP-tagged Cx31 in human embryonic kidney (HEK)-293 cells, we demonstrated that the two connexins were able to co-assemble in vitro in the same junction plaque. Together, our data indicate that a genetic interaction between these two connexin genes can lead to hearing loss.     

Regulation of Connexin43 Oligomerization is Saturable

We have used connexin constructs containing a C-terminal di-lysine-based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL) transfected into HeLa cells to study early events in connexin oligomerization. Using this approach, we found that Cx43-HKKSL stably expressed at moderate levels by HeLa cells was retained in the ER and prevented from oligomerization. However, Cx43-HKKSL stably overexpressed by HeLa cells escaped from the ER and localized to a perinuclear region of the cell that included the Golgi apparatus. Overexpressed Cx43-HKKSL oligomerized into hexamers and also formed Triton X-100 insoluble, intracellular complexes that resembled gap junctions. Thus, the ability of HeLa cells to inhibit Cx43 oligomerization was saturable. HeLa cells stably overexpressing Cx43-HKKSL may provide a useful model system to evaluate pharmacologic agents and/or cDNAs encoding chaperones with the potential to regulate initial steps in Cx43 oligomerization.     

Regulation of Gap Junction Coupling Through the Neuronal Connexin Cx35 by Nitric Oxide and cGMP

Gap-junctional coupling among neurons is subject to regulation by a number of neurotransmitters including nitric oxide. We studied the mechanisms by which NO regulates coupling in cells expressing Cx35, a connexin expressed in neurons throughout the central nervous system. NO donors caused potent uncoupling of HeLa cells stably transfected with Cx35. This effect was mimicked by Bay 21-4272, an activator of guanylyl cyclase. A pharmacological analysis indicated that NO-induced uncoupling involved both PKG-dependent and PKG-independent pathways. PKA was involved in both pathways, suggesting that PKG-dependent uncoupling may be indirect. In vitro, PKG phosphorylated Cx35 at three sites: Ser110, Ser276, and Ser289. A mutational analysis indicated that phosphorylation on Ser110 and Ser276, sites previously shown also to be phosphorylated by PKA, had a significant influence on regulation. Ser289 phosphorylation had very limited effects. We conclude that NO can regulate coupling through Cx35 and that regulation is indirect in HeLa cells.     

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. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

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. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.     

Differential Oligomerization of Endoplasmic Reticulum-Retained Connexin43/Connexin32 Chimeras

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.     

Properties of gap junction channels formed by Cx46 alone and in combination with Cx50

Gap junctions formed of connexin46 (Cx46) and connexin50 (Cx50) in lens fiber cells are crucial for maintaining lens transparency. We determined the functional properties of homotypic Cx46, heterotypic Cx46/Cx50, and heteromeric Cx46/Cx50 channels in a communication-deficient neuroblastoma (N2A) cell line, using dual whole-cell recordings. N2A cultures were stably and/or transiently transfected with Cx46, Cx50, and green fluorescent protein (EGFP). The macroscopic voltage sensitivity of homotypic Cx46 conformed to the two-state model (Boltzmann parameters: G(min) = 0.11, V(0) = +/- 48.1 mV, gating charge = 2). Cx46 single channels showed a main-state conductance of 140 +/- 8 pS and multiple subconductance states ranging from < or =10 pS to 60 pS. Conservation of homotypic properties in heterotypic Cx46/Cx50 cell pairs allowed the determination of a positive relative gating polarity for the dominant gating mechanisms in Cx46 and Cx50. Observed gating properties were consistent with a second gating mechanism in Cx46 connexons. Moreover, rectification was observed in heterotypic cell pairs. Some cell pairs in cultures simultaneously transfected with Cx46 and Cx50 exhibited junctional properties not observed in other preparations, suggesting the formation of heteromeric channels. We conclude that different combinations of Cx46 and Cx50 within gap junction channels lead to unique biophysical properties.     

Cx43/beta-gal inhibits Cx43 transport in the Golgi apparatus

A connexin construct consisting of bacterial beta-galactosidase fused to the C-terminus of connexin43 (Cx43/beta-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/beta-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/beta-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/beta-gal were assembled into a sub-hexameric complex, and that Cx43/beta-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/beta-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.     

Cx43/β-Gal Inhibits Cx43 Transport in the Golgi Apparatus

A connexin construct consisting of bacterial β-galactosidase fused to the C-terminus of connexin43 (Cx43/β-gal) was used to examine Cx43 assembly in NIH 3T3 cells. Cx43/β-gal is retained in a perinuclear compartment and inhibits Cx43 transport to the cell surface. The intracellular connexin pool trapped by Cx43/β-gal was retained in a compartment that co-localized with a medial Golgi apparatus marker by immunofluorescence microscopy and that was readily disassembled by treatment with brefeldin A. Further analysis by sucrose gradient fractionation showed that Cx43 and Cx43/β-gal were assembled into a sub-hexameric complex, and that Cx43/β-gal expression also inhibited Cx43 assembly into hemichannels. While this is consistent with Cx43 hemichannel assembly in the trans Golgi network (TGN), these data also suggest that the dominant negative effect of Cx43/β-gal on Cx43 trafficking may reflect a putative sub-hexameric assembly intermediate formed in the Golgi apparatus.     

Connexin 43 hemichannels mediate the Ca2+ influx induced by extracellular alkalinization

Although alkaline pH is known to trigger Ca(2+) influx in diverse cells, no pH-sensitive Ca(2+) channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca(2+)/Mg(2+) concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca(2+) concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La(3+)). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca(2+) levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca(2+) and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca(2+) permeable, suggesting that Ca(2+) influx through Cx43 HCs could account for the elevation in intracellular Ca(2+) elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca(2+) influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.     

Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells

In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G(1)/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G(1). Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G(1) and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G(1)/S checkpoint.     

Mouse Horizontal Cells do not Express Connexin26 or Connexin36

Gap junctions between neurons function as electrical synapses, and are present in all layers of mammalian and teleost retina. These synapses are largest and most prominent between horizontal cells where they function to increase the receptive field of a single neuron beyond the width of its dendrites. Receptive field size and the extent of gap junctional coupling between horizontal cells is regulated by ambient light levels and may mediate light/dark adaptation. Furthermore, teleost horizontal cell gap junction hemichannels may facilitate a mechanism of feedback inhibition between horizontal cells and cone photoreceptors. As a prelude to using mouse genetic models to study horizontal cell gap junctions and hemichannels, we sought to determine the connexin complement of mouse horizontal cells. Cx36, Cx37, Cx43, Cx45 and Cx57 mRNA could be detected in mouse retina by RT-PCR. Microscopy was used to further examine the distribution of Cx26 and Cx36. Cx26 immunofluorescence and a β-gal reporter under regulatory control of the Cx36 promoter did not colocalize with a horizontal cell marker, indicating that these genes are not expressed by horizontal cells. The identity of the connexin(s) forming electrical synapses between mouse horizontal cells and the connexin that may form hemichannels in the horizontal cell telodendria remains unknown.     

Molecular cloning, functional analysis, and RNA expression analysis of connexin45.6: a zebrafish cardiovascular connexin

In the vertebrate cardiovascular system, gap junctions function in intercellular communication essential for both the coordinated propagation of the heartbeat and the control of vasomotor responses in the vascular system. Connexins, the protein subunits of gap junctions, are coded by a multigene family. In this study, a connexin gene (zfCx45.6), which exhibits 53% amino acid identity to chick Cx42, was cloned from zebrafish genomic DNA. With the use of the LN54 radiation hybrid panel, zfCx45.6 was mapped to zebrafish linkage group 9. Northern blots and RT-PCR revealed the presence of zfCx45.6 mRNA in the embryo before 2 h postfertilization (hpf) and then again beginning at about 12 hpf, after which time no major changes in relative expression levels were detected. In the adult, zfCx45.6 mRNA continued to be detected in the heart, as well as the brain, liver, and ovary, but not the lens. Whole mount in situ hybridization revealed zfCx45.6 mRNA was expressed at high levels in the major vessels of the entire embryo and in both the atrium and ventricle of the adult heart. Expression of zfCx45.6 channels in paired Xenopus oocytes produced high levels of intercellular coupling that was voltage sensitive. With the previous isolation of zebrafish Cx43 and Cx43.4, zebrafish orthologues have now been isolated for three of the four connexins expressed in the mammalian cardiovascular system.     

Connexin 32 mutations from X-linked Charcot-Marie-Tooth disease patients: functional defects and dominant negative effects.

We have characterized the function of connexin (Cx) 32 gene mutations found in X-linked dominant Charcot-Marie-Tooth disease with respect to their ability to form functional gap junctions among themselves and to inactivate wild-type Cx32 by a dominant negative mechanism. We prepared four types of Cx32 mutant cDNAs and transfected them into HeLa cells, which do not show detectable levels of gap junctional intercellular communication (GJIC), nor expression of any connexins examined. Cells transfected with the wild-type Cx32 gene, but not those transfected with three different base substitution mutations (i.e. Cys 60 to Phe, Val 139 to Met, and Arg 215 to Trp), restored GJIC. Unexpectedly, in cells transfected with a nonsense mutant at codon 220, there was also restored GJIC. When we double-transfected these mutant constructs into the HeLa cells that had already been transfected with the wild-type Cx32 gene and thus were GJIC proficient, three base substitution mutants inhibited GJIC, suggesting that these three mutants can eliminate the function of wild-type Cx32 in a dominant negative manner. The nonsense mutation at codon 220 did not show such a dominant negative effect. Since both mutant and wild-type Cx32 mRNAs were detected, but only poor Cx32 protein expression at cell-cell contact areas was observed in the double transfectants, it is suggested that certain mutants form nonfunctional chimeric connexons with wild-type connexins, which are not properly inserted into the cytoplasmic membrane.