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.     

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.     

Characterization of the internal IRES element of the zebrafish connexin55.5 reveals functional implication of the polypyrimidine tract binding protein

Background  

Connexin55.5 (Cx55.5) is a gap junction protein with horizontal cell-restricted expression in zebrafish accumulating at dendritic sites within the receptor-horizontal cell complex in form of hemichannels where light-dependent plasticity occurs. This connexin is the first example of a gap junction protein processed to form two protein isoforms from a monocistronic message by an IRES mediated process. The nuclear occurrence of a carboxy-terminal fragment of this protein provides evidence that this gap junction protein may participate in a putative cytoplasmic to nuclear signal transfer.     

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.     

Adenosine-triphosphate-sensitive K+channel (Kir6.1): A novel phosphospecific interaction partner of connexin 43 (Cx43)

Connexin 43 (Cx43) is a phosphoprotein expressed in a wide variety of cells. Cx43 and adenosine-triphosphate-sensitive K⁺channels [K⁺(ATP)] are part of same signaling pathway that regulates cell survival during stress and ischemia preconditioning. Molecular mechanism for their coordinated role in ischemia/hypoxia preconditioning is not well known. Using pull down, co-immunoprecipitation assays and co-localization studies we provide evidence, for the first time that Kir6.1, a K⁺(ATP) channel protein component, can interact with Cx43. Further we show that the interaction was phospho-specific such that Cx43 phosphorylated at serine 262 (S262) interacted with Kir6.1 in preference to the unphosphorylated form of Cx43. Introduction of phospho-deficient mutation at serine 262 (S262A) in Cx43 completely abolished the interaction. Our data provide an interesting lead about a possible partnership between Cx43 and Kir6.1, which will help in better understanding their role in ischemia/hypoxia preconditioning.     

Divalent regulation and intersubunit interactions of human Connexin26 (Cx26) hemichannels

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca²⁺ and Mg²⁺ of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca²⁺. To effect hemichannel closure, the apparent affinity of Ca²⁺ (0.33 mM) is higher than for Mg²⁺ (1.8 mM). Hemichannel closure is accelerated by physiological Ca²⁺ concentrations, but non-physiological concentrations of extracellular Mg²⁺ are required for this effect. Our recent report provided evidence that extracellular Ca²⁺ facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca²⁺ compared with the D50-K61 interaction.     

Mutations in connexin43 (GJA1) perturb bone growth in zebrafish fins

Mechanisms that regulate the size and shape of bony structures are largely unknown. The molecular identification of the fin length mutant short fin (sof), which causes defects in the length of bony fin ray segments, may provide insights regarding the regulation of bone growth. In this report, we demonstrate that the sof phenotype is caused by mutations in the connexin43 (cx43) gene. This conclusion is supported by genetic mapping, reduced expression of cx43 in the original sof allele (sofb123), identification of missense mutations in three ENU-induced alleles, and by demonstration of partially abrogated cx43 function in sofb123 embryos. Expression of cx43 was identified in cells flanking the germinal region of newly growing segments as well as in the osteoblasts at segment boundaries. This pattern of cx43 expression in cells lateral to new segment growth is consistent with a model where cx43-expressing cells represent a biological ruler that measures segment size. This report identifies the first gene identification for a fin length mutation (sof) as well as the first connexin mutations in zebrafish, and therefore reveals a critical role for local cell-cell communication in the regulation of bone size and growth.