Expression of connexin genes in the human retina

Goran Söhl,Klaus Willecke,Norbert Kociok,Antonia Joussen

doi:10.1186/1471-2415-10-27

Abstract

BackgroundGap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues. Each channel is composed of 12 protein subunits, termed connexins (Cx). In the mouse retina, Cx43 could be localized mostly between astroglial cells whereas expression of Cx36, Cx45 and Cx57 genes has been detected in different neuronal subtypes. In the human retina, however, the expression pattern of connexin genes is largely unknown.MethodsNorthern blot hybridizations, RT-PCR as well as immunofluorescence analyses helped to explore at least partially the expression pattern of the following human connexin genes GJD2 (hCx36), GJC1 (hCx45), GJA9 (hCx59) and GJA10 (hCx62) in the human retina.ResultsHere we report that Northern blot hybridization signals of the orthologuous hCx36 and hCx45 were found in human retinal RNA. Immunofluorescence signals for both connexins could be located in both inner and outer plexiform layer (IPL, OPL). Expression of a third connexin gene denoted as GJA10 (Cx62) was also detected after Northern blot hybridization in the human retina. Interestingly, its gene structure is similar to that of Gja10 (mCx57) being expressed in mouse horizontal cells. RT-PCR analysis suggested that an additional exon of about 25 kb further downstream, coding for 12 amino acid residues, is spliced to the nearly complete reading frame on exon2 of GJA10 (Cx62). Cx59 mRNA, however, with high sequence identity to zebrafish Cx55.5 was only weakly detectable by RT-PCR in cDNA of human retina.ConclusionIn contrast to the neuron-expressed connexin genes Gjd2 coding for mCx36, Gjc1 coding for mCx45 and Gja10 coding for mCx57 in the mouse, a subset of 4 connexin genes, including the unique GJA9 (Cx59) and GJA10 (Cx62), could be detected at least as transcript isoforms in the human retina. First immunofluorescence analyses revealed a staining pattern of hCx36 and hCx45 expression both in the IPL and OPL, partially reminiscent to that in the mouse, although additional post-mortem material is needed to further explore their sublamina-specific distribution. Appropriate antibodies against Cx59 and Cx62 protein will clarify expression of these proteins in future studies.

Highlights

Gap junction channels allow direct metabolically and electrical coupling between adjacent cells in various mammalian tissues
Results from a phylogenetic alignment, indicated that hCx59 (GJA9) might belong to a group of connexin open reading frames (ORFs) of relatively high molecular weight having its closest sequence relationship to zebrafish Cx55.5, with which it was tightly clustered. Both connexin genes are further aligned to a subgroup containing mCx57 (Gja10), porcine Cx60 [55] and hCx62 (GJA10)
In former years research on gap junction mediated signal transmission and its modulation in the mammalian retina focused on three aspects: (i) Coupling of photoreceptor cells and the improvement of signal-to-noise ratio under various light conditions. (ii) Horizontal cell coupling with respect to lateral inhibition and (iii) AIION-cone bipolar cell coupling transferring rod-mediated signals into the cone pathway under mesopic light conditions [42,43]

Summary

Results

(GJA9) and hCx62 (GJA10) Deduced from comparisons of connexin gene pairs, human Cx62 (GJA10) is most likely the orthologuous connexin gene to mouse Cx57 (Gja10) After application of downstream primer DSP3, -4, -5 and -6, the corresponding RT-PCR amplicons failed to emerge in each case (data not shown), indicating that neither the C-terminus (25 amino acid residues) is encoded on exon (as predicted by [41]), nor that the hypothetical transcript isoforms, deduced from data base predictions, are transcribed in figure 2. 25 kb further downstream of hCx62 exon2 At this position, the same 12 amino acid residues [NMLLELSSIMKK] expressed in mCx57 are encoded, coinciding with a proper splice acceptor site (figure 2). Expression of the hCx62 as an uninterrupted coding region on exon ( including 63 amino acid residues downstream of the splice donor site) in human retinal cDNA could not be confirmed. The overall abundance and distribution of the punctate immunopositive staining patterns representing Cx36 and Cx45 protein in the human retina are reminiscent to the corresponding immunofluorescence signals detected in the mouse retina [25,28]

Conclusion

Methods

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