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HB Nguyen, SL Mills, J O'Brien; Connexins 35 and 34.7 Differ in Function and DIstribution in the Hybrid Bass Retina . Invest. Ophthalmol. Vis. Sci. 2002;43(13):726.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: In bass retina, gap junctions contain two closely related connexins of the gamma group, Cx35 and Cx34.7. This contrasts with the situation in mammalian retina in which Cx36, the gene homologue of Cx35, is the only member of this group expressed. Since gap junctions in neural circuits show a variety of responses to transmitters such as dopamine and nitric oxide, we hypothesized that the two bass connexins are modulated in different ways by second messengers and/or are involved in different neural circuits. Methods: We used immunofluorescence labeling to analyze the distribution of Cx35 and Cx34.7 in hybrid bass retina. We expressed the cloned Cx35 and Cx34.7 cDNAs in HeLa cells and measured gap junctional coupling by intracellular injection of Neurobiotin and visualization with Cy3-streptavidin. Protein kinase A activity in the cultures was modulated by application of cAMP analogs that either activate (Sp-8-cpt-cAMPS) or inhibit (Rp-8-cpt-cAMPS) PKA. Results: A monoclonal antibody to Cx35 labeled gap junctions in both the inner and outer plexiform layers. In the inner plexiform layer, Cx35 labeling occurred in two bands corresponding to the ON and OFF sublaminae. There were abundant small plaques (<2 µm) and sparse large plaques up to 5 µm in length in both sublaminae. In contrast, a polyclonal antiserum to Cx34.7 labeled only minute plaques in the outer plexiform layer. Double label experiments revealed that Cx35 and Cx34.7 do not co-localize, although plaques were in very close proximity in the OPL. Triple label experiments showed that all Cx34.7 and the majority of Cx35 plaques were clustered below cone pedicles. HeLa cells transfected with Cx35 transferred Neurobiotin with a coupling rate 50% higher than untransfected cells, while Cx34.7-transfected cells showed a 30% reduction in coupling. The coupling rate in Cx35-transfected cells was increased by PKA inhibitor and decreased by PKA activator such that an approximately four-fold range of modulation was measured. In contrast, coupling rate was not modulated by cAMP analogs in either Cx34.7-transfected cells or control cells. Conclusions: Cx35 and Cx34.7 proteins occupy different locations in the retina. While Cx35 is widespread, Cx34.7 appears only in a restricted portion of the outer plexiform layer beneath cone pedicles. Furthermore, cAMP-dependent pathways modulate Cx35 and Cx34.7 differently. This suggests that in bass, different connexin types may endow neuronal gap junctions with distinct responses to cAMP.
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