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Z. Sun, D. G. McMahon; Contribution of Connexin Isoforms to Native Hemichannel Currents in Zebrafish Retinal Horizontal Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3281.
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© ARVO (1962-2015); The Authors (2016-present)
In zebrafish retina, Cx52.6 and Cx55.5 are expressed specifically in horizontal cells (HCs). In our previous work, expression of Cx52.6 and Cx55.5 in adult zebrafish was transiently blocked using various anti-sense morpholinos (MOs). We found that decreased expression of different connexin isoforms resulted in distinct effects on native hemichannel currents. The MO against Cx55.5 (anti-Cx55.5-MO-I) that was used in previous work was designed to target the first 25 nucleotides of the cx55.5 coding sequence, which is highly homologous to that of the cx52.6 gene. To test a possibility of non-specific effect of anti-Cx55.5-MO-I, in this study, we tested the effect of a second MO against Cx55.5 (anti-Cx55.5-MO-II), which targets the cx55.5 gene at 25 nucleotides within the 5’ untranslated region and shares no sequence similarity with that of cx52.6.
For MO-mediated connexin knockdown, lissamine-tagged anti-Cx55.5-MO-II was injected into the vitreous space of one eye of zebrafish followed by electroporation. Whole cell patch-clamp recording was used to characterize hemichannel currents in untreated and MO-treated HCs four days post-transfection. For immunocytochemistry, primary antibodies against Cx52.6 and Cx55.5 were used at 1:800 and 1:4000, respectively. Specimens were visualized by confocal microscopy.
Four days after electroporation, anti-Cx55.5-MO-II was successfully introduced into multiple layers of the dorsal retina and the Cx55.5 expression decreased significantly, whereas the Cx52.6 expression was almost unaffected. In the Ca2+-free solution, outward hemichannel currents of control HCs elicited by depolarization to +60 mV from 0 mV averaged 337 ± 63 pA (n=6), whereas inward hemichannel currents induced by hyperpolarization to -60 mV averaged 32 ± 4 (n=6) pA. Compared to untreated HCs, the outward and inward hemichannel currents of MO-treated HCs decreased substantially to 31 ± 3 pA (n=4) and 9 ± 2 pA (n = 4), respectively.
Compared to the experiments using anti-Cx55.5-MO-I, similar results were obtained when anti-Cx55.5-MO-II was used, further supporting that MOs against Cx55.5 can be successfully introduced into adult zebrafish, that suppression of Cx55.5 expression can be achieved four days after transfection, and that both outward and inward hemichannel currents are dependent on the Cx55.5 isoform.
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