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Z. Sun, M. Kamermans, D. G. McMahon; Connexin Gene Dependence of Hemi-Gap-Junction Channel Currents in Zebrafish Horizontal Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1040.
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© ARVO (1962-2015); The Authors (2016-present)
Several connexin (Cx) genes are expressed in zebrafish retina, including Cx52.6 and Cx55.5, which are specifically expressed in retinal horizontal cells (HCs). We have previously recorded both outward and inward hemi-gap-junction (HGJ) channel currents elicited from cultured zebrafish HCs. In this study, we examined the relationship between Cx expression and hemichannel current properties in HCs isolated from either Cx55.5 null mutant zebrafish or wild-type (WT) zebrafish treated with anti-Cx55.5 morpholino oligonucleotides (MOs).
Conventional whole-cell voltage clamp was applied to the solitary HCs isolated from zebrafish retinas. In immunocytochemistry (ICC) experiments, primary antibodies against Cx52.6 and Cx55.5 were used at 1:800 and 1:4000 dilutions respectively, and visualized using confocal microscopy. For the MO-mediated Cx knockdown experiments, lissamine tagged anti-Cx55.5 MO was injected into the vitreous space of one eye followed by electroporation. Electroporated retinas were isolated four days later.
Cultured HCs from WT zebrafish were immunoreactive to Cx52.6 and Cx55.5 antibodies with punctate staining on the cell body and dendritic processes. In contrast, Cx55.5 and Cx52.6 immunostaining were almost eliminated in HCs isolated from Cx55.5 mutant zebrafish. Cultured HCs from mutant zebrafish exhibited a significant decrease in inward hemichannel currents evoked at negative potentials but an increase in outward hemichannel currents evoked at positive potentials. We also transiently blocked Cx55.5 expression using MOs. Preliminary data showed that the protein level of Cx55.5 four days after electroporation decreased significantly, while the Cx52.6 protein level was almost unaffected. Interestingly, under these conditions, both outward and inward hemichannel currents were significantly reduced.
Our data show that both Cx52.6 and Cx55.5 proteins are expressed in the cultured WT HCs, and suggest that normal function of both inward and outward hemichannel currents depends on Cx55.5 expression in the zebrafish retina.
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