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Z. Sun, D.-Q. Zhang, M. Kamermans, D. McMahon; Hemi Gap Junction Channels in Zebrafish Horizontal Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3045.
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It has been proposed that hemi gap junction (HGJ) channels of horizontal cells (HCs) formed by connexins (Cxs) participate in the negative feedback from HCs to cones in the vertebrate retina. In this study, we sought to examine the properties of HGJ channels in cultured zebrafish HCs and to test further the possible physiological function and molecular basis of these hemichannel currents.
Solitary HCs were isolated from zebrafish retinas and macroscopic currents were recorded with a conventional whole-cell voltage clamp mode. During the recordings, potassium channels of HCs were blocked by adding potassium channel blockers into intracellular and extracellular solutions. Primary antibodies of rabbit anti-zfCx52.6 (1:800) and rabbit anti-zfCx55.5 (1:4000) were used for immunohistochemistry (ICC) experiments in cultured HCs and the images were visualized using confocal microscopy at excitation wavelengths of 488 nm for Alexa 488.
In this study, both outward and inward hemichannel currents were elicited from the cultured zebrafish HCs at positive and negative voltages respectively. The inward currents induced at negative voltages were physiologically and pharmacologically distinct from outward currents. Increasing the external [Ca2+] suppressed the outward and inward currents with different sensitivities. The Ca2+ dose-response curves yielded a half-maximal inhibitory concentration (IC50) of 0.69 mM for outward currents and 1.58 mM for inward currents. Application of 200 µM quinine enhanced outward currents without affecting inward currents. Cobalt ions have been reported as an inhibitor of negative feedback in the outer retina. We found that external application of 100 µM Co2+ suppressed both outward and inward currents, reducing the outward currents to 46 ± 5% of control (P < 0.001, n = 6) and the inward currents to 53 ± 6% of control (P < 0.001, n = 6). Furthermore, ICC results demonstrated that both zfCx52.6 and zfCx55.5 antibodies labeled HCs with punctuate staining on the cell body and dendritic membranes.
Taken together, our data demonstrate that the distinct inward and outward HGJ currents of zebrafish HCs are both blocked by Co2+ , a blocker of cone/HC feedback, and that both zfCx52.6 and zfCx55.5 proteins are expressed on the HCs in our recording conditions. Future studies are needed to determine if specific Cx subunits mediate these distinct hemichannel currents.
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