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C. Paillart, K. Zhang, T. Rebrik, W. Baehr, J. Korenbrot; Differential expression of CNG channels in fish photoreceptors . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1348.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Rod and cone photoreceptors respond to light with changes in membrane conductance that reflect the activity of cyclic–nucleotide gated channels (CNG channels). A Ca++–dependent, diffusible factor exists in single cone photoreceptors isolated from a fish retina, that modulates CNG channels. As part of a strategy to identify this endogenous factor, we have cloned the rod and cone CNG channels from the fish. Methods:A cDNA library was prepared from striped bass retina mRNA and screened using bovine, chicken (alpha subunit) and mouse (beta subunit) cone photoreceptors CNG channel as probes. Western blot procedure was carried out using antipeptide antibodies in striped bass retina or transfected HEK cells. We performed northern blot and in situ hybridization experiments to specify the presence and find the cellular localization of mRNA transcripts. Single–cell RT–PCR was conducted on striped bass single photoreceptors after their in vitro isolation, using primers specific for each clone. Results:We obtained three full–length cDNA clones, termed Bass A, Bass B and Bass C. Alignment of deduced first two amino acid sequences revealed an overall 61% identity to mammalian rod, cone, and olfactory CNG channel alpha subunits, while Bass C grouped with CNG beta subunits. Specific antibodies identified Bass A as a 65kDa protein and Bass B as a 75kDa protein in striped bass retina and Bass A as a 75kDa protein and Bass B as a 80kDa protein in transfected HEK cells. By retinal RNA blotting, we found one mRNA coding for Bass A (3.4kb) and two mRNAs coding for Bass B (1.8kb and 2.9kb). By in situ hybridization, mRNA transcripts were localized in the photoreceptor layer (ONL), but we could not distinguish rods from cones. Single–cell RT–PCR eliminated this ambiguity as we found that Bass A (and Bass C) is expressed both in single– and twin–cone photoreceptors and Bass B is not expressed in cone photoreceptors. Conclusions:Three different cDNA clones encoding cGMP–gated channel alpha and beta subunits have been characterized from the striped bass retina. Bass A and Bass C (CNGA3 and CNGB3) are expressed in both types of cone photoreceptors and Bass B (CNGA1) is expressed in the rod photoreceptors.
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