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G Matthews, D Henry, S Burke; Cyclic Nucleotide-gated Channels in Retinal Bipolar Neurons . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2917.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Cyclic nucleotide-gated (CNG) channels are involved in a number of physiological functions, possibly including the glutamate response of ON bipolar cells. At least three different genes encode α subunits of CNG channels, but the isoform expressed in bipolar cells has not yet been identified. Thus, we used molecular biological and anatomical approaches to characterize CNG channel subtypes of retinal bipolar cells. Methods: Molecular characterization of CNG channel α subunits expressed in goldfish retina was carried out using RT-PCR. The cellular expression pattern was established with in situ hybridization in intact retina and single isolated cells. The identity of the isoform expressed in particular cell types was then confirmed using single-cell PCR. Physiological experiments were performed using calcium imaging in single bipolar cells. Results: Single-cell RT-PCR from individual cones identified the CNG channel α subunit of goldfish cones. The encoded protein is ∼66% identical to the α subunit of human cone photoreceptors, CNGA3. In situ hybridization in retina sections and in isolated cells confirmed the expression of this channel in goldfish cones. In addition, specific hybridization of the cone-selective probe was observed in ∼50% of cells in the inner nuclear layer (including bipolar and amacrine cells) and in ganglion cells. A similar pattern of in situ hybridization was also observed in mouse retina, using a probe specific for mouse CNGA3. In situ hybridization in single cells showed that ON bipolar cells express the cone CNG channel in goldfish retina. In isolated goldfish bipolar cells, single-cell RT-PCR confirmed the presence of the cone α subunit and identified an additional CNG channel, which was most similar to mammalian olfactory CNG channels. In a subset of isolated goldfish bipolar cells filled with fura-2, glutamate silenced spontaneous calcium action potentials, presumably by activating the cascade underlying the ON response. The phosphodiesterase inhibitor, IBMX, reversed this effect of glutamate, but had no effect on bipolar cells that did not respond to glutamate. Conclusion: In situ hybridization and single-cell RT-PCR demonstrated that the cone CNG channel is expressed in goldfish bipolar cells. The same is likely true in mammalian retina, as indicated by in situ hybridization in mouse retina. A second CNG channel isoform was observed in single-cell PCR in goldfish bipolar cells, but it is not yet clear whether this isoform represents a ß subunit or an alternative α subunit. The reversal of glutamate's action by IBMX is consistent with a role for cyclic nucleotides and CNG channels in the glutamate response of ON bipolar cells.
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