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P. Sulaiman, R. M. Feddersen, Y. Xu, N. Vardi; Ret-PCP2: A New PCP2 Splice Variant in Retinal on Bipolar Cells Modulates Their Light Response. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5971.
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© ARVO (1962-2015); The Authors (2016-present)
ON bipolar cells respond to glutamate using the metabotropic glutamate receptor mGluR6 and the heterotrimeric G-protein Go. The alpha subunit of Go strongly interacts with Purkinje cell protein 2 (PCP2), thought to be specific to cerebellar Purkinje and retinal rod bipolar cells. In the cerebellum, two different PCP2 RNA transcripts were described, but their function is still unknown. Here we identified the PCP2 isoform in retina and studied its contribution to the light response.
RACE PCR was used to identify the PCP2 transcript in the retina. Western blotting and immunolabelling with anti-PCP2 (antibody raised against the N-terminus) were performed to test expression and localize the protein. Electroretinograms were recorded in the wild type and PCP2 knockout mice to compare their electroretinogram b-waves.
RACE PCR and high resolution Northern blotting revealed that retina expresses a new longer form of PCP2 transcript, thence called ret-PCP2. All three transcripts express exons 2-4, but they differ in their first exon. In retina, the first exon is located upstream of the other splice variants. Consistent with the predicted size of ret-PCP2, Western blotting identified a protein of 15 kDa in retina and 13 kDa in cerebellum. Immunolabeling of retinal slices of transgenic mouse expressing GFP in all ON bipolar cells with anti-PCP2 and anti-PKCα (a rod bipolar cell marker) showed that ret-PCP2 is expressed not only by rod bipolar cells, but also by certain types of ON cone bipolar cells. Notably, ON bipolar cells stratifying at 60-80% of the inner plexiform layer (border with inner nuclear layer is defined as 0%) were unlabeled for PCP2. Staining in ON bipolar dendrites and axons was equally strong. Light responses of the ON bipolar cells were tested by recording the electroretinogram b-wave. Dark-adapted mice stimulated with a saturating flash gave a large b-wave whose rising phase and the initial fast falling phase were similar in wild type and PCP2-null mice. However, the following slow falling phase was significantly slower in the PCP2-null mouse than in the wild type: at 150 ms following the peak, the wild type b-wave declined to 8.5% above the baseline (peak of b-wave is 100%) (n=3), while that of the PCP2-null mouse declined only to 17.0% (n=3; t-test, p<0.005)
Our results show that a new splice variant of PCP2 is present in the majority of retinal ON bipolar cells, and they suggest that PCP2 facilitates the falling phase of the light ON-response.
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