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Tatiana N. Sidorova, David J. Calkins; TRPV Subunit Interactions in the Rodent Retina: Implications for Neurodegeneration in Glaucoma. Invest. Ophthalmol. Vis. Sci. 2012;53(14):6600.
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The transient receptor potential TRP vanilloid (TRPV) channel subfamily (TRPV1-TRPV6) are voltage-gated ion channels important for cellular Ca2+ homeostasis. TRPV subunits can form functional homomeric and heteromeric complexes, with electrophysiological properties that vary depending on subunit composition. Both TRPV1 and TRPV4 have been implicated in disease processes relevant to glaucoma. Here we examine their interaction in rodent retina as a basis for further understanding their roles in degenerative processes.
Co-immunoprecipitation and immunoblotting with specific antibodies were used to determine the interaction of endogenous TRPV1 and TRPV4 proteins in whole rat brain and retina protein extracts. Subcellular localization of proteins was determined by fluorescent immunohistochemistry in rat whole eye sections and whole-mount retina, followed by confocal fluorescence microscopy. Antibodies specific to TRPV1 (R&D System) and TRPV4 (Alomone) were applied together with SMI-31 (Covance), an antibody against phosphorylated neurofilament, a marker for RGCs.
Immmunoprecipitation of TRPV1 immuno complex from whole retina and brain extracts resulted in a band at 90 kDa when immunoblotted with antibodies against TRPV4. Similarly, a TRPV1 band 85 kDa was observed following immunoprecipitation of TRPV4 from retinal and brain extracts. TRPV1 and TRPV4 immunoreactivity co-localized within SMI-31-positive RGCs in whole eye sections and whole-mount retina. TRPV4 localized to the nuclear membrane of all RGCs and labeling appeared uniform. TRPV1 localized to the nuclear membrane and the cytoplasm in RGCs, with intense TRPV1 label observed in some RGCs.
Co-immunoprecipitation and immunohistochemistry indicate that TRPV1 and TRPV4 form a heteromeric protein complex in RGCs. The differences in distribution of TRPV1 and TRPV4 immunoreactivity in the RGC layer suggest functionally different TRPV channels are likely to contribute to RGC signaling in response to disease-relevant stressors in glaucoma.
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