title: Plasma membrane localization and function of TRPC1 is dependent on its interaction with beta-tubulin in retinal epithelium cells.
abstract: Mammalian homologues of the Drosophila canonical Transient Receptor Potential (TRPC) protein have been proposed to encode the store-operated Ca2+ influx (SOC) channel(s). This study examines the role of TRPC1 in the SOC mechanism of retinal cells. htrpc1 transcript was detected in bovine retinal and in human adult retinal pigment epithelial (ARPE) cells. Western blot analysis also confirmed the expression of TRPC1 protein in neuronal cells including retina and ARPE cells. To determine the role of TRPC1 protein in retinal cells, TRPC1 was recombinantly expressed in ARPE cells and changes in intracellular Ca2+ were analyzed. ARPE cells stably transfected with htrp1 cDNA displayed 2-fold higher Ca2+ influx with no significant increase in the basal influx. Consistent with this the overexpressed TRPC1 protein was localized in the plasma membrane region of ARPE cells. Interestingly, both bovine retinal tissues and ARPE cells showed that TRPC1 protein co-localizes and could be co-immunoprecipitated with beta-tubulin. Disruption of tubulin by colchicine significantly decreased both plasma membrane staining of the TRPC1 protein and Ca2+ influx in ARPE cells. These results suggest that TRPC1 channel protein is expressed in retinal cells, further, targeting/retention of the TRPC1 protein to the plasma membrane in retinal cells is mediated via its interaction with beta-tubulin.
authors.affiliation: Department of Biochemistry and Molecular Biology, School of Medicine & Health Sciences, University of North Dakota, Grand Forks, ND 58201, USA.
authors.forename: Sunitha
authors.initials: S
authors.lastname: Bollimuntha
authors.forename: Eric
authors.initials: E
authors.lastname: Cornatzer
authors.forename: Brij B
authors.initials: BB
authors.lastname: Singh
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D018929:Cell Culture Techniques; D002462:Cell Membrane / Q000378:metabolism*; D016257:Fura-2 / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D018613:Microscopy, Confocal; D010857:Pigment Epithelium of Eye / Q000378:metabolism*; D012160:Retina / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050052:TRPC Cation Channels; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection; D014404:Tubulin / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D015220:Calcium Channels; D050052:TRPC Cation Channels; D014404:Tubulin; C096351:transient receptor potential cation channel, subfamily C, member 1; D019284:Thapsigargin; D002118:Calcium; D016257:Fura-2