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.

Fig. 1

Expression of TRPC1 in retinal cells. RT-PCR products (∼ 500-base pair region) amplified from mRNA isolated from fresh bovine retina (A) or cultured ARPE cells (B). TRPC specific forward and reverse primers were used along with 0.5 μg of mRNA for 25 cycles. C, D: Detection of TRPC1 protein (from bovine heart, liver, kidney, brain, retina, and ARPE cells) using anti-TRPC1 antibodies. Conditions for SDS-polyacrylamide gel electrophoresis and western blot analysis were the same as described in Singh et al. (2001). The TRPC1 protein is indicated by an arrow. E, F,G: Localization of TRPC1 in ARPE cells detected using anti-TRPC1 antibody followed by incubation with rhodamine-linked anti-rabbit IgG (F). No fluorescence was detected when the primary antibody was not added (E). Localization in ARPE cells over expressing TRPC1 detected using anti-HA antibodies (G)

Fig. 2

Interaction of TRPC1 with β-tubulin. A: Yeast two-hybrid interactions between the N (aa 1-349) or C-terminus (aa 643-793) of TRPC1 (NTRPC1 and CTRPC1) and β-tubulin protein. Quantitative β-gal assay were 18 and 25 for NTRPC1/tubulin and for the positive controls, 0 indicates no β-gal activity. B: Proteins identified by mass spectrometer, TRPC1-specific immunoprecipitate was trypsined and individual peptides were sequenced and matched against the protein data bank.

Fig. 3

Immunoprecipitation and co-localization of TRPC1 with tubulin. A: Co-immunoprecipitation of TRPC1 with the cytoskeletal protein tubulin. Crude membranes prepared from ARPE cells transiently expressing HA-TRPC1 were solubilized and immunoprecipitation was performed as described in experimental procedures. Proteins in the IP were detected using SDS PAGE and immunoblotting. Antibodies used for IP and IB are indicated in the figure. Control IPs is shown by crude membranes of HA-TRPC1 expressing ARPE cells. B: Co-localization of HA-TRPC1 with endogenous tubulin proteins in ARPE cells. Anti-HA antibody, anti tubulin antibody, and FITC or rhodamine-conjugated secondary antibodies were used to detect transiently expressed HA-TRPC1 and endogenous tubulin in ARPE cells. Overlay of the images is shown in the right panel (yellow signal). Confocal pictures of the transiently expressing HA-TRPC1 and tubulin proteins in ARPE cells treated with colchicine (lower panels). Arrows in the images show the protein localization.

Fig. 4

Colchicine treatment decrease thapsigargin stimulated TRPC1 activity. Ca²⁺ influx were measured in Tg-stimulated cells in a Ca²⁺-free buffer, followed by addition of 1 mM Ca²⁺ to the medium. A, C: Fluorescence traces in either control ARPE cells (A) or colchine-treated ARPE cells (B) stimulated with Tg. B, D: Bar graphs showing the relative Ca²⁺ influx in the absence and in the presence of extracellular Ca²⁺. “*” indicate values that are significantly different from that of the respective control condition (P < 0.02, number of cells is indicated in each case). Internal Ca²⁺ release and basal Ca²⁺ influx were not altered by colchine treatment.