Abstract
Large-conductance (BKCa type) Ca2+-activated K+ channels encoded by the Slo1 gene and various canonical transient receptor potential channels (TRPCs) are coexpressed in many cell types, including podocytes (visceral epithelial cells) of the renal glomerulus. In this study, we show by coimmunoprecipitation and GST pull-down assays that BKCa channels can associate with endogenous TRPC3 and TRPC6 channels in differentiated cells of a podocyte cell line. Both types of TRPC channels colocalize with Slo1 in podocytes and in human embryonic kidney (HEK) 293T cells transiently coexpressing the TRPC channels with Slo1. In HEK293T cells, coexpression of TRPC6 increased surface expression of a Slo1 subunit splice variant (Slo1VEDEC) that is typically retained in intracellular compartments, as assessed by cell-surface biotinylation assays and confocal microscopy. Corresponding currents through BKCa channels were also increased with TRPC6 coexpression, as assessed by whole-cell and excised inside-out patch recordings. By contrast, coexpression of TRPC3 had no effect on the surface expression of BKCa channels in HEK293T cells or on the amplitudes of currents in whole cells or excised patches. In podocytes, small interfering RNA knockdown of endogenous TRPC6 reduced steady-state surface expression of endogenous Slo1 channels, but knockdown of TRPC3 had no effect. TRPC6, but not TRPC3 knockdown also reduced voltage-evoked outward current through podocyte BKCa channels. These data indicate that TRPC6 and TRPC3 channels can bind to Slo1, and this colocalization may allow them to serve as a source of Ca2+ for the activation of BKCa channels. TRPC6 channels also play a role in the regulation of surface expression of a subset of podocyte BKCa channels.
Footnotes
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This work was supported by a Grant to Enhance and Advance Research (GEAR) from the University of Houston. The monoclonal antibody against Slo1 (clone L6/60) was obtained from the University of California Davis/National Institute of Neurological Disorders and Stroke/National Institute of Mental Health NeuroMab Facility, supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grant U24-NS050606].
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ABBREVIATIONS: BKCa channels, large-conductance Ca2+-activated K+ channels; HEDTA, N-hydroxy-EDTA; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride; Slo1, pore-forming subunit of large-conductance Ca2+-activated K+ channels; TRPC, canonical transient receptor potential channels; WGA, wheat germ agglutinin; HEK, human embryonic kidney; siRNA, small interfering RNA; FITC, fluorescein isothiocyanate.
- Received September 9, 2008.
- Accepted December 3, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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