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This Article Full Text Full Text (PDF) All Versions of this Article: 293/5/F1476    most recent 00186.2007v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (2) Citing Articles via Google Scholar Google Scholar Articles by Goel, M. Articles by Schilling, W. P. Search for Related Content PubMed PubMed Citation Articles by Goel, M. Articles by Schilling, W. P.

Vasopressin-induced membrane trafficking of TRPC3 and AQP2 channels in cells of the rat renal collecting duct

¹Rammelkamp Center for Education and Research, MetroHealth Medical Center, and ²Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio

Submitted 17 April 2007 ; accepted in final form 8 August 2007

The canonical transient receptor potential channels TRPC3 and TRPC6 are abundantly expressed along with the water channel aquaporin-2 (AQP2) in principal cells of the cortical and medullary collecting duct. Although TRPC3 is selectively localized to the apical membrane and TRPC6 is found in both the apical and basolateral domains, immunofluorescence is often observed in the cytoplasm, suggesting that TRPC3 and TRPC6 may exist in intracellular vesicles and may shuttle to and from the membrane in response to receptor stimulation. To test this hypothesis, the effect of arginine-vasopressin (AVP) on the subcellular distribution of TRPC3, TRPC6, and AQP2 was examined in the rat kidney and in cultured cell lines from the cortical (M1) and inner medullary (IMCD-3) collecting duct. Immunofluorescence analysis revealed that TRPC3, but not TRPC6, colocalized with AQP2 in intracellular vesicles. AVP caused the insertion and accumulation of TRPC3 and AQP2 in the apical membrane but had no effect on the subcellular distribution of TRPC6. TRPC3, but not TRPC6, coimmunoprecipitated with AQP2 from both medulla and M1 and IMCD-3 cell lysates. Apical-to-basolateral transepithelial ⁴⁵Ca²⁺ flux in polarized IMCD-3 cell monolayers was stimulated by diacylglycerol analogs or by the purinergic receptor agonist ATP but not by thapsigargin. Stimulated ⁴⁵Ca²⁺ flux was increased by overexpression of TRPC3 and attenuated by a dominant-negative TRPC3 construct. Furthermore, ⁴⁵Ca²⁺ flux was greatly reduced by the pyrazole-derivative BTP2, a known inhibitor of TRPC3 channels. These results demonstrate that 1) TRPC3 and TRPC6 exist in different vesicle populations, 2) TRPC3 physically associates with APQ2 and shuttles to the apical membrane in response to AVP, and 3) TRPC3 is responsible for transepithelial Ca²⁺ flux in principal cells of the renal collecting duct.

ion channels; renal nephron; immunoprecipitation; immunofluorescence; subcellular localization; Ca²⁺ flux

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