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J. Biol. Chem., Vol. 280, Issue 13, 12908-12916, April 1, 2005

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Apical Localization of a Functional TRPC3/TRPC6-Ca²⁺-Signaling Complex in Polarized Epithelial Cells

ROLE IN APICAL Ca²⁺ INFLUX^*

Bidhan C. Bandyopadhyay, William D. Swaim, Xibao Liu, Robert S. Redman, Randen L. Patterson¶, and Indu S. Ambudkar||

From the Secretory Physiology Section, Gene Therapy and Therapeutics Branch, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, Oral Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, D. C. 20422, and the ^¶Department of Biology, Pennsylvania State University, University Park, Pennsylvania 16802

Receptor-coupled [Ca²⁺]_i increase is initiated in the apical region of epithelial cells and has been associated with apically localized Ca²⁺-signaling proteins. However, localization of Ca²⁺ channels that are regulated by such Ca²⁺-signaling events has not yet been established. This study examines the localization of TRPC channels in polarized epithelial cells and demonstrates a role for TRPC3 in apical Ca²⁺ uptake. Endogenously and exogenously expressed TRPC3 was localized apically in polarized Madin-Darby canine kidney cells (MDCK) and salivary gland epithelial cells. In contrast, TRPC1 was localized basolaterally, whereas TRPC6 was detected in both locations. Localization of G_q/11, inositol 1,4,5-trisphosphate receptor-3, and phospholipase C1 and -2 was also predominantly apical. TRPC3 co-immunoprecipitated with endogenous TRPC6, phospholipase Cs, G_q/11, inositol 1,4,5-trisphosphate receptor-3, and syntaxin 3 but not with TRPC1. Furthermore, 1-oleoyl-2-acetyl-sn-glycerol (OAG)-stimulated apical ⁴⁵Ca²⁺ uptake was higher in TRPC3-MDCK cells compared with control (MDCK) cells. Bradykinin-stimulated apical ⁴⁵Ca²⁺ uptake and transepithelial ⁴⁵Ca²⁺ flux were also higher in TRPC3-expressing cells. Consistent with this, OAG induced [Ca²⁺]_i increase in the apical, but not basal, region of TRPC3-MDCK cells that was blocked by EGTA addition to the apical medium. Most importantly, (i) TRPC3 was detected in the apical region of rat submandibular gland ducts, whereas TRPC6 was present in apical as well as basolateral regions of ducts and acini; and (ii) OAG stimulated Ca²⁺ influx into dispersed ductal cells. These data demonstrate functional localization of TRPC3/TRPC6 channels in the apical region of polarized epithelial cells. In salivary gland ducts this could contribute to the regulation of salivary [Ca²⁺] and secretion.

Received for publication, August 31, 2004 , and in revised form, November 29, 2004.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| To whom correspondence should be addressed: Bldg. 10, Rm. N-113, National Institutes of Health, Bethesda, MD 20892. Tel.: 301-496-5298; Fax: 301-402-1228; E-mail: indu.ambudkar{at}nih.gov.

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