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J. Biol. Chem., Vol. 281, Issue 49, 37566-37575, December 8, 2006

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Polycystin-2 Cation Channel Function Is under the Control of Microtubular Structures in Primary Cilia of Renal Epithelial Cells^*

Qiang Li¹², Nicolás Montalbetti¹, Yuliang Wu, Arnolt Ramos^¶, Malay K. Raychowdhury^¶, Xing-Zhen Chen, Supported by the Canadian Institutes for Health Research, the Alberta Heritage Foundation for Medical Research, and the Kidney Foundation of Canada³, and Horacio F. Cantiello, Funded by the Polycystic Kidney Disease Foundation^¶4

From the Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada, the Laboratorio de Canales Iónicos, Departamento de Fisicoquímica y Química Analítica, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires Buenos Aires 1113, Argentina, and the ^¶Renal Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts 02129

Mutations in the gene encoding polycystin-2 (PC2) result in autosomal dominant polycystic kidney disease and defects in left-right asymmetry during embryogenesis. PC2 is a TRP-type Ca²⁺-permeable non-selective cation channel, which is expressed in kidney and other organs. PC2 is present and functional in microtubule-containing primary cilia of renal epithelial cells. However, no information is yet available as to whether PC2 interacts with microtubules. Here, we assessed the role of microtubular dynamics in regulating PC2 channel function in primary cilia. Isolated ciliary membranes from LLC-PK1 epithelial cells were reconstituted in a lipid bilayer system. The acute addition of the microtubular disrupter colchicine (15 µM) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 µM) increased ciliary PC2 channel activity. The further addition of -tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, -tubulin and GTP had no effect on in vitro translated PC2. Using the yeast two-hybrid assay, we found that PC2 interacts with the microtubule-dependent motor kinesin-2 subunit KIF3A, a protein involved in polycystic kidney disease. The interaction occurred through the carboxyl termini domain of both proteins, which was further confirmed by in vitro glutathione S-transferase pull-down and dot blot overlay assays. Co-immunoprecipitation experiments showed that PC2 and KIF3A are in the same complex in native HEK293, Madin-Darby canine kidney cells (MDCK), and LLC-PK1 cells. Immunofluorescent staining also showed substantial PC2 and KIF3A co-localization in primary cilia of renal epithelial cells. The data indicate that microtubular organization regulates PC2 function, which may explain, among others, the regulatory role of PC2 in the sensory function of primary cilia.

Received for publication, April 17, 2006 , and in revised form, August 28, 2006.

^* 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.

¹ Both authors contributed equally to this work.

² A recipient of the Kidney Foundation of Canada Biomedical Fellowship.

³ To whom correspondence may be addressed. E-mail: xzchen{at}ualberta.ca. ⁴ To whom correspondence may be addressed: Renal Unit, Massachusetts General Hospital East, 149 13th St., Charlestown, MA 02129. E-mail: cantiello{at}helix.mgh.harvard.edu.

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