Polycystin-2 cation channel function is under the control of microtubular structures in primary cilia of renal epithelial cells.
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 Ca(2+)-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 mum) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 mum) increased ciliary PC2 channel activity. The further addition of alpha-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, alpha-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.
Li
Qiang
Q
Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Montalbetti
Nicolás
N
Wu
Yuliang
Y
Ramos
Arnolt
A
Raychowdhury
Malay K
MK
Chen
Xing-Zhen
XZ
Cantiello
Horacio F
HF
D000818:Animals; D002460:Cell Line; D002923:Cilia / Q000378:metabolism; D003078:Colchicine / Q000494:pharmacology; D004285:Dogs; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000187:drug effects / Q000378:metabolism*; D016547:Kinesins / Q000235:genetics / Q000378:metabolism; D018374:LLC-PK1 Cells; D008051:Lipid Bilayers / Q000378:metabolism; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D017239:Paclitaxel / Q000494:pharmacology; D016891:Polycystic Kidney, Autosomal Dominant / Q000235:genetics / Q000378:metabolism; D013552:Swine; D050396:TRPP Cation Channels / Q000378:metabolism*; D014404:Tubulin / Q000494:pharmacology; D020798:Two-Hybrid System Techniques
D003078:Colchicine; D016547:Kinesins; D008051:Lipid Bilayers; D017239:Paclitaxel; D050396:TRPP Cation Channels; D014404:Tubulin; C087415:KIF3A protein, human