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

Abstract

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 M) rapidly abolished, whereas the addition of the microtubular stabilizer paclitaxel (taxol, 15 M) 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.

Specific α- and β-Tubulin Isotypes Optimize the Functions of Sensory Cilia inCaenorhabditis elegans

“…In particular, tubulins can play an important role in linking channel proteins with the cytoskeleton (Li et al 2006a;Bounoutas et al 2009). Many of the neurons in which we observed sensory defects are thought to sense mechanosensory stimuli, including the nose-touch neurons OLQ, ASH, and FLP (Kaplan and Horvitz 1993) (Left) For each genotype, the the entire set of rays is shown.…”

LRRC50, a Conserved Ciliary Protein Implicated in Polycystic Kidney Disease

“…It is surprising that an axonemal dynein-associated protein might underlie the regulation of both types of apical structures, and we therefore speculate LRRC50 might have additional and/or converged functions in vertebrates. Interestingly, polycystin 2 function was recently found to be regulated by both microtubular organization involving IFT component KIF3A 16 and the BASIC RESEARCH www.jasn.org actin cytoskeleton via anchoring with actin bundling protein ␣-actinin. 38 Furthermore, myosin VIIa was shown to be a common component of cilia and renal microvilli in mice.…”

“…Cilia are now perceived as important cellular antennae, and mechanisms underlying ciliogenesis and cilia maintenance have recently become a major focus of research, including systematic bioinformatic screens to define a comprehensive ciliary proteome or ciliome. 4,[11][12][13][14][15][16] In search for novel genes essential for ciliary motility, we performed a forward genetic N-ethyl-N-nitrosourea (ENU) screen in zebrafish. Here, we report the isolation of a mutant with ciliary dyskinesia that develops proliferative kidney cysts.…”

c-Myc Signalling in the Genetic Mechanism of Polycystic Kidney Disease

“…Notably, kidneys in this mouse model displayed substantial increase in c-Myc expression (96). The motor protein kinesin family 3A, Kif3a, mediates intraflagellar transport in the primary cilium and interacts with PC2 (98). Renal inactivation of Kif3a causes loss of primary cilia and formation of renal tubular cysts (60).…”