Malay K. Raychowdhury

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 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 α-tubulin plus GTP also stimulated PC2 channel activity in ciliary membranes. However, α-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.

Vasopressin receptor-mediated functional signaling pathway in primary cilia of renal epithelial cells

The primary cilium of renal epithelial cells is a nonmotile sensory organelle, implicated in mechanosensory transduction signals. Recent studies from our laboratory indicate that renal epithelial primary cilia display abundant channel activity; however, the presence and functional role of specific membrane receptors in this organelle are heretofore unknown. Here, we determined a functional signaling pathway associated with the type 2 vasopressin receptor (V2R) in primary cilia of renal epithelial cells. Besides their normal localization on basolateral membrane, V2R was expressed in primary cilia of LLC-PK(1) renal epithelial cells. The presence of V2R in primary cilia was determined by spontaneous fluorescence of a V2R-gfp chimera and confirmed by immunocytochemical analysis of wild-type LLC-PK(1) cells stained with anti-V2R antibodies and in LLC-PK(1) cells overexpressing the V2R-Flag, with anti-Flag antibody. Ciliary V2R colocalized with adenylyl cyclase (AC) type V/VI in all cell types tested. Functional coupling of the receptors with AC was confirmed by measurement of cAMP production in isolated cilia and by testing AVP-induced cation-selective channel activity either in reconstituted lipid bilayers or subjected to membrane-attached patch clamping. Addition of either 10 microM AVP (trans) or forskolin (cis) in the presence but not the absence of ATP (1 mM, cis) stimulated cation-selective channel activity in ciliary membranes. This channel activity was reduced by addition of the PKA inhibitor PKI. The data provide the first demonstration for the presence of V2R in primary cilia of renal epithelial cells, and a functional cAMP-signaling pathway, which targets ciliary channel function and may help control the sensory function of the primary cilium.

Cation channel activity of mucolipin-1 : the effect of calcium

Mucolipidosis type IV (MLIV) is a rare, neurogenetic disorder characterized by developmental abnormalities of the brain, and impaired neurological, ophthalmological, and gastric function. Considered a lysosomal disease, MLIV is characterized by the accumulation of large vacuoles in various cell types. Recent evidence indicates that MLIV is caused by mutations in MCOLN1, the gene that encodes mucolipin-1 (ML1), a 65-kDa protein showing sequence homology and topological similarities with polycystin-2 and other transient receptor potential (TRP) channels. In this report, our observations on the channel properties of ML1, and molecular pathophysiology of MLIV are reviewed and expanded. Our studies have shown that ML1 is a multiple sub-conductance, non-selective cation channel. MLIV-causing mutations result in functional differences in the channel protein. In particular, the V446L and ΔF408 mutations retain channel function but have interesting functional differences with regards to pH dependence and Ca2+ transport. While the wild-type protein is inhibited by Ca2+ transport, mutant ML1 is not. Atomic force microscopy imaging of ML1 channels shows that changes in pH modify the aggregation and size of the ML1 channels, which has an impact on vesicular fusogenesis. The new evidence provides support for a novel role of ML1 cation channels in vesicular acidification and normal endosomal function.

Morphological and Electrical Properties of Human Trophoblast Choriocarcinoma, BeWo Cells

The syncytiotrophoblast of the human placenta arises from fusion of stem cells called cytotrophoblasts. The molecular mechanisms associated with cell fusion and syncytiation of cytotrophoblastic cells remain largely unknown. In the present study, we investigated the morphological and electrical properties of BeWo cells, a human choriocarcinoma-derived trophoblast cell model, with several features of the human cytotrophoblast. Cultured cells tended to cluster, but only fused into small, multinucleated syncytia in the presence of cAMP (72 h). The morphological features of both the actin and microtubular cytoskeletons indicated that within 72 h of constant exposure to cAMP, intracellular cortical actin cytoskeleton disappeared, which was the most prominent inducing factor of multi-nucleation. The presence of the cation channel protein, polycystin-2 (PC2), a TRP-type cation channel, associated with placental ion transport in term human syncytiotrophoblast, co-localised with acetylated tubulin in midbodies, but was found non-functional under any conditions. Different electrical phenotypes were observed among control BeWo cells, where only 26% (8 of 31 cells) displayed a voltage-dependent outwardly rectifying conductance. Most quiescent BeWo cells had, however, a low, slightly outwardly rectifying basal whole cell conductance. Acute exposure to intracellular cAMP (<15 min) increased the whole cell conductance by 122%, from 0.72 nS/cell to 1.60 nS/cell, and eliminated the voltage-regulated conductance. The encompassed evidence indicates that the early events in BeWo cell fusion and syncytiation occur by cAMP-associated changes in ionic conductance but not morphological changes associated to chronic exposure to the second messenger. This suggests a tight regulation, and important contribution of cation conductances in cytotrophoblastic cells prior to syncytiation.