TRPP2 and TRPV4 Form an EGF-Activated Calcium Permeable Channel at the Apical Membrane of Renal Collecting Duct Cells

Abstract

ObjectiveRegulation of apical calcium entry is important for the function of principal cells of the collecting duct. However, the molecular identity and the regulators of the transporter/channel, which is responsible for apical calcium entry and what factors regulate the calcium conduction remain unclear.Methods and ResultsWe report that endogenous TRPP2 and TRPV4 assemble to form a 23-pS divalent cation-permeable non-selective ion channel at the apical membrane of renal principal cells of the collecting duct. TRPP2\TRPV4 channel complex was identified by patch-clamp, immunofluorescence and co-immunprecipitation studies in both principal cells that either possess normal cilia (cilia (+)) or in which cilia are absent (cilia (-)). This channel has distinct biophysical and pharmacological and regulatory profiles compared to either TRPP2 or TRPV4 channels. The rate of occurrence detected by patch clamp was higher in cilia (-) compared to cilia (+) cells. In addition, shRNA knockdown of TRPP2 increased the prevalence of TRPV4 channel activity while knockdown of TRPV4 resulted in TRPP2 activity and knockdown of both proteins vastly decreased the 23-pS channel activity. Epidermal growth factor (EGF) stimulated TRPP2\TRPV4 channel through the EGF receptor (EGFR) tyrosine kinase-dependent signaling. With loss of cilia, apical EGF treatment resulted in 64-fold increase in channel activity in cilia (-) but not cilia (+) cells. In addition EGF increased cell proliferation in cilia (-) cell that was dependent upon TRPP2\TRPV4 channel mediated increase in intracellular calcium.ConclusionWe conclude that in the absence of cilia, an EGF activated TRPP2\TRPV4 channel may play an important role in increased cell proliferation and cystogenesis.

Epidermal growth factor-induced proliferation of collecting duct cells from Oak Ridge polycystic kidney mice involves activation of Na+/H+ exchanger

“…Therefore, we performed immunofluorescence study in orpk cells to determine the localization of the NHE isoforms in collecting duct cell lines. Similar to a previous report (41), EGFR (green) was predominantly localized in the intracellular compartment and at the basolateral membrane in cilia (ϩ) cells but was apically localized in cilia (Ϫ) cells ( Fig. 2A, yellow arrow pointing apical EGFR).…”

“…We also found that EGFR and NHE-1 were both localized at the apical surface in cilia (Ϫ) cells compared with the cilia (ϩ) cells by immunofluorescence (Fig. 2), which supports findings from previous reports (4,18,41). Interestingly, EGF stimulated NHE activities in cilia (Ϫ) cells compared with cilia (ϩ) cells (Fig.…”

TRPV4 activation mediates flow-induced nitric oxide production in the rat thick ascending limb

“…Nonetheless, it has been suggested that TRPP2, present in the primary cilia, can form heterodimers with TRPV4 to function as a mechanosensitive channel. In this regard, TRPV4 has been suggested to be an essential component of the TRPP2-TRPV4 heterodimer (31,69). In fact, depletion of TRPV4 abolishes the ability of renal epithelial cells to sense luminal flow (31).…”

Molecular Pathways and Therapies in Autosomal-Dominant Polycystic Kidney Disease

“…This flow-mediated increase in cell calcium is diminished if either PC1 or PC2 are absent or nonfunctional (8, 87,150). Recent studies suggest that PC2 forms multimeres with either TRPV4 or with TRPC1, which form cation channels with distinct biophysical properties (2, 65,157). The functional consequences of PC2 partnering with other TRP channels in terms of normal flow-mediated calcium responses or in PKD have yet to be determined.…”