Regulation of Polycystin-2 TRPP2 Associated Cation Currents in the Renal Epithelial Cell Line LLC-PK1 by the Calcium-Sensing Receptor

Abstract

Polycystin-2 (PC2, TRPP2) is a nonselective cation channel that contributes to Ca2+ transport and cell signaling in renal epithelia and other tissues. Little is known however, as to how Ca2+ triggers regulatory mechanisms that control PC2 channel function. In this study, we explored the effect external Ca2+ has on endogenous PC2 channel function in wild type LLC-PK1 renal epithelial cells. Whole cell currents were obtained with the voltage clamping technique at different external Ca2+ concentrations, and observed that the basal whole cell conductance in normal Ca2+ (1.2 mM), decreased by 30.2% in zero (nominal) Ca2+, while it increased by 38% in the presence of high external Ca2+ (6.2 mM). Exposure to high Ca2+ also was associated with PC2 relocation to the plasma membrane. The high Ca2+-induced whole cell currents were inhibited by intracellular dialysis with active, but not denatured, anti-PC2 antibody. To confirm the possible role of a Ca2+ sensing receptor (CaR) on the external Ca2+ modulation of PC2 currents, other maneuvers were also tested, including the CaR agonist spermine, which stimulated the whole cell currents under Ca2+-free conditions, and gentamicin, which also increased the PC2-associated whole-cell currents in normal external Ca2+. Conversely, intracellular dialysis with ketamine used to inhibit the CaR, eliminated both the high Ca2+, and gentamicin-induced whole cell currents of LLC-PK1 cells. The presence of CaR was confirmed by immunocytochemistry as well as its external Ca2+ dependent colocalization with PC2. The data support a novel mechanism for the regulation of PC2 by external Ca2+, involving the Ca2+-sensing receptor. This regulation by Ca2+ may have important implications to the physiology of renal epithelial cells.