High calcium transport by Polycystin-2 (TRPP2) induces channel clustering and oscillatory currents

Abstract

The regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. Polycystin-2 (PC2, TRPP2), a member of the TRP channel family (Transient Potential Receptor), is a Ca2+ permeable non-selective cation channel. Previous studies from our laboratory demonstrated that physiological concentrations of Ca2+ do not regulate in vitro translated PC2 (PC2iv) channel activity. However, the issue as to PC2′s Ca2+ permeability and regulation remain ill-defined, in particular because Ca2+ transport is usually observed in the presence of other ionic gradients. In this study, we assessed Ca2+ transport by PC2iv in a lipid bilayer reconstitution system in a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans) in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline and ethanolamine lipid mixtures. Reconstituted PC2iv showed spontaneous Ca2+ currents in both lipid mixtures, with a maximum conductance of 63 ± 13 pS (n = 19) and 105 pS ± 9.8 (n = 9), respectively. In both cases, we best fitted the experimental data with the Goldman-Hodgkin-Katz equation, observing a reversal potential (Vrev ∼ −27 mV) consistent with strict Ca2+ selectivity. The R742X mutated PC2 (PC2R742X), lacking the carboxy terminal domain of the channel showed no differences with wild type PC2. Interestingly, we also observed the onset of spontaneous Ca2+ current oscillations whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the ionic oscillations were highly dependent on the applied voltage, the imposed Ca2+ gradient, and the presence of high Ca2+, which induced PC2 channel clustering as observed by atomic force microscopy (AFM). We also used the QuB suite to kinetically model the PC2 channel Ca2+ oscillations based on the presence of subconductance states in the channel. The encompassed evidence supports a high Ca2+ permeability by PC2, and a novel oscillatory mechanism dependent on the presence of Ca2+ and phospholipids that provides the first evidence for the relation between stochasticity and deterministic processes mediated by ion channels.