High Calcium Transport by Polycystin-2 (TRPP2) Induces Channel Clustering and Coordinated Oscillatory Behavior

Abstract

AbstractThe 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 this study, we assessed Ca2+ transport by PC2iv, in a lipid bilayer reconstitution system in the presence of a high Ca2+ gradient (CaCl2 100 mM cis, CaCl2 10 mM trans). PC2iv channel reconstitution was conducted in the presence of either 3:7 or 7:3 1-palmitoyl-2-oleoyl-choline (POPC) and ethanolamine (POPE) 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, experimental data were best fitted with the Goldman-Hodgkin-Katz equation, showing 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, spontaneous Ca2+ current oscillations were observed whenever PC2-containing samples were reconstituted in the 3:7, but not 7:3 POPC:POPE lipid mixture. The amplitude and frequency of the 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 data provide new evidence to support a high Ca2+ permeability by PC2, and a novel regulatory feedback mechanism dependent on the presence of Ca2+ and phospholipids on its function.Statement of SignificanceThe regulation by Ca2+ of Ca2+-permeable ion channels represents an important mechanism in the control of cell function. The Transient Potential Receptor channel Polycystin-2 (TRPP2, PC2), is a Ca2+ permeable non-selective cation channel. Ca2+ transport by PC2 has largely been inferred by changes in reversal potential. This study provides experimental evidence on the Ca2+-transporting capabilities of PC2 in high Ca2+ that is modulated by lipids and generates a novel phenomenon of oscillatory currents by channel clustering and multiple subconductance behavior. PC2 can be self-regulated by feedback mechanisms, which are independent of external regulatory proteins. This oscillatory behavior, previously unknown for a single channel species, depend on the presence of Ca2+ interaction sites as have been postulated for the channel protein.