Equilibrium Ion Selectivity of Human TPC2 Studied using a Plasma Membrane-Targeted Human TPC2 and Spermine Block

Abstract

The permeation pathway of hTPC2 (two-pore channel 2) was studied using a plasma membrane-targeted hTPC2 (hTPC2PM) and the tetravalent cation spermine. We found that intracellular spermine blocks hTPC2PM (Kd=1.5mM) and displays intrinsic voltage sensitivity under zero extracellular Na+ condition. Extracellular Na+ decreases the observed binding energy of spermine, which allowed the determination of equilibrium dissociation constant of the responsible binding site. The contribution of extracellular ions to the effective valence of spermine block suggested that this binding site is located within the voltage field, likely at the selectivity filter external to the spermine-binding site. By analyzing the shifts in spermine binding energy, we arrived at the following sequence of equilibrium ion selectivity (Kd) of this binding site: Li+ (0.72mM) < Na+ (3.7mM) < Ca2+ (9.2mM) < K+ (26mM) < Cs+ (140mM), which suggests a linear decrease in ion binding energy as a function of ionic radius for the tested monovalent ions. This result is contrasted with the selectivity sequence determined using bi-ionic reversal potential, Na+ (1) ≥ Li+ (0.94) ≫ Ca2+ (0.04) ≥ K+ (0.03) ≥ Cs+ (0.02), and suggests that, in addition to equilibrium selectivity, kinetic factors may also be fundamental to the generation of Na+ selectivity in hTPC2.