Ca2+/H+ exchange in the plasma membrane of Arabidopsis thaliana leaves

A large number of plant Ca²⁺/H⁺ exchangers have been identified in endomembranes, but far fewer have been studied for Ca²⁺/H⁺ exchange in plasma membrane so far. To investigate the Ca²⁺/H⁺ exchange in plasma membrane here, inside-out plasma membrane vesicles were isolated from Arabidopsis thaliana leaves using aqueous two-phase partitioning method. Ca²⁺/H⁺ exchange in plasma membrane vesicles was measured by Ca²⁺-dependent dissipation of a pre-established pH gradient. The results showed that transport mediated by the Ca²⁺/H⁺ exchange was optimal at pH 7.0, and displayed transport specificity for Ca²⁺ with saturation kinetics at K _m = 47 μM. Sulfate and vanadate inhibited pH gradient across vesicles and decreased the Ca²⁺-dependent transport of H⁺ out of vesicles significantly. When the electrical potential across plasma membrane was dissipated with valinomycin and potassium, the rate of Ca²⁺/H⁺ exchange increased comparing to control without valinomycin effect, suggesting that the Ca²⁺/H⁺ exchange generated a membrane potential (interior negative), i.e. that the stoichiometric ratio for the exchange is greater than 2H⁺:Ca²⁺. Eosin Y, a Ca²⁺-ATPase inhibitor, drastically inhibited Ca²⁺/H⁺ exchange in plasma membrane as it does for the purified Ca²⁺-ATPase in proteoliposomes, indicating that measured Ca²⁺/H⁺ exchange activity is mainly due to a plasma membrane Ca²⁺ pump. These suggest that calcium (Ca²⁺) is transported out of Arabidopsis cells mainly through a Ca²⁺-ATPase-mediated Ca²⁺/H⁺ exchange system that is driven by the proton-motive force from the plasma membrane H⁺-ATPase.