Calcium current activated by depletion of calcium stores in Xenopus oocytes

Abstract

Ca2+ currents activated by depletion of Ca2+ stores in Xenopus oocytes were studied with a two-electrode voltage clamp. Buffering of cytosolic Ca2+ with EGTA and MeBAPTA abolished I(Cl(Ca)) and unmasked a current in oocytes that was activated by InsP3 or ionomycin in minutes and by thapsigargin or the chelators themselves over hours. At -60 mV in 10 mM extracellular CaCl2, the current was typically around -90 or -160 nA in oocytes loaded with EGTA or MeBAPTA, respectively. This current was judged to be a Ca2+ -selective current for the following reasons; (as) it was inwardly rectifying and reversed at membrane potentials usually more positive than +40 mV; (b) it was dependent on extracellular [CaCl2] with K(m) = 11.5 mM; (c) it was highly selective for Ca2+ against monovalent cations Na+ K+, because replacing Na+ and K+ by N-methyl-D-glucammonium did not reduce the amplitude or voltage dependence of the current significantly; and (d) Ca2+, Sr2+, and Ba2+ currents had similar instantaneous conductances, but Sr2+ and Ba2+ currents appeared to inactivate more strongly than Ca2+. This Ca2+ current was blocked by metal ions with the following potency sequence; Mg2+ << Ni2+ ≃ Co2+ ≃ Mn2+ < Cd2+ << Zn2+ << La3+. It was also inhibited by niflumic acid, which is commonly used to block I(Cl(Ca)). PMA partially inhibited the Ca2+ current, and this effect was mostly abolished by calphostin C, indicating that the Ca2+ current is sensitive to protein kinase C. These results are the first detailed electrophysiological characterization of depletion-activated Ca2+ current in nondialyzed cells. Because exogenous molecules and channels are easy to introduce into oocytes and the distortions in measuring I(Cl(Ca)) can now be bypassed, oocytes are now a superior system in which to analyze the activation mechanisms of capacitative Ca2+ influx.