UC San Diego

Ca(2+) currents activated by depletion of Ca(2+) stores in Xenopus oocytes were studied with a two-electrode voltage clamp. Buffering of cytosolic Ca(2+) with EGTA and MeBAPTA abolished I(Cl(Ca)) and unmasked a current in oocytes that was activated by InsP(3) or ionomycin in minutes and by thapsigargin or the chelators themselves over hours. At -60 mV in 10 mM extracellular CaCl(2), the current was typically around -90 or -160 nA in oocytes loaded with EGTA or MeBAPTA, respectively. This current was judged to be a Ca(2+)-selective current for the following reasons: (a) it was inwardly rectifying and reversed at membrane potentials usually more positive than +40 mV; (b) it was dependent on extracellular [CaCl(2)] with K(m) = 11.5 mM; (c) it was highly selective for Ca(2+) against monovalent cations Na(+) and 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) Ca(2+), Sr(2+), and Ba(2+) currents had similar instantaneous conductances, but Sr(2+) and Ba(2+) currents appeared to inactivate more strongly than Ca(2+). This Ca(2+) current was blocked by metal ions with the following potency sequence: Mg(2+) << Ni(2+) approximately Co(2+) approximately Mn(2+) < Cd(2+) << Zn(2+) << La(3+). It was also inhibited by niflumic acid, which is commonly used to block I(Cl(Ca)). PMA partially inhibited the Ca(2+) current, and this effect was mostly abolished by calphostin C, indicating that the Ca(2+) current is sensitive to protein kinase C. These results are the first detailed electrophysiological characterization of depletion-activated Ca(2+) 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 Ca(2+) influx.