PHARMACOLOGY OF A CAPACITATIVE CA2+ ENTRY IN XENOPUS OOCYTES

Abstract We have characterized pharmacological properties of inositol trisphosphate (InsP 3 )-mediated calcium entry pathway in Xenopus oocytes via activation of Ca 2+ -dependent Cl − channels ( I Cl,Ca ) as a sensitive indicator for increase in cytosolic [Ca 2+ ]. This type of Ca 2+ entry mechanism is known as a capacitative Ca 2+ entry (CCE). Voltage-clamped oocytes were maintained in Ca 2+ -free medium and injected with InsP 3 which depleted the InsP 3 -sensitive Ca 2+ stores. 10–20 min later, the oocytes were exposed, at 2–3 min intervals, to 5 mM Ca 2+ -containing medium for 5–10 s which evoked repeated inward Cl − current. No effect of external Ca 2+ was apparent before InsP 3 injection. To determine the pharmacological characteristics of CCE, oocytes were incubated with various chemical agents in Ca 2+ -free solution and exposed to Ca 2+ again in presence of the chemical. It was found that organic Ca 2+ channel blockers were relatively ineffective in blocking CCE while the inorganic Ca 2+ channel blocker La 3+ was most efficient in blocking the current. Attempts to measure conductance increase when the Cl − channels were blocked during activation of Ca 2+ influx were unsuccessful. Therefore we tested the hypothesis that the Ca 2+ influx is mediated via a CaH transporter. Lowering the external pH (to pH 6.5) or application of the protonophore carbonylcyanide p -trifluoromethoxyphenyl hydrazone ( EC 50 = 2 × 10 −8 M) effectively blocked CCE. Since CaH countertransport in the plasma membrane is coupled to Ca 2+ extrusion by Ca-ATPase in vascular smooth muscle we suggest that the capacitative Ca 2+ entry in Xenopus oocytes may possibly arise from slippage of plasma membrane Ca-ATPase coupled to proton countertransport, a mechanism reported in a variety of cells. Ca 2+ slippage may arise from the large Ca 2+ gradient produced by the Ca 2+ depletion protocol.