Pharmacology of a capacitative Ca²⁺ entry in Xenopus oocytes

We have characterized pharmacological properties of inositol trisphosphate (InsP₃)-mediated calcium entry pathway in Xenopus oocytes via activation of Ca²⁺-dependent Cl^- channels (I(Cl,Ca)) as a sensitive indicator for increase in cytosolic [Ca²⁺]. This type of Ca²⁺ entry mechanism is known as a capacitative Ca²⁺ entry (CCE). Voltage-clamped oocytes were maintained in Ca²⁺-free medium and injected with InsP₃ which depleted the InsP₃-sensitive Ca²⁺ stores. 10-20 min later, the oocytes were exposed, at 2-3 min intervals, to 5 mM Ca²⁺-containing medium for 5-10 s which evoked repeated inward Cl^- current. No effect of external Ca²⁺ was apparent before InsP₃ injection. To determine the pharmacological characteristics of CCE, oocytes were incubated with various chemical agents in Ca²⁺-free solution and exposed to Ca²⁺ again in presence of the chemical. It was found that organic Ca²⁺ channel blockers were relatively ineffective in blocking CCE while the inorganic Ca²⁺ channel blocker La³⁺ was most efficient in blocking the current. Attempts to measure conductance increase when the Cl^- channels were blocked during activation of Ca²⁺ influx were unsuccessful. Therefore we tested the hypothesis that the Ca²⁺ 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 (EC₅₀ = 2 x 10^-8 M) effectively blocked CCE. Since Ca-H countertransport in the plasma membrane is coupled to Ca²⁺ extrusion by Ca-ATPase in vascular smooth muscle we suggest that the capacitative Ca²⁺ entry in Xenopus occytes may possibly arise from slippage of plasma membrane Ca-ATPase coupled to proton countertransport, a mechanism reported in a variety of cells. Ca²⁺ slippage may arise from the large Ca²⁺ gradient produced by the Ca²⁺ depletion protocol.