Further characteristics of the Ca2+-inactivated Cl- channel in Xenopus laevis oocytes

Removal of extracellular Ca2+ activates ion channels in the plasma membrane of defolliculated oocytes of the South Africa clawed toad Xenopus laevis. At present, there is controversy about the nature of the Ca2+-inactivated ion channels. Recently, we identified one of these channels as a Ca2+-inactivated Cl- channel (CaIC) using single channel analysis. In this work we confirm and extend previous observations on the CaIC by presenting a decisive extension of the regulation and inhibition profile. CaIC current is reversibly blocked by the divalent and trivalent cations Zn2+ (half-maximal blocker concentration, K-1/2 = 8 mu M), Cu2+ (K-1/2 = 120 mu M) and Gd3+ (K-1/2 = 20 mu M). Furthermore, CaIC is inhibited by the specific Cl- channel blocker NPPB (K-1/2 approximate to 3 mu M). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 mu M) but insensitive to its high affinity analogue benzamil (100 mu M). An investigation of the pH-dependence of the CaIC revealed a reduction of currents in the acidic range. Using simultaneous measurements of membrane current (I-m), conductance (G(m)) and capacitance (C-m) we demonstrate that Ca2+ removal leads to instant activation of CaIC already present in the plasma membrane. Since C-m remains constant upon Ca2+ depletion while I-m and G(m) increase drastically, no exocytotic transport of CaIC from intracellular pools and functional insertion into the plasma membrane is involved in the large CaIC currents. A detailed overview of applicable blockers is given. These blockers are useful when oocytes are utilized as an expression system for foreign proteins whose investigations require Ca2+-free solutions and disturbances by CaIC currents are unwanted. We further compare and discuss our results with data of Ca2+-inactivated cation channels reported by other groups.