Further Characteristics of the Ca2+-inactivated Cl− Channel in Xenopus laevis Oocytes

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, K1/2= 8 μm), Cu2+ (K1/2= 120 μm) and Gd3+ (K1/2= 20 μm). Furthermore, CaIC is inhibited by the specific Cl− channel blocker NPPB (K1/2≈ 3 μm). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 μm) but insensitive to its high affinity analogue benzamil (100 μm). 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Membrane Biology Springer Journals

Further Characteristics of the Ca2+-inactivated Cl− Channel in Xenopus laevis Oocytes

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Publisher
Springer-Verlag
Copyright
Copyright © Inc. by 1999 Springer-Verlag New York
Subject
Life Sciences; Biochemistry, general; Human Physiology
ISSN
0022-2631
eISSN
1432-1424
D.O.I.
10.1007/s002329900594
Publisher site
See Article on Publisher Site

Abstract

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, K1/2= 8 μm), Cu2+ (K1/2= 120 μm) and Gd3+ (K1/2= 20 μm). Furthermore, CaIC is inhibited by the specific Cl− channel blocker NPPB (K1/2≈ 3 μm). Interestingly, CaIC-mediated currents are further sensitive to the cation channel inhibitor amiloride (500 μm) but insensitive to its high affinity analogue benzamil (100 μm). 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.

Journal

The Journal of Membrane BiologySpringer Journals

Published: Nov 15, 1999

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