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Article

Hubert H. Kerschbaum^*, and Michael D. Cahalan^*

From the ^* Department of Physiology and Biophysics, University of California, Irvine, California 92697; and Department of Animal Physiology, University of Salzburg, Institute for Zoology, A-5020 Salzburg, Austria

We used whole-cell recording to characterize ion permeation, rectification, and block of monovalent current through calcium release-activated calcium (CRAC) channels in Jurkat T lymphocytes. Under physiological conditions, CRAC channels exhibit a high degree of selectivity for Ca²⁺, but can be induced to carry a slowly declining Na⁺ current when external divalent ions are reduced to micromolar levels. Using a series of organic cations as probes of varying size, we measured reversal potentials and calculated permeability ratios relative to Na⁺, P_X/P_Na, in order to estimate the diameter of the conducting pore. Ammonium (NH₄⁺) exhibited the highest relative permeability (P_NH4/P_Na = 1.37). The largest permeant ion, tetramethylammonium with a diameter of 0.55 nm, had P_TMA/P_Na of 0.09. N-methyl-D-glucamine (0.50 x 0.64 x 1.20 nm) was not measurably permeant. In addition to carrying monovalent current, NH₄⁺ reduced the slow decline of monovalent current ("inactivation") upon lowering [Ca²⁺]_o. This kinetic effect of extracellular NH₄⁺ can be accounted for by an increase in intracellular pH (pH_i), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pH_i reduced monovalent and divalent current amplitudes through CRAC channels with a pK_a of 6.8. In several channel types, Mg²⁺ has been shown to produce rectification by a voltage-dependent block mechanism. Mg²⁺ removal from the pipette solution permitted large outward monovalent currents to flow through CRAC channels while also increasing the channel's relative Cs⁺ conductance and eliminating the inactivation of monovalent current. Boltzmann fits indicate that intracellular Mg²⁺ contributes to inward rectification by blocking in a voltage-dependent manner, with a z product of 1.88. Ca²⁺ block from the outside was also found to be voltage dependent with z of 1.62. These experiments indicate that the CRAC channel, like voltage-gated Ca²⁺ channels, achieves selectivity for Ca²⁺ by selective binding in a large pore with current–voltage characteristics shaped by internal Mg²⁺.

Key Words: calcium channel • CRAC channel • I_CRAC • ion selectivity • inward rectification

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