Monovalent Permeability, Rectification, and Ionic Block of Store-operated Calcium Channels in Jurkat T Lymphocytes

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 × 0.64 × 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 (pHi), since raising intracellular pH above 8 reduced the extent of inactivation. In addition, decreasing pHi reduced monovalent and divalent current amplitudes through CRAC channels with a pKa 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 zδ product of 1.88. Ca2+ block from the outside was also found to be voltage dependent with zδ 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+.