1. Whole-cell patch clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study the biophysical properties of a calcium current activated by depletion of intracellular calcium stores in rat peritoneal mast cells. 2. Calcium influx through an inward calcium release-activated calcium current (I(CRAC)) was induced by three independent mechanisms that result in store depletion: intracellular infusion of inositol 1,4,5-trisphosphate (InsP3) or extracellular application of ionomycin (active depletion), and intracellular infusion of calcium chelators (ethylene glycol bis-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)) to prevent reuptake of leaked-out calcium into the stores (passive depletion). 3. The activation of I(CRAC) induced by active store depletion has a short delay (4-14 s) following intracellular infusion of InsP3 or extracellular application of ionomycin. It has a monoexponential time course with a time constant of 20-30 s and, depending on the complementary Ca 2+ buffer, a mean normalized amplitude (at 0 mV) of 0.6 pA pF-1 (with EGTA) and 1.1 pA pF-1 (with BAPTA). 4. After full activation of I(CRAC) by InsP3 in the presence of EGTA (10 mm), hyperpolarizing pulses to - 100 mV induced an instantaneous inward current that decayed by 64% within 50 ms. This inactivation is probably mediated by [Ca 2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (I 0 mm) was only 30 %. 5. The amplitude of I(CRAC) was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (K(D)) of 3.3 mm. Inward currents were non-saturating up to - 200 mV. 6. The selectivity of I(CRAC) for Ca 2+ was assessed by using fura-2 as the dominant intracellular buffer (at a concentration of 2 mm) and relating the absolute changes in the calcium-sensitive fluorescence (390 nm excitation) with the calcium current integral. This relationship was almost identical to the one determined for Ca2+ influx through voltage-activated calcium currents in chromaffin cells, suggesting a similar selectivity. Replacing Na+ and K+ by N-methyl-D-glucamine (with Ca 2+ ions as exclusive charge carriers) reduced the amplitude of I(CRAC) by only 9% further suggesting a high specificity for Ca 2+ ions. 7. The current amplitude was not greatly affected by variations of external Mg2+ in the range of 0-12 mm. Even at 12 mm Mg2+ the current amplitude was reduced by only 23 %. 8. I(CRAC) was dose-dependently inhibited by Cd2+. The concentration-response relationship for Cd 2+ Could be described by a Michaelis-Menten function with an apparent K(D) of 0-24 mm and a Hill coefficient of 1. 9. All other tested divalent ions also dose-dependently and reversibly inhibited I(CRAC). The order of potency was determined by the relative blocking efficacy of 1 mm of the respective ions: Ba2+ almost-equal-to Sr2+ < Ni2+ < Mn 2+ almost-equal-to Co2+ almost-equal-to Be2+ < Cd 2+ < Zn2+. The trivalent ion La 3+ was the most potent blocker of I(CRAC). 10. I(CRAC) excluded monovalent ions in the presence of divalent ions. Complete removal of divalent ions typically resulted in a triphasic conductance change: an initial decrease in the calcium current, an abrupt increase in inward current with modest inward rectification due to passage of monovalent ions, and a subsequent decrease in total current with a linear current-voltage relationship. At the same time, these changes were accompanied by a shift in the reversal potential from > + 50 to 0 mV. 11. While all the features of I(CRAC) are compatible with an ion channel mechanism, there was no significant increase in current noise associated with its activation. 12. Our results suggest that the calcium current activated by depletion of intracellular calcium stores is a highly selective pathway for calcium entry into mast cells and may constitute one of the mechanisms underlying the plateau phase of elevated cytosolic calcium concentration following receptor-mediated release of intracellular calcium.
