Calcium release‐activated calcium current in rat mast cells.

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 (ICRAC) 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 ICRAC 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 Ca2+ 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 ICRAC 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 [Ca2+]i, since the decrease of inward current in the presence of the fast Ca2+ buffer BAPTA (10 mM) was only 30%. 5. The amplitude of ICRAC was dependent on the extracellular Ca2+ concentration with an apparent dissociation constant (KD) of 3.3 mM. Inward currents were nonsaturating up to ‐200 mV. 6. The selectivity of ICRAC for Ca2+ 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 Ca2+ ions as exclusive charge carriers) reduced the amplitude of ICRAC by only 9% further suggesting a high specificity for Ca2+ 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. ICRAC was dose‐dependently inhibited by Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)