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)
Hoth
M
M
Department of Membrane Biophysics, Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany.
Penner
R
R
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000187:drug effects / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002414:Cations, Monovalent / Q000378:metabolism / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D016257:Fura-2; D007293:Inosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D008564:Membrane Potentials / Q000502:physiology; D010529:Peritoneal Cavity / Q000166:cytology; D051381:Rats; D017208:Rats, Wistar
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002413:Cations, Divalent; D002414:Cations, Monovalent; D002614:Chelating Agents; D007293:Inosine Triphosphate; D002118:Calcium; D016257:Fura-2