Markus Hoth

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)

NON-SPECIFIC EFFECTS OF CALCIUM ENTRY ANTAGONISTS IN MAST CELLS

Calcium entry in non-excitable cells occurs through calcium-selective currents activated secondarily to store depletion and/or through non-selective cation channels (e.g., receptor- or second-messenger-activated channels). The driving force for calcium influx can be modified by chloride or potassium channels, which set the membrane potential of cells. Together, these conductances determine the extent of calcium entry. Mast cells are an excellent model system for studying calcium influx, because calcium-release-activated calcium currents (ICRAC), second-messenger-activated non-selective currents and chloride currents are present in these cells. Whole-cell patch-clamp recordings were used to test the Effects of the commonly used calcium entry blockers econazole and SK&F 96365, as well as the antiallergic and anti-inflammatory drugs tenidap, ketotifen and cromolyn on these channels. All tested drugs blocked the three different channel types with a similar order of magnitude (IC50 values ranging from micromolar to millimolar). Hence, these drugs cannot be used to discriminate between different calcium entry mechanisms.

Calcium influx and its control by calcium release

Changes in the concentration of intracellular Ca2+ are crucial for signal transduction in virtually every cell. In the past year, more of the diversity of receptor-mediated Ca2+ influx mechanisms has been shown, and it has been disclosed that one of the most effective Ca2+ influx pathways, known as capacitative Ca2+ entry, occurs via Ca(2+)-selective ion channels in the plasma membrane that are activated following depletion of intracellular Ca2+ stores. Although the exact activation mechanism of capacitative Ca2+ entry still remains a mystery, the identification of plasma membrane currents following store depletion and the characterization of their biophysical properties opens the possibility of unraveling the features and molecular components of the phenomenon of capacitative Ca2+ entry.

Calcium and barium permeation through calcium release-activated calcium (CRAC) channels

A Ca2+ current activated by store depletion has been described recently in several cell types and has been termed ICRAC (for Ca2+ release-activated Ca2+ current). In this paper, the Ca2+ and Ba2+ permeability of CRAC channels is investigated in mast cells, rat basophilic leukaemia cells (RBL) and human T-lymphocytes (Jurkat). The selectivity of CRAC channels for Ca2+ over monovalent cations is identical in all three cell types and is at least as high as that of voltage-operated Ca2+ (VOC) channels in the various tissues tested. The amplitude of Ba2+ currents relative to Ca2+ currents (IBa/ICa) through CRAC channels was found to be strongly dependent on the membrane potential and was much smaller in Jurkat cells compared to mast and RBL cells. An anomalous mole-fraction behavior was observed at very negative membrane potentials in all three cell types when using different mixtures of external Ca2+ and Ba2+. In contrast to VOC channels, the anomalous mole-fraction effect was not observed at potentials positive to−20 mV.

Multiple mechanisms of manganese-induced quenching of fura-2 fluorescence in rat mast cells.

Whole-cell patch-clamp recordings of membrane currents and fura-2 measurements of free intracellular calcium concentration ([Ca2+]i) were used to study Mn2+ influx in rat peritoneal mast cells. The calcium-selective current, activated by depletion of intracellular calcium stores (ICRAC for calcium release-activated calcium current), supports a small but measurable Mn2+ current. In the presence of intracellular BAPTA, a Mn2+ current through ICRAC was recorded in isotonic MnCl2 (100 mM) without a significant quenching of fura-2 fluorescence. Its amplitude was 10% of that measured in physiological solution containing 10 mM Ca2+. However, following store depletion, a significant quenching of fura-2 fluorescence could be measured only when intracellular BAPTA was omitted, so that all the incoming Mn2+ could be captured by the fluorescent dye. Two other ionic currents activated by receptor stimulation also induced Mn2+ quenching of fura-2 fluorescence: a small current through non-specific cation channels of 50-pS unitary conductance and a distinct cationic current of large amplitude. In addition to these influx mechanisms, Mn2+ was taken up into calcium stores and was subsequently co-released with Ca2+ by Ca2+-mobilizing agonists.

Swapping of functional domains in voltage-gated K+ channels

Functionally significant properties of domains in the amino acid sequence of potassium (K+) channel-forming proteins have been investigated by constructing chimeric K+ channels. The N-terminal domain of ShA2 channels was responsible for the fast inactivation (IkA) and also determined a shift in the threshold of activation whereas the membrane domain determined the timecourse of slow inactivation. The binding site for dendrotoxin (DTX), but not for mast cell degranulating peptide (MCDP), is completely located on the loop between the membrane spanning segments S5 and S6 in RCK1 channels. A certain part of this region which has recently been designated as a narrow part of the pore was found to be not responsible for the differences in the single-channel current amplitude between RCK4 and RCK2 K+ channels. Interchange of the C-terminal domain did not influence activation or inactivation of the channels.