CALCIUM RELEASE-ACTIVATED CALCIUM CURRENT IN RAT MAST-CELLS
CALCIUM RELEASE-ACTIVATED CALCIUM CURRENT IN RAT MAST-CELLS
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DOI：
10.1113/jphysiol.1993.sp019681
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发表时间：
1993-06-01
影响因子：
5.5
通讯作者：
PENNER, R
中科院分区：
文献类型：
--
作者：
HOTH, M;PENNER, R
1.采用全细胞膜片钳记录膜电流和fura-2测定细胞内游离钙离子浓度（[Ca 2 +]i），研究大鼠腹腔肥大细胞内钙库耗竭激活的钙电流的生物物理特性.通过内向钙释放激活的钙电流（I（CRAC））的钙内流由三种独立的机制诱导，导致钙池耗竭：细胞内输注1，4，5-三磷酸肌醇（InsP 3）或细胞外应用离子霉素（主动耗竭）和钙螯合剂的细胞内输注（乙二醇双-N，N，N '，N'-四乙酸（EGTA）或1，2-双（2-氨基苯氧基）乙烷-N，N，N '，N'-四乙酸（BAPTA））以防止漏出的钙再摄取到储存中（被动消耗）。在细胞内输注InsP 3或细胞外应用离子霉素后，由主动储存耗尽诱导的I（CRAC）的激活具有短暂的延迟（4-14 s）。它有一个单指数的时间过程，时间常数为20-30秒，取决于互补的Ca 2+缓冲液中，平均归一化幅度（在0 mV时）为0.6 pA pF-1（使用EGTA）和1.1 pA pF-1（使用BAPTA）。在EGTA（10 mm）存在下，InsP 3完全激活I（CRAC）后，超极化脉冲至-100 mV诱导瞬时内向电流，在50 ms内衰减64%。 2+]i，因为在快速Ca 2+缓冲剂BAPTA（I 0 mm）的存在下，内向电流仅降低30%。I（CRAC）的幅度依赖于细胞外Ca 2+浓度，表观解离常数（K（D））为3.3 mm。内向电流不饱和高达-200 mV。通过使用Fura-2作为主要的细胞内缓冲液（浓度为2 mM）并将钙敏感荧光（390 nm激发）的绝对变化与钙电流积分相关联来评估I（CRAC）对Ca 2+的选择性。这种关系是几乎相同的一个确定的钙离子内流通过电压激活的钙电流在嗜铬细胞，表明类似的选择性。用N-甲基-D-葡萄糖胺（含Ca）取代Na+和K+ 2+离子作为唯一的电荷载体）使I（CRAC）的幅度仅降低9%，进一步表明对Ca 2+离子的高特异性。在0-12 mm范围内，Mg ~（2+）浓度的变化对电流幅值的影响不大，即使在12 mm Mg 2+使电流振幅仅降低23%。8. I（CRAC）呈剂量依赖性地被Cd ~（2+）抑制。Cd 2+的浓度-反应关系可用米氏函数描述，表观K（D）为0-24 mm，Hill系数为1.9。所有其他测试的二价离子也剂量依赖性和可逆地抑制I（CRAC）。根据各离子1 mm的相对阻断效能，确定了其作用顺序为：Ba 2+几乎等于Sr 2 + <Ni 2 + < Mn 2+几乎等于Co 2+几乎等于Be 2 + < Cd 2+ <Zn 2+。三价离子La 3+是I（CRAC）最有效的阻滞剂。10. I（CRAC）在二价离子存在下排除单价离子。完全去除二价离子通常会导致三相电导变化：钙电流的初始降低，由于单价离子的通过而导致内向电流的突然增加和适度的内向整流，以及随后的总电流的线性电流-电压关系的降低。同时，这些变化伴随着逆转电位从>+50到0 mV的偏移。虽然I（CRAC）的所有功能都与离子通道机制兼容，但与其激活相关的电流噪声没有显着增加。我们的研究结果表明，细胞内钙库的耗竭激活的钙电流是一个高度选择性的钙进入肥大细胞的途径，并可能构成的机制之一的高原期升高的胞浆钙浓度受体介导的细胞内钙释放。
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.