MEASUREMENT OF REVERSAL POTENTIAL OF NA+-CA2+ EXCHANGE CURRENT IN SINGLE GUINEA-PIG VENTRICULAR CELLS
MEASUREMENT OF REVERSAL POTENTIAL OF NA+-CA2+ EXCHANGE CURRENT IN SINGLE GUINEA-PIG VENTRICULAR CELLS
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DOI：
10.1113/jphysiol.1989.sp017530
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发表时间：
1989-03-01
影响因子：
5.5
通讯作者：
NOMA, A
中科院分区：
文献类型：
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作者：
EHARA, T;MATSUOKA, S;NOMA, A
1.为了将Na+或Ca 2+诱导的电流鉴定为Na+-Ca 2+交换电流并确定Na+-Ca 2+交换的化学计量，在宽范围的外部Na+（[Na+]o）或Ca 2+（[Ca 2 +]o）浓度下测量反转电位。采用全细胞电压钳技术结合内灌注技术，在酶促分离的豚鼠心室肌细胞上记录了Na+或Ca ~（2+）诱导的电流。2.在内部溶液中存在10-40 mM-Na+和55-803 nM-Ca 2+的情况下，[Ca 2 +]o从0.1增加到0.5-20 nM或[Na+]o从30增加到50-140 nM诱导与膜电导增加相关的额外电流。这些额外电流的逆转电位是从在不存在和存在Na+-Ca 2+交换阻滞剂Ni 2+（2 mM）的情况下获得的电流-电压（I-V）关系的相互作用确定的。3.外溶液中的Ba ~（2+）不能诱导额外电流，但抑制背景电导，在0 mV左右具有反转电位。因此，将1 mM-Ba 2+添加到所有外部溶液中，使得在施加Ca 2+或Ni 2+期间背景电流的变化最小化。4.在存在和不存在Ni 2+的情况下，检查[Ca 2 +]o和Ca 2+诱导电流的幅度之间的关系。Lineweaver-Burk分析表明，Ni ~（2+）对该电流的作用可能是竞争性和非竞争性的混合抑制。5.在施加Ca ~（2+）时，Ca ~（2+）诱导的外向电流随时间衰减，导致I-V关系向正电位方向移动。通过使用BAPTA（1，2-双（邻氨基苯氧基）乙烷-N，N，N“，N”-四乙酸）或更高浓度的EGTA增加内部Ca 2+缓冲液的容量来抑制该电流衰减。结果表明，[Ca ~（2+）]i，至少在细胞膜下，由于通过Na ~+-Ca ~（2+）交换的离子通量而改变，并且控制细胞内的离子浓度是测量Na ~+-Ca ~（2+）交换的逆转电位的先决条件。6.当膜电位固定在3 Na+：1Ca ~（2+）交换的平衡电位时，抑制了维持电流和I-V关系的移动。在这些条件下，Ca ~（2+）或Na ~（+）诱导电流的Ni ~（2+）敏感成分显示出与0.2-20 mM [Ca ~（2+）]o和39-140 mM [Na ~（+）]o离子条件下的理论平衡电位一致的反转电位。7.我们的结论是，外部-Ca ~（2+）-或外部-Na ~+-诱导电流是由Na ~+-Ca ~（2+）交换系统产生的，化学计量比为3 Na ~+：1Ca ~（2+）。
1. To identify the Na+- or Ca2+-induced current as Na+-Ca2+ exchange current and to determine the stoichiometry of the Na+-Ca2+ exchange, the reversal potential was measured in a wide range of external Na+ ([Na+]o) or Ca2+ ([Ca2+]o) concentrations. The Na+- or Ca2+-induced current was recorded in single ventricular cells enzymatically dispersed from guinea-pig hearts, using the technique of whole-cell voltage clamp combined with internal perfusion. 2. In the presence of 10-40 mM-Na+ and 55-803 nM-Ca2+ in the internal solution, an increase of [Ca2+]o from 0.1 to 0.5-20 nM or an increase of [Na+]o from 30 to 50-140 nM induced an extra current associated with an increase in membrane conductance. The reversal potential of these extra currents was determined from an interaction of the current-voltage (I-V) relations obtained in the absence and presence of a Na+-Ca2+ exchange blocker, Ni2+ (2 mM). 3. Ba2+ in the external solution failed to induce the extra current, but inhibited the background conductance having a reversal potential at around 0 mV. Thus, 1 mM-Ba2+ was added to all external solutions, so that a change in the background current was minimized during application of Ca2+ or Ni2+. 4. The relation between [Ca2+]o and amplitude of the Ca2+-induced current was examined in the presence and absence of Ni2+. Lineweaver-Burk analysis revealed that the action of Ni2+ on the extra current might be a mixed type of competitive and non-competitive inhibition. 5. During the application of Ca2+, the Ca2+-induced outward current decayed in a time-dependent manner, resulting in a shift of the I-V relations towards positive potentials. This current decay was inhibited by increasing the capacity of the internal Ca2+-buffer, using BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N'',N''-tetraacetic acid) or higher concentrations of EGTA. The result indicates that [Ca2+]i, at least under the cell membrane, changes due to ion fluxes through the Na+-Ca2+ exchange and that control of the ion concentrations within the cell is prerequisite for measuring the reversal potential of the Na+-Ca2+ exchange. 6. The shift of both the holding current and the I-V relations during stimulation of the exchange was suppressed, when the membrane potential was clamped at the equilibrium potential of 3Na+:1Ca2+ exchange. Under these conditions, the Ni2+-sensitive component of the Ca2+- or Na+-induced current showed reversal potentials which were in agreement with theoretical equilibrium potentials under the ionic conditions of 0.2-20 mM [Ca2+]o and 39-140 mM [Na+]o. 7. We conclude that the external-Ca2+- or external-Na+-induced current is generated by the Na+-Ca2+ exchange system and that the stoichiometry is 3Na+:1Ca2+.