Ishihara Keiko, Ehara Tsuguhisa
Department of Physiology, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan.
J Physiol. 2004 Apr 1;556(Pt 1):61-78. doi: 10.1113/jphysiol.2003.055434. Epub 2004 Jan 14.
The strong inward rectifier K(+) current, I(K1), shows significant outward current amplitude in the voltage range near the reversal potential and thereby causes rapid repolarization at the final phase of cardiac action potentials. However, the mechanism that generates the outward I(K1) is not well understood. We recorded currents from the inside-out patches of HEK 293T cells that express the strong inward rectifier K(+) channel Kir2.1 and studied the blockage of the currents caused by cytoplasmic polyamines, namely, spermine and spermidine. The outward current-voltage (I-V) relationships of Kir2.1, obtained with 5-10 microm spermine or 10-100 microm spermidine, were similar to the steady-state outward I-V relationship of I(K1), showing a peak at a level that is approximately 20 mV more positive than the reversal potential, with a negative slope at more positive voltages. The relationships exhibited a plateau or a double-hump shape with 1 microm spermine/spermidine or 0.1 microm spermine, respectively. In the chord conductance-voltage relationships, there were extra conductances in the positive voltage range, which could not be described by the Boltzmann relations fitting the major part of the relationships. The extra conductances, which generated most of the outward currents in the presence of 5-10 microm spermine or 10-100 microm spermidine, were quantitatively explained by a model that considered two populations of Kir2.1 channels, which were blocked by polyamines in either a high-affinity mode (Mode 1 channel) or a low-affinity mode (Mode 2 channel). Analysis of the inward tail currents following test pulses indicated that the relief from the spermine block of Kir2.1 consisted of an exponential component and a virtually instantaneous component. The fractions of the two components nearly agreed with the fractions of the blockages in Mode 1 and Mode 2 calculated by the model. The estimated proportion of Mode 1 channels to total channels was 0.9 with 0.1-10 microm spermine, 0.75 with 1-100 microm spermidine, and between 0.75 and 0.9 when spermine and spermidine coexisted. An interaction of spermine/spermidine with the channel at an intracellular site appeared to modify the equilibrium of the two conformational channel states that allow different modes of blockage. Our results suggest that the outward I(K1) is primarily generated by channels with lower affinities for polyamines. Polyamines may regulate the amplitude of the outward I(K1), not only by blocking the channels but also by modifying the proportion of channels that show different sensitivities to the polyamine block.
强内向整流钾电流（I(K1)）在接近反转电位的电压范围内呈现出显著的外向电流幅度，从而在心脏动作电位的终末阶段引起快速复极化。然而，产生外向I(K1)的机制尚不清楚。我们记录了表达强内向整流钾通道Kir2.1的HEK 293T细胞内面向外膜片钳记录的电流，并研究了细胞质多胺（即精胺和亚精胺）对电流的阻断作用。用5 - 10 μM精胺或10 - 100 μM亚精胺获得的Kir2.1外向电流-电压（I-V）关系与I(K1)的稳态外向I-V关系相似，在比反转电位正约20 mV的水平处出现峰值，在更正的电压处斜率为负。当分别用1 μM精胺/亚精胺或0.1 μM精胺时，关系呈现出平台或双峰形状。在弦电导-电压关系中，正电压范围内存在额外电导，这无法用拟合关系主要部分的玻尔兹曼关系来描述。在存在5 - 10 μM精胺或10 - 100 μM亚精胺时产生大部分外向电流的额外电导，通过一个考虑了两种Kir2.1通道群体的模型进行了定量解释，这两种通道群体分别以高亲和力模式（模式1通道）或低亲和力模式（模式2通道）被多胺阻断。对测试脉冲后的内向尾电流分析表明，Kir2.1从精胺阻断中的解除由一个指数成分和一个几乎瞬时的成分组成。这两个成分的比例与模型计算的模式1和模式2中的阻断比例几乎一致。估计模式1通道占总通道的比例在0.1 - 10 μM精胺时为0.9，在1 - 100 μM亚精胺时为0.75，当精胺和亚精胺共存时在0.75和0.9之间。精胺/亚精胺在细胞内位点与通道的相互作用似乎改变了允许不同阻断模式的两种构象通道状态的平衡。我们的结果表明，外向I(K1)主要由对多胺亲和力较低的通道产生。多胺可能不仅通过阻断通道，还通过改变对多胺阻断表现出不同敏感性的通道比例来调节外向I(K1)的幅度。
