Molecular mechanisms of the slow deactivation of HERG channel

人I_通道的成孔亚基<Kr>由HERG编码。我们研究了第六跨膜段（S6）对HERG通道慢失活的影响以及S6与已知参与减缓失活的氨基末端区域之间的可能关系。我们还研究了失活率的变化是否会影响各种药物对HERG通道的影响。(1)用Phe、Tyr或Trp取代S6中647位的Ile导致失活动力学减慢。这个残基就在门控铰链Gly旁边，当钾通道打开时，内螺旋在门控铰链处弯曲。可能是铰链附近的大体积芳香残基干扰了HERG通道的关闭。(2)缺失WT和I647 F的氨基末端区域以产生WTΔ2-354和I647 F Δ2-354。当比较WT、I647 F及其氨基端缺失突变体的失活动力学时，I647 F Δ2-354的失活动力学与WT、I647 F Δ2-354的失活动力学之间存在差异。 ...更多信息 比I647 F快，但比WTΔ2-354慢。这一结果表明，氨基末端区域和S6通过不同的机制影响失活动力学。(3)在I647 F、I647 Y和I647 W中，各种药物的作用降低，表现出失活动力学减慢。为了研究失活动力学本身的变化是否是药物作用降低的原因，研究了它们对I647 F Δ2-354的作用。I647 F Δ2-354的电流被药物抑制显著小于I647 F，但显著大于WT。因此，虽然较慢的失活动力学可能是部分负责的药物的效果降低，I647的突变可能会影响药物的结合，除了改变失活动力学。(4)我们不能研究需要多少氨基末端来减缓失活动力学，因为通过WT和氨基末端缺失突变体的串联构建体没有观察到可检测的电流。少

The pore-forming subunit of human I_<Kr> channel is encoded by HERG. We have investigated influence of the sixth transmembrane segment (S6) on the slow deactivation of HERG channel and possible relationship between the S6 and the ammo-terminal region that is known to be involved in slowing the deactivation. We have also investigated whether changes in the deactivation rate affect the effects of various drugs on the HERG channel. (1) Substitution of Ile at 647 in the S6 with Phe, Tyr or Trp resulted in slowing of the deactivation kinetics. This residue is just next to the gating hinge Gly at which the inner helices bend when the potassium channels open. It might be possible that the bulky aromatic residue at the site next to the hinge interferes closing of the HERG channel. (2) Amino-terminal region of WT and I647F was deleted to generate WTΔ2-354 and I647FΔ2-354. When deactivation kinetics of WT, I647F and their amino-terminal deletion mutants were compared, I647FΔ2-354 deactivated mar … More kedly faster than I647F, but slower than WTΔ2-354. This result suggests that the amino-terminal region and the S6 affect the deactivation kinetics through different mechanisms. (3) Effects of various drugs were reduced in I647F, I647Y and I647W that exhibited slowing of the deactivation kinetics. To investigate whether the change in deactivation kinetics itself is responsible for the reduced effects of the drugs, their effects on I647FΔ2-354 were studied. Currents of I647FΔ2-354 were inhibited by the drugs significantly less than I647F, but markedly greater than WT. Thus, although the slower deactivation kinetics might be partly responsible for the reduced effects of the drugs, the mutation of I647 probably affects the binding of the drugs besides changing the deactivation kinetics. (4) We could not investigate how many amino-termini are needed to slow the deactivation kinetics, since no detectable currents were observed through tandem constructs of WT and the amino-terminal deletion mutants. Less