Molecular mechanisms of the slow deactivation of HERG channel

人类I_<Kr>通道的成孔亚基由HERG编码。我们研究了第六跨膜段（S6）对HERG通道缓慢失活的影响，以及S6与已知参与减慢失活的氨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通道的作用。(1)用苯丙氨酸、酪氨酸或色氨酸取代S6中647位的Ile可减缓失活动力学。当钾离子通道打开时，这个残基就在内螺旋弯曲的门状铰链Gly旁边。这可能是由于靠近铰链的位置上的大量芳香残基干扰了HERG通道的关闭。(2)删除WT和I647F的氨基末端区域，生成WTΔ2-354和I647FΔ2-354。当比较WT、I647F及其氨基末端缺失突变体的失活动力学时，I647FΔ2-354失活的速度比I647F快，但比WTΔ2-354慢。这表明氨基端区和S6通过不同的机制影响失活动力学。(3)各种药物对I647F、I647Y和I647W的作用减弱，失活动力学减慢。为了研究失活动力学本身的变化是否导致了药物效果的降低，研究了它们对I647FΔ2-354的影响。药物对I647FΔ2-354电流的抑制作用明显小于I647F，但明显大于WT。因此，虽然药物失活动力学的减慢可能是药物作用降低的部分原因，但I647的突变除了改变失活动力学外，还可能影响药物的结合。(4)我们无法研究需要多少个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