Identification of the activation gate of HERG K^+ channel

1. 野生型HERG K^+通道在去极化时打开，在超极化时不打开。我们发现S6上的I655点突变使HERG通道在超极化时打开。进一步的诱变分析表明，I655被带电荷或极性氨基酸残基取代会导致类似的变化（由此产生的突变体在超极化时打开）。另一方面，HERG K^+通道似乎需要655处的疏水残基才能像野生型通道一样在去极化时打开。G657在S6上的点突变得到了类似但不完全相同的结果。当带电残基（谷氨酸除外）或极性残基存在于657位点时，突变体通道在超极化时打开。在657位残基的情况下，HERG K^+通道似乎需要少量残基如657位的丙氨酸或丝氨酸才能打开。在I655和G657突变体通道中，K^+离子的选择性在超极化打开时失去。虽然机制尚不清楚，但在超极化时打开突变通道似乎涉及离子选择性过滤器的运动。当HERG WT通道的S4-S5连接体上的3个带电残基被中和后，在超极化时，观察到突变体通道有明显的向内电流，除除极化时有向外电流。然而，在I655和G657突变体中，带电残基的中和并没有引起任何改变。据报道，当K^+通道打开时，甘氨酸铰链处的内螺旋弯曲。用丙氨酸取代HERG中相应的甘氨酸表明甘氨酸铰链参与WT HERG通道的打开，但不参与I655和G657突变体通道。

1. Wild type HERG K^+ channel opens upon depolarization and does not open upon hyperpolarization. We have found that a point mutation at I655 on the S6 makes the HERG channel open upon hyperpolarization. Further mutagenesis analyses showed that substitution of I655 with charged or polar amino acid residues resulted in the similar change (The resultant mutants opened upon hyperpolarization). On the other hand, HERG K^+ channel seemed to require hydrophobic residues at 655 to open upon depolarization like the wild type channel.2. Similar but not identical results were obtained with point mutations of G657 on the S6. When charged residues (except glutamatic acid) or polar residues were present at 657, the mutant channels opened upon hyperpolarization. In the case of the residue at 657, HERG K^+ channel seemed to require a small residue such as alanine or serine at 657 to open upon depolarization.3. Selectivity for K^+ ion was lost in the I655 and G657 mutant channels that open upon hyperpolarization. Although the mechanism is not known, opening of the mutant channels upon hyperpolarization seemed to involve the movement of the ion selectivity filter.4. When the three charged residues on the S4-S5 linker of HERG WT channel were neutralized, upon hyperpolarization, obvious inward currents flowing through the mutant channel were observed, in addition to outward currents on depolarization. However, the neutralization of the charged residues did not cause any change in the I655 and G657 mutants that open upon hyperpolarization.5. It has been reported that the inner helices bend at the glycine hinge, when K^+ channel opens. Substitution of the corresponding glycine in HERG with alanine suggested that the glycine hinge is involved in the opening of the WT HERG channel, but not in the I655 and G657 mutant channels.