Identification of the activation gate of HERG K^+ channel

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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.