発表者： 松原 一樹（第５学年）
Title:
Interaction of an antiarrhythmic drug bepridil with human Kv1.5 channel through specific amino acids within the pore region (progress report).
Interaction of an antiarrhythmic drug bepridil with human Kv1.5 channel through specific amino acids within the pore region (progress report).
Abstract:
Because human Kv1.5 (hKv1.5) channels are highly expressed in cardiac atria but are scarce in ventricle, pharmacological blockade of hKv1.5 has been regarded as an effective strategy for prevention and treatment of reentry-based atrial tachyarrhythmia such as atrial fibrillation. Although the antiarrhythmic drug bepridil has been reported to inhibit hKv1.5 channel, there is little information as to the amino acids in the pore region of hKv1.5 which bepridil interacts with. Thus, this study was undertaken to examine the effect of bepridil on hKv1.5 channel and to elucidate the underlying molecular determinants. Site-directed mutagenesis was carried out to introduce single point mutants (T462C, T479A, T480A, A501V, I502A, V505A, I508A, L510A, V512A and V516A) into hKv1.5 cDNA by using a QuikChange Ⅱ XL kit. Whole-cell patch-clamp technique was used to record membrane currents from hKv1.5 wild type and mutant channels heterologously expressed in Chinese hamster ovary cells. Bepridil concentration-dependently blocked hKv1.5 current. In addition, bepridil-induced current block gradually progressed during the depolarizing pulse, suggesting that bepridil preferentially block the channels as an open-channel blocker. Moreover, the degree of current block by bepridil was significantly attenuated in I502A, L510A and V516A, but not in other mutants. Our results indicate that, several amino acids within the S6 domain (Ile502, Leu510 and Val516) are critically involved in bepridil-induced inhibitory effect on hKv1.5 channel. The analysis of other mutant channels (H463C, R487V and A509G) and the docking simulation are scheduled to be conducted.
Because human Kv1.5 (hKv1.5) channels are highly expressed in cardiac atria but are scarce in ventricle, pharmacological blockade of hKv1.5 has been regarded as an effective strategy for prevention and treatment of reentry-based atrial tachyarrhythmia such as atrial fibrillation. Although the antiarrhythmic drug bepridil has been reported to inhibit hKv1.5 channel, there is little information as to the amino acids in the pore region of hKv1.5 which bepridil interacts with. Thus, this study was undertaken to examine the effect of bepridil on hKv1.5 channel and to elucidate the underlying molecular determinants. Site-directed mutagenesis was carried out to introduce single point mutants (T462C, T479A, T480A, A501V, I502A, V505A, I508A, L510A, V512A and V516A) into hKv1.5 cDNA by using a QuikChange Ⅱ XL kit. Whole-cell patch-clamp technique was used to record membrane currents from hKv1.5 wild type and mutant channels heterologously expressed in Chinese hamster ovary cells. Bepridil concentration-dependently blocked hKv1.5 current. In addition, bepridil-induced current block gradually progressed during the depolarizing pulse, suggesting that bepridil preferentially block the channels as an open-channel blocker. Moreover, the degree of current block by bepridil was significantly attenuated in I502A, L510A and V516A, but not in other mutants. Our results indicate that, several amino acids within the S6 domain (Ile502, Leu510 and Val516) are critically involved in bepridil-induced inhibitory effect on hKv1.5 channel. The analysis of other mutant channels (H463C, R487V and A509G) and the docking simulation are scheduled to be conducted.
