Voltage-sensor trapping in Hv1 channel (CiHv1).

Abstract

Previous studies on monomeric N264R showed that the OFF-gating current is completely trapped and that, from a kinetic point of view, this charge displacement takes a long time to return. Currently, the molecular mechanism governing this entrapment of charge remains unknown. In order to understand the OFF-gating charge trapping mechanism, we performed patch-clamp in its inside-out configuration using Xenopus laevis oocytes as a heterologous expression system, combined with molecular dynamics and kinetic modeling. Our results shows that the ΔNΔC D160N mutant does not have a trapped charge at the OFF-gating charge; but, when the N264R mutation is added to this mutant, the OFF-gating charge trapping appears in a diminished state. This is possibly because the residue in position 160, which is also the selectivity filter of the channel, interacts with the arginines of the voltage sensor. In addition, the 2GBI blocker possesses a guanidinium group which also presents an arginine in its side chain. This inhibitor generates an effect similar to ΔNΔC N264R OFF-gating charge displacement. This suggests that arginine is required for charge trapping. By performing mutations at position 160 it was found that hydrophobicity near that position obstructs the movement of the voltage sensor exerting an effect similar to that of the aromatic rings of 2GBI. Furthermore, when ΔpH is increased and especially when acidity is increased on the intracellular side, the OFF-gating charge is also trapped. We performed molecular dynamics simulations on ΔNΔC N264R, and our results suggest that the positive charge of arginine “push-up” the arginines 258 and 261 and form a stable salt-bridge over time with D160. Finally, our results confirm that for the gating charge to be trapped it is necessary to change the electrostatic configuration on the intracellular side near position 264.