Effect of S5P α‐helix charge mutants on inactivation of hERG K⁺ channels

Abstract

The ether-à-go-go (EAG) family of voltage-gated K + channels contains three subfamilies, EAG, ether-à-go-go related (ERG) and ether-à-go-go like (ELK). The human ether-à-go-go related gene (hERG) K + channel has been of significant interest because loss of function in the hERG channel is associated with a markedly increased risk of cardiac arrhythmias. The hERG channel has unusual kinetics with slow activation and deactivation but very rapid and voltage-dependent inactivation. The outer pore region of the hERG K + channel is predicted to be different from that of other members of the voltage-gated K + channel family. HERG has a much longer linker between the fifth transmembrane domain (SS) and the pore helix (S5P linker) compared to other families of voltage-gated K + channels (43 amino acids compared to 14-23 amino acids). Further, the S5P linker contains an amphipathic α-helix that in hERG channels probably interacts with the mouth of the pore to modulate inactivation. The human EAG and rat ELK2 channels (hEAG and rELK2) show reduced or no inactivation in comparison to hERG channels, yet both channels are predicted to contain a similarly long S5P linker to that of hERG. In this study, we have constructed a series of chimaeric channels consisting of the S1-S6 of hERG but with the S5P α-helical region of either hEAG or rELK2, and one consisting of the S1-S6 of rELK2 but with the S5P α-helical region of hERG to investigate the role of the S5P linker in inactivation. Our studies show that charged residues on the α-helix of the S5P linker contribute significantly to the differences in inactivation characteristics of the EAG family channels. Further, individually mutating each of the hydrophilic residues on the S5P α-helix of hERG to a charged residue had significant effects on the voltage dependence of inactivation and the two residues with the greatest affect when mutated to a lysine, N588 and Q592, both lie on the same face of the S5P α -helix. We suggest that inactivation of hERG involves the interaction of this face of the S5P α-helix with a charged residue on the remainder of the outer pore domain of the channel.

Novel characteristics of a trafficking-defective G572R-hERG channel linked to hereditary long QT syndrome

“…Our results agree with most previous reports on hERG in the S5/P region (G572S, I593R, P596R, G601S, G604S, Y611H, V612L and T613M), which were shown to induce trafficking deficiency (9,28,29). Interestingly, N588D causes a loss of function with the voltage dependence of inactivation shifted to more negative potentials (30). However, the N588K mutation involving the same residue leads to a gain of function produced by positive shifts in the voltage dependence of hERG current inactivation (31).…”

The Pore Domain Outer Helix Contributes to Both Activation and Inactivation of the hERG K+ Channel

“…Mutagenesis was carried out using the megaprimer PCR method as previously described in detail (28). Mutant constructs were confirmed by bi-directional sequencing.…”

“…All data are shown as means Ϯ S.E. Measurements of steady state activation and inactivation were performed as previously described (28,30). Steady state activation and inactivation data were analyzed using a simple twostate Boltzmann function,…”

The M1P1 Loop of TASK3 K2P Channels Apposes the Selectivity Filter and Influences Channel Function

“…Two-electrode Voltage Clamp Recording-Xenopus laevis oocytes were prepared exactly as described previously (30). All experiments were approved by the Animal Ethics Committee of the University of Sydney.…”