Modification of hERG1 channel gating by Cd2+

Abstract

Each of the four subunits in a voltage-gated potassium channel has a voltage sensor domain (VSD) that is formed by four transmembrane helical segments (S1–S4). In response to changes in membrane potential, intramembrane displacement of basic residues in S4 produces a gating current. As S4 moves through the membrane, its basic residues also form sequential electrostatic interactions with acidic residues in immobile regions of the S2 and S3 segments. Transition metal cations interact with these same acidic residues and modify channel gating. In human ether-á-go-go–related gene type 1 (hERG1) channels, Cd2+ coordinated by D456 and D460 in S2 and D509 in S3 induces a positive shift in the voltage dependence of activation of ionic currents. Here, we characterize the effects of Cd2+ on hERG1 gating currents in Xenopus oocytes using the cut-open Vaseline gap technique. Cd2+ shifted the half-point (V1/2) for the voltage dependence of the OFF gating charge–voltage (QOFF-V) relationship with an EC50 of 171 µM; at 0.3 mM, V1/2 was shifted by +50 mV. Cd2+ also induced an as of yet unrecognized small outward current (ICd-out) upon repolarization in a concentration- and voltage-dependent manner. We propose that Cd2+ and Arg residues in the S4 segment compete for interaction with acidic residues in S2 and S3 segments, and that the initial inward movement of S4 associated with membrane repolarization displaces Cd2+ in an outward direction to produce ICd-out. Co2+, Zn2+, and La3+ at concentrations that caused ∼+35-mV shifts in the QOFF-V relationship did not induce a current similar to ICd-out, suggesting that the binding site for these cations or their competition with basic residues in S4 differs from Cd2+. New Markov models of hERG1 channels were developed that describe gating currents as a noncooperative two-phase process of the VSD and can account for changes in these currents caused by extracellular Cd2+.

Stabilization of the Activated hERG Channel Voltage Sensor by Depolarization Involves the S4-S5 Linker

“…This observation demonstrates that voltage sensor return is less energetically favorable than pore closure upon repolarization and demonstrates the important role for voltage sensor relaxation is stabilizing the hERG activated voltage sensor and limiting its return. This finding is consistent with a previous observation of the dissociation between voltage sensor return and pore closure caused by a pharmacological activator compound (29). Furthermore, our data also provide supporting functional evidence for the recent cryo-EM structural prediction of the related eag channel, which captures the channel in a state in which the pore gate is closed, but the voltage sensor is in the activated configuration (6).…”

“…Data were fitted to a double exponential function (n ¼ 6, 5, and 4, respectively). step depolarization to þ60 mV, which highlights the complex kinetics associated with charge transit across the membrane as has been reported previously in these channels (11,(28)(29)(30)(31). Both on-and off-gating currents display pronounced fast and slow phases of decay.…”

C-Linker Accounts for Differential Sensitivity of ERG1 and ERG2 K+ Channels to RPR260243-Induced Slow Deactivation

“…Although our current findings indicate that differential sensitivity to the allosteric effects of RPR on channel gating can be swapped between hERG1 and rERG2 by mutating the few key nonconserved residues in the C-linker, the structural basis of RPR-induced slowing of hERG1 deactivation remains elusive. We previously reported that RPR has no effect on the kinetics, magnitude, or voltage dependence of hERG1 gating currents (Abbruzzese et al, 2010), indicating that the compound dissociates movement of the voltage sensor domain from the opening and closing of the activation gate. RPR could directly or by an allosteric mechanism stabilize the association between the PAS cap and the C-linker or interfere with the coupling between the S4/S5 linkers and the C-terminal region of the S6 segments (Ferrer et al, 2006).…”

“…Procedures used for the surgical removal of ovarian lobes from Xenopus laevis and isolation of oocytes was approved by the University of Utah Institutional Animal Care and Use Committee and performed as described previously (Abbruzzese et al, 2010). Single oocytes were injected with 5-40 ng cRNA encoding WT or mutant ERG subunits and studied 2-7 days later.…”

The Link between Inactivation and High-Affinity Block of hERG1 Channels

“…Oocytes were enzymatically isolated from ovarian lobes excised from Xenopus laevis using procedures as described previously (Abbruzzese et al, 2010) and approved by the University of Utah Institutional Animal Care and Use Committee. Single oocytes were injected with 5-30 ng of hERG1 cRNA and incubated for 1-4 days before use in voltage-clamp experiments.…”