Human ether-a-go-go related gene (hERG) channel gating is associated with slow activation, yet the mechanistic basis for this is unclear. Here, we examine the effects of mutation of a unique glycine residue (G546) in the S4-S5 linker on voltage sensor movement and its coupling to pore gating. Substitution of G546 with residues possessing different physicochemical properties shifted activation gating by ∼-50 mV (with the exception of G546C). With the activation shift taken into account, the time constant of activation was also accelerated, suggesting a stabilization of the closed state by ∼1.6-4.3 kcal/mol (the energy equivalent of one to two hydrogen bonds). Predictions of the α-helical content of the S4-S5 linker suggest that the presence of G546 in wild-type hERG provides flexibility to the helix. Deactivation gating was affected differentially by the G546 substitutions. G546V induced a pronounced slow component of closing that was voltage-independent. Fluorescence measurements of voltage sensor movement in G546V revealed a slow component of voltage sensor return that was uncoupled from charge movement, suggesting a direct effect of the mutation on voltage sensor movement. These data suggest that G546 plays a critical role in channel gating and that hERG channel closing involves at least two independently modifiable reconfigurations of the voltage sensor.

hERG K(+) channel function is vital for normal cardiac rhythm, yet the mechanisms underlying the unique biophysical characteristics of the channel, such as slow activation and deactivation gating, are incompletely understood. The S4-S5 linker is thought to transduce voltage sensor movement to opening of the pore gate, but may also integrate signals from cytoplasmic domains. Previously, we showed that substitutions of G546 within the S4-S5 linker destabilize the closed state of the channel. Here, we present results of a glycine-scan in the background of 546L. We demonstrate site-specific restoration of WT-like activation which suggests that flexibility in the N-terminal portion of the S4-S5 linker is critical for the voltage dependence of hERG channel activation. In addition, we show that the voltage dependence of deactivation, which was recently shown to be left-shifted from that of activation due to voltage sensor mode-shift, is also modulated by the S4-S5 linker. The G546L mutation greatly attenuated the coupling of voltage sensor mode-shift to the pore gate without altering the mode-shift itself. Indeed, all of the S4-S5 linker mutations tested similarly reduced coupling of the mode-shift to the pore gate. These data demonstrate a key role for S4-S5 linker in the unique activation and deactivation gating of hERG channels. Furthermore, uncoupling of the mode-shift to the pore by S4-S5 linker mutations parallels the effects of mutations in the N-terminus suggestive of functional interactions between the two regions.

Extracellular acidosis shifts hERG channel activation to more depolarized potentials and accelerates channel deactivation; however, the mechanisms underlying these effects are unclear. External divalent cations, e.g., Ca(2+) and Cd(2+), mimic these effects and coordinate within a metal ion binding pocket composed of three acidic residues in hERG: D456 and D460 in S2 and D509 in S3. A common mechanism may underlie divalent cation and proton effects on hERG gating. Using two-electrode voltage clamp, we show proton sensitivity of hERG channel activation (pKa = 5.6), but not deactivation, was greatly reduced in the presence of Cd(2+) (0.1 mM), suggesting a common binding site for the Cd(2+) and proton effect on activation and separable effects of protons on activation and deactivation. Mutational analysis confirmed that D509 plays a critical role in the pH dependence of activation, as shown previously, and that cooperative actions involving D456 and D460 are also required. Importantly, neutralization of all three acidic residues abolished the proton-induced shift of activation, suggesting that the metal ion binding pocket alone accounts for the effects of protons on hERG channel activation. Voltage-clamp fluorimetry measurements demonstrated that protons shifted the voltage dependence of S4 movement to more depolarized potentials. The data indicate a site and mechanism of action for protons on hERG activation gating; protonation of D456, D460 and D509 disrupts interactions between these residues and S4 gating charges to destabilize the activated configuration of S4.

Van Slyke AC, Cheng YM, Mafi P, Allard CR, Hull CM, Shi YP, Claydon TW. Proton block of the pore underlies the inhibition of hERG cardiac K ϩ channels during acidosis.

Background: The Mustardé otoplasty is a commonly used procedure for the correction of the prominent ear deformity. Complication rates related to suture extrusion and long-term outcomes are variable in the literature. The study’s purpose was to examine the efficacy and safety of the Mustardé otoplasty and its resource utilization, using an “iron triangle” methodology incorporating quality, time, and cost. Methods: Retrospective data were collected on patients under 18 years who underwent primary Mustardé otoplasty between 2009 and 2018. Patient demographics, intraoperative details, complications, follow-up, and satisfaction were collected and analyzed. Results: There were 119 Mustardé otoplasties performed on 68 patients, with a median follow-up of 72 weeks (24–476 weeks). In total, 51 of the 68 patients underwent bilateral procedures. The median operative time was 95 minutes (31–133 minutes), translating to a facility case cost of $2046. A total of 24 complications were reported in 17 patients. Minor complications included the following: suture extrusion (n = 20), hematoma (n = 1), and suture abscess (n = 1). Major complications included reoperation (n = 2). The series had a revision rate of 1.7% (n = 2). No additional procedures were documented at other hospitals in the province. The majority (97%) of ear outcomes demonstrated both patient and surgeon satisfaction. Conclusions: The Mustardé otoplasty demonstrated a high efficacy in the correction of the prominent ear, with low reoperation rates and high patient and surgeon satisfaction. The procedure demonstrated intriguing results in resource utilization, with brief operative times, a “knife and fork” supply chain, and minimal overall case costs. This technique qualifies as a good, fast, and cheap outpatient otoplasty option.