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

Abstract

Slow deactivation of hERG channels is critical for preventing cardiac arrhythmia yet the mechanistic basis for the slow gating transition is unclear. Here, we characterized the temporal sequence of events leading to voltage sensor stabilization upon membrane depolarization. Progressive increase in step depolarization duration slowed voltage-sensor return in a biphasic manner (t fast ¼ 34 ms, t slow ¼ 2.5 s). The faster phase of voltage-sensor return slowing correlated with the kinetics of pore opening. The slower component occurred over durations that exceeded channel activation and was consistent with voltage sensor relaxation. The S4-S5 linker mutation, G546L, impeded the faster phase of voltage sensor stabilization without attenuating the slower phase, suggesting that the S4-S5 linker is important for communications between the pore gate and the voltage sensor during deactivation. These data also demonstrate that the mechanisms of pore gate-opening-induced and relaxation-induced voltage-sensor stabilization are separable. Deletion of the distal N-terminus (D2-135) accelerated off-gating current, but did not influence the relative contribution of either mechanism of stabilization of the voltage sensor. Lastly, we characterized mode-shift behavior in hERG channels, which results from stabilization of activated channel states. The apparent mode-shift depended greatly on recording conditions. By measuring slow activation and deactivation at steady state we found the ''true'' mode-shift to be~15 mV. Interestingly, the ''true'' mode-shift of gating currents was~40 mV, much greater than that of the pore gate. This demonstrates that voltage sensor return is less energetically favorable upon repolarization than pore gate closure. We interpret this to indicate that stabilization of the activated voltage sensor limits the return of hERG channels to rest. The data suggest that this stabilization occurs as a result of reconfiguration of the pore gate upon opening by a mechanism that is influenced by the S4-S5 linker, and by a separable voltage-sensor intrinsic relaxation mechanism.

Modulation of hERG K+ Channel Deactivation by Voltage Sensor Relaxation

“…The measured observation is that the voltage sensor required stronger repolarization to return to its resting position and to close the pore gate than was required to activate it ( Figure 3A). Similar hysteresis behavior has been reported in a broad collection of channels including sodium channels of the squid giant axon and NaChBac (Bezanilla et al, 1982;Kuzmenkin et al, 2004), calcium channels (Brum and Rios, 1987;Brum et al, 1988;Shirokov et al, 1992), potassium channels, such as KcSA (Tilegenova et al, 2017), Shaker (Haddad and Blunck, 2011;Lacroix et al, 2011;Labro et al, 2012;Priest et al, 2013), Kv1.2 (Labro et al, 2012), Kv3.1 (Labro et al, 2015), Kv7.2/7.3 (Corbin-Leftwich et al, 2016), Kv11.1 (hERG) (Piper et al, 2003;Tan et al, 2012;Hull et al, 2014;Goodchild et al, 2015;Thouta et al, 2017;Shi et al, 2019), Kv12.1 (Dierich et al, 2018), and HCN channels (Elinder et al, 2006;Bruening-Wright and Larsson, 2007). In HCN channels, the term mode-shift has been used to describe the hysteresis in the voltage-dependence of activation and deactivation in response to prolonged depolarization (Elinder et al, 2006), and the terms hysteresis and mode-shift are often used to describe the separation between the voltagedependence of Kv channel activation and deactivation.…”

“…When voltage steps of physiological duration were applied to measure activation and deactivation, hERG channels showed a profound ionic hysteresis with the voltage-dependence of ionic current activation and deactivation separated by~65 mV on the voltage axis. However, longer voltage step durations that allow the slow transitions to reach steady-state, produced an ionic hysteresis of~15 mV (Thouta et al, 2017). Similar experiments capturing pseudo steady-state gating charge movement revealed a voltage sensor hysteresis of~40 mV, much greater than that of ionic current through the pore.…”

“…In doing so, more energy is required to return the voltage sensor than to activate it, thus inducing a hyperpolarizing-shift in the voltage-dependence of deactivation compared to that for activation, and a slowing of the deactivation kinetics. Recent studies suggest a role for the N-terminus in stabilizing the activated voltage sensor in a relaxed state (Tan et al, 2012;Goodchild et al, 2015;Thouta et al, 2017). As an important contributor to slow deactivation, voltage sensor relaxation could perhaps act as a master-switch that is influenced by mutations within the transmembrane core, as well as the N-terminus.…”

“…hERG channel gating displays a prominent hysteresis in the voltage-dependencies of activation and deactivation of both the movement of the voltage sensor (voltage sensor mode-shift) and the gating of the pore (ionic mode-shift). Interestingly, modeshift in hERG channels occurs on a physiological time scale suggesting that the dynamic switching of voltage-dependencies of activation and deactivation gating may contribute to the amplitude and timing of the repolarizing I Kr current during the cardiac action potential (Piper et al, 2003;Tan et al, 2012;Goodchild et al, 2015;Thouta et al, 2017;Shi et al, 2019). In Shaker and Kv1.2 channels, the time-dependence of voltage sensor stabilization and mode-shift were measured by applying depolarizing steps of increasing duration and observing the progressive slowing of charge return (Lacroix et al, 2011;Labro et al, 2012).…”

“…In Kv3.1 channels a very rapid component of charge return that kinetically precedes pore gate opening was observed, and this was attributed to an ultra-fast relaxation mechanism in these channels (Labro et al, 2015). In hERG channels, increasing depolarization duration slows charge return (Goodchild and Fedida, 2014;Goodchild et al, 2015;Thouta et al, 2017;Shi et al, 2019). The slowing of voltage sensor return showed a biphasic dependence upon depolarizing step duration similar to that in Shaker and Kv1.2 channels, with a fast phase (tau = 34 ms) and a slower component (tau = 2.5 s) (Thouta et al, 2017).…”