Extracellular protons accelerate hERG channel deactivation by destabilizing voltage sensor relaxation

Abstract

hERG channels underlie the delayed-rectifier K+ channel current (IKr), which is crucial for membrane repolarization and therefore termination of the cardiac action potential. hERG channels display unusually slow deactivation gating, which contributes to a resurgent current upon repolarization and may protect against post-depolarization–induced arrhythmias. hERG channels also exhibit robust mode shift behavior, which reflects the energetic separation of activation and deactivation pathways due to voltage sensor relaxation into a stable activated state. The mechanism of relaxation is unknown and likely contributes to slow hERG channel deactivation. Here, we use extracellular acidification to probe the structural determinants of voltage sensor relaxation and its influence on the deactivation gating pathway. Using gating current recordings and voltage clamp fluorimetry measurements of voltage sensor domain dynamics, we show that voltage sensor relaxation is destabilized at pH 6.5, causing an ∼20-mV shift in the voltage dependence of deactivation. We show that the pH dependence of the resultant loss of mode shift behavior is similar to that of the deactivation kinetics acceleration, suggesting that voltage sensor relaxation correlates with slower pore gate closure. Neutralization of D509 in S3 also destabilizes the relaxed state of the voltage sensor, mimicking the effect of protons, suggesting that acidic residues on S3, which act as countercharges to S4 basic residues, are involved in stabilizing the relaxed state and slowing deactivation kinetics. Our findings identify the mechanistic determinants of voltage sensor relaxation and define the long-sought mechanism by which protons accelerate hERG deactivation.

Modulation of hERG K+ Channel Deactivation by Voltage Sensor Relaxation

“…Accessibility studies have also revealed that D460 and D509 stabilize the activated state (Liu et al, 2003), which is consistent with observations that neutralization of any of the acidic charges accelerates deactivation kinetics, likely by disrupting electrostatic interactions with S4 basic residues (Liu et al, 2003;Fernandez et al, 2005;Piper et al, 2008;Shi et al, 2014). Interestingly, disruption of the electrostatic pairing involving D509, either by protonation or alanine substitution, destabilizes the relaxed state of the voltage sensor leading to the loss of hysteresis and accelerated deactivation (Shi et al, 2019). This suggests that external acidic residues form stabilizing electrostatic interactions that are critical in recruiting the voltage sensor into the relaxed conformation and that these consequently control pore closure.…”

“…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.…”

“…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).…”

“…The Relaxed State of the Voltage Sensor Is Destabilized by Extracellular Protons hERG channel deactivation kinetics are sensitive to changes in the extracellular proton concentration and numerous studies have shown that acidosis accelerates deactivation rate (Anumonwo et al, 1999;Bérubé et al, 1999;Jiang et al, 1999;Terai et al, 2000;Bett and Rasmusson, 2003;Du et al, 2010;Zhou and Bett, 2010;Du et al, 2011;Van Slyke et al, 2012;Shi et al, 2014;Shi et al, 2019). pH reductions within the physiological range (as low as pH 6.5) accelerated both the fast and the slow components of deactivation with a pKa of~6.8 (Bett and Rasmusson, 2003;Shi et al, 2019) with little effect channel conductance, and other voltage-dependent gating parameters (Jiang et al, 1999;Bett and Rasmusson, 2003;Shi et al, 2019). Until recently, the mechanism by which protons modulate deactivation was unknown although studies had ruled out involvement of extracellular histidine residues (Jiang et al, 1999;Van Slyke et al, 2012), the N-terminus, and P/C-type inactivation, and shown the action to be mediated by an extracellular site (Jiang et al, 1999).…”