Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

Abstract

BackgroundhERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening.Methods and FindingsTetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the V½ of activation to −27.5±2.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with V½ ,1 = −37.8±1.7 mV, and V½ ,2 = 43.5±7.9 mV. The first phase, V½ ,1, was ∼20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V½ = −18.3±1.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V½ = −34.4±1.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarization (V½ = −20.6±1.2, k = 11.4 mV) and L520C quenching during depolarization (V½ = −26.8±1.0, k = 13.3 mV) matched the respective voltage dependencies of hERG ionic tails, and deactivation time constants from −40 to −110 mV, suggesting they detected pore-S4 rearrangements related to ionic current flow during pore opening and closing.ConclusionThe data indicate: 1) that rapid environmental changes occur at the outer end of S4 in hERG channels that underlie channel activation gating, and 2) that secondary slower changes reflect channel pore opening during sustained depolarizations, and channel closing upon repolarization. 3) No direct evidence was obtained of conformational changes related to inactivation from fluorophores attached at the outer end of S4.

Dual Effect of Phosphatidyl (4,5)-Bisphosphate PIP2 on Shaker K+ Channels

“…Protocol for Shaker-IR-A359C-C445V Ion Currents and Voltage-clamp Fluorimetry-Oocytes were prepared as previously reported (29). Oocytes were placed in a bath chamber that was perfused with control ND96 bath solution containing (in mmol/liter), 96 NaCl, 3 KCl, 1 MgCl 2 , 2 CaCl 2 , and 5 HEPES, titrated to pH 7.4 with NaOH.…”

“…After 30 min of labeling, oocytes were stored in ND96 solution in the dark until voltageclamped. Fluorimetry was performed using a Nikon TE300 inverted microscope with Epi-Fluorescence attachment and a 9124b Electron Tubes photomultiplier tube (PMT) module (Cairn Research, Kent, UK) as was described previously (EsSalah-Lamoureux et al, 29). To minimize fluorophore bleaching, a Uniblitz computer-controlled shutter (Vincent Associates, Ottawa, ON, Canada) was used, and opened shortly prior to application of voltage clamp pulses.…”

The neutral, hydrophobic isoleucine at position I521 in the extracellular S4 domain of hERG contributes to channel gating equilibrium

“…In all hERG constructs used in this study, the two extracellular cysteines in the S1-S2 linker, C445 and C449, were mutated to valines to avoid inadvertent labeling of these cysteines by reactive agents, and thus hERG C445V:C449V:I521 is referred to as wild-type (WT) C-less. We have previously shown that the WT C-less channel has only minor effects on channel gating (8).…”

Molecular motions that shape the cardiac action potential: Insights from voltage clamp fluorometry

“…Further work showed that two endogenous cysteines in the S1eS2 linker distorted the signals reported by the S4-labeled positions (Es-Salah-Lamoureux et al, 2010). Removing these endogenous cysteines enabled cleaner measure of VSD activation.…”