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Sequence of gating charge movement and pore gating in HERG activation and deactivation pathways.

ABSTRACT: KV11.1 voltage-gated K(+) channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor movement, and also by a mode-shift after activation. To test these ideas, we compared the kinetics and voltage dependence of ionic activation and deactivation with gating charge movement. At 0 mV, gating charge moved ?threefold faster than ionic current, which suggests the presence of additional slow transitions downstream of charge movement in the physiological activation pathway. A significant voltage sensor mode-shift was apparent by 24 ms at +60 mV in gating currents, and return of charge closely tracked pore closure after pulses of 100 and 300 ms duration. A deletion of the N-terminus PAS domain, mutation R4AR5A or the LQT2-causing mutation R56Q gave faster-deactivating channels that displayed an attenuated mode-shift of charge. This indicates that charge movement is perturbed by N- and C-terminus interactions, and that these domain interactions stabilize the open state and limit the rate of charge return. We conclude that slow on-gating charge movement can only partly account for slow hERG ionic activation, and that the rate of pore closure has a limiting role in the slow return of gating charges.

SUBMITTER: Goodchild SJ

PROVIDER: S-EPMC4375626 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Sequence of gating charge movement and pore gating in HERG activation and deactivation pathways.

Goodchild Samuel J SJ Macdonald Logan C LC Fedida David D

Biophysical journal 20150301 6

KV11.1 voltage-gated K(+) channels are noted for unusually slow activation, fast inactivation, and slow deactivation kinetics, which tune channel activity to provide vital repolarizing current during later stages of the cardiac action potential. The bulk of charge movement in human ether-a-go-go-related gene (hERG) is slow, as is return of charge upon repolarization, suggesting that the rates of hERG channel opening and, critically, that of deactivation might be determined by slow voltage sensor ...[more]

PMID: 25809256

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Project description: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 V((1/2)) 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-V(ON), with V((1/2)) (,1) = -37.8+/-1.7 mV, and V((1/2)) (,2) = 43.5+/-7.9 mV. The first phase, V((1/2)) (,1), was approximately 20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-V((1/2)) = -18.3+/-1.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V((1/2)) = -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 (V((1/2)) = -20.6+/-1.2, k = 11.4 mV) and L520C quenching during depolarization (V((1/2)) = -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.

Dataset Information

Sequence of gating charge movement and pore gating in HERG activation and deactivation pathways.

Publications

Sequence of gating charge movement and pore gating in HERG activation and deactivation pathways.

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