Dataset Information

Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

ABSTRACT:

Background

hERG 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 findings

Tetramethylrhodamine-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.

Conclusion

THE 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.

SUBMITTER: Es-Salah-Lamoureux Z

PROVIDER: S-EPMC2878317 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

Es-Salah-Lamoureux Zeineb Z Fougere Robert R Xiong Ping Yu PY Robertson Gail A GA Fedida David D

PloS one 20100528 5

<h4>Background</h4>hERG 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.<h4>Methods and findings</h4>Tetramethylrhodamine-5-maleim ...[more]

PMID: 20526358

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Project description:Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1-R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.

Dataset Information

Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

Background

Methods and findings

Conclusion

Publications

Fluorescence-tracking of activation gating in human ERG channels reveals rapid S4 movement and slow pore opening.

Similar Datasets

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