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hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

Wynia-Smith SL, Gillian-Daniel AL, Satyshur KA, Robertson GA - J. Gen. Physiol. (2008)

Bottom Line: We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states.In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating.Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706, USA.

ABSTRACT
Human ether-à-go-go-related gene (hERG) channels mediate cardiac repolarization and bind drugs that can cause acquired long QT syndrome and life-threatening arrhythmias. Drugs bind in the vestibule formed by the S6 transmembrane domain, which also contains the activation gate that traps drugs in the vestibule and contributes to their efficacy of block. Although drug-binding residues have been identified, we know little about the roles of specific S6 residues in gating. We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states. Energy-minimized molecular models based on the crystal structures of rKv1.2 (open state) and MlotiK1 and KcsA (closed state) provided structural contexts for evaluating mutant residues. The majority of mutations slowed deactivation, shifted conductance voltage curves to more negative potentials, or conferred a constitutive conductance over voltages that normally cause the channel to close. At the most intracellular extreme of the S6 region, Q664, Y667, and S668 were especially sensitive and together formed a ringed domain that occludes the pore in the closed state model. In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating. Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself. Overall, the study indicated that S6 mutagenesis disrupts the energetics primarily of channel closing and identified several residues critical for this process in the native channel.

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Summary of kinetics and equilibrium measurements. (A) Fold change from control for average deactivation time constants measured at −100 mV. Mutants are aligned vertically with residues at the top of the graph representing those closest to the selectivity filter, and residues at the bottom representing those closest to the intracellular face of the pore. (B) Fold change from control for activation time constants measured at +40 mV. Mutants are represented vertically as in A. (C) Change in V1/2 compared with control. *, P < 0.05.
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fig7: Summary of kinetics and equilibrium measurements. (A) Fold change from control for average deactivation time constants measured at −100 mV. Mutants are aligned vertically with residues at the top of the graph representing those closest to the selectivity filter, and residues at the bottom representing those closest to the intracellular face of the pore. (B) Fold change from control for activation time constants measured at +40 mV. Mutants are represented vertically as in A. (C) Change in V1/2 compared with control. *, P < 0.05.

Mentions: In general, deactivation was more sensitive than activation to S6 cysteine mutagenesis. As summarized in Fig. 7, deactivation was slowed by more than 10-fold over control (Fig. 7 A and Table I), compared with less than twofold change in activation (Fig. 7 B and Table III). In all but two mutants with large perturbations, deactivation was slowed. The exceptions were N658C and G657C, the latter exhibiting properties consistent with those previously reported for G657A (Hardman et al., 2007). Differences in deactivation between the cysteine mutants and control at nearly every position were statistically significant (Table I). Steady-state conductance voltage curves showed a concomitant shift for some of the mutants (Fig. 7 C and Table IV), but overall there was a lack of correspondence between kinetic changes and V1/2, not unexpected from a channel with gating between more than two states (see Discussion).


hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

Wynia-Smith SL, Gillian-Daniel AL, Satyshur KA, Robertson GA - J. Gen. Physiol. (2008)

Summary of kinetics and equilibrium measurements. (A) Fold change from control for average deactivation time constants measured at −100 mV. Mutants are aligned vertically with residues at the top of the graph representing those closest to the selectivity filter, and residues at the bottom representing those closest to the intracellular face of the pore. (B) Fold change from control for activation time constants measured at +40 mV. Mutants are represented vertically as in A. (C) Change in V1/2 compared with control. *, P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2571969&req=5

fig7: Summary of kinetics and equilibrium measurements. (A) Fold change from control for average deactivation time constants measured at −100 mV. Mutants are aligned vertically with residues at the top of the graph representing those closest to the selectivity filter, and residues at the bottom representing those closest to the intracellular face of the pore. (B) Fold change from control for activation time constants measured at +40 mV. Mutants are represented vertically as in A. (C) Change in V1/2 compared with control. *, P < 0.05.
Mentions: In general, deactivation was more sensitive than activation to S6 cysteine mutagenesis. As summarized in Fig. 7, deactivation was slowed by more than 10-fold over control (Fig. 7 A and Table I), compared with less than twofold change in activation (Fig. 7 B and Table III). In all but two mutants with large perturbations, deactivation was slowed. The exceptions were N658C and G657C, the latter exhibiting properties consistent with those previously reported for G657A (Hardman et al., 2007). Differences in deactivation between the cysteine mutants and control at nearly every position were statistically significant (Table I). Steady-state conductance voltage curves showed a concomitant shift for some of the mutants (Fig. 7 C and Table IV), but overall there was a lack of correspondence between kinetic changes and V1/2, not unexpected from a channel with gating between more than two states (see Discussion).

Bottom Line: We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states.In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating.Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Wisconsin-Madison School of Medicine and Public Health, Madison, WI 53706, USA.

ABSTRACT
Human ether-à-go-go-related gene (hERG) channels mediate cardiac repolarization and bind drugs that can cause acquired long QT syndrome and life-threatening arrhythmias. Drugs bind in the vestibule formed by the S6 transmembrane domain, which also contains the activation gate that traps drugs in the vestibule and contributes to their efficacy of block. Although drug-binding residues have been identified, we know little about the roles of specific S6 residues in gating. We introduced cysteine mutations into the hERG channel S6 domain and measured mutational effects on the steady-state distribution and kinetics of transitions between the closed and open states. Energy-minimized molecular models based on the crystal structures of rKv1.2 (open state) and MlotiK1 and KcsA (closed state) provided structural contexts for evaluating mutant residues. The majority of mutations slowed deactivation, shifted conductance voltage curves to more negative potentials, or conferred a constitutive conductance over voltages that normally cause the channel to close. At the most intracellular extreme of the S6 region, Q664, Y667, and S668 were especially sensitive and together formed a ringed domain that occludes the pore in the closed state model. In contrast, mutation of S660, more than a full helical turn away and corresponding by alignment to a critical Shaker gate residue (V478), had little effect on gating. Multiple substitutions of chemically distinct amino acids at the adjacent V659 suggested that, upon closing, the native V659 side chain moves into a hydrophobic pocket but likely does not form the occluding gate itself. Overall, the study indicated that S6 mutagenesis disrupts the energetics primarily of channel closing and identified several residues critical for this process in the native channel.

Show MeSH
Related in: MedlinePlus