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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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V659 couples voltage sensing and gating. (A) Conductance–voltage relation for V659C mutant fit with a Boltzmann equation reveals constitutive conductance over negative potentials and shallower slope (z). (B) Scaled V659C tail currents evoked at −100 mV after a pulse to +60 mV show dramatically slowed deactivation. Dotted line indicates zero current level. (C) Plot of time constants obtained from fits to deactivating currents on a log scale plotted as a function of voltage for V659C. (D) Steady-state current versus voltage relations for a variety of substitutions made at V659. Mutations to A, F, or T were fit with a standard Boltzmann function and show constitutive conductance and shallower slope. V659I shows an intermediate phenotype that was fit with a double Boltzmann equation (see Materials and methods). n = 3–5 for each data point. (E) Scaled V659 mutant tail currents evoked at −100 mV after pulses to +60 mV. (F) Average deactivation time constants on a log scale versus voltage for V659 mutants. n = 3–5 for each data point. (G) Deactivation time constants versus side chain volume for V659 mutants. Amino acids with side chains significantly larger or smaller than the native valine show slowed deactivation. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles above the line at the top of the y axis at appropriate side chain volume. Side chain volume obtained from Zamyatnin (1972). (H) Deactivation time constant versus hydrophobicity of side chains substituted at V659. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles at the top of the y axis at the x axis location of appropriate hydrophobicity. Hydrophobicity scale adapted from Black and Mould (1991).
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fig3: V659 couples voltage sensing and gating. (A) Conductance–voltage relation for V659C mutant fit with a Boltzmann equation reveals constitutive conductance over negative potentials and shallower slope (z). (B) Scaled V659C tail currents evoked at −100 mV after a pulse to +60 mV show dramatically slowed deactivation. Dotted line indicates zero current level. (C) Plot of time constants obtained from fits to deactivating currents on a log scale plotted as a function of voltage for V659C. (D) Steady-state current versus voltage relations for a variety of substitutions made at V659. Mutations to A, F, or T were fit with a standard Boltzmann function and show constitutive conductance and shallower slope. V659I shows an intermediate phenotype that was fit with a double Boltzmann equation (see Materials and methods). n = 3–5 for each data point. (E) Scaled V659 mutant tail currents evoked at −100 mV after pulses to +60 mV. (F) Average deactivation time constants on a log scale versus voltage for V659 mutants. n = 3–5 for each data point. (G) Deactivation time constants versus side chain volume for V659 mutants. Amino acids with side chains significantly larger or smaller than the native valine show slowed deactivation. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles above the line at the top of the y axis at appropriate side chain volume. Side chain volume obtained from Zamyatnin (1972). (H) Deactivation time constant versus hydrophobicity of side chains substituted at V659. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles at the top of the y axis at the x axis location of appropriate hydrophobicity. Hydrophobicity scale adapted from Black and Mould (1991).

Mentions: A distinct but equally dramatic phenotype was observed for mutations at V659. The V659C G-V relationship exhibited a marked reduction in slope and less pronounced saturation at the voltage extremes (Fig. 3 A, red). Like Q664, Y667, and S668, V659C also exhibited a constitutive conductance that was abolished by CnErg-1 application (Fig. S6, which is available at http://www.jgp.org/cgi/content/full/jgp.200810083/DC1). The V659C mutation had little effect on the activation time constant (Table III), but slowed deactivation dramatically (Fig. 3, B and C).


hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

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

V659 couples voltage sensing and gating. (A) Conductance–voltage relation for V659C mutant fit with a Boltzmann equation reveals constitutive conductance over negative potentials and shallower slope (z). (B) Scaled V659C tail currents evoked at −100 mV after a pulse to +60 mV show dramatically slowed deactivation. Dotted line indicates zero current level. (C) Plot of time constants obtained from fits to deactivating currents on a log scale plotted as a function of voltage for V659C. (D) Steady-state current versus voltage relations for a variety of substitutions made at V659. Mutations to A, F, or T were fit with a standard Boltzmann function and show constitutive conductance and shallower slope. V659I shows an intermediate phenotype that was fit with a double Boltzmann equation (see Materials and methods). n = 3–5 for each data point. (E) Scaled V659 mutant tail currents evoked at −100 mV after pulses to +60 mV. (F) Average deactivation time constants on a log scale versus voltage for V659 mutants. n = 3–5 for each data point. (G) Deactivation time constants versus side chain volume for V659 mutants. Amino acids with side chains significantly larger or smaller than the native valine show slowed deactivation. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles above the line at the top of the y axis at appropriate side chain volume. Side chain volume obtained from Zamyatnin (1972). (H) Deactivation time constant versus hydrophobicity of side chains substituted at V659. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles at the top of the y axis at the x axis location of appropriate hydrophobicity. Hydrophobicity scale adapted from Black and Mould (1991).
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fig3: V659 couples voltage sensing and gating. (A) Conductance–voltage relation for V659C mutant fit with a Boltzmann equation reveals constitutive conductance over negative potentials and shallower slope (z). (B) Scaled V659C tail currents evoked at −100 mV after a pulse to +60 mV show dramatically slowed deactivation. Dotted line indicates zero current level. (C) Plot of time constants obtained from fits to deactivating currents on a log scale plotted as a function of voltage for V659C. (D) Steady-state current versus voltage relations for a variety of substitutions made at V659. Mutations to A, F, or T were fit with a standard Boltzmann function and show constitutive conductance and shallower slope. V659I shows an intermediate phenotype that was fit with a double Boltzmann equation (see Materials and methods). n = 3–5 for each data point. (E) Scaled V659 mutant tail currents evoked at −100 mV after pulses to +60 mV. (F) Average deactivation time constants on a log scale versus voltage for V659 mutants. n = 3–5 for each data point. (G) Deactivation time constants versus side chain volume for V659 mutants. Amino acids with side chains significantly larger or smaller than the native valine show slowed deactivation. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles above the line at the top of the y axis at appropriate side chain volume. Side chain volume obtained from Zamyatnin (1972). (H) Deactivation time constant versus hydrophobicity of side chains substituted at V659. Nonfunctional mutants (V659D, V659K, V659N, and V659P) are included as open triangles at the top of the y axis at the x axis location of appropriate hydrophobicity. Hydrophobicity scale adapted from Black and Mould (1991).
Mentions: A distinct but equally dramatic phenotype was observed for mutations at V659. The V659C G-V relationship exhibited a marked reduction in slope and less pronounced saturation at the voltage extremes (Fig. 3 A, red). Like Q664, Y667, and S668, V659C also exhibited a constitutive conductance that was abolished by CnErg-1 application (Fig. S6, which is available at http://www.jgp.org/cgi/content/full/jgp.200810083/DC1). The V659C mutation had little effect on the activation time constant (Table III), but slowed deactivation dramatically (Fig. 3, B and C).

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