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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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A661C and I663C primarily affect activation time course. (A) Conductance versus voltage relations for A661C and I663C. (B) Current traces of slowly activating mutant channels A661C and I663C. Traces were evoked by a depolarizing pulse to +40 mV from a holding potential of −80 mV. Dashed line indicates zero current level. (C) Time constants of activation over the range of voltages tested. Activation traces as in B were fit with a single exponential function, ignoring initial sigmoidicity so as to capture the kinetics of the final closed to open transition. n = 8–10 for each data point. The activation time constant is voltage dependent and slower over the range of voltages. (D) Cross-sectional membrane view showing A661 (orange) and I663 (green) side chains based on the closed-state MlotiK1 homology model. (E) View from the cytoplasm of the closed-state MlotiK1 homology model, illustrating the proximity of A661 and I663 side chains from neighboring subunits. (F) Close-up view shows A661 and I663 side chain interaction in the closed state. (G) Cross-sectional membrane view of A661 and I663 in the open-state rKv1.2 homology model. (H) View of A661 and I663 from the cytoplasm in the open state. (I) Close-up view of A661 and I663 in open state.
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fig5: A661C and I663C primarily affect activation time course. (A) Conductance versus voltage relations for A661C and I663C. (B) Current traces of slowly activating mutant channels A661C and I663C. Traces were evoked by a depolarizing pulse to +40 mV from a holding potential of −80 mV. Dashed line indicates zero current level. (C) Time constants of activation over the range of voltages tested. Activation traces as in B were fit with a single exponential function, ignoring initial sigmoidicity so as to capture the kinetics of the final closed to open transition. n = 8–10 for each data point. The activation time constant is voltage dependent and slower over the range of voltages. (D) Cross-sectional membrane view showing A661 (orange) and I663 (green) side chains based on the closed-state MlotiK1 homology model. (E) View from the cytoplasm of the closed-state MlotiK1 homology model, illustrating the proximity of A661 and I663 side chains from neighboring subunits. (F) Close-up view shows A661 and I663 side chain interaction in the closed state. (G) Cross-sectional membrane view of A661 and I663 in the open-state rKv1.2 homology model. (H) View of A661 and I663 from the cytoplasm in the open state. (I) Close-up view of A661 and I663 in open state.

Mentions: Supplemental material includes six figures, several PDB files, one table, and a document detailing methods for the construction of the KcsA-based homology model. Figs. S1–S4 show views of the KcsA-based homology model of the closed state for comparison with the MlotiK1-based homology models shown in the paper. Specifically, Fig. S1 illustrates the native side chain locations of constitutively conducting mutants (compare with Fig. 2). Fig. S2 shows the location of the native V659 side chain (compare with Fig. 4). Fig. S3 shows locations of native A661 and I663 side chains (compare with Fig. 5). Fig. S4 displays the locations of the native G657 and N658 side chains. Fig. S5 shows deactivating tail currents for S6 mutants in wild-type background, demonstrating similar phenotypes to those in the S620T background. Table S1 presents the average deactivation time constants derived from a bi-exponential fit for these mutants. Fig. S6 demonstrates reduction of constitutive conductance in V659 after CnErg-1 application. PDB files containing the coordinates for each of the homology models (based on KcsA, rKv1.2, and MlotiK1) are included. The online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200810083/DC1.


hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

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

A661C and I663C primarily affect activation time course. (A) Conductance versus voltage relations for A661C and I663C. (B) Current traces of slowly activating mutant channels A661C and I663C. Traces were evoked by a depolarizing pulse to +40 mV from a holding potential of −80 mV. Dashed line indicates zero current level. (C) Time constants of activation over the range of voltages tested. Activation traces as in B were fit with a single exponential function, ignoring initial sigmoidicity so as to capture the kinetics of the final closed to open transition. n = 8–10 for each data point. The activation time constant is voltage dependent and slower over the range of voltages. (D) Cross-sectional membrane view showing A661 (orange) and I663 (green) side chains based on the closed-state MlotiK1 homology model. (E) View from the cytoplasm of the closed-state MlotiK1 homology model, illustrating the proximity of A661 and I663 side chains from neighboring subunits. (F) Close-up view shows A661 and I663 side chain interaction in the closed state. (G) Cross-sectional membrane view of A661 and I663 in the open-state rKv1.2 homology model. (H) View of A661 and I663 from the cytoplasm in the open state. (I) Close-up view of A661 and I663 in open state.
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Related In: Results  -  Collection

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fig5: A661C and I663C primarily affect activation time course. (A) Conductance versus voltage relations for A661C and I663C. (B) Current traces of slowly activating mutant channels A661C and I663C. Traces were evoked by a depolarizing pulse to +40 mV from a holding potential of −80 mV. Dashed line indicates zero current level. (C) Time constants of activation over the range of voltages tested. Activation traces as in B were fit with a single exponential function, ignoring initial sigmoidicity so as to capture the kinetics of the final closed to open transition. n = 8–10 for each data point. The activation time constant is voltage dependent and slower over the range of voltages. (D) Cross-sectional membrane view showing A661 (orange) and I663 (green) side chains based on the closed-state MlotiK1 homology model. (E) View from the cytoplasm of the closed-state MlotiK1 homology model, illustrating the proximity of A661 and I663 side chains from neighboring subunits. (F) Close-up view shows A661 and I663 side chain interaction in the closed state. (G) Cross-sectional membrane view of A661 and I663 in the open-state rKv1.2 homology model. (H) View of A661 and I663 from the cytoplasm in the open state. (I) Close-up view of A661 and I663 in open state.
Mentions: Supplemental material includes six figures, several PDB files, one table, and a document detailing methods for the construction of the KcsA-based homology model. Figs. S1–S4 show views of the KcsA-based homology model of the closed state for comparison with the MlotiK1-based homology models shown in the paper. Specifically, Fig. S1 illustrates the native side chain locations of constitutively conducting mutants (compare with Fig. 2). Fig. S2 shows the location of the native V659 side chain (compare with Fig. 4). Fig. S3 shows locations of native A661 and I663 side chains (compare with Fig. 5). Fig. S4 displays the locations of the native G657 and N658 side chains. Fig. S5 shows deactivating tail currents for S6 mutants in wild-type background, demonstrating similar phenotypes to those in the S620T background. Table S1 presents the average deactivation time constants derived from a bi-exponential fit for these mutants. Fig. S6 demonstrates reduction of constitutive conductance in V659 after CnErg-1 application. PDB files containing the coordinates for each of the homology models (based on KcsA, rKv1.2, and MlotiK1) are included. The online supplemental material is available at http://www.jgp.org/cgi/content/full/jgp.200810083/DC1.

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