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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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Constitutively conducting S6 mutants. (A) Conductance–voltage relations for constitutively conducting S6 mutants Q664C, Y667C, and S668C illustrate steady-state current at negative potentials where the channels are normally closed (n = 3–10). Tail currents were normalized as described in Materials and methods to produce G/Gmax. Points are means ± SEM. (B) Scaled tail currents evoked at −100 mV after a step to +60 mV show constitutively conducting mutant channels deactivate more slowly than control channels. Dotted line indicates zero current level. (C) Average deactivation time constant (see Materials and methods) as a function of voltage for constitutive conductors. (D–G) Homology modeling of constitutively conducting mutants reveals a domain of clustered amino acid side chains. Backbone α-helix is represented as gray ribbon; native side chains of affected residues are represented as space-filled spheres, with Q664 in green, Y667 in orange, and S668 in purple. S1–S4 domains have been omitted from the model views for clarity. (D) Model of hERG based on rKv1.2 (open state) in membrane cross-sectional view. (E) Model of hERG based on MlotiK1 (closed state) in membrane cross-sectional view. (F) Model of hERG based on Kv1.2 (open state) viewed from the cytosol. (G) Model of hERG based on MlotiK1 (closed state) viewed from the cytosol. (H) Amplitude of slow and fast time constants of deactivation for constitutively conducting mutants. (I) Application of CnErg-1 reduces the level of hERG constitutive conductance in Q664C, Y667C, and S668C.
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fig2: Constitutively conducting S6 mutants. (A) Conductance–voltage relations for constitutively conducting S6 mutants Q664C, Y667C, and S668C illustrate steady-state current at negative potentials where the channels are normally closed (n = 3–10). Tail currents were normalized as described in Materials and methods to produce G/Gmax. Points are means ± SEM. (B) Scaled tail currents evoked at −100 mV after a step to +60 mV show constitutively conducting mutant channels deactivate more slowly than control channels. Dotted line indicates zero current level. (C) Average deactivation time constant (see Materials and methods) as a function of voltage for constitutive conductors. (D–G) Homology modeling of constitutively conducting mutants reveals a domain of clustered amino acid side chains. Backbone α-helix is represented as gray ribbon; native side chains of affected residues are represented as space-filled spheres, with Q664 in green, Y667 in orange, and S668 in purple. S1–S4 domains have been omitted from the model views for clarity. (D) Model of hERG based on rKv1.2 (open state) in membrane cross-sectional view. (E) Model of hERG based on MlotiK1 (closed state) in membrane cross-sectional view. (F) Model of hERG based on Kv1.2 (open state) viewed from the cytosol. (G) Model of hERG based on MlotiK1 (closed state) viewed from the cytosol. (H) Amplitude of slow and fast time constants of deactivation for constitutively conducting mutants. (I) Application of CnErg-1 reduces the level of hERG constitutive conductance in Q664C, Y667C, and S668C.

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)

Constitutively conducting S6 mutants. (A) Conductance–voltage relations for constitutively conducting S6 mutants Q664C, Y667C, and S668C illustrate steady-state current at negative potentials where the channels are normally closed (n = 3–10). Tail currents were normalized as described in Materials and methods to produce G/Gmax. Points are means ± SEM. (B) Scaled tail currents evoked at −100 mV after a step to +60 mV show constitutively conducting mutant channels deactivate more slowly than control channels. Dotted line indicates zero current level. (C) Average deactivation time constant (see Materials and methods) as a function of voltage for constitutive conductors. (D–G) Homology modeling of constitutively conducting mutants reveals a domain of clustered amino acid side chains. Backbone α-helix is represented as gray ribbon; native side chains of affected residues are represented as space-filled spheres, with Q664 in green, Y667 in orange, and S668 in purple. S1–S4 domains have been omitted from the model views for clarity. (D) Model of hERG based on rKv1.2 (open state) in membrane cross-sectional view. (E) Model of hERG based on MlotiK1 (closed state) in membrane cross-sectional view. (F) Model of hERG based on Kv1.2 (open state) viewed from the cytosol. (G) Model of hERG based on MlotiK1 (closed state) viewed from the cytosol. (H) Amplitude of slow and fast time constants of deactivation for constitutively conducting mutants. (I) Application of CnErg-1 reduces the level of hERG constitutive conductance in Q664C, Y667C, and S668C.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2571969&req=5

fig2: Constitutively conducting S6 mutants. (A) Conductance–voltage relations for constitutively conducting S6 mutants Q664C, Y667C, and S668C illustrate steady-state current at negative potentials where the channels are normally closed (n = 3–10). Tail currents were normalized as described in Materials and methods to produce G/Gmax. Points are means ± SEM. (B) Scaled tail currents evoked at −100 mV after a step to +60 mV show constitutively conducting mutant channels deactivate more slowly than control channels. Dotted line indicates zero current level. (C) Average deactivation time constant (see Materials and methods) as a function of voltage for constitutive conductors. (D–G) Homology modeling of constitutively conducting mutants reveals a domain of clustered amino acid side chains. Backbone α-helix is represented as gray ribbon; native side chains of affected residues are represented as space-filled spheres, with Q664 in green, Y667 in orange, and S668 in purple. S1–S4 domains have been omitted from the model views for clarity. (D) Model of hERG based on rKv1.2 (open state) in membrane cross-sectional view. (E) Model of hERG based on MlotiK1 (closed state) in membrane cross-sectional view. (F) Model of hERG based on Kv1.2 (open state) viewed from the cytosol. (G) Model of hERG based on MlotiK1 (closed state) viewed from the cytosol. (H) Amplitude of slow and fast time constants of deactivation for constitutively conducting mutants. (I) Application of CnErg-1 reduces the level of hERG constitutive conductance in Q664C, Y667C, and S668C.
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