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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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Topology and alignment of hERG and related channels. (A) Energy-minimized models of hERG in membrane cross section in the closed (left) and open (right) states based on crystal structures of MlotiK1 and rKv1.2 (see text). The S4-S5 linker in shadow and S5 and S6 in gray ribbons from two of the four subunits are shown. S6 residues highlighted are, top-to-bottom: F656 (blue), V659 (red), S660 (yellow; aligns with V478 in Shaker), Q664 (green), Y667 (orange), and S668 (purple). (B) Schematic of one hERG subunit. α-helical transmembrane domains S1 through S6 along with the pore helix are represented as cylinders. S6 domains from each of the four subunits form the ion conduction pathway. The region of mutagenic scan of S6 is bracketed. CNBD, cyclic nucleotide binding domain; PAS, per-arnt-sim domain. (C) Alignment of hERG, MlotiK1, and KcsA used in homology modeling. Selectivity filter and region of mutagenic scan in hERG is boxed, as is rKv1.2 residue analogous to Shaker residue V478, corresponding to the region of the activation gate (Liu et al., 1997; Kitaguchi et al., 2004).
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fig1: Topology and alignment of hERG and related channels. (A) Energy-minimized models of hERG in membrane cross section in the closed (left) and open (right) states based on crystal structures of MlotiK1 and rKv1.2 (see text). The S4-S5 linker in shadow and S5 and S6 in gray ribbons from two of the four subunits are shown. S6 residues highlighted are, top-to-bottom: F656 (blue), V659 (red), S660 (yellow; aligns with V478 in Shaker), Q664 (green), Y667 (orange), and S668 (purple). (B) Schematic of one hERG subunit. α-helical transmembrane domains S1 through S6 along with the pore helix are represented as cylinders. S6 domains from each of the four subunits form the ion conduction pathway. The region of mutagenic scan of S6 is bracketed. CNBD, cyclic nucleotide binding domain; PAS, per-arnt-sim domain. (C) Alignment of hERG, MlotiK1, and KcsA used in homology modeling. Selectivity filter and region of mutagenic scan in hERG is boxed, as is rKv1.2 residue analogous to Shaker residue V478, corresponding to the region of the activation gate (Liu et al., 1997; Kitaguchi et al., 2004).

Mentions: Compounds that block hERG enter the pore from the cytosol through the open activation gate (Snyders and Chaudhary, 1996; Zhou et al., 1998). Residue F656 (see Fig. 1 A, blue) in the S6 transmembrane domain is critical for drug block, as mutagenesis at this site reduces the block by several compounds by several orders of magnitude (Lees-Miller et al., 2000; Mitcheson et al., 2000a, 2000b). An S4-S5 linker mutation that allows reactivation of current at hyperpolarized voltages alleviates hERG block, indicating that drugs are trapped in the vestibule by a gate that regulates the permeant path (Mitcheson et al., 2000b). Determining the structural and functional details of S6 will be important in understanding why hERG channels are blocked with high affinity by such a wide range of drugs.


hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

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

Topology and alignment of hERG and related channels. (A) Energy-minimized models of hERG in membrane cross section in the closed (left) and open (right) states based on crystal structures of MlotiK1 and rKv1.2 (see text). The S4-S5 linker in shadow and S5 and S6 in gray ribbons from two of the four subunits are shown. S6 residues highlighted are, top-to-bottom: F656 (blue), V659 (red), S660 (yellow; aligns with V478 in Shaker), Q664 (green), Y667 (orange), and S668 (purple). (B) Schematic of one hERG subunit. α-helical transmembrane domains S1 through S6 along with the pore helix are represented as cylinders. S6 domains from each of the four subunits form the ion conduction pathway. The region of mutagenic scan of S6 is bracketed. CNBD, cyclic nucleotide binding domain; PAS, per-arnt-sim domain. (C) Alignment of hERG, MlotiK1, and KcsA used in homology modeling. Selectivity filter and region of mutagenic scan in hERG is boxed, as is rKv1.2 residue analogous to Shaker residue V478, corresponding to the region of the activation gate (Liu et al., 1997; Kitaguchi et al., 2004).
© Copyright Policy
Related In: Results  -  Collection

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

fig1: Topology and alignment of hERG and related channels. (A) Energy-minimized models of hERG in membrane cross section in the closed (left) and open (right) states based on crystal structures of MlotiK1 and rKv1.2 (see text). The S4-S5 linker in shadow and S5 and S6 in gray ribbons from two of the four subunits are shown. S6 residues highlighted are, top-to-bottom: F656 (blue), V659 (red), S660 (yellow; aligns with V478 in Shaker), Q664 (green), Y667 (orange), and S668 (purple). (B) Schematic of one hERG subunit. α-helical transmembrane domains S1 through S6 along with the pore helix are represented as cylinders. S6 domains from each of the four subunits form the ion conduction pathway. The region of mutagenic scan of S6 is bracketed. CNBD, cyclic nucleotide binding domain; PAS, per-arnt-sim domain. (C) Alignment of hERG, MlotiK1, and KcsA used in homology modeling. Selectivity filter and region of mutagenic scan in hERG is boxed, as is rKv1.2 residue analogous to Shaker residue V478, corresponding to the region of the activation gate (Liu et al., 1997; Kitaguchi et al., 2004).
Mentions: Compounds that block hERG enter the pore from the cytosol through the open activation gate (Snyders and Chaudhary, 1996; Zhou et al., 1998). Residue F656 (see Fig. 1 A, blue) in the S6 transmembrane domain is critical for drug block, as mutagenesis at this site reduces the block by several compounds by several orders of magnitude (Lees-Miller et al., 2000; Mitcheson et al., 2000a, 2000b). An S4-S5 linker mutation that allows reactivation of current at hyperpolarized voltages alleviates hERG block, indicating that drugs are trapped in the vestibule by a gate that regulates the permeant path (Mitcheson et al., 2000b). Determining the structural and functional details of S6 will be important in understanding why hERG channels are blocked with high affinity by such a wide range of drugs.

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