Limits...
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.

Show MeSH

Related in: MedlinePlus

G657C and N658C affect both activation and deactivation. (A) Conductance versus voltage relations for G657C and N658C. An empirical double Boltzmann relation was required to fit the curve for G657C (See Materials and methods). (B) Exemplar G657C and N658C currents evoked at +40 mV from a holding potential of −80 mV. (C) Voltage dependence of G657C and N658C activation time constants. (D) Exemplar deactivation traces at −100 mV after a pulse to +40 mV. (E) Average deactivation time constants versus voltage. (F) Closed-state homology model of the closed state shows G657 (blue) and N658 (red) located next to each other in the vestibule. (G) A view from the cytoplasm shows the side chains' proximity to one another. (H) Open-state homology model cross-sectional membrane view shows G657 and N658 at the level of the S4-S5 linker. (I) View of G657 and N658 from the cytoplasm in the closed state.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: G657C and N658C affect both activation and deactivation. (A) Conductance versus voltage relations for G657C and N658C. An empirical double Boltzmann relation was required to fit the curve for G657C (See Materials and methods). (B) Exemplar G657C and N658C currents evoked at +40 mV from a holding potential of −80 mV. (C) Voltage dependence of G657C and N658C activation time constants. (D) Exemplar deactivation traces at −100 mV after a pulse to +40 mV. (E) Average deactivation time constants versus voltage. (F) Closed-state homology model of the closed state shows G657 (blue) and N658 (red) located next to each other in the vestibule. (G) A view from the cytoplasm shows the side chains' proximity to one another. (H) Open-state homology model cross-sectional membrane view shows G657 and N658 at the level of the S4-S5 linker. (I) View of G657 and N658 from the cytoplasm in the closed state.

Mentions: Adjacent residues G657C and N658C exhibited distinct G-V characteristics but similar kinetic phenotypes. The G-V curve was positively shifted for N658C and required a double Boltzmann fit for G657C (Fig. 6 A and Table IV). Both showed an increase in apparent rate of activation (Fig. 6, B and C, and Table III) and deactivation (Fig. 6, D and E, and Table I). The similar phenotypes exhibited by these cysteine mutants likely relate more to their linkage as adjacent residues because there are no obvious common side chain interactions in the molecular models (Fig. 6, F–I). Like V659, these residues correspond to the region critical for gating in Shaker channels (Fig. 1 C), and the phenotypes indicate that mutations on all sides of the helix in this region have considerable impact on gating.


hERG gating microdomains defined by S6 mutagenesis and molecular modeling.

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

G657C and N658C affect both activation and deactivation. (A) Conductance versus voltage relations for G657C and N658C. An empirical double Boltzmann relation was required to fit the curve for G657C (See Materials and methods). (B) Exemplar G657C and N658C currents evoked at +40 mV from a holding potential of −80 mV. (C) Voltage dependence of G657C and N658C activation time constants. (D) Exemplar deactivation traces at −100 mV after a pulse to +40 mV. (E) Average deactivation time constants versus voltage. (F) Closed-state homology model of the closed state shows G657 (blue) and N658 (red) located next to each other in the vestibule. (G) A view from the cytoplasm shows the side chains' proximity to one another. (H) Open-state homology model cross-sectional membrane view shows G657 and N658 at the level of the S4-S5 linker. (I) View of G657 and N658 from the cytoplasm in the closed state.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: G657C and N658C affect both activation and deactivation. (A) Conductance versus voltage relations for G657C and N658C. An empirical double Boltzmann relation was required to fit the curve for G657C (See Materials and methods). (B) Exemplar G657C and N658C currents evoked at +40 mV from a holding potential of −80 mV. (C) Voltage dependence of G657C and N658C activation time constants. (D) Exemplar deactivation traces at −100 mV after a pulse to +40 mV. (E) Average deactivation time constants versus voltage. (F) Closed-state homology model of the closed state shows G657 (blue) and N658 (red) located next to each other in the vestibule. (G) A view from the cytoplasm shows the side chains' proximity to one another. (H) Open-state homology model cross-sectional membrane view shows G657 and N658 at the level of the S4-S5 linker. (I) View of G657 and N658 from the cytoplasm in the closed state.
Mentions: Adjacent residues G657C and N658C exhibited distinct G-V characteristics but similar kinetic phenotypes. The G-V curve was positively shifted for N658C and required a double Boltzmann fit for G657C (Fig. 6 A and Table IV). Both showed an increase in apparent rate of activation (Fig. 6, B and C, and Table III) and deactivation (Fig. 6, D and E, and Table I). The similar phenotypes exhibited by these cysteine mutants likely relate more to their linkage as adjacent residues because there are no obvious common side chain interactions in the molecular models (Fig. 6, F–I). Like V659, these residues correspond to the region critical for gating in Shaker channels (Fig. 1 C), and the phenotypes indicate that mutations on all sides of the helix in this region have considerable impact on gating.

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