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The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation.

Ng CA, Hunter MJ, Perry MD, Mobli M, Ke Y, Kuchel PW, King GF, Stock D, Vandenberg JI - PLoS ONE (2011)

Bottom Line: Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics.Alanine mutants in the helical region had less dramatic phenotypes.We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

ABSTRACT
The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

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Point mutations within the N–terminal tail alter deactivation rates of hERG channels.Examples of point mutations that either slow (D16A, blue trace in left panel) or enhance (R5A, red trace in right panel) rates of deactivation compared to WT hERG (black traces) are shown in panel A. Rates of deactivation are represented by the decay in tail currents recorded at −120 mV following a step to +40 mV. Current traces were normalized to peak tail current to aid comparison. B. Mean ± SEM rates of deactivation (τfast) plotted against the total electrochemical driving force –(ΔG0–zgEF) for channel deactivation. When compared at an equivalent driving force of –30 kJmol–1 (dotted line) four mutant channels exhibited altered deactivation rates compared with WT hERG (open squares, n = 11). Two mutations, P10A (green circles, n = 14) and D16A (blue triangles, n = 10), produced channels that were slower than WT (left panel), while R5A (red diamonds, n = 8) and G6A mutant channels (orange inverted triangles, n = 7) deactivated faster than WT hERG (right panel).
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pone-0016191-g005: Point mutations within the N–terminal tail alter deactivation rates of hERG channels.Examples of point mutations that either slow (D16A, blue trace in left panel) or enhance (R5A, red trace in right panel) rates of deactivation compared to WT hERG (black traces) are shown in panel A. Rates of deactivation are represented by the decay in tail currents recorded at −120 mV following a step to +40 mV. Current traces were normalized to peak tail current to aid comparison. B. Mean ± SEM rates of deactivation (τfast) plotted against the total electrochemical driving force –(ΔG0–zgEF) for channel deactivation. When compared at an equivalent driving force of –30 kJmol–1 (dotted line) four mutant channels exhibited altered deactivation rates compared with WT hERG (open squares, n = 11). Two mutations, P10A (green circles, n = 14) and D16A (blue triangles, n = 10), produced channels that were slower than WT (left panel), while R5A (red diamonds, n = 8) and G6A mutant channels (orange inverted triangles, n = 7) deactivated faster than WT hERG (right panel).

Mentions: To probe the role of individual residues within the N–terminal tail, native residues from P2–E23 were individually replaced with alanine, or with valine in the case of A9. Measured rates for fast (τfast) and slow (τslow) components of deactivation, in addition to the relative contributions of these components are given in Table S2. At negative potentials, the fast component accounted for the majority of deactivation (>80%). This parameter was therefore used to compare WT and mutant channels. Several of the mutations introduced small (less than ±10 mV), and statistically significant shifts in the voltage dependence of channel activation when compared to WT hERG (Table S1). Accordingly, the effects of each mutation on deactivation rate were compared at an equivalent driving force of −30 kJmol–1 (as indicated in Fig. 5). In Fig. 6, the effect of alanine mutants on deactivation rates are classified into those that were unchanged (grey bars), faster (red bars) and slower (blue bars) compared to WT.


The N-terminal tail of hERG contains an amphipathic α-helix that regulates channel deactivation.

Ng CA, Hunter MJ, Perry MD, Mobli M, Ke Y, Kuchel PW, King GF, Stock D, Vandenberg JI - PLoS ONE (2011)

Point mutations within the N–terminal tail alter deactivation rates of hERG channels.Examples of point mutations that either slow (D16A, blue trace in left panel) or enhance (R5A, red trace in right panel) rates of deactivation compared to WT hERG (black traces) are shown in panel A. Rates of deactivation are represented by the decay in tail currents recorded at −120 mV following a step to +40 mV. Current traces were normalized to peak tail current to aid comparison. B. Mean ± SEM rates of deactivation (τfast) plotted against the total electrochemical driving force –(ΔG0–zgEF) for channel deactivation. When compared at an equivalent driving force of –30 kJmol–1 (dotted line) four mutant channels exhibited altered deactivation rates compared with WT hERG (open squares, n = 11). Two mutations, P10A (green circles, n = 14) and D16A (blue triangles, n = 10), produced channels that were slower than WT (left panel), while R5A (red diamonds, n = 8) and G6A mutant channels (orange inverted triangles, n = 7) deactivated faster than WT hERG (right panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020963&req=5

pone-0016191-g005: Point mutations within the N–terminal tail alter deactivation rates of hERG channels.Examples of point mutations that either slow (D16A, blue trace in left panel) or enhance (R5A, red trace in right panel) rates of deactivation compared to WT hERG (black traces) are shown in panel A. Rates of deactivation are represented by the decay in tail currents recorded at −120 mV following a step to +40 mV. Current traces were normalized to peak tail current to aid comparison. B. Mean ± SEM rates of deactivation (τfast) plotted against the total electrochemical driving force –(ΔG0–zgEF) for channel deactivation. When compared at an equivalent driving force of –30 kJmol–1 (dotted line) four mutant channels exhibited altered deactivation rates compared with WT hERG (open squares, n = 11). Two mutations, P10A (green circles, n = 14) and D16A (blue triangles, n = 10), produced channels that were slower than WT (left panel), while R5A (red diamonds, n = 8) and G6A mutant channels (orange inverted triangles, n = 7) deactivated faster than WT hERG (right panel).
Mentions: To probe the role of individual residues within the N–terminal tail, native residues from P2–E23 were individually replaced with alanine, or with valine in the case of A9. Measured rates for fast (τfast) and slow (τslow) components of deactivation, in addition to the relative contributions of these components are given in Table S2. At negative potentials, the fast component accounted for the majority of deactivation (>80%). This parameter was therefore used to compare WT and mutant channels. Several of the mutations introduced small (less than ±10 mV), and statistically significant shifts in the voltage dependence of channel activation when compared to WT hERG (Table S1). Accordingly, the effects of each mutation on deactivation rate were compared at an equivalent driving force of −30 kJmol–1 (as indicated in Fig. 5). In Fig. 6, the effect of alanine mutants on deactivation rates are classified into those that were unchanged (grey bars), faster (red bars) and slower (blue bars) compared to WT.

Bottom Line: Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics.Alanine mutants in the helical region had less dramatic phenotypes.We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

View Article: PubMed Central - PubMed

Affiliation: Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, New South Wales, Australia.

ABSTRACT
The cytoplasmic N-terminal domain of the human ether-a-go-go related gene (hERG) K+ channel is critical for the slow deactivation kinetics of the channel. However, the mechanism(s) by which the N-terminal domain regulates deactivation remains to be determined. Here we show that the solution NMR structure of the N-terminal 135 residues of hERG contains a previously described Per-Arnt-Sim (PAS) domain (residues 26-135) as well as an amphipathic α-helix (residues 13-23) and an initial unstructured segment (residues 2-9). Deletion of residues 2-25, only the unstructured segment (residues 2-9) or replacement of the α-helix with a flexible linker all result in enhanced rates of deactivation. Thus, both the initial flexible segment and the α-helix are required but neither is sufficient to confer slow deactivation kinetics. Alanine scanning mutagenesis identified R5 and G6 in the initial flexible segment as critical for slow deactivation. Alanine mutants in the helical region had less dramatic phenotypes. We propose that the PAS domain is bound close to the central core of the channel and that the N-terminal α-helix ensures that the flexible tail is correctly orientated for interaction with the activation gating machinery to stabilize the open state of the channel.

Show MeSH
Related in: MedlinePlus