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Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels.

Perry MD, Wong S, Ng CA, Vandenberg JI - J. Gen. Physiol. (2013)

Bottom Line: Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating.Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices.Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.

ABSTRACT
Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed "activation" and "inactivation," located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 "voltage-sensor" helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4-S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

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Scanning mutagenesis of the Kv11.1 channel S4 helix. (A–C) Shifts, relative to WT, in log(Keq,0) values for alanine (A), serine (B), and tryptophan (C) mutagenesis scans of S4 residues spanning from Gly522 to Lys538. Data are presented as means ± SEM for n = 3–20 cells (see Table S1). Dashed lines indicate Δlog(Keq,0) > ±0.5 log units, which has been shown previously to be the minimum perturbation required to derive an accurate Φ-value (Cymes et al., 2002; Wang et al., 2011). Mutations that fulfill this minimum requirement are indicated by closed bars, whereas mutations with Δlog(Keq,0) < 0.5 log units are indicated by open bars. *, mutant channels that failed to express or expressed poorly.
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fig2: Scanning mutagenesis of the Kv11.1 channel S4 helix. (A–C) Shifts, relative to WT, in log(Keq,0) values for alanine (A), serine (B), and tryptophan (C) mutagenesis scans of S4 residues spanning from Gly522 to Lys538. Data are presented as means ± SEM for n = 3–20 cells (see Table S1). Dashed lines indicate Δlog(Keq,0) > ±0.5 log units, which has been shown previously to be the minimum perturbation required to derive an accurate Φ-value (Cymes et al., 2002; Wang et al., 2011). Mutations that fulfill this minimum requirement are indicated by closed bars, whereas mutations with Δlog(Keq,0) < 0.5 log units are indicated by open bars. *, mutant channels that failed to express or expressed poorly.

Mentions: To obtain an accurate estimate of a Φ-value from a REFER plot, one or more mutations of a given residue must cause a sufficient perturbation to the transition pathway. We, both previously (Wang et al., 2011) and in Fig. S2, and others (Cymes et al., 2002; Fersht and Sato, 2004) have established a Δlog(Keq,0) of greater than or equal to ±0.5 as a reasonable cutoff criterion. Based on these criteria, we used REFER analysis to perform an extensive investigation of the role of the S4 helix during Kv11.1 channel inactivation. To optimize our chances of finding mutant channels within the S4 helix that were amenable to REFER analysis, we performed three separate mutagenesis scans on residues Gly522 to Lys538. Each residue was mutated to alanine (Fig. 2 A), serine (Fig. 2 B), or tryptophan (Fig. 2 C). From these scans, several residues stood out as having at least one mutation with a Δlog(Keq,0) of greater than ±0.5 log units, namely, L529A/S, L530S/W, R531A/S, L532S, V535A/W/S, and R537W (Fig. 2, closed bars, and Table S1).


Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels.

Perry MD, Wong S, Ng CA, Vandenberg JI - J. Gen. Physiol. (2013)

Scanning mutagenesis of the Kv11.1 channel S4 helix. (A–C) Shifts, relative to WT, in log(Keq,0) values for alanine (A), serine (B), and tryptophan (C) mutagenesis scans of S4 residues spanning from Gly522 to Lys538. Data are presented as means ± SEM for n = 3–20 cells (see Table S1). Dashed lines indicate Δlog(Keq,0) > ±0.5 log units, which has been shown previously to be the minimum perturbation required to derive an accurate Φ-value (Cymes et al., 2002; Wang et al., 2011). Mutations that fulfill this minimum requirement are indicated by closed bars, whereas mutations with Δlog(Keq,0) < 0.5 log units are indicated by open bars. *, mutant channels that failed to express or expressed poorly.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3753607&req=5

fig2: Scanning mutagenesis of the Kv11.1 channel S4 helix. (A–C) Shifts, relative to WT, in log(Keq,0) values for alanine (A), serine (B), and tryptophan (C) mutagenesis scans of S4 residues spanning from Gly522 to Lys538. Data are presented as means ± SEM for n = 3–20 cells (see Table S1). Dashed lines indicate Δlog(Keq,0) > ±0.5 log units, which has been shown previously to be the minimum perturbation required to derive an accurate Φ-value (Cymes et al., 2002; Wang et al., 2011). Mutations that fulfill this minimum requirement are indicated by closed bars, whereas mutations with Δlog(Keq,0) < 0.5 log units are indicated by open bars. *, mutant channels that failed to express or expressed poorly.
Mentions: To obtain an accurate estimate of a Φ-value from a REFER plot, one or more mutations of a given residue must cause a sufficient perturbation to the transition pathway. We, both previously (Wang et al., 2011) and in Fig. S2, and others (Cymes et al., 2002; Fersht and Sato, 2004) have established a Δlog(Keq,0) of greater than or equal to ±0.5 as a reasonable cutoff criterion. Based on these criteria, we used REFER analysis to perform an extensive investigation of the role of the S4 helix during Kv11.1 channel inactivation. To optimize our chances of finding mutant channels within the S4 helix that were amenable to REFER analysis, we performed three separate mutagenesis scans on residues Gly522 to Lys538. Each residue was mutated to alanine (Fig. 2 A), serine (Fig. 2 B), or tryptophan (Fig. 2 C). From these scans, several residues stood out as having at least one mutation with a Δlog(Keq,0) of greater than ±0.5 log units, namely, L529A/S, L530S/W, R531A/S, L532S, V535A/W/S, and R537W (Fig. 2, closed bars, and Table S1).

Bottom Line: Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating.Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices.Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Cardiology and Biophysics Division, Victor Chang Cardiac Research Institute, Darlinghurst, NSW 2010, Australia.

ABSTRACT
Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed "activation" and "inactivation," located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 "voltage-sensor" helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4-S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

Show MeSH
Related in: MedlinePlus