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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.

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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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Families of mutations at important Kv11.1 channel S4 residues. (A and B) Shifts in log(Keq,0), relative to WT, for families of mutations at charged (A) or hydrophobic (B) residues that were identified as important determinants for inactivation gating from our mutagenesis scans of the S4 helix. Mutations are indicated by single–amino acid code on the y axis. Data are presented as means ± SEM for n = 4–14 cells (see Table S1). Mutations that cause a significant perturbation to inactivation, measured as Δlog(Keq,0) > ±0.5 log units relative to WT, 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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fig3: Families of mutations at important Kv11.1 channel S4 residues. (A and B) Shifts in log(Keq,0), relative to WT, for families of mutations at charged (A) or hydrophobic (B) residues that were identified as important determinants for inactivation gating from our mutagenesis scans of the S4 helix. Mutations are indicated by single–amino acid code on the y axis. Data are presented as means ± SEM for n = 4–14 cells (see Table S1). Mutations that cause a significant perturbation to inactivation, measured as Δlog(Keq,0) > ±0.5 log units relative to WT, 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: Although it is possible to derive Φ-values from the individual mutant channels listed above (see Table S1), it is important to note that the temporal information they provide is based on the effect of the specific mutation in perturbing the transition pathway, and may not necessarily reflect the role of the native amino acid residue per se. For example, any single mutation could affect two or more different processes that, by coincidence, cancel each other out to give a Φ-value between 0 and 1 (see Materials and methods). However, this type of coincidental combination will not occur with every mutation at that position. Therefore, a better estimation of Φ-values can be derived from plots of log(kinact,0) versus log(Keq,0) for a family of mutations at a single residue or region of the channel (Grosman et al., 2000). We therefore interrogated S4 helix residues identified from our scanning mutagenesis in more detail by obtaining families of mutations at each position (Fig. 3). Mutation of charged residues Arg531 (to Gln, Thr, and Asn) or Arg537 (to Gln, Asp, and Thr) did not produce any further mutant channels with adequate perturbations to Δlog(Keq,0) (Wang et al., 2011, and Fig. 3 A). The relative tolerance of neutral or polar side chains at these positions, as well as at Lys525, Arg528, Arg534, and Lys538 (Table S1 and Wang et al., 2011), suggests that the positively charged side chains are not critical to the inactivation gating transition. We therefore concentrated on the hydrophobic residues: Leu529, Leu530, Leu532, and Val535, which were further mutated to threonine, histidine, proline, asparagine, and glutamine. In the case of Val535, we also mutated this residue to glycine, leucine, isoleucine, methionine, and tyrosine. Perturbations to Δlog(Keq,0) of greater than ±0.5 log units were observed for several mutant channels at all positions, with the exception of Leu532 (Fig. 3 B). Hydrophobic residues Leu530 and Val535 were particularly sensitive to mutation; i.e., tyrosine, methionine, glycine, proline, histidine, asparagine, and threonine mutations at Val535 all exhibited a Δlog(Keq,0) of >0.5 log units compared with WT, whereas only the relatively conserved hydrophobic side chains of leucine and isoleucine produced smaller perturbations (Fig. 3 B). The relative intolerance of these hydrophobic residues to mutations that do not maintain their hydrophobic side chain properties indicates their importance in mediating open-to-inactivated transition.


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)

Families of mutations at important Kv11.1 channel S4 residues. (A and B) Shifts in log(Keq,0), relative to WT, for families of mutations at charged (A) or hydrophobic (B) residues that were identified as important determinants for inactivation gating from our mutagenesis scans of the S4 helix. Mutations are indicated by single–amino acid code on the y axis. Data are presented as means ± SEM for n = 4–14 cells (see Table S1). Mutations that cause a significant perturbation to inactivation, measured as Δlog(Keq,0) > ±0.5 log units relative to WT, 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

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fig3: Families of mutations at important Kv11.1 channel S4 residues. (A and B) Shifts in log(Keq,0), relative to WT, for families of mutations at charged (A) or hydrophobic (B) residues that were identified as important determinants for inactivation gating from our mutagenesis scans of the S4 helix. Mutations are indicated by single–amino acid code on the y axis. Data are presented as means ± SEM for n = 4–14 cells (see Table S1). Mutations that cause a significant perturbation to inactivation, measured as Δlog(Keq,0) > ±0.5 log units relative to WT, 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: Although it is possible to derive Φ-values from the individual mutant channels listed above (see Table S1), it is important to note that the temporal information they provide is based on the effect of the specific mutation in perturbing the transition pathway, and may not necessarily reflect the role of the native amino acid residue per se. For example, any single mutation could affect two or more different processes that, by coincidence, cancel each other out to give a Φ-value between 0 and 1 (see Materials and methods). However, this type of coincidental combination will not occur with every mutation at that position. Therefore, a better estimation of Φ-values can be derived from plots of log(kinact,0) versus log(Keq,0) for a family of mutations at a single residue or region of the channel (Grosman et al., 2000). We therefore interrogated S4 helix residues identified from our scanning mutagenesis in more detail by obtaining families of mutations at each position (Fig. 3). Mutation of charged residues Arg531 (to Gln, Thr, and Asn) or Arg537 (to Gln, Asp, and Thr) did not produce any further mutant channels with adequate perturbations to Δlog(Keq,0) (Wang et al., 2011, and Fig. 3 A). The relative tolerance of neutral or polar side chains at these positions, as well as at Lys525, Arg528, Arg534, and Lys538 (Table S1 and Wang et al., 2011), suggests that the positively charged side chains are not critical to the inactivation gating transition. We therefore concentrated on the hydrophobic residues: Leu529, Leu530, Leu532, and Val535, which were further mutated to threonine, histidine, proline, asparagine, and glutamine. In the case of Val535, we also mutated this residue to glycine, leucine, isoleucine, methionine, and tyrosine. Perturbations to Δlog(Keq,0) of greater than ±0.5 log units were observed for several mutant channels at all positions, with the exception of Leu532 (Fig. 3 B). Hydrophobic residues Leu530 and Val535 were particularly sensitive to mutation; i.e., tyrosine, methionine, glycine, proline, histidine, asparagine, and threonine mutations at Val535 all exhibited a Δlog(Keq,0) of >0.5 log units compared with WT, whereas only the relatively conserved hydrophobic side chains of leucine and isoleucine produced smaller perturbations (Fig. 3 B). The relative intolerance of these hydrophobic residues to mutations that do not maintain their hydrophobic side chain properties indicates their importance in mediating open-to-inactivated transition.

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