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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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Homology model of the Kv11.1 channel. View parallel to the membrane of an amplified region of a Kv11.1 channel homology model created using the Kv1.2/2.1 chimera crystal structure (Long et al., 2007) as a template, according to the alignment shown in Fig. S3. The amplified region is indicated by the boxed region of the entire four-subunit homology model shown in the inset. Hydrophobic residues on the S4 helix (Leu529, Leu532, and Val535) of one subunit (sub1; shown in green, with residues colored orange) face toward hydrophobic residues on the S5 helix (Ile560, Leu564, and Ile567) of the neighboring subunit (sub2; shown in blue, with residues colored purple). The arrow represents a cavity filled by lipid in the Kv1.2/2.1 crystal structure.
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fig9: Homology model of the Kv11.1 channel. View parallel to the membrane of an amplified region of a Kv11.1 channel homology model created using the Kv1.2/2.1 chimera crystal structure (Long et al., 2007) as a template, according to the alignment shown in Fig. S3. The amplified region is indicated by the boxed region of the entire four-subunit homology model shown in the inset. Hydrophobic residues on the S4 helix (Leu529, Leu532, and Val535) of one subunit (sub1; shown in green, with residues colored orange) face toward hydrophobic residues on the S5 helix (Ile560, Leu564, and Ile567) of the neighboring subunit (sub2; shown in blue, with residues colored purple). The arrow represents a cavity filled by lipid in the Kv1.2/2.1 crystal structure.

Mentions: When viewed on a Kv11.1 channel homology model, based on the crystal structure of a Kv1.2/2.1 channel chimera (Long et al., 2007), the side chains of the hydrophobic residues Leu529, Leu532, and Val535 face toward hydrophobic residues on the S5 helix of the neighboring subunit (Fig. 9). This raises the question of whether the S4 helix and S5 helix are energetically coupled via an intersubunit hydrophobic interaction. To experimentally test for an energetic coupling between hydrophobic residues on the S4 helix (Leu529, Leu530, Leu532, or Val535) and hydrophobic residues on the S5 helix (Ile560, Leu564, or Ile567), or a nonhydrophobic residue on the S5P linker (Asp591), we combined perturbing mutations of residues within each helix (Fig. 10). If an energetic coupling exists between two residues, we would predict that the perturbations caused by the individual mutations, measured as Δlog(Keq,0) relative to WT, would not be additive when combined in the double mutant (i.e., ΔΔGmut1+mut2 < ΔΔGmut1 + ΔΔGmut2; see Materials and methods). Conversely, if the perturbations caused by the two single mutants were additive when combined in the double mutant, the two residues are not energetically coupled (i.e., ΔΔGmut1+mut2 = ΔΔGmut1 + ΔΔGmut2). For example, the schematic in Fig. 10 A represents double mutant cycle analysis between two residues: Leu529 on the S4 helix and Ile560 on the S5 helix. It is clear that the perturbations caused by the two mutations, L529S (ΔΔGmut1) and I560A (ΔΔGmut2), are not additive when combined in the double mutant, L529S + I560A (ΔΔGmut1+mut2). In fact, the ΔΔGmut1+mut2 of the double mutant is significantly less than the theoretical additive value (ΔΔGmut1 + ΔΔGmut2) (Fig. 10 B; P < 0.05; two-tailed Welch’s t test), indicating that Leu529 on the S4 helix and Ile560 on the S5 helix are, at least in part, energetically coupled. Similarly, combining the same S4 helix mutation, L529S, with a different S5 helix mutation, I567A, resulted in a ΔΔGmut1+mut2, which was significantly less than ΔΔGmut1 + ΔΔGmut2 (Fig. 10 B; P < 0.05; two-tailed Welch’s t test), suggesting that these two residues are also energetically coupled. In contrast, combining L529S with either L564A (S5 helix) or D591K (S5P linker) resulted in a ΔΔGmut1+mut2 ≈ ΔΔGmut1 + ΔΔGmut2 (Fig. 10 B; P = NS; two-tailed Welch’s t test), suggesting that Leu529 is not energetically coupled with either Leu564 (S5) or Asp591 (S5P).


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)

Homology model of the Kv11.1 channel. View parallel to the membrane of an amplified region of a Kv11.1 channel homology model created using the Kv1.2/2.1 chimera crystal structure (Long et al., 2007) as a template, according to the alignment shown in Fig. S3. The amplified region is indicated by the boxed region of the entire four-subunit homology model shown in the inset. Hydrophobic residues on the S4 helix (Leu529, Leu532, and Val535) of one subunit (sub1; shown in green, with residues colored orange) face toward hydrophobic residues on the S5 helix (Ile560, Leu564, and Ile567) of the neighboring subunit (sub2; shown in blue, with residues colored purple). The arrow represents a cavity filled by lipid in the Kv1.2/2.1 crystal structure.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig9: Homology model of the Kv11.1 channel. View parallel to the membrane of an amplified region of a Kv11.1 channel homology model created using the Kv1.2/2.1 chimera crystal structure (Long et al., 2007) as a template, according to the alignment shown in Fig. S3. The amplified region is indicated by the boxed region of the entire four-subunit homology model shown in the inset. Hydrophobic residues on the S4 helix (Leu529, Leu532, and Val535) of one subunit (sub1; shown in green, with residues colored orange) face toward hydrophobic residues on the S5 helix (Ile560, Leu564, and Ile567) of the neighboring subunit (sub2; shown in blue, with residues colored purple). The arrow represents a cavity filled by lipid in the Kv1.2/2.1 crystal structure.
Mentions: When viewed on a Kv11.1 channel homology model, based on the crystal structure of a Kv1.2/2.1 channel chimera (Long et al., 2007), the side chains of the hydrophobic residues Leu529, Leu532, and Val535 face toward hydrophobic residues on the S5 helix of the neighboring subunit (Fig. 9). This raises the question of whether the S4 helix and S5 helix are energetically coupled via an intersubunit hydrophobic interaction. To experimentally test for an energetic coupling between hydrophobic residues on the S4 helix (Leu529, Leu530, Leu532, or Val535) and hydrophobic residues on the S5 helix (Ile560, Leu564, or Ile567), or a nonhydrophobic residue on the S5P linker (Asp591), we combined perturbing mutations of residues within each helix (Fig. 10). If an energetic coupling exists between two residues, we would predict that the perturbations caused by the individual mutations, measured as Δlog(Keq,0) relative to WT, would not be additive when combined in the double mutant (i.e., ΔΔGmut1+mut2 < ΔΔGmut1 + ΔΔGmut2; see Materials and methods). Conversely, if the perturbations caused by the two single mutants were additive when combined in the double mutant, the two residues are not energetically coupled (i.e., ΔΔGmut1+mut2 = ΔΔGmut1 + ΔΔGmut2). For example, the schematic in Fig. 10 A represents double mutant cycle analysis between two residues: Leu529 on the S4 helix and Ile560 on the S5 helix. It is clear that the perturbations caused by the two mutations, L529S (ΔΔGmut1) and I560A (ΔΔGmut2), are not additive when combined in the double mutant, L529S + I560A (ΔΔGmut1+mut2). In fact, the ΔΔGmut1+mut2 of the double mutant is significantly less than the theoretical additive value (ΔΔGmut1 + ΔΔGmut2) (Fig. 10 B; P < 0.05; two-tailed Welch’s t test), indicating that Leu529 on the S4 helix and Ile560 on the S5 helix are, at least in part, energetically coupled. Similarly, combining the same S4 helix mutation, L529S, with a different S5 helix mutation, I567A, resulted in a ΔΔGmut1+mut2, which was significantly less than ΔΔGmut1 + ΔΔGmut2 (Fig. 10 B; P < 0.05; two-tailed Welch’s t test), suggesting that these two residues are also energetically coupled. In contrast, combining L529S with either L564A (S5 helix) or D591K (S5P linker) resulted in a ΔΔGmut1+mut2 ≈ ΔΔGmut1 + ΔΔGmut2 (Fig. 10 B; P = NS; two-tailed Welch’s t test), suggesting that Leu529 is not energetically coupled with either Leu564 (S5) or Asp591 (S5P).

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