Li-Smerin Y et al. (2000), alpha-helical structural elements within the vo...

XB-ART-11736

J Gen Physiol 2000 Jan 01;1151:33-50. doi: 10.1085/jgp.115.1.33.

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alpha-helical structural elements within the voltage-sensing domains of a K(+) channel.

Li-Smerin Y, Hackos DH, Swartz KJ.

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Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.

???displayArticle.pubmedLink??? 10613917
???displayArticle.pmcLink??? PMC1887781
???displayArticle.link??? J Gen Physiol
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Species referenced: Xenopus laevis
Genes referenced: kcnb1 tbx2

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	Figure 1. Topology and sequence alignment of voltage-gated K+ channels. (A) Putative topology of a single subunit of the voltage-gated K+ channel with six putative transmembrane segments. Top is extracellular and the bottom is intracellular. In the tetramer, the coassembly of S5–S6 segments form the pore domain. The first four transmembrane segments form a single voltage-sensing domain with four of these domains surrounding the central pore domain. (B) Sequence alignment between the four classes of voltage-gated K+ channels in a region spanning four putative transmembrane segments (S1–S4) and linkers. Black bars above the sequence represent the approximate positions of the four transmembrane segments as indicated by the Kyte-Doolittle hydrophobicity analysis. Residue numbering is for the drk1 K+ channel. Yellow highlighting indicates similarity to drk1. Bold letters indicate residues that are highly conserved in all the voltage-gated K+ channels. Red arrows mark three highly conserved proline residues.
	Figure 2. Measurement of the effects of mutations on channel gating properties. (A) Current traces obtained from two-electrode voltage-clamp recordings for the wild-type (middle) and two mutant drk1 K+ (I297, left; D262A, right) channels. Current families were elicited by voltage pulses given in 5-mV increments. Tail currents carried by 50 mM Rb+ were elicited by repolarization to appropriate voltages. For I297A, holding voltage was −110 mV, tail voltage was −90 mV, and test pulses start at −90 mV. For the wild-type channel, holding voltage was −80 mV, tail voltage was −50 mV, and test pulses start at −60 mV. For D262A, holding voltage was −80 mV, tail voltage was −10 mV, and test pulses start at −15 mV. (B) Voltage-activation relations for the three channels in A. Symbols are normalized tail current amplitude measured 2–3 ms after repolarization. Solid lines are single Boltzmann fits to the data. The parameters derived from fitting are: V50 = −59.6 mV and z = 3.5 for I297A, V50 = −2.1 mV and z = 2.5 for wild type, and V50 = +36.9 mV and z = 2.2 for D262A.
	Figure 3. Distribution of free energy perturbations in channel gating. (A) The bar graph plots the absolute value of ΔΔG0 for all mutations studied. Data are mean ± SEM from Table . The solid line superimposed on the \|ΔΔG0\| plot is a windowed hydrophobicity index calculated using the Kyte-Doolittle scale (Kyte and Doolittle 1982) with a 17-residue window. (B) Sliding window analysis for mean ΔΔG0 (black line) and hydrophobicity index (gray line). Both use a 17-residue sliding window. Letters and numbers indicate the wild-type residues and their positions, respectively.
	Figure 8. Periodicity of gating perturbations in the S4 segment. (A) Amino acid sequence of the S4 segment in the drk1 K+ channel with bars indicating the length used for the Fourier transform analysis. (B) Power spectra of \|ΔΔG0\| values are for either 19 (top) or 13 (bottom) residues. The two left spectra were calculated using all \|ΔΔG0\| values for the indicated stretch. The two right spectra were calculated after setting the ΔΔG0 values for the basic residues to the mean \|ΔΔG0\| of all other residues in the indicated stretch. (C) Helical wheel diagram containing 19 residues (from Q293 to T311) viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 3.4 kcal mol−1. (D) Helical net diagram for 19 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 4. Periodicity in the distribution of free energy perturbations. Plot of actual values of ΔΔG0 from Table for all positions. Letters and numbers indicate the wild-type residues and their positions, respectively. Dotted lines mark ± 1 kcal mol−1. Open bars mark the approximate limits of the four transmembrane segments defined by hydrophobicity analysis.
	Figure 5. Periodicity of gating perturbations in the S1 segment. (A) Amino acid sequence of the S1 segment in the drk1 K+ channel. The bar indicates the stretch used for Fourier transform analysis. (B) The power spectrum of the \|ΔΔG0\| values for this stretch, where P(ω) is plotted as a function of angular frequency (ω). The primary peak of power spectrum occurs at 106°. (C) Helical wheel diagram of these 23 residues viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 4.8 kcal mol−1. (D) Helical net diagram for 23 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 6. Periodicity of gating perturbations in the S2 Segment. (A) Amino acid sequence of the S2 segment in the drk1 K+ channel with bars indicating the lengths used for the Fourier transform analysis. (B) Power spectra of \|ΔΔG0\| values are for either 18 (left) or 23 (right) residues. The primary peak in the power spectra occurs at 102° for the short stretch and at 103° for the long sequence. Gray line in right power spectrum was calculated using data from Monks et al. 1999 for positions in Shaker K+ channel equivalent to 224–246 in the drk1 K+ channel. P(ω) for the Shaker data was scaled down by factor of 8.5 for comparison and has a dominant peak at 109°. (C) Helical wheel diagram containing 23 residues (from Q224 to S246) viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 5.3 kcal mol−1. (D) Helical net diagram for 23 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 7. Periodicity of gating perturbations in the S3 segment. (A) Amino acid sequence of the S3 segment in the drk1 K+ channel with bars indicating the length used for the Fourier transform analysis. (B) Power spectra of \|ΔΔG0\| values are for either 15 (left) or 23 (right) residues. The primary peak of power spectrum occurs at 122° for the short stretch. The spectrum for the longer length of sequence contains a peak at 120°, but has greater complexity with many additional peaks. (C) Helical wheel diagram containing 23 residues (from F253 to L275) viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 2.6 kcal mol−1. (D) Helical net diagram for 23 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 9. Windowed α-periodicity analysis for S1–S4. (A) Windowed α-periodicity index analysis with a 13-residue sliding window (black line). (B) Windowed α-periodicity index analysis with a 17-residue sliding window (black line). The superimposed gray line in both A and B is a windowed hydrophobicity index calculated using the Kyte-Doolittle scale (Kyte and Doolittle 1982) with a 17-residue window. Dashed lines in both A and B mark α-PI = 2.0. α-PI ≥ 2.0 suggest α-helical secondary structure (Cornette et al. 1987; Rees et al. 1989b). The arrows mark positions in the S1–S2 and S3–S4 linkers with high α-PI values.
	Figure 10. Periodicity of gating perturbations in the S1–S2 linker. (A) Amino acid sequence of the S1–S2 linker in the drk1 K+ channel. The bar indicates the stretch used for Fourier transform analysis. (B) The power spectrum of the \|ΔΔG0\| values for this stretch, where P(ω) is plotted as a function of angular frequency (ω). The primary peak of power spectrum occurs at 103°. (C) Helical wheel diagram of these 17 residues viewed from the NH2 terminus. Large shaded circles indicate positions with \|ΔΔG0\| > 0.5 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| ≤ 0.5 kcal mol−1.
	Figure 11. Periodicity of gating perturbations in the S3–S4 linker. (A) Amino acid sequence of the S3–S4 linker in the drk1 K+ channel. The bar indicates the stretch used for Fourier transform analysis. (B) The power spectrum of the \|ΔΔG0\| values for this stretch, where P(ω) is plotted as a function of angular frequency (ω). The primary peak of power spectrum occurs at 104°. (C) Helical wheel diagram of these 17 residues viewed from the NH2 terminus. Large shaded circles indicate positions with \|ΔΔG0\| > 0.5 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| ≤ 0.5 kcal mol−1.
	Figure 12. Secondary structure and packing orientation of S1 to S4. (A) Membrane-folding model for a voltage-gated K+ channel showing four membrane spanning α-helices (black cylinders) and two possible α-helices (gray cylinders) in the extracellular linkers. The black and gray helices comprise the voltage-sensing domains, while the blue region (S5 through S6) forms the pore domain. (B) Possible arrangement of transmembrane helices in the tetrameric voltage-gated K+ channel shown schematically. Gray helices in the S1–S2 and S3–S4 linkers are shown lying on the surface of the four transmembrane helices of the voltage-sensing domain. (C) Helical wheel diagrams for S1–S4 arranged to allow for appropriate protein–lipid and protein–protein interfaces and to account for previously proposed electrostatic interactions (marked by red dotted line). Electrostatic interactions are for E239 in S2 and D262 in S3 with K305 in S4 and for E229 in S2 with R302 and R299 in S4 (see discussion). The helical wheel diagrams (same as used in Fig. 5Fig. 6Fig. 7Fig. 8) are shown as viewed from the extracellular side of the channel. Vector sums all point towards the interior of the complex, although this was not a constraint used in making the arrangement. (D) S1 to S4 segments presented as a bundle of four helices viewed from the extracellular side with the approximate position of the interface with pore domain indicated. Each helix is tilted relative to the membrane plane and to each other to maximize the presence of residues with ΔΔG < 1 kcal mol−1 on the surface and minimize the presence of residues with ΔΔG ≥ 1 kcal mol−1 on the surface. The relative position of each segment is the same as in C.

References [+] :

Aggarwal, Contribution of the S4 segment to gating charge in the Shaker K+ channel. 1996, Pubmed, Xenbase

	Figure 1. Topology and sequence alignment of voltage-gated K+ channels. (A) Putative topology of a single subunit of the voltage-gated K+ channel with six putative transmembrane segments. Top is extracellular and the bottom is intracellular. In the tetramer, the coassembly of S5–S6 segments form the pore domain. The first four transmembrane segments form a single voltage-sensing domain with four of these domains surrounding the central pore domain. (B) Sequence alignment between the four classes of voltage-gated K+ channels in a region spanning four putative transmembrane segments (S1–S4) and linkers. Black bars above the sequence represent the approximate positions of the four transmembrane segments as indicated by the Kyte-Doolittle hydrophobicity analysis. Residue numbering is for the drk1 K+ channel. Yellow highlighting indicates similarity to drk1. Bold letters indicate residues that are highly conserved in all the voltage-gated K+ channels. Red arrows mark three highly conserved proline residues.
	Figure 2. Measurement of the effects of mutations on channel gating properties. (A) Current traces obtained from two-electrode voltage-clamp recordings for the wild-type (middle) and two mutant drk1 K+ (I297, left; D262A, right) channels. Current families were elicited by voltage pulses given in 5-mV increments. Tail currents carried by 50 mM Rb+ were elicited by repolarization to appropriate voltages. For I297A, holding voltage was −110 mV, tail voltage was −90 mV, and test pulses start at −90 mV. For the wild-type channel, holding voltage was −80 mV, tail voltage was −50 mV, and test pulses start at −60 mV. For D262A, holding voltage was −80 mV, tail voltage was −10 mV, and test pulses start at −15 mV. (B) Voltage-activation relations for the three channels in A. Symbols are normalized tail current amplitude measured 2–3 ms after repolarization. Solid lines are single Boltzmann fits to the data. The parameters derived from fitting are: V50 = −59.6 mV and z = 3.5 for I297A, V50 = −2.1 mV and z = 2.5 for wild type, and V50 = +36.9 mV and z = 2.2 for D262A.
	Figure 3. Distribution of free energy perturbations in channel gating. (A) The bar graph plots the absolute value of ΔΔG0 for all mutations studied. Data are mean ± SEM from Table . The solid line superimposed on the \|ΔΔG0\| plot is a windowed hydrophobicity index calculated using the Kyte-Doolittle scale (Kyte and Doolittle 1982) with a 17-residue window. (B) Sliding window analysis for mean ΔΔG0 (black line) and hydrophobicity index (gray line). Both use a 17-residue sliding window. Letters and numbers indicate the wild-type residues and their positions, respectively.
	Figure 8. Periodicity of gating perturbations in the S4 segment. (A) Amino acid sequence of the S4 segment in the drk1 K+ channel with bars indicating the length used for the Fourier transform analysis. (B) Power spectra of \|ΔΔG0\| values are for either 19 (top) or 13 (bottom) residues. The two left spectra were calculated using all \|ΔΔG0\| values for the indicated stretch. The two right spectra were calculated after setting the ΔΔG0 values for the basic residues to the mean \|ΔΔG0\| of all other residues in the indicated stretch. (C) Helical wheel diagram containing 19 residues (from Q293 to T311) viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 3.4 kcal mol−1. (D) Helical net diagram for 19 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 4. Periodicity in the distribution of free energy perturbations. Plot of actual values of ΔΔG0 from Table for all positions. Letters and numbers indicate the wild-type residues and their positions, respectively. Dotted lines mark ± 1 kcal mol−1. Open bars mark the approximate limits of the four transmembrane segments defined by hydrophobicity analysis.
	Figure 5. Periodicity of gating perturbations in the S1 segment. (A) Amino acid sequence of the S1 segment in the drk1 K+ channel. The bar indicates the stretch used for Fourier transform analysis. (B) The power spectrum of the \|ΔΔG0\| values for this stretch, where P(ω) is plotted as a function of angular frequency (ω). The primary peak of power spectrum occurs at 106°. (C) Helical wheel diagram of these 23 residues viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 4.8 kcal mol−1. (D) Helical net diagram for 23 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 6. Periodicity of gating perturbations in the S2 Segment. (A) Amino acid sequence of the S2 segment in the drk1 K+ channel with bars indicating the lengths used for the Fourier transform analysis. (B) Power spectra of \|ΔΔG0\| values are for either 18 (left) or 23 (right) residues. The primary peak in the power spectra occurs at 102° for the short stretch and at 103° for the long sequence. Gray line in right power spectrum was calculated using data from Monks et al. 1999 for positions in Shaker K+ channel equivalent to 224–246 in the drk1 K+ channel. P(ω) for the Shaker data was scaled down by factor of 8.5 for comparison and has a dominant peak at 109°. (C) Helical wheel diagram containing 23 residues (from Q224 to S246) viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 5.3 kcal mol−1. (D) Helical net diagram for 23 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 7. Periodicity of gating perturbations in the S3 segment. (A) Amino acid sequence of the S3 segment in the drk1 K+ channel with bars indicating the length used for the Fourier transform analysis. (B) Power spectra of \|ΔΔG0\| values are for either 15 (left) or 23 (right) residues. The primary peak of power spectrum occurs at 122° for the short stretch. The spectrum for the longer length of sequence contains a peak at 120°, but has greater complexity with many additional peaks. (C) Helical wheel diagram containing 23 residues (from F253 to L275) viewed from the extracellular side of the membrane. Large shaded circles indicate positions with \|ΔΔG0\| ≥ 1 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| < 1.0 kcal mol−1. \|ΔΔG0\| values were plotted as vectors on the helical wheel (small open circles); scale is 0–6.9 kcal mol−1. The sum of the \|ΔΔG0\| vectors, represented by the solid circle, has a magnitude of 2.6 kcal mol−1. (D) Helical net diagram for 23 residues with top as extracellular and bottom as intracellular. Open and shaded circles are as in C.
	Figure 9. Windowed α-periodicity analysis for S1–S4. (A) Windowed α-periodicity index analysis with a 13-residue sliding window (black line). (B) Windowed α-periodicity index analysis with a 17-residue sliding window (black line). The superimposed gray line in both A and B is a windowed hydrophobicity index calculated using the Kyte-Doolittle scale (Kyte and Doolittle 1982) with a 17-residue window. Dashed lines in both A and B mark α-PI = 2.0. α-PI ≥ 2.0 suggest α-helical secondary structure (Cornette et al. 1987; Rees et al. 1989b). The arrows mark positions in the S1–S2 and S3–S4 linkers with high α-PI values.
	Figure 10. Periodicity of gating perturbations in the S1–S2 linker. (A) Amino acid sequence of the S1–S2 linker in the drk1 K+ channel. The bar indicates the stretch used for Fourier transform analysis. (B) The power spectrum of the \|ΔΔG0\| values for this stretch, where P(ω) is plotted as a function of angular frequency (ω). The primary peak of power spectrum occurs at 103°. (C) Helical wheel diagram of these 17 residues viewed from the NH2 terminus. Large shaded circles indicate positions with \|ΔΔG0\| > 0.5 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| ≤ 0.5 kcal mol−1.
	Figure 11. Periodicity of gating perturbations in the S3–S4 linker. (A) Amino acid sequence of the S3–S4 linker in the drk1 K+ channel. The bar indicates the stretch used for Fourier transform analysis. (B) The power spectrum of the \|ΔΔG0\| values for this stretch, where P(ω) is plotted as a function of angular frequency (ω). The primary peak of power spectrum occurs at 104°. (C) Helical wheel diagram of these 17 residues viewed from the NH2 terminus. Large shaded circles indicate positions with \|ΔΔG0\| > 0.5 kcal mol−1 and large open circles indicate positions with \|ΔΔG0\| ≤ 0.5 kcal mol−1.
	Figure 12. Secondary structure and packing orientation of S1 to S4. (A) Membrane-folding model for a voltage-gated K+ channel showing four membrane spanning α-helices (black cylinders) and two possible α-helices (gray cylinders) in the extracellular linkers. The black and gray helices comprise the voltage-sensing domains, while the blue region (S5 through S6) forms the pore domain. (B) Possible arrangement of transmembrane helices in the tetrameric voltage-gated K+ channel shown schematically. Gray helices in the S1–S2 and S3–S4 linkers are shown lying on the surface of the four transmembrane helices of the voltage-sensing domain. (C) Helical wheel diagrams for S1–S4 arranged to allow for appropriate protein–lipid and protein–protein interfaces and to account for previously proposed electrostatic interactions (marked by red dotted line). Electrostatic interactions are for E239 in S2 and D262 in S3 with K305 in S4 and for E229 in S2 with R302 and R299 in S4 (see discussion). The helical wheel diagrams (same as used in Fig. 5Fig. 6Fig. 7Fig. 8) are shown as viewed from the extracellular side of the channel. Vector sums all point towards the interior of the complex, although this was not a constraint used in making the arrangement. (D) S1 to S4 segments presented as a bundle of four helices viewed from the extracellular side with the approximate position of the interface with pore domain indicated. Each helix is tilted relative to the membrane plane and to each other to maximize the presence of residues with ΔΔG < 1 kcal mol−1 on the surface and minimize the presence of residues with ΔΔG ≥ 1 kcal mol−1 on the surface. The relative position of each segment is the same as in C.