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

Li-Smerin Y, Hackos DH, Swartz KJ - J. Gen. Physiol. (2000)

Bottom Line: Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
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.

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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.
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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.

Mentions: Two power spectra calculated for S3 are shown in Fig. 7 B. As with S1 and S2, a conserved proline residue (P268) is present in S3, but in this case the proline is located at the center of the segment, as collectively defined by the hydrophobicity analysis and the distribution of gating perturbations (Fig. 1 and Fig. 3). We therefore initially examined the power spectrum for only 15 residues beginning with F253A and ending at L267A, just before the conserved proline at 268. This power spectrum shows a major peak at 122°, near the edge of the α-helical frequency, with an α-PI of 1.8. The α-PI for a 13-residue window in this region is 2.2 (see Fig. 9). The spectrum for a COOH-terminally extended segment past the conserved P268 (F253 to L275) is more complex. A peak is still observed within the helix frequency (120°), but it is no longer dominant, and there are many other peaks observed at both lower and higher frequencies. The helical wheel diagram presented in Fig. 7 C shows that the mutations with /ΔΔG0/ ≥ 1 kcal mol−1 (shaded circles) distribute over a large portion of the wheel. However, the net diagram in Fig. 7 D shows a cluster of residues on one face of the helix where mutations have only small effects on gating. The sum of the individual /ΔΔG0/ vectors was 2.6 kcal mol−1, slightly smaller than the largest individual /ΔΔG0/ (3 kcal mol−1 for A265). The sum vector points to the side containing conserved residues (Fig. 1 B; Durell et al. 1998; Monks et al. 1999), including a highly conserved aspartate (D262). While our results for the internal or NH2-terminal two-thirds of S3 are consistent with α-helical structure, they are ambiguous for the COOH-terminal part of S3.


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

Li-Smerin Y, Hackos DH, Swartz KJ - J. Gen. Physiol. (2000)

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.
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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.
Mentions: Two power spectra calculated for S3 are shown in Fig. 7 B. As with S1 and S2, a conserved proline residue (P268) is present in S3, but in this case the proline is located at the center of the segment, as collectively defined by the hydrophobicity analysis and the distribution of gating perturbations (Fig. 1 and Fig. 3). We therefore initially examined the power spectrum for only 15 residues beginning with F253A and ending at L267A, just before the conserved proline at 268. This power spectrum shows a major peak at 122°, near the edge of the α-helical frequency, with an α-PI of 1.8. The α-PI for a 13-residue window in this region is 2.2 (see Fig. 9). The spectrum for a COOH-terminally extended segment past the conserved P268 (F253 to L275) is more complex. A peak is still observed within the helix frequency (120°), but it is no longer dominant, and there are many other peaks observed at both lower and higher frequencies. The helical wheel diagram presented in Fig. 7 C shows that the mutations with /ΔΔG0/ ≥ 1 kcal mol−1 (shaded circles) distribute over a large portion of the wheel. However, the net diagram in Fig. 7 D shows a cluster of residues on one face of the helix where mutations have only small effects on gating. The sum of the individual /ΔΔG0/ vectors was 2.6 kcal mol−1, slightly smaller than the largest individual /ΔΔG0/ (3 kcal mol−1 for A265). The sum vector points to the side containing conserved residues (Fig. 1 B; Durell et al. 1998; Monks et al. 1999), including a highly conserved aspartate (D262). While our results for the internal or NH2-terminal two-thirds of S3 are consistent with α-helical structure, they are ambiguous for the COOH-terminal part of S3.

Bottom Line: Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Physiology and Biophysics Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus