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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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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.
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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.

Mentions: The voltage-gated K+ channels comprise a large family of tetrameric membrane proteins that open and close in response to changes in membrane voltage. Based on hydrophobicity analysis, each subunit in the tetramer contains six putative transmembrane segments, termed S1 through S6 (Fig. 1 A). The central pore domain, which contains the K+ selective ion conduction pathway, is formed by the assembly of the S5 through S6 regions (MacKinnon and Miller 1989; MacKinnon and Yellen 1990; Hartmann et al. 1991; MacKinnon 1991; Yellen et al. 1991; Yool and Schwarz 1991; Liman et al. 1992; Heginbotham et al. 1994; Ranganathan et al. 1996; Armstrong and Hille 1998). The KcsA K+ channel, a tetrameric membrane protein of known three-dimensional structure, is a relatively simple prokaryotic K+ channel with two transmembrane segments in each subunit that are homologous to S5–S6 in voltage-gated K+ channels (Schrempf et al. 1995; Doyle et al. 1998). Both sequence homology and the conservation of pore-blocking toxin receptors suggests that the structure of the pore domain of voltage-gated channels is likely to be similar to that of KcsA (Schrempf et al. 1995; Doyle et al. 1998; MacKinnon et al. 1998). Thus, both S5 and S6 are undoubtedly membrane spanning α-helices with the S5–S6 linker, the most conserved region of all K+ channels, forming a short pore helix and the selectivity filter.


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

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

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1887781&req=5

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.
Mentions: The voltage-gated K+ channels comprise a large family of tetrameric membrane proteins that open and close in response to changes in membrane voltage. Based on hydrophobicity analysis, each subunit in the tetramer contains six putative transmembrane segments, termed S1 through S6 (Fig. 1 A). The central pore domain, which contains the K+ selective ion conduction pathway, is formed by the assembly of the S5 through S6 regions (MacKinnon and Miller 1989; MacKinnon and Yellen 1990; Hartmann et al. 1991; MacKinnon 1991; Yellen et al. 1991; Yool and Schwarz 1991; Liman et al. 1992; Heginbotham et al. 1994; Ranganathan et al. 1996; Armstrong and Hille 1998). The KcsA K+ channel, a tetrameric membrane protein of known three-dimensional structure, is a relatively simple prokaryotic K+ channel with two transmembrane segments in each subunit that are homologous to S5–S6 in voltage-gated K+ channels (Schrempf et al. 1995; Doyle et al. 1998). Both sequence homology and the conservation of pore-blocking toxin receptors suggests that the structure of the pore domain of voltage-gated channels is likely to be similar to that of KcsA (Schrempf et al. 1995; Doyle et al. 1998; MacKinnon et al. 1998). Thus, both S5 and S6 are undoubtedly membrane spanning α-helices with the S5–S6 linker, the most conserved region of all K+ channels, forming a short pore helix and the selectivity filter.

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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