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Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel.

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

Bottom Line: We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors.Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel.Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Hanatoxin inhibits voltage-gated K(+) channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K(+) channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.

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Effects of multiple substitutions at position 274 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and two mutant drk1 K+ channels by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to five cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with Kd values of 103 nM, 4.4 μM, and 61.6 μM for WT, F274G, and F274R, respectively. The equation assumes four equivalent and independent toxin binding sites per channel. (B) Normalized Kd values for 14 substitutions at the position 274. Mean ± SEM (n = 3–15) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 3.7 K, 3.6 R, 2.8 E, 2.6 S, 2.6 D, 2.4 G, 1.8 C, 1.6 A, 1.5 M, 1.5 P, 1.5 Y, 1.4 W, 1.4 V, and 1.2 I.
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Figure 3: Effects of multiple substitutions at position 274 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and two mutant drk1 K+ channels by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to five cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with Kd values of 103 nM, 4.4 μM, and 61.6 μM for WT, F274G, and F274R, respectively. The equation assumes four equivalent and independent toxin binding sites per channel. (B) Normalized Kd values for 14 substitutions at the position 274. Mean ± SEM (n = 3–15) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 3.7 K, 3.6 R, 2.8 E, 2.6 S, 2.6 D, 2.4 G, 1.8 C, 1.6 A, 1.5 M, 1.5 P, 1.5 Y, 1.4 W, 1.4 V, and 1.2 I.

Mentions: Fig. 3 shows the results for multiple substitutions at F274, where mutation to Ala was previously reported to reduce Hanatoxin binding affinity by ∼25-fold. The 14 substitutions made at F274 include 8 hydrophobic residues (Cys, Ala, Met, Pro, Tyr, Trp, Val, Ile), 2 basic residues (Lys, Arg), 2 acidic residues (Glu, Asp), Ser, and Gly. Fig. 3 A shows the dependence of In/In0 on toxin concentration for the wild-type and two mutant channels, F274G and F274R. For the wild-type channel, the data were well described by a model assuming four equivalent and independent binding sites for Hanatoxin on the drk1 K+ channel with a Kd of 103 nM for toxin binding to each site (see materials and methods). The two mutants, F274G and F274R, greatly reduced the toxin binding affinity with Kd values of 4.4 and 61.6 μM, respectively. The normalized Hanatoxin Kd values for all 14 substitutions at position 274 are summarized in Fig. 3 B. One prominent trend in the data is that substitutions with hydrophobic amino acids cause the smallest perturbation in Hanatoxin binding energy. The range of ΔΔG values [ΔΔG = −RT ln (Kdwt/Kdmut)] for hydrophobic residues (Ile, Val, Trp, Tyr, Pro, Met, Ala, Cys) is 1.2–1.8 kcal mol−1, while the range of ΔΔG values for nonhydrophobic residues (Gly, Asp, Ser, Glu, Arg, Lys) is 2.4–3.7 kcal mol−1. Although the hydrophobic residues caused the smallest perturbations, even these perturbations were quite significant, implying a very close and specific interaction with the toxin. These results are consistent with an intimate hydrophobic interaction between F274 and the toxin. It is also interesting that at position 274, substitutions with either Lys or Arg produce the largest perturbations, with ΔΔG values of 3.7 and 3.6 kcal mol−1, respectively. One possibility is that F274 is positioned close to a basic toxin residue in the channel-toxin complex (see discussion).


Localization and molecular determinants of the Hanatoxin receptors on the voltage-sensing domains of a K(+) channel.

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

Effects of multiple substitutions at position 274 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and two mutant drk1 K+ channels by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to five cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with Kd values of 103 nM, 4.4 μM, and 61.6 μM for WT, F274G, and F274R, respectively. The equation assumes four equivalent and independent toxin binding sites per channel. (B) Normalized Kd values for 14 substitutions at the position 274. Mean ± SEM (n = 3–15) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 3.7 K, 3.6 R, 2.8 E, 2.6 S, 2.6 D, 2.4 G, 1.8 C, 1.6 A, 1.5 M, 1.5 P, 1.5 Y, 1.4 W, 1.4 V, and 1.2 I.
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Figure 3: Effects of multiple substitutions at position 274 on Hanatoxin binding affinity. (A) Concentration dependence for inhibition of the wild-type and two mutant drk1 K+ channels by Hanatoxin. In/In0 is the value of I/I0 measured in the plateau phase at negative voltages (e.g., Fig. 2 C). Symbols are experimental data for the mean ± SEM of three to five cells. Solid lines correspond to In/In0 = (1 − P)4, where P = [toxin]/([toxin] + Kd), with Kd values of 103 nM, 4.4 μM, and 61.6 μM for WT, F274G, and F274R, respectively. The equation assumes four equivalent and independent toxin binding sites per channel. (B) Normalized Kd values for 14 substitutions at the position 274. Mean ± SEM (n = 3–15) for each mutant channel. The corresponding ΔΔG values are (kcal mol−1): 3.7 K, 3.6 R, 2.8 E, 2.6 S, 2.6 D, 2.4 G, 1.8 C, 1.6 A, 1.5 M, 1.5 P, 1.5 Y, 1.4 W, 1.4 V, and 1.2 I.
Mentions: Fig. 3 shows the results for multiple substitutions at F274, where mutation to Ala was previously reported to reduce Hanatoxin binding affinity by ∼25-fold. The 14 substitutions made at F274 include 8 hydrophobic residues (Cys, Ala, Met, Pro, Tyr, Trp, Val, Ile), 2 basic residues (Lys, Arg), 2 acidic residues (Glu, Asp), Ser, and Gly. Fig. 3 A shows the dependence of In/In0 on toxin concentration for the wild-type and two mutant channels, F274G and F274R. For the wild-type channel, the data were well described by a model assuming four equivalent and independent binding sites for Hanatoxin on the drk1 K+ channel with a Kd of 103 nM for toxin binding to each site (see materials and methods). The two mutants, F274G and F274R, greatly reduced the toxin binding affinity with Kd values of 4.4 and 61.6 μM, respectively. The normalized Hanatoxin Kd values for all 14 substitutions at position 274 are summarized in Fig. 3 B. One prominent trend in the data is that substitutions with hydrophobic amino acids cause the smallest perturbation in Hanatoxin binding energy. The range of ΔΔG values [ΔΔG = −RT ln (Kdwt/Kdmut)] for hydrophobic residues (Ile, Val, Trp, Tyr, Pro, Met, Ala, Cys) is 1.2–1.8 kcal mol−1, while the range of ΔΔG values for nonhydrophobic residues (Gly, Asp, Ser, Glu, Arg, Lys) is 2.4–3.7 kcal mol−1. Although the hydrophobic residues caused the smallest perturbations, even these perturbations were quite significant, implying a very close and specific interaction with the toxin. These results are consistent with an intimate hydrophobic interaction between F274 and the toxin. It is also interesting that at position 274, substitutions with either Lys or Arg produce the largest perturbations, with ΔΔG values of 3.7 and 3.6 kcal mol−1, respectively. One possibility is that F274 is positioned close to a basic toxin residue in the channel-toxin complex (see discussion).

Bottom Line: We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors.Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel.Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.

View Article: PubMed Central - PubMed

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

ABSTRACT
Hanatoxin inhibits voltage-gated K(+) channels by modifying the energetics of activation. We studied the molecular determinants and physical location of the Hanatoxin receptors on the drk1 voltage-gated K(+) channel. First, we made multiple substitutions at three previously identified positions in the COOH terminus of S3 to examine whether these residues interact intimately with the toxin. We also examined a region encompassing S1-S3 using alanine-scanning mutagenesis to identify additional determinants of the toxin receptors. Finally, guided by the structure of the KcsA K(+) channel, we explored whether the toxin interacts with the peripheral extracellular surface of the pore domain in the drk1 K(+) channel. Our results argue for an intimate interaction between the toxin and the COOH terminus of S3 and suggest that the Hanatoxin receptors are confined within the voltage-sensing domains of the channel, at least 20-25 A away from the central pore axis.

Show MeSH
Related in: MedlinePlus