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An electrostatic potassium channel opener targeting the final voltage sensor transition.

Börjesson SI, Elinder F - J. Gen. Physiol. (2011)

Bottom Line: However, molecular details for the interaction between PUFA and ion channels are not well understood.In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect.Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, Sweden.

ABSTRACT
Free polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of voltage-gated ion channels. As an important consequence thereof, PUFAs can suppress epileptic seizures and cardiac arrhythmia. However, molecular details for the interaction between PUFA and ion channels are not well understood. In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect. Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect. Because different voltage-gated K channels have different charge profiles, this implies channel-specific PUFA effects. The identified site and the pharmacological mechanism will potentially be very useful in future drug design of small-molecule compounds specifically targeting neuronal and cardiac excitability.

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Effect of gating charge mutations on DHA sensitivity. (A) Data for WT-IR. Structure of the Shaker K channel in the open state (based on the Kv1.2/2.1 chimera), with residues R362, R365, R368, and R371 in blue (left). One VSD and part of the pore domain are shown. Current traces at −40 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −43.5 and −53.2 mV, and s = 11.3 mV. (B) Data for R362−. Residue R362 are shown in red, and residues R365, R368, and R371 are shown in blue (left). Current traces at −35 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −36.8 and −34.9 mV, and s = 25.9 mV. (C) Data for R362+. Residues R362, R365, R368, and R371 are shown in blue (left). Current traces at −45 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −45.9 and −58.3 mV, and s = 13.1 mV. (D) Data for A359+. Residues A359, R362, R365, R368, and R371 are shown in blue (left). Current traces at −5 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −9.5 and −33.0 mV, and s = 18.1 mV. (E) Summary of experimental data for gating charge mutants. Data are expressed as mean ± SEM (n = 4–9).
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fig7: Effect of gating charge mutations on DHA sensitivity. (A) Data for WT-IR. Structure of the Shaker K channel in the open state (based on the Kv1.2/2.1 chimera), with residues R362, R365, R368, and R371 in blue (left). One VSD and part of the pore domain are shown. Current traces at −40 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −43.5 and −53.2 mV, and s = 11.3 mV. (B) Data for R362−. Residue R362 are shown in red, and residues R365, R368, and R371 are shown in blue (left). Current traces at −35 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −36.8 and −34.9 mV, and s = 25.9 mV. (C) Data for R362+. Residues R362, R365, R368, and R371 are shown in blue (left). Current traces at −45 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −45.9 and −58.3 mV, and s = 13.1 mV. (D) Data for A359+. Residues A359, R362, R365, R368, and R371 are shown in blue (left). Current traces at −5 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −9.5 and −33.0 mV, and s = 18.1 mV. (E) Summary of experimental data for gating charge mutants. Data are expressed as mean ± SEM (n = 4–9).

Mentions: The above mentioned structural model, where R1 moves from a position ∼16 Å away from the PUFA charge to a position only ∼6 Å away during the opening step, implies that the positive charge at R1 is critical for the effect of PUFAs. If the PUFAs interact electrostatically with R1 to promote opening, a negative charge at R1 would instead be expected to reduce or even reverse the PUFA effect. To alter the charge of R1 without altering the size of the side chain, we changed the arginine to a cysteine and modified with differently charged MTS reagents (Fig. 6 C): negatively charged MTSES− (mutant called R362−; Fig. 7 B) and positively charged MTSET+ (mutant called R362+; Fig. 7 C). These experiments were performed at pH 9.0 to promote a negative charge of the carboxyl group of DHA, avoiding changes in the charge of the DHA (pKa = 7.4; Börjesson et al., 2008) as a result of possible changes in local pH caused by the differently charged MTS reagents. In the same investigation, we showed that introducing three positive charges close to the voltage sensor only affected the local pH with 0.3 pH units.


An electrostatic potassium channel opener targeting the final voltage sensor transition.

Börjesson SI, Elinder F - J. Gen. Physiol. (2011)

Effect of gating charge mutations on DHA sensitivity. (A) Data for WT-IR. Structure of the Shaker K channel in the open state (based on the Kv1.2/2.1 chimera), with residues R362, R365, R368, and R371 in blue (left). One VSD and part of the pore domain are shown. Current traces at −40 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −43.5 and −53.2 mV, and s = 11.3 mV. (B) Data for R362−. Residue R362 are shown in red, and residues R365, R368, and R371 are shown in blue (left). Current traces at −35 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −36.8 and −34.9 mV, and s = 25.9 mV. (C) Data for R362+. Residues R362, R365, R368, and R371 are shown in blue (left). Current traces at −45 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −45.9 and −58.3 mV, and s = 13.1 mV. (D) Data for A359+. Residues A359, R362, R365, R368, and R371 are shown in blue (left). Current traces at −5 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −9.5 and −33.0 mV, and s = 18.1 mV. (E) Summary of experimental data for gating charge mutants. Data are expressed as mean ± SEM (n = 4–9).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3105513&req=5

fig7: Effect of gating charge mutations on DHA sensitivity. (A) Data for WT-IR. Structure of the Shaker K channel in the open state (based on the Kv1.2/2.1 chimera), with residues R362, R365, R368, and R371 in blue (left). One VSD and part of the pore domain are shown. Current traces at −40 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −43.5 and −53.2 mV, and s = 11.3 mV. (B) Data for R362−. Residue R362 are shown in red, and residues R365, R368, and R371 are shown in blue (left). Current traces at −35 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −36.8 and −34.9 mV, and s = 25.9 mV. (C) Data for R362+. Residues R362, R365, R368, and R371 are shown in blue (left). Current traces at −45 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −45.9 and −58.3 mV, and s = 13.1 mV. (D) Data for A359+. Residues A359, R362, R365, R368, and R371 are shown in blue (left). Current traces at −5 mV (middle) and G-V curve fitted to Eq. 2 (right). V1/2 = −9.5 and −33.0 mV, and s = 18.1 mV. (E) Summary of experimental data for gating charge mutants. Data are expressed as mean ± SEM (n = 4–9).
Mentions: The above mentioned structural model, where R1 moves from a position ∼16 Å away from the PUFA charge to a position only ∼6 Å away during the opening step, implies that the positive charge at R1 is critical for the effect of PUFAs. If the PUFAs interact electrostatically with R1 to promote opening, a negative charge at R1 would instead be expected to reduce or even reverse the PUFA effect. To alter the charge of R1 without altering the size of the side chain, we changed the arginine to a cysteine and modified with differently charged MTS reagents (Fig. 6 C): negatively charged MTSES− (mutant called R362−; Fig. 7 B) and positively charged MTSET+ (mutant called R362+; Fig. 7 C). These experiments were performed at pH 9.0 to promote a negative charge of the carboxyl group of DHA, avoiding changes in the charge of the DHA (pKa = 7.4; Börjesson et al., 2008) as a result of possible changes in local pH caused by the differently charged MTS reagents. In the same investigation, we showed that introducing three positive charges close to the voltage sensor only affected the local pH with 0.3 pH units.

Bottom Line: However, molecular details for the interaction between PUFA and ion channels are not well understood.In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect.Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, Sweden.

ABSTRACT
Free polyunsaturated fatty acids (PUFAs) modulate the voltage dependence of voltage-gated ion channels. As an important consequence thereof, PUFAs can suppress epileptic seizures and cardiac arrhythmia. However, molecular details for the interaction between PUFA and ion channels are not well understood. In this study, we have localized the site of action for PUFAs on the voltage-gated Shaker K channel by introducing positive charges on the channel surface, which potentiated the PUFA effect. Furthermore, we found that PUFA mainly affects the final voltage sensor movement, which is closely linked to channel opening, and that specific charges at the extracellular end of the voltage sensor are critical for the PUFA effect. Because different voltage-gated K channels have different charge profiles, this implies channel-specific PUFA effects. The identified site and the pharmacological mechanism will potentially be very useful in future drug design of small-molecule compounds specifically targeting neuronal and cardiac excitability.

Show MeSH
Related in: MedlinePlus