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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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Effects of DHA and MTSEA+ at pH 7.4 on WT-IR and three mutations. Graphs display representative G-V curves with current traces as insets for voltages corresponding to 10% of Gmax in control solution. Eq. 2 is used for the fit. (A) Data for WT-IR. 70 µM DHA shifts the control curve for WT-IR with −4.7 mV (left). V1/2 = −42.8 and −47.5 mV, and s = 16.1 mV. MTSEA+ modification does not shift the control curve (middle). V1/2 = −38.9 and −38.9 mV, and s = 7.4 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −39.7 and −45.4 mV, and s = 8.2 mV. (B) Data for F416C. 70 µM DHA shifts the control curve for F416C with −2.8 mV (left). V1/2 = −45.7 and −48.5 mV, and s = 14.0 mV. MTSEA+ modification shifts the control curve with +28.2 mV (middle). V1/2 = −46.2 and −18.0 mV, and s = 14.0 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −18.0 and −21.8 mV, and s = 14.0 mV. (C) Data for I360C. 70 µM DHA shifts the control curve for I360C with −3.1 mV (left). V1/2 = −33.5 and −36.6 mV, and s = 8.7 mV. MTSEA+ modification shifts the control curve with +8.8 mV (middle). V1/2 = −34.7 and −25.9 mV, and s = 12.4 mV. The DHA-induced shift is increased to −8.5 mV after modification (right). V1/2 = −27.3 and −35.8 mV, and s = 16.1 mV. (D) Data for I325C. 70 µM DHA shifts the control curve for I325C with −2.9 mV (left). V1/2 = −40.1 and −43.0 mV, and s = 17.2 mV. MTSEA+ modification shifts the control curve with +16.9 (middle). V1/2 = −39.4 and −22.5 mV, and s = 13.7 mV. The DHA-induced shift is increased to −4.4 after modification (right). V1/2 = −22.6 and −27.0 mV, and s = 13.5 mV.
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fig3: Effects of DHA and MTSEA+ at pH 7.4 on WT-IR and three mutations. Graphs display representative G-V curves with current traces as insets for voltages corresponding to 10% of Gmax in control solution. Eq. 2 is used for the fit. (A) Data for WT-IR. 70 µM DHA shifts the control curve for WT-IR with −4.7 mV (left). V1/2 = −42.8 and −47.5 mV, and s = 16.1 mV. MTSEA+ modification does not shift the control curve (middle). V1/2 = −38.9 and −38.9 mV, and s = 7.4 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −39.7 and −45.4 mV, and s = 8.2 mV. (B) Data for F416C. 70 µM DHA shifts the control curve for F416C with −2.8 mV (left). V1/2 = −45.7 and −48.5 mV, and s = 14.0 mV. MTSEA+ modification shifts the control curve with +28.2 mV (middle). V1/2 = −46.2 and −18.0 mV, and s = 14.0 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −18.0 and −21.8 mV, and s = 14.0 mV. (C) Data for I360C. 70 µM DHA shifts the control curve for I360C with −3.1 mV (left). V1/2 = −33.5 and −36.6 mV, and s = 8.7 mV. MTSEA+ modification shifts the control curve with +8.8 mV (middle). V1/2 = −34.7 and −25.9 mV, and s = 12.4 mV. The DHA-induced shift is increased to −8.5 mV after modification (right). V1/2 = −27.3 and −35.8 mV, and s = 16.1 mV. (D) Data for I325C. 70 µM DHA shifts the control curve for I325C with −2.9 mV (left). V1/2 = −40.1 and −43.0 mV, and s = 17.2 mV. MTSEA+ modification shifts the control curve with +16.9 (middle). V1/2 = −39.4 and −22.5 mV, and s = 13.7 mV. The DHA-induced shift is increased to −4.4 after modification (right). V1/2 = −22.6 and −27.0 mV, and s = 13.5 mV.

Mentions: If the cysteine substitution of a residue induces only marginal changes in the side chain properties, the mutation will probably not alter the PUFA effect. Such cysteine mutants would thus incorrectly be interpreted as distant to the PUFA action site. Therefore, to further explore the critical area for PUFA action, we introduced a charge to each of the investigated residues. This charge can have three different effects on PUFA efficiency. (1) A positive charge close to the PUFA action site will increase the local pH and consequently deprotonate the PUFA molecule (Börjesson et al., 2008). This in turn will increase the PUFA effect on the channel’s voltage dependence (Fig. 2 C). (2) A positive charge close to the PUFA action site will increase PUFA affinity and thereby increase PUFA effects at concentrations below saturating effects. (3) A positive charge located in the voltage sensor S4 moving toward the PUFA molecule promotes larger effects of the PUFA molecule on the voltage sensor movement. These three components can also work together, and we do not aim at quantifying the impact of each component. What is important is that in all three cases, high-impact residues should be close to the PUFA action site. A change in PUFA potency, independent of the underlying mechanism, is therefore our readout to indentify high-impact residues. To experimentally change the charge of each mutated cysteine, we modified the cysteine with the positively charged cysteine-specific reagent MTSEA+. This modification of a residue that is close to the voltage sensor will electrostatically shift the G-V curve in a positive direction along the voltage axis, in addition to any steric effect on channel activation. The closer to the voltage sensor, the larger the expected electrostatic effect is (Elinder et al., 2001a; Broomand and Elinder, 2008). For these experiments, we used pH 7.4 because the charge-induced effects are expected to be larger close to the apparent pKa value. Control experiments showed that WT-IR was not affected by MTSEA+ (Fig. 3 A).


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

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

Effects of DHA and MTSEA+ at pH 7.4 on WT-IR and three mutations. Graphs display representative G-V curves with current traces as insets for voltages corresponding to 10% of Gmax in control solution. Eq. 2 is used for the fit. (A) Data for WT-IR. 70 µM DHA shifts the control curve for WT-IR with −4.7 mV (left). V1/2 = −42.8 and −47.5 mV, and s = 16.1 mV. MTSEA+ modification does not shift the control curve (middle). V1/2 = −38.9 and −38.9 mV, and s = 7.4 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −39.7 and −45.4 mV, and s = 8.2 mV. (B) Data for F416C. 70 µM DHA shifts the control curve for F416C with −2.8 mV (left). V1/2 = −45.7 and −48.5 mV, and s = 14.0 mV. MTSEA+ modification shifts the control curve with +28.2 mV (middle). V1/2 = −46.2 and −18.0 mV, and s = 14.0 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −18.0 and −21.8 mV, and s = 14.0 mV. (C) Data for I360C. 70 µM DHA shifts the control curve for I360C with −3.1 mV (left). V1/2 = −33.5 and −36.6 mV, and s = 8.7 mV. MTSEA+ modification shifts the control curve with +8.8 mV (middle). V1/2 = −34.7 and −25.9 mV, and s = 12.4 mV. The DHA-induced shift is increased to −8.5 mV after modification (right). V1/2 = −27.3 and −35.8 mV, and s = 16.1 mV. (D) Data for I325C. 70 µM DHA shifts the control curve for I325C with −2.9 mV (left). V1/2 = −40.1 and −43.0 mV, and s = 17.2 mV. MTSEA+ modification shifts the control curve with +16.9 (middle). V1/2 = −39.4 and −22.5 mV, and s = 13.7 mV. The DHA-induced shift is increased to −4.4 after modification (right). V1/2 = −22.6 and −27.0 mV, and s = 13.5 mV.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
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fig3: Effects of DHA and MTSEA+ at pH 7.4 on WT-IR and three mutations. Graphs display representative G-V curves with current traces as insets for voltages corresponding to 10% of Gmax in control solution. Eq. 2 is used for the fit. (A) Data for WT-IR. 70 µM DHA shifts the control curve for WT-IR with −4.7 mV (left). V1/2 = −42.8 and −47.5 mV, and s = 16.1 mV. MTSEA+ modification does not shift the control curve (middle). V1/2 = −38.9 and −38.9 mV, and s = 7.4 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −39.7 and −45.4 mV, and s = 8.2 mV. (B) Data for F416C. 70 µM DHA shifts the control curve for F416C with −2.8 mV (left). V1/2 = −45.7 and −48.5 mV, and s = 14.0 mV. MTSEA+ modification shifts the control curve with +28.2 mV (middle). V1/2 = −46.2 and −18.0 mV, and s = 14.0 mV. The DHA-induced shift is not affected by MTSEA+ modification (right). V1/2 = −18.0 and −21.8 mV, and s = 14.0 mV. (C) Data for I360C. 70 µM DHA shifts the control curve for I360C with −3.1 mV (left). V1/2 = −33.5 and −36.6 mV, and s = 8.7 mV. MTSEA+ modification shifts the control curve with +8.8 mV (middle). V1/2 = −34.7 and −25.9 mV, and s = 12.4 mV. The DHA-induced shift is increased to −8.5 mV after modification (right). V1/2 = −27.3 and −35.8 mV, and s = 16.1 mV. (D) Data for I325C. 70 µM DHA shifts the control curve for I325C with −2.9 mV (left). V1/2 = −40.1 and −43.0 mV, and s = 17.2 mV. MTSEA+ modification shifts the control curve with +16.9 (middle). V1/2 = −39.4 and −22.5 mV, and s = 13.7 mV. The DHA-induced shift is increased to −4.4 after modification (right). V1/2 = −22.6 and −27.0 mV, and s = 13.5 mV.
Mentions: If the cysteine substitution of a residue induces only marginal changes in the side chain properties, the mutation will probably not alter the PUFA effect. Such cysteine mutants would thus incorrectly be interpreted as distant to the PUFA action site. Therefore, to further explore the critical area for PUFA action, we introduced a charge to each of the investigated residues. This charge can have three different effects on PUFA efficiency. (1) A positive charge close to the PUFA action site will increase the local pH and consequently deprotonate the PUFA molecule (Börjesson et al., 2008). This in turn will increase the PUFA effect on the channel’s voltage dependence (Fig. 2 C). (2) A positive charge close to the PUFA action site will increase PUFA affinity and thereby increase PUFA effects at concentrations below saturating effects. (3) A positive charge located in the voltage sensor S4 moving toward the PUFA molecule promotes larger effects of the PUFA molecule on the voltage sensor movement. These three components can also work together, and we do not aim at quantifying the impact of each component. What is important is that in all three cases, high-impact residues should be close to the PUFA action site. A change in PUFA potency, independent of the underlying mechanism, is therefore our readout to indentify high-impact residues. To experimentally change the charge of each mutated cysteine, we modified the cysteine with the positively charged cysteine-specific reagent MTSEA+. This modification of a residue that is close to the voltage sensor will electrostatically shift the G-V curve in a positive direction along the voltage axis, in addition to any steric effect on channel activation. The closer to the voltage sensor, the larger the expected electrostatic effect is (Elinder et al., 2001a; Broomand and Elinder, 2008). For these experiments, we used pH 7.4 because the charge-induced effects are expected to be larger close to the apparent pKa value. Control experiments showed that WT-IR was not affected by MTSEA+ (Fig. 3 A).

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