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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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Strategy to determine the PUFA action site. (A) Sequence of segment S3–S6 for the Shaker K channel. //, the extracellular linkers are omitted; *, tested residues. Underlined residues mark helical transmembrane segments. (B) Structures of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). View from the extracellular side (left). Only one VSD and part of the pore domain are shown. View from the membrane side (right) as indicated by the arrow in the left panel. Only one VSD and the closest pore domain from another subunit are shown. Selectivity filter regions from all four subunits are displayed in cyan. The blue residues are the four most extracellular gating charges in S4 (R362, R365, R368, and R371). Red residues are explored in the present investigation. (C) Only the negatively charged form of the carboxyl group affects the voltage sensor. The effect is pH dependent. The introduction of a fixed positive charge close to the PUFA changes the local pH, deprotonates the carboxyl group, and potentiates the PUFA effect on the voltage sensor. The closer the charge is to the PUFA, the larger the potentiation is.
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fig2: Strategy to determine the PUFA action site. (A) Sequence of segment S3–S6 for the Shaker K channel. //, the extracellular linkers are omitted; *, tested residues. Underlined residues mark helical transmembrane segments. (B) Structures of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). View from the extracellular side (left). Only one VSD and part of the pore domain are shown. View from the membrane side (right) as indicated by the arrow in the left panel. Only one VSD and the closest pore domain from another subunit are shown. Selectivity filter regions from all four subunits are displayed in cyan. The blue residues are the four most extracellular gating charges in S4 (R362, R365, R368, and R371). Red residues are explored in the present investigation. (C) Only the negatively charged form of the carboxyl group affects the voltage sensor. The effect is pH dependent. The introduction of a fixed positive charge close to the PUFA changes the local pH, deprotonates the carboxyl group, and potentiates the PUFA effect on the voltage sensor. The closer the charge is to the PUFA, the larger the potentiation is.

Mentions: A PUFA bound to the channel has an apparent pKa value close to 7.4 (Börjesson et al., 2008). An alteration in the local pH close to the active PUFA alters the effect on the channel’s voltage sensitivity by altering the proportion of deprotonated charged PUFA (Fig. 2 C; see also triple mutant in Börjesson et al., 2008). The local pH in turn depends on the fixed charges close to the PUFA. To alter the charge of the substituted cysteines, we used the cysteine-specific thiol reagents 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA+), [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+), and sodium [2-sulfonatoethyl] methanethiosulfonate (MTSES−; Toronto Research Chemicals). The MTS reagents were applied continuously to the bath solution using a gravity-driven perfusion system. The reagents were applied until full modification was achieved (typically 100 µM MTSEA+, 100 µM MTSET+, or 1 mM MTSES− for 200 s). The modification was assayed functionally in two-electrode voltage-clamped oocytes. The main substance used to introduce a positive charge at each mutated cysteine was MTSEA+. The reason for using the smaller MTSEA+ instead of the larger MTSET+ is that we wanted a more defined charge localization and less steric effects than what is obtained from the larger reagents. MTSEA+ can, in contrast to MTSET+ and MTSES−, pass the cell membrane (Holmgren et al., 1996) and was therefore first tested on Shaker WT-IR to identify possible interference from MTSEA+ in the cytoplasm. 100 µM MTSEA+ applied for >480 s had no effect on Shaker WT-IR (Table I). MTSEA+ experiments were done at pH 7.4 because a higher pH would already from the beginning push the PUFAs toward a deprotonated state, and no further effect will be seen upon modification.


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

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

Strategy to determine the PUFA action site. (A) Sequence of segment S3–S6 for the Shaker K channel. //, the extracellular linkers are omitted; *, tested residues. Underlined residues mark helical transmembrane segments. (B) Structures of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). View from the extracellular side (left). Only one VSD and part of the pore domain are shown. View from the membrane side (right) as indicated by the arrow in the left panel. Only one VSD and the closest pore domain from another subunit are shown. Selectivity filter regions from all four subunits are displayed in cyan. The blue residues are the four most extracellular gating charges in S4 (R362, R365, R368, and R371). Red residues are explored in the present investigation. (C) Only the negatively charged form of the carboxyl group affects the voltage sensor. The effect is pH dependent. The introduction of a fixed positive charge close to the PUFA changes the local pH, deprotonates the carboxyl group, and potentiates the PUFA effect on the voltage sensor. The closer the charge is to the PUFA, the larger the potentiation is.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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

fig2: Strategy to determine the PUFA action site. (A) Sequence of segment S3–S6 for the Shaker K channel. //, the extracellular linkers are omitted; *, tested residues. Underlined residues mark helical transmembrane segments. (B) Structures of the Shaker K channel in an open state (based on the Kv1.2/2.1 chimera; Long et al., 2007). View from the extracellular side (left). Only one VSD and part of the pore domain are shown. View from the membrane side (right) as indicated by the arrow in the left panel. Only one VSD and the closest pore domain from another subunit are shown. Selectivity filter regions from all four subunits are displayed in cyan. The blue residues are the four most extracellular gating charges in S4 (R362, R365, R368, and R371). Red residues are explored in the present investigation. (C) Only the negatively charged form of the carboxyl group affects the voltage sensor. The effect is pH dependent. The introduction of a fixed positive charge close to the PUFA changes the local pH, deprotonates the carboxyl group, and potentiates the PUFA effect on the voltage sensor. The closer the charge is to the PUFA, the larger the potentiation is.
Mentions: A PUFA bound to the channel has an apparent pKa value close to 7.4 (Börjesson et al., 2008). An alteration in the local pH close to the active PUFA alters the effect on the channel’s voltage sensitivity by altering the proportion of deprotonated charged PUFA (Fig. 2 C; see also triple mutant in Börjesson et al., 2008). The local pH in turn depends on the fixed charges close to the PUFA. To alter the charge of the substituted cysteines, we used the cysteine-specific thiol reagents 2-aminoethyl methanethiosulfonate hydrochloride (MTSEA+), [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+), and sodium [2-sulfonatoethyl] methanethiosulfonate (MTSES−; Toronto Research Chemicals). The MTS reagents were applied continuously to the bath solution using a gravity-driven perfusion system. The reagents were applied until full modification was achieved (typically 100 µM MTSEA+, 100 µM MTSET+, or 1 mM MTSES− for 200 s). The modification was assayed functionally in two-electrode voltage-clamped oocytes. The main substance used to introduce a positive charge at each mutated cysteine was MTSEA+. The reason for using the smaller MTSEA+ instead of the larger MTSET+ is that we wanted a more defined charge localization and less steric effects than what is obtained from the larger reagents. MTSEA+ can, in contrast to MTSET+ and MTSES−, pass the cell membrane (Holmgren et al., 1996) and was therefore first tested on Shaker WT-IR to identify possible interference from MTSEA+ in the cytoplasm. 100 µM MTSEA+ applied for >480 s had no effect on Shaker WT-IR (Table I). MTSEA+ experiments were done at pH 7.4 because a higher pH would already from the beginning push the PUFAs toward a deprotonated state, and no further effect will be seen upon modification.

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