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Alkanols inhibit voltage-gated K(+) channels via a distinct gating modifying mechanism that prevents gate opening.

Martínez-Morales E, Kopljar I, Snyders DJ, Labro AJ - Sci Rep (2015)

Bottom Line: Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition.Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening.Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, 2610, Belgium.

ABSTRACT
Alkanols are small aliphatic compounds that inhibit voltage-gated K(+) (K(v)) channels through a yet unresolved gating mechanism. K(v) channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker K(v) channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type K(v) channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state.

No MeSH data available.


Related in: MedlinePlus

1-BuOH and 4-AP do not compete for inhibiting Shaker-IR.(A) Sequentially recorded IK of Shaker-IR in control condition and after steady-state inhibition by 50 mM 1-BuOH and 30 μM 4-AP. Finally, instead of washing the 30 μM 4-AP out, a mixture of 30 μM 4-AP plus 50 mM 1-BuOH was added and the amount of IK inhibition was determined. (B) Bar chart shows the average reduction in IK ± S.E.M. (n = 7) after applying 50 mM 1-BuOH, 30 μM 4-AP and the mixture of both compounds (30 μM 4-AP plus 50 mM 1-BuOH). The percentage of IK inhibition was calculated by normalizing the steady-state IK in presence of drug to the IK amplitude in control conditions. The expected reduction in IK for an allotopic and syntopic model was calculated as described in Material and Methods. Note, the experimentally obtained value differed only statistically from the predicted value of a syntopic model (*p < 0.05).
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f6: 1-BuOH and 4-AP do not compete for inhibiting Shaker-IR.(A) Sequentially recorded IK of Shaker-IR in control condition and after steady-state inhibition by 50 mM 1-BuOH and 30 μM 4-AP. Finally, instead of washing the 30 μM 4-AP out, a mixture of 30 μM 4-AP plus 50 mM 1-BuOH was added and the amount of IK inhibition was determined. (B) Bar chart shows the average reduction in IK ± S.E.M. (n = 7) after applying 50 mM 1-BuOH, 30 μM 4-AP and the mixture of both compounds (30 μM 4-AP plus 50 mM 1-BuOH). The percentage of IK inhibition was calculated by normalizing the steady-state IK in presence of drug to the IK amplitude in control conditions. The expected reduction in IK for an allotopic and syntopic model was calculated as described in Material and Methods. Note, the experimentally obtained value differed only statistically from the predicted value of a syntopic model (*p < 0.05).

Mentions: Although alkanols and 4-AP exert a similar effect at the gating current level, they may act through different binding sites. Whereas the binding site of 4-AP partially overlaps with that of internal pore blockers2627, alkanols have been proposed to target the electromechanical coupling that is located outside the K+ pore. To test whether 4-AP and 1-BuOH have structurally different binding sites, we performed drug competition experiments using IC50 concentrations of 4-AP (30 μM) and 1-BuOH (50 mM). After establishing approximately 50% steady-state IK inhibition with 4-AP, we applied a mixture of 30 μM 4-AP and 50 mM 1-BuOH. This mixture resulted in 78.7 ± 4.1% (n = 7) inhibition of IK (Fig. 6), thus yielding an additional inhibition of 29% in IK amplitude compared to each compound separately.


Alkanols inhibit voltage-gated K(+) channels via a distinct gating modifying mechanism that prevents gate opening.

Martínez-Morales E, Kopljar I, Snyders DJ, Labro AJ - Sci Rep (2015)

1-BuOH and 4-AP do not compete for inhibiting Shaker-IR.(A) Sequentially recorded IK of Shaker-IR in control condition and after steady-state inhibition by 50 mM 1-BuOH and 30 μM 4-AP. Finally, instead of washing the 30 μM 4-AP out, a mixture of 30 μM 4-AP plus 50 mM 1-BuOH was added and the amount of IK inhibition was determined. (B) Bar chart shows the average reduction in IK ± S.E.M. (n = 7) after applying 50 mM 1-BuOH, 30 μM 4-AP and the mixture of both compounds (30 μM 4-AP plus 50 mM 1-BuOH). The percentage of IK inhibition was calculated by normalizing the steady-state IK in presence of drug to the IK amplitude in control conditions. The expected reduction in IK for an allotopic and syntopic model was calculated as described in Material and Methods. Note, the experimentally obtained value differed only statistically from the predicted value of a syntopic model (*p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663795&req=5

f6: 1-BuOH and 4-AP do not compete for inhibiting Shaker-IR.(A) Sequentially recorded IK of Shaker-IR in control condition and after steady-state inhibition by 50 mM 1-BuOH and 30 μM 4-AP. Finally, instead of washing the 30 μM 4-AP out, a mixture of 30 μM 4-AP plus 50 mM 1-BuOH was added and the amount of IK inhibition was determined. (B) Bar chart shows the average reduction in IK ± S.E.M. (n = 7) after applying 50 mM 1-BuOH, 30 μM 4-AP and the mixture of both compounds (30 μM 4-AP plus 50 mM 1-BuOH). The percentage of IK inhibition was calculated by normalizing the steady-state IK in presence of drug to the IK amplitude in control conditions. The expected reduction in IK for an allotopic and syntopic model was calculated as described in Material and Methods. Note, the experimentally obtained value differed only statistically from the predicted value of a syntopic model (*p < 0.05).
Mentions: Although alkanols and 4-AP exert a similar effect at the gating current level, they may act through different binding sites. Whereas the binding site of 4-AP partially overlaps with that of internal pore blockers2627, alkanols have been proposed to target the electromechanical coupling that is located outside the K+ pore. To test whether 4-AP and 1-BuOH have structurally different binding sites, we performed drug competition experiments using IC50 concentrations of 4-AP (30 μM) and 1-BuOH (50 mM). After establishing approximately 50% steady-state IK inhibition with 4-AP, we applied a mixture of 30 μM 4-AP and 50 mM 1-BuOH. This mixture resulted in 78.7 ± 4.1% (n = 7) inhibition of IK (Fig. 6), thus yielding an additional inhibition of 29% in IK amplitude compared to each compound separately.

Bottom Line: Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition.Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening.Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Molecular Biophysics, Physiology and Pharmacology, Department of Biomedical Sciences, University of Antwerp, Antwerp, 2610, Belgium.

ABSTRACT
Alkanols are small aliphatic compounds that inhibit voltage-gated K(+) (K(v)) channels through a yet unresolved gating mechanism. K(v) channels detect changes in the membrane potential with their voltage-sensing domains (VSDs) that reorient and generate a transient gating current. Both 1-Butanol (1-BuOH) and 1-Hexanol (1-HeOH) inhibited the ionic currents of the Shaker K(v) channel in a concentration dependent manner with an IC50 value of approximately 50 mM and 3 mM, respectively. Using the non-conducting Shaker-W434F mutant, we found that both alkanols immobilized approximately 10% of the gating charge and accelerated the deactivating gating currents simultaneously with ionic current inhibition. Thus, alkanols prevent the final VSD movement(s) that is associated with channel gate opening. Applying 1-BuOH and 1-HeOH to the Shaker-P475A mutant, in which the final gating transition is isolated from earlier VSD movements, strengthened that neither alkanol affected the early VSD movements. Drug competition experiments showed that alkanols do not share the binding site of 4-aminopyridine, a drug that exerts a similar effect at the gating current level. Thus, alkanols inhibit Shaker-type K(v) channels via a unique gating modifying mechanism that stabilizes the channel in its non-conducting activated state.

No MeSH data available.


Related in: MedlinePlus