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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

Impact of alkanols on IG recordings of Shaker-IR-W434F.(A) Representative IG recordings of Shaker-IR-W434F recorded in control conditions with the pulse protocol shown on top. Note that prolonging the depolarization at +20 mV gradually slowed down IGdeac upon repolarization to −120 mV. (B) Superposition of Shaker-IR-W434F steady-state IG recordings in control condition (gray) and in presence of 100 mM (dark gray) and 300 mM (black) 1-BuOH. Inset shows a scale up view of IGac. Note the gradual acceleration in IGdeac upon application of higher concentrations of 1-BuOH. (C) Superposition of steady-state IG recordings in control condition (gray) and in presence of 3 mM (dark gray) and 30 mM (black) 1-HeOH. (D) Concentration-response curves obtained by plotting the weighted τIGdeac at −90 mV (obtained from IGdeac recordings shown in panel A and B) as a function of 1-BuOH (circles, n = 10) or 1-HeOH (triangles, n = 6) concentration. (E) Concentration-response curves obtained by plotting the normalized charge movement, which was determined by integrating the steady-state IGac recordings and normalizing the calculated charge to the total charge moved in control condition, as a function of alkanol concentration.
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f3: Impact of alkanols on IG recordings of Shaker-IR-W434F.(A) Representative IG recordings of Shaker-IR-W434F recorded in control conditions with the pulse protocol shown on top. Note that prolonging the depolarization at +20 mV gradually slowed down IGdeac upon repolarization to −120 mV. (B) Superposition of Shaker-IR-W434F steady-state IG recordings in control condition (gray) and in presence of 100 mM (dark gray) and 300 mM (black) 1-BuOH. Inset shows a scale up view of IGac. Note the gradual acceleration in IGdeac upon application of higher concentrations of 1-BuOH. (C) Superposition of steady-state IG recordings in control condition (gray) and in presence of 3 mM (dark gray) and 30 mM (black) 1-HeOH. (D) Concentration-response curves obtained by plotting the weighted τIGdeac at −90 mV (obtained from IGdeac recordings shown in panel A and B) as a function of 1-BuOH (circles, n = 10) or 1-HeOH (triangles, n = 6) concentration. (E) Concentration-response curves obtained by plotting the normalized charge movement, which was determined by integrating the steady-state IGac recordings and normalizing the calculated charge to the total charge moved in control condition, as a function of alkanol concentration.

Mentions: The IK measurements only report on the final opening of the channel gate, which is an end state in the activation pathway from closed to open. From IG analysis it has been reported that the VSD traverses at least one non-conducting activated state before the channel gate opens. Channel gate opening subsequently slows down VSD deactivation1015, which can be visualized by gradually prolonging the duration of the depolarizing pre-pulse (Fig. 3A). Thus, to assess whether 1-BuOH and 1-HeOH affect transitions early in the activation pathway, i.e. before the channel gate opened, we tested the effect of both compounds on the IG recordings of the non-conducting Shaker-IR pore mutant W434F25. During wash-in of both 1-BuOH and 1-HeOH we noted a concentration-dependent acceleration of the deactivating (IGdeac) gating currents (Fig. 3B,C). Plotting the time constant of VSD deactivation (τIGdeac, obtained by fitting the decaying phase of IGdeac) as a function of 1-BuOH or 1-HeOH concentration yielded concentration-response curves with IC50 values of 67 ± 1 mM (n = 10) and 3.0 ± 0.4 mM (n = 6), and Hill coefficients of 1.3 ± 0.4 and 1.6 ± 0.3, respectively (Fig. 3D).


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)

Impact of alkanols on IG recordings of Shaker-IR-W434F.(A) Representative IG recordings of Shaker-IR-W434F recorded in control conditions with the pulse protocol shown on top. Note that prolonging the depolarization at +20 mV gradually slowed down IGdeac upon repolarization to −120 mV. (B) Superposition of Shaker-IR-W434F steady-state IG recordings in control condition (gray) and in presence of 100 mM (dark gray) and 300 mM (black) 1-BuOH. Inset shows a scale up view of IGac. Note the gradual acceleration in IGdeac upon application of higher concentrations of 1-BuOH. (C) Superposition of steady-state IG recordings in control condition (gray) and in presence of 3 mM (dark gray) and 30 mM (black) 1-HeOH. (D) Concentration-response curves obtained by plotting the weighted τIGdeac at −90 mV (obtained from IGdeac recordings shown in panel A and B) as a function of 1-BuOH (circles, n = 10) or 1-HeOH (triangles, n = 6) concentration. (E) Concentration-response curves obtained by plotting the normalized charge movement, which was determined by integrating the steady-state IGac recordings and normalizing the calculated charge to the total charge moved in control condition, as a function of alkanol concentration.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Impact of alkanols on IG recordings of Shaker-IR-W434F.(A) Representative IG recordings of Shaker-IR-W434F recorded in control conditions with the pulse protocol shown on top. Note that prolonging the depolarization at +20 mV gradually slowed down IGdeac upon repolarization to −120 mV. (B) Superposition of Shaker-IR-W434F steady-state IG recordings in control condition (gray) and in presence of 100 mM (dark gray) and 300 mM (black) 1-BuOH. Inset shows a scale up view of IGac. Note the gradual acceleration in IGdeac upon application of higher concentrations of 1-BuOH. (C) Superposition of steady-state IG recordings in control condition (gray) and in presence of 3 mM (dark gray) and 30 mM (black) 1-HeOH. (D) Concentration-response curves obtained by plotting the weighted τIGdeac at −90 mV (obtained from IGdeac recordings shown in panel A and B) as a function of 1-BuOH (circles, n = 10) or 1-HeOH (triangles, n = 6) concentration. (E) Concentration-response curves obtained by plotting the normalized charge movement, which was determined by integrating the steady-state IGac recordings and normalizing the calculated charge to the total charge moved in control condition, as a function of alkanol concentration.
Mentions: The IK measurements only report on the final opening of the channel gate, which is an end state in the activation pathway from closed to open. From IG analysis it has been reported that the VSD traverses at least one non-conducting activated state before the channel gate opens. Channel gate opening subsequently slows down VSD deactivation1015, which can be visualized by gradually prolonging the duration of the depolarizing pre-pulse (Fig. 3A). Thus, to assess whether 1-BuOH and 1-HeOH affect transitions early in the activation pathway, i.e. before the channel gate opened, we tested the effect of both compounds on the IG recordings of the non-conducting Shaker-IR pore mutant W434F25. During wash-in of both 1-BuOH and 1-HeOH we noted a concentration-dependent acceleration of the deactivating (IGdeac) gating currents (Fig. 3B,C). Plotting the time constant of VSD deactivation (τIGdeac, obtained by fitting the decaying phase of IGdeac) as a function of 1-BuOH or 1-HeOH concentration yielded concentration-response curves with IC50 values of 67 ± 1 mM (n = 10) and 3.0 ± 0.4 mM (n = 6), and Hill coefficients of 1.3 ± 0.4 and 1.6 ± 0.3, respectively (Fig. 3D).

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