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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

Inhibition of Shaker-IR by 1-BuOH and 1-HeOH.(A) Representative IK recordings of Shaker-IR in control condition (left) and in presence of 100 mM 1-BuOH (right) elicited by applying depolarization steps from a -80 mV holding potential (pulse protocols are shown on top). (B) IK recordings of Shaker-IR obtained in control conditions (left) and in presence of 3 mM 1-HeOH (right). (C) Steady-state IK recordings (elicited with a voltage step from -80 mV to 0 mV) upon wash-in of different concentrations of 1-BuOH (left) and 1-HeOH (right). Establishment of channel inhibition was monitored by repetitive pulsing to 0 mV. (D) Concentration-response curves obtained by plotting the normalized steady-state IK amplitude at 0 mV, determined from IK recordings as shown in panel C, as a function of 1-BuOH (circles, n = 5) or 1-HeOH (triangles, n = 7) concentration. Solid lines represent the average fit with a Hill function.
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f1: Inhibition of Shaker-IR by 1-BuOH and 1-HeOH.(A) Representative IK recordings of Shaker-IR in control condition (left) and in presence of 100 mM 1-BuOH (right) elicited by applying depolarization steps from a -80 mV holding potential (pulse protocols are shown on top). (B) IK recordings of Shaker-IR obtained in control conditions (left) and in presence of 3 mM 1-HeOH (right). (C) Steady-state IK recordings (elicited with a voltage step from -80 mV to 0 mV) upon wash-in of different concentrations of 1-BuOH (left) and 1-HeOH (right). Establishment of channel inhibition was monitored by repetitive pulsing to 0 mV. (D) Concentration-response curves obtained by plotting the normalized steady-state IK amplitude at 0 mV, determined from IK recordings as shown in panel C, as a function of 1-BuOH (circles, n = 5) or 1-HeOH (triangles, n = 7) concentration. Solid lines represent the average fit with a Hill function.

Mentions: Alkanols are classified in short chain (up to 5 carbonyls, C1 to C5) or long chain (C6 – C22) 1-alcohols24. In this study, 1-BuOH and 1-HeOH were chosen as representative compounds of a short and long chain alkanol. Their effect was tested on both the ionic (IK) and gating (IG) currents of the fast (N-type) inactivation removed Shaker-IR channel. At the IK level, Shaker-IR was inhibited by both 1-BuOH and 1-HeOH in a concentration-dependent manner (Fig. 1A–C). For 1-BuOH a concentration-response curve was obtained with an IC50 value of 51.8 ± 5.9 mM (n = 5) and a Hill coefficient of 0.92 ± 0.04 (Fig. 1D). 1-HeOH had a slightly higher affinity and yielded a concentration-response curve with an IC50 value of 2.7 ± 0.2 mM (n = 7) and a Hill coefficient of 1.13 ± 0.22 (Fig. 1D). Monitoring the development of IK inhibition and analyzing the remaining steady-state IK amplitude upon application of 50 mM 1-BuOH or 3 mM 1-HeOH (IC50 concentrations) indicated that: (1) the IK inhibition developed rapidly and was fully reversible upon wash-out of both alkanols, and (2) both alkanols did not induce major alterations in the voltage dependence of channel opening nor the time constants of channel activation (τIKac) and deactivation (τIKdeac) (Fig. 2 and Table 1). An apparent channel inactivation behavior or rising phase in the deactivating (IKdeac) tail current (i.e. a hooked tail), which are typical hallmarks for an open channel blocker, were not observed (Fig. 1A–C). Thus, 1-BuOH and 1-HeOH inhibited the IK amplitude without affecting the kinetics, and both compounds achieved this through a mechanism most likely different from open channel block, as proposed previously4.


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)

Inhibition of Shaker-IR by 1-BuOH and 1-HeOH.(A) Representative IK recordings of Shaker-IR in control condition (left) and in presence of 100 mM 1-BuOH (right) elicited by applying depolarization steps from a -80 mV holding potential (pulse protocols are shown on top). (B) IK recordings of Shaker-IR obtained in control conditions (left) and in presence of 3 mM 1-HeOH (right). (C) Steady-state IK recordings (elicited with a voltage step from -80 mV to 0 mV) upon wash-in of different concentrations of 1-BuOH (left) and 1-HeOH (right). Establishment of channel inhibition was monitored by repetitive pulsing to 0 mV. (D) Concentration-response curves obtained by plotting the normalized steady-state IK amplitude at 0 mV, determined from IK recordings as shown in panel C, as a function of 1-BuOH (circles, n = 5) or 1-HeOH (triangles, n = 7) concentration. Solid lines represent the average fit with a Hill function.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Inhibition of Shaker-IR by 1-BuOH and 1-HeOH.(A) Representative IK recordings of Shaker-IR in control condition (left) and in presence of 100 mM 1-BuOH (right) elicited by applying depolarization steps from a -80 mV holding potential (pulse protocols are shown on top). (B) IK recordings of Shaker-IR obtained in control conditions (left) and in presence of 3 mM 1-HeOH (right). (C) Steady-state IK recordings (elicited with a voltage step from -80 mV to 0 mV) upon wash-in of different concentrations of 1-BuOH (left) and 1-HeOH (right). Establishment of channel inhibition was monitored by repetitive pulsing to 0 mV. (D) Concentration-response curves obtained by plotting the normalized steady-state IK amplitude at 0 mV, determined from IK recordings as shown in panel C, as a function of 1-BuOH (circles, n = 5) or 1-HeOH (triangles, n = 7) concentration. Solid lines represent the average fit with a Hill function.
Mentions: Alkanols are classified in short chain (up to 5 carbonyls, C1 to C5) or long chain (C6 – C22) 1-alcohols24. In this study, 1-BuOH and 1-HeOH were chosen as representative compounds of a short and long chain alkanol. Their effect was tested on both the ionic (IK) and gating (IG) currents of the fast (N-type) inactivation removed Shaker-IR channel. At the IK level, Shaker-IR was inhibited by both 1-BuOH and 1-HeOH in a concentration-dependent manner (Fig. 1A–C). For 1-BuOH a concentration-response curve was obtained with an IC50 value of 51.8 ± 5.9 mM (n = 5) and a Hill coefficient of 0.92 ± 0.04 (Fig. 1D). 1-HeOH had a slightly higher affinity and yielded a concentration-response curve with an IC50 value of 2.7 ± 0.2 mM (n = 7) and a Hill coefficient of 1.13 ± 0.22 (Fig. 1D). Monitoring the development of IK inhibition and analyzing the remaining steady-state IK amplitude upon application of 50 mM 1-BuOH or 3 mM 1-HeOH (IC50 concentrations) indicated that: (1) the IK inhibition developed rapidly and was fully reversible upon wash-out of both alkanols, and (2) both alkanols did not induce major alterations in the voltage dependence of channel opening nor the time constants of channel activation (τIKac) and deactivation (τIKdeac) (Fig. 2 and Table 1). An apparent channel inactivation behavior or rising phase in the deactivating (IKdeac) tail current (i.e. a hooked tail), which are typical hallmarks for an open channel blocker, were not observed (Fig. 1A–C). Thus, 1-BuOH and 1-HeOH inhibited the IK amplitude without affecting the kinetics, and both compounds achieved this through a mechanism most likely different from open channel block, as proposed previously4.

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