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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

Alkanols inhibited IK without affecting the kinetics.(A) Monitoring the inhibition in IK during application of 50 mM 1-BuOH (top panel) or 3 mM 1-HeOH (bottom panel) indicated that the IK inhibition developed rapidly with a time constant of 5.2 ± 1.2 s (n = 8) and 3.7 ± 0.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. The IK inhibition was fully reversible upon wash-out of both alkanols and the current recovery was relatively fast yielding time constants of 7.6 ± 2.1 s (n = 8) and 4.8 ± 1.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. (B) Normalized peak current versus voltage relationships, obtained from pulse protocols shown in Fig. 1A, in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). (C) Normalized conduction versus voltage GV curves in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). (D) Time constants of IK activation (τIKac) and deactivation (τIKdeac) in control conditions (open symbols) and in presence of 50 mM 1-BuOH (top panel, n = 7) or 3 mM 1-HeOH (bottom panel, n = 8).
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f2: Alkanols inhibited IK without affecting the kinetics.(A) Monitoring the inhibition in IK during application of 50 mM 1-BuOH (top panel) or 3 mM 1-HeOH (bottom panel) indicated that the IK inhibition developed rapidly with a time constant of 5.2 ± 1.2 s (n = 8) and 3.7 ± 0.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. The IK inhibition was fully reversible upon wash-out of both alkanols and the current recovery was relatively fast yielding time constants of 7.6 ± 2.1 s (n = 8) and 4.8 ± 1.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. (B) Normalized peak current versus voltage relationships, obtained from pulse protocols shown in Fig. 1A, in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). (C) Normalized conduction versus voltage GV curves in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). (D) Time constants of IK activation (τIKac) and deactivation (τIKdeac) in control conditions (open symbols) and in presence of 50 mM 1-BuOH (top panel, n = 7) or 3 mM 1-HeOH (bottom panel, n = 8).

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)

Alkanols inhibited IK without affecting the kinetics.(A) Monitoring the inhibition in IK during application of 50 mM 1-BuOH (top panel) or 3 mM 1-HeOH (bottom panel) indicated that the IK inhibition developed rapidly with a time constant of 5.2 ± 1.2 s (n = 8) and 3.7 ± 0.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. The IK inhibition was fully reversible upon wash-out of both alkanols and the current recovery was relatively fast yielding time constants of 7.6 ± 2.1 s (n = 8) and 4.8 ± 1.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. (B) Normalized peak current versus voltage relationships, obtained from pulse protocols shown in Fig. 1A, in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). (C) Normalized conduction versus voltage GV curves in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). (D) Time constants of IK activation (τIKac) and deactivation (τIKdeac) in control conditions (open symbols) and in presence of 50 mM 1-BuOH (top panel, n = 7) or 3 mM 1-HeOH (bottom panel, n = 8).
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f2: Alkanols inhibited IK without affecting the kinetics.(A) Monitoring the inhibition in IK during application of 50 mM 1-BuOH (top panel) or 3 mM 1-HeOH (bottom panel) indicated that the IK inhibition developed rapidly with a time constant of 5.2 ± 1.2 s (n = 8) and 3.7 ± 0.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. The IK inhibition was fully reversible upon wash-out of both alkanols and the current recovery was relatively fast yielding time constants of 7.6 ± 2.1 s (n = 8) and 4.8 ± 1.7 s (n = 9) for 1-BuOH and 1-HeOH respectively. (B) Normalized peak current versus voltage relationships, obtained from pulse protocols shown in Fig. 1A, in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). (C) Normalized conduction versus voltage GV curves in control conditions (open symbols) and presence of 50 mM 1-BuOH (top panel, n = 5) or 3 mM 1-HeOH (bottom panel, n = 6). Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). (D) Time constants of IK activation (τIKac) and deactivation (τIKdeac) in control conditions (open symbols) and in presence of 50 mM 1-BuOH (top panel, n = 7) or 3 mM 1-HeOH (bottom panel, n = 8).
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