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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

Alkanol-dependent activation of Shaker-IR-P475A.(A) Steady-state IK recordings of Shaker-IR-P475A in control condition (black), 100 mM (blue), and 300 mM (red) 1-BuOH elicited using the pulse protocol shown on top. (B) Steady-state IK recordings obtained in control condition (black), 10 mM (blue) and 30 mM 1-HeOH (red). In presence of alkanols the currents activated markedly faster and current inactivation was more pronounced. Insets show scale up views of the deactivating (IKdeac) tail currents.
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f7: Alkanol-dependent activation of Shaker-IR-P475A.(A) Steady-state IK recordings of Shaker-IR-P475A in control condition (black), 100 mM (blue), and 300 mM (red) 1-BuOH elicited using the pulse protocol shown on top. (B) Steady-state IK recordings obtained in control condition (black), 10 mM (blue) and 30 mM 1-HeOH (red). In presence of alkanols the currents activated markedly faster and current inactivation was more pronounced. Insets show scale up views of the deactivating (IKdeac) tail currents.

Mentions: Applying 1-BuOH or 1-HeOH to the Shaker-IR-P475A mutant resulted in a concentration-dependent increase in IK and an acceleration of τ IKac (Fig. 7A,B), which is in agreement with previous data obtained in the Shaw2 channel22. With higher concentrations of 1-BuOH or 1-HeOH the typical conduction versus voltage GV curves, which were determined from normalizing the deactivation tail current of activation protocols (Fig. 8A), appeared to become steeper and to shift slightly towards more hyperpolarized potentials (Fig. 8B, Table 1). However, concomitantly with the accelerated τIKac kinetics, also the inactivation process became more pronounced and the peak IK amplitude started to decrease at higher alkanol concentrations (Fig. 7A,B). Therefore, the small hyperpolarizing shift and steepening of the GV curves could be an apparent effect due to the accelerated channel inactivation. To test this possibility, we determined the normalized conduction G from the peak outward currents using the Goldman-Hodgkin-Katz current equation. The GV curves obtained with this approach, which should be less sensitive to inactivation, were in presence of alkanols similar to those in control conditions (Fig. 8B). Thus, although both compounds resulted in IK activation, neither 1-BuOH nor 1-HeOH affected the voltage dependence of channel opening substantially. To evaluate if the pronounced channel inactivation behavior reflects in fact open channel block, we examined IKdeac more closely. In contrast to what is expected with open channel block, the IKdeac recordings did not cross nor did they display a noticeable hook (Fig. 7A,B). In fact, the τIKdeac kinetics accelerated markedly which suggested that also the accelerated channel inactivation was due to gating modification. All these effects were fully reversible upon wash-out of both alkanols.


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)

Alkanol-dependent activation of Shaker-IR-P475A.(A) Steady-state IK recordings of Shaker-IR-P475A in control condition (black), 100 mM (blue), and 300 mM (red) 1-BuOH elicited using the pulse protocol shown on top. (B) Steady-state IK recordings obtained in control condition (black), 10 mM (blue) and 30 mM 1-HeOH (red). In presence of alkanols the currents activated markedly faster and current inactivation was more pronounced. Insets show scale up views of the deactivating (IKdeac) tail currents.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Alkanol-dependent activation of Shaker-IR-P475A.(A) Steady-state IK recordings of Shaker-IR-P475A in control condition (black), 100 mM (blue), and 300 mM (red) 1-BuOH elicited using the pulse protocol shown on top. (B) Steady-state IK recordings obtained in control condition (black), 10 mM (blue) and 30 mM 1-HeOH (red). In presence of alkanols the currents activated markedly faster and current inactivation was more pronounced. Insets show scale up views of the deactivating (IKdeac) tail currents.
Mentions: Applying 1-BuOH or 1-HeOH to the Shaker-IR-P475A mutant resulted in a concentration-dependent increase in IK and an acceleration of τ IKac (Fig. 7A,B), which is in agreement with previous data obtained in the Shaw2 channel22. With higher concentrations of 1-BuOH or 1-HeOH the typical conduction versus voltage GV curves, which were determined from normalizing the deactivation tail current of activation protocols (Fig. 8A), appeared to become steeper and to shift slightly towards more hyperpolarized potentials (Fig. 8B, Table 1). However, concomitantly with the accelerated τIKac kinetics, also the inactivation process became more pronounced and the peak IK amplitude started to decrease at higher alkanol concentrations (Fig. 7A,B). Therefore, the small hyperpolarizing shift and steepening of the GV curves could be an apparent effect due to the accelerated channel inactivation. To test this possibility, we determined the normalized conduction G from the peak outward currents using the Goldman-Hodgkin-Katz current equation. The GV curves obtained with this approach, which should be less sensitive to inactivation, were in presence of alkanols similar to those in control conditions (Fig. 8B). Thus, although both compounds resulted in IK activation, neither 1-BuOH nor 1-HeOH affected the voltage dependence of channel opening substantially. To evaluate if the pronounced channel inactivation behavior reflects in fact open channel block, we examined IKdeac more closely. In contrast to what is expected with open channel block, the IKdeac recordings did not cross nor did they display a noticeable hook (Fig. 7A,B). In fact, the τIKdeac kinetics accelerated markedly which suggested that also the accelerated channel inactivation was due to gating modification. All these effects were fully reversible upon wash-out of both alkanols.

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