Limits...
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

Biophysical properties of Shaker-IR-P475A upon alkanol application.(A) Representative IKac recordings of Shaker-IR-P475A in control conditions, 100 mM 1-BuOH and 10 mM 1-HeOH, elicited using the pulse protocol shown on top. (B) Conduction vs. voltage GV curves of Shaker-IR-P475A in control conditions (white circles), 100 mM 1-BuOH (blue circles), 300 mM 1-BuOH (red cricles), 10 mM 1-HeOH (blue triangles), and 30 mM 1-HeOH (red triangles). GV curves displayed in the left panel were obtained by normalizing tail current amplitudes. Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). Right panel displays the GV curves determined from analyzing the peak outward currents. (C) Panels from left to right show the τIKac values of Shaker-IR-P475A upon increasing 1-BuOH concentrations with the left most panel showing the values in control conditions. The fast and slow τIKac components are represented with open symbols and the weighted τIKac with filled symbols. Note that the contribution of the fast τIKac component increased upon higher 1-BuOH concentrations; compare weighted τIKac values in 30 mM (yellow symbols) and 100 mM (blue symbols). In presence of 300 mM 1-BuOH (red symbols) only the fast component could be resolved and IKac was approximated with a single exponential function. (D) Plot shows the weighted τIKac and τIKdeac values in control conditions (white) and in presence of 10 mM (gray, n = 4), 30 mM (yellow, n = 5), 100 mM (blue, n = 8), and 300 mM (red, n = 4) 1-BuOH. (E) Plot shows the effect of 1 mM (gray, n = 7), 3 mM (yellow, n = 9), 10 mM (blue, n = 5), and 30 mM (red, n = 5) 1-HeOH on the weighted τIKac and τIKdeac kinetics. (F) Concentration-response curves obtained by plotting the weighted τIKac at +100 mV as a function of 1-BuOH (black circles, n = 10) and 1-HeOH (gray triangles, n = 6) concentration. Solid lines represent the fit with a Hill equation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663795&req=5

f8: Biophysical properties of Shaker-IR-P475A upon alkanol application.(A) Representative IKac recordings of Shaker-IR-P475A in control conditions, 100 mM 1-BuOH and 10 mM 1-HeOH, elicited using the pulse protocol shown on top. (B) Conduction vs. voltage GV curves of Shaker-IR-P475A in control conditions (white circles), 100 mM 1-BuOH (blue circles), 300 mM 1-BuOH (red cricles), 10 mM 1-HeOH (blue triangles), and 30 mM 1-HeOH (red triangles). GV curves displayed in the left panel were obtained by normalizing tail current amplitudes. Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). Right panel displays the GV curves determined from analyzing the peak outward currents. (C) Panels from left to right show the τIKac values of Shaker-IR-P475A upon increasing 1-BuOH concentrations with the left most panel showing the values in control conditions. The fast and slow τIKac components are represented with open symbols and the weighted τIKac with filled symbols. Note that the contribution of the fast τIKac component increased upon higher 1-BuOH concentrations; compare weighted τIKac values in 30 mM (yellow symbols) and 100 mM (blue symbols). In presence of 300 mM 1-BuOH (red symbols) only the fast component could be resolved and IKac was approximated with a single exponential function. (D) Plot shows the weighted τIKac and τIKdeac values in control conditions (white) and in presence of 10 mM (gray, n = 4), 30 mM (yellow, n = 5), 100 mM (blue, n = 8), and 300 mM (red, n = 4) 1-BuOH. (E) Plot shows the effect of 1 mM (gray, n = 7), 3 mM (yellow, n = 9), 10 mM (blue, n = 5), and 30 mM (red, n = 5) 1-HeOH on the weighted τIKac and τIKdeac kinetics. (F) Concentration-response curves obtained by plotting the weighted τIKac at +100 mV as a function of 1-BuOH (black circles, n = 10) and 1-HeOH (gray triangles, n = 6) concentration. Solid lines represent the fit with a Hill equation.

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)

Biophysical properties of Shaker-IR-P475A upon alkanol application.(A) Representative IKac recordings of Shaker-IR-P475A in control conditions, 100 mM 1-BuOH and 10 mM 1-HeOH, elicited using the pulse protocol shown on top. (B) Conduction vs. voltage GV curves of Shaker-IR-P475A in control conditions (white circles), 100 mM 1-BuOH (blue circles), 300 mM 1-BuOH (red cricles), 10 mM 1-HeOH (blue triangles), and 30 mM 1-HeOH (red triangles). GV curves displayed in the left panel were obtained by normalizing tail current amplitudes. Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). Right panel displays the GV curves determined from analyzing the peak outward currents. (C) Panels from left to right show the τIKac values of Shaker-IR-P475A upon increasing 1-BuOH concentrations with the left most panel showing the values in control conditions. The fast and slow τIKac components are represented with open symbols and the weighted τIKac with filled symbols. Note that the contribution of the fast τIKac component increased upon higher 1-BuOH concentrations; compare weighted τIKac values in 30 mM (yellow symbols) and 100 mM (blue symbols). In presence of 300 mM 1-BuOH (red symbols) only the fast component could be resolved and IKac was approximated with a single exponential function. (D) Plot shows the weighted τIKac and τIKdeac values in control conditions (white) and in presence of 10 mM (gray, n = 4), 30 mM (yellow, n = 5), 100 mM (blue, n = 8), and 300 mM (red, n = 4) 1-BuOH. (E) Plot shows the effect of 1 mM (gray, n = 7), 3 mM (yellow, n = 9), 10 mM (blue, n = 5), and 30 mM (red, n = 5) 1-HeOH on the weighted τIKac and τIKdeac kinetics. (F) Concentration-response curves obtained by plotting the weighted τIKac at +100 mV as a function of 1-BuOH (black circles, n = 10) and 1-HeOH (gray triangles, n = 6) concentration. Solid lines represent the fit with a Hill equation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f8: Biophysical properties of Shaker-IR-P475A upon alkanol application.(A) Representative IKac recordings of Shaker-IR-P475A in control conditions, 100 mM 1-BuOH and 10 mM 1-HeOH, elicited using the pulse protocol shown on top. (B) Conduction vs. voltage GV curves of Shaker-IR-P475A in control conditions (white circles), 100 mM 1-BuOH (blue circles), 300 mM 1-BuOH (red cricles), 10 mM 1-HeOH (blue triangles), and 30 mM 1-HeOH (red triangles). GV curves displayed in the left panel were obtained by normalizing tail current amplitudes. Solid lines represent the average fit with a Boltzmann equation (V1/2 and slope factor values are provided in Table 1). Right panel displays the GV curves determined from analyzing the peak outward currents. (C) Panels from left to right show the τIKac values of Shaker-IR-P475A upon increasing 1-BuOH concentrations with the left most panel showing the values in control conditions. The fast and slow τIKac components are represented with open symbols and the weighted τIKac with filled symbols. Note that the contribution of the fast τIKac component increased upon higher 1-BuOH concentrations; compare weighted τIKac values in 30 mM (yellow symbols) and 100 mM (blue symbols). In presence of 300 mM 1-BuOH (red symbols) only the fast component could be resolved and IKac was approximated with a single exponential function. (D) Plot shows the weighted τIKac and τIKdeac values in control conditions (white) and in presence of 10 mM (gray, n = 4), 30 mM (yellow, n = 5), 100 mM (blue, n = 8), and 300 mM (red, n = 4) 1-BuOH. (E) Plot shows the effect of 1 mM (gray, n = 7), 3 mM (yellow, n = 9), 10 mM (blue, n = 5), and 30 mM (red, n = 5) 1-HeOH on the weighted τIKac and τIKdeac kinetics. (F) Concentration-response curves obtained by plotting the weighted τIKac at +100 mV as a function of 1-BuOH (black circles, n = 10) and 1-HeOH (gray triangles, n = 6) concentration. Solid lines represent the fit with a Hill equation.
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