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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

Biophysical properties of Shaker-IR-W434F upon alkanol application.(A) Representative IGac recordings of Shaker-IR-W434F in control condition (left) and in presence of 300 mM 1-BuOH (right) elicited using the pulse protocols shown on top. (B) Representative IGdeac recordings elicited with the deactivation pulse protocols shown on top; in control conditions (left) and in presence of 100 mM 1-BuOH (right). Inter-sweep holding potential was −90 mV and the depolarizing pre- and post-pulse to 0 mV were 15 ms in duration. (C) Charge vs. voltage QV curves in control condition (white circles, n = 10) and in presence of 300 mM 1-BuOH (black circles, n = 5) or 30 mM 1-HeOH (black triangles, n = 4) were created by plotting the normalized charge (obtained from integrating IGac recordings from pulse protocols shown in panel A) as a function of voltage. Curves shown are the average fit to a Boltzmann equation. (D) Time constants of VSD activation (τIGac) in control condition (white diamonds, n = 8) and in presence of 100 mM (gray diamonds, n = 3) or 300 mM (black diamonds, n = 5) 1-BuOH. For VSD deactivation the weighted τIGdeac kinetics are shown. Note the gradual acceleration in τIGdeac between control (white circles), 100 mM 1-BuOH (gray circles) and 300 mM 1-BuOH (black circles). (E) Panel shows the voltage-dependent τIGac kinetics in control condition (white squares, n = 7) and in presence of 10 mM (gray squares, n = 3) or 30 mM (black squares, n = 4) 1-HeOH. Similar to 1-BuOH the τIGdeac kinetics accelerated in presence of 10 mM (gray triangles, n = 3) and 30 mM 1-HeOH (black triangles, n = 4), control conditions (white triangles).
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f4: Biophysical properties of Shaker-IR-W434F upon alkanol application.(A) Representative IGac recordings of Shaker-IR-W434F in control condition (left) and in presence of 300 mM 1-BuOH (right) elicited using the pulse protocols shown on top. (B) Representative IGdeac recordings elicited with the deactivation pulse protocols shown on top; in control conditions (left) and in presence of 100 mM 1-BuOH (right). Inter-sweep holding potential was −90 mV and the depolarizing pre- and post-pulse to 0 mV were 15 ms in duration. (C) Charge vs. voltage QV curves in control condition (white circles, n = 10) and in presence of 300 mM 1-BuOH (black circles, n = 5) or 30 mM 1-HeOH (black triangles, n = 4) were created by plotting the normalized charge (obtained from integrating IGac recordings from pulse protocols shown in panel A) as a function of voltage. Curves shown are the average fit to a Boltzmann equation. (D) Time constants of VSD activation (τIGac) in control condition (white diamonds, n = 8) and in presence of 100 mM (gray diamonds, n = 3) or 300 mM (black diamonds, n = 5) 1-BuOH. For VSD deactivation the weighted τIGdeac kinetics are shown. Note the gradual acceleration in τIGdeac between control (white circles), 100 mM 1-BuOH (gray circles) and 300 mM 1-BuOH (black circles). (E) Panel shows the voltage-dependent τIGac kinetics in control condition (white squares, n = 7) and in presence of 10 mM (gray squares, n = 3) or 30 mM (black squares, n = 4) 1-HeOH. Similar to 1-BuOH the τIGdeac kinetics accelerated in presence of 10 mM (gray triangles, n = 3) and 30 mM 1-HeOH (black triangles, n = 4), control conditions (white triangles).

Mentions: To determine the kinetics and voltage dependence of VSD activation, we applied incremental depolarizing voltage steps starting from a constant hyperpolarized initial voltage (activation protocol, Fig. 4A). To characterize VSD deactivation adequately, a deactivation pulse protocol was used (Fig. 4B). Integrating the IGac recordings, obtained in control conditions and after steady-state 1-BuOH and 1-HeOH modification, yielded charge vs. voltage QV curves (Fig. 4C). Interestingly, the QV curves determined in presence of 1-BuOH or 1-HeOH displayed V1/2 and slope factor values similar as in control condition (Table 1). This indicated that neither alkanol affected the voltage dependence of the remaining gating charge movement. As noted during the wash-in protocol (Fig. 3B,C), both alkanols accelerated τIGdeac without markedly altering the IGac kinetics (τIGac, Fig. 4D,E). Thus, both alkanols accelerated τIGdeac and immobilized approximately 10% of the gating charge movement but did not affect the voltage dependence of the early VSD movements. These observations indicated that in presence of 1-BuOH or 1-HeOH the Shaker channel is able to reach the non-conducting activated state but it cannot pass the subunit-cooperative step leading to channel gate opening. Accordingly, the τIGdeac values in presence of saturating alkanol concentrations should corresponded to τIGdeac in control conditions when the activating pre-pulse is very short and channels only reach the non-conducting activated state. In control conditions τIGdeac amounted at −120 mV to 0.32 ± 0.03 ms (n = 6) upon a brief 0.5 ms depolarization, determined from pulse protocols shown in Fig. 3A. In presence of 300 mM 1-BuOH or 30 mM 1-HeOH τIGdeac at −120 mV were 0.48 ± 0.08 ms (n = 4) and 0.53 ± 0.10 ms (n = 4) respectively (Fig. 4D,E), which are indeed similar to the value in control conditions.


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-W434F upon alkanol application.(A) Representative IGac recordings of Shaker-IR-W434F in control condition (left) and in presence of 300 mM 1-BuOH (right) elicited using the pulse protocols shown on top. (B) Representative IGdeac recordings elicited with the deactivation pulse protocols shown on top; in control conditions (left) and in presence of 100 mM 1-BuOH (right). Inter-sweep holding potential was −90 mV and the depolarizing pre- and post-pulse to 0 mV were 15 ms in duration. (C) Charge vs. voltage QV curves in control condition (white circles, n = 10) and in presence of 300 mM 1-BuOH (black circles, n = 5) or 30 mM 1-HeOH (black triangles, n = 4) were created by plotting the normalized charge (obtained from integrating IGac recordings from pulse protocols shown in panel A) as a function of voltage. Curves shown are the average fit to a Boltzmann equation. (D) Time constants of VSD activation (τIGac) in control condition (white diamonds, n = 8) and in presence of 100 mM (gray diamonds, n = 3) or 300 mM (black diamonds, n = 5) 1-BuOH. For VSD deactivation the weighted τIGdeac kinetics are shown. Note the gradual acceleration in τIGdeac between control (white circles), 100 mM 1-BuOH (gray circles) and 300 mM 1-BuOH (black circles). (E) Panel shows the voltage-dependent τIGac kinetics in control condition (white squares, n = 7) and in presence of 10 mM (gray squares, n = 3) or 30 mM (black squares, n = 4) 1-HeOH. Similar to 1-BuOH the τIGdeac kinetics accelerated in presence of 10 mM (gray triangles, n = 3) and 30 mM 1-HeOH (black triangles, n = 4), control conditions (white triangles).
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Related In: Results  -  Collection

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f4: Biophysical properties of Shaker-IR-W434F upon alkanol application.(A) Representative IGac recordings of Shaker-IR-W434F in control condition (left) and in presence of 300 mM 1-BuOH (right) elicited using the pulse protocols shown on top. (B) Representative IGdeac recordings elicited with the deactivation pulse protocols shown on top; in control conditions (left) and in presence of 100 mM 1-BuOH (right). Inter-sweep holding potential was −90 mV and the depolarizing pre- and post-pulse to 0 mV were 15 ms in duration. (C) Charge vs. voltage QV curves in control condition (white circles, n = 10) and in presence of 300 mM 1-BuOH (black circles, n = 5) or 30 mM 1-HeOH (black triangles, n = 4) were created by plotting the normalized charge (obtained from integrating IGac recordings from pulse protocols shown in panel A) as a function of voltage. Curves shown are the average fit to a Boltzmann equation. (D) Time constants of VSD activation (τIGac) in control condition (white diamonds, n = 8) and in presence of 100 mM (gray diamonds, n = 3) or 300 mM (black diamonds, n = 5) 1-BuOH. For VSD deactivation the weighted τIGdeac kinetics are shown. Note the gradual acceleration in τIGdeac between control (white circles), 100 mM 1-BuOH (gray circles) and 300 mM 1-BuOH (black circles). (E) Panel shows the voltage-dependent τIGac kinetics in control condition (white squares, n = 7) and in presence of 10 mM (gray squares, n = 3) or 30 mM (black squares, n = 4) 1-HeOH. Similar to 1-BuOH the τIGdeac kinetics accelerated in presence of 10 mM (gray triangles, n = 3) and 30 mM 1-HeOH (black triangles, n = 4), control conditions (white triangles).
Mentions: To determine the kinetics and voltage dependence of VSD activation, we applied incremental depolarizing voltage steps starting from a constant hyperpolarized initial voltage (activation protocol, Fig. 4A). To characterize VSD deactivation adequately, a deactivation pulse protocol was used (Fig. 4B). Integrating the IGac recordings, obtained in control conditions and after steady-state 1-BuOH and 1-HeOH modification, yielded charge vs. voltage QV curves (Fig. 4C). Interestingly, the QV curves determined in presence of 1-BuOH or 1-HeOH displayed V1/2 and slope factor values similar as in control condition (Table 1). This indicated that neither alkanol affected the voltage dependence of the remaining gating charge movement. As noted during the wash-in protocol (Fig. 3B,C), both alkanols accelerated τIGdeac without markedly altering the IGac kinetics (τIGac, Fig. 4D,E). Thus, both alkanols accelerated τIGdeac and immobilized approximately 10% of the gating charge movement but did not affect the voltage dependence of the early VSD movements. These observations indicated that in presence of 1-BuOH or 1-HeOH the Shaker channel is able to reach the non-conducting activated state but it cannot pass the subunit-cooperative step leading to channel gate opening. Accordingly, the τIGdeac values in presence of saturating alkanol concentrations should corresponded to τIGdeac in control conditions when the activating pre-pulse is very short and channels only reach the non-conducting activated state. In control conditions τIGdeac amounted at −120 mV to 0.32 ± 0.03 ms (n = 6) upon a brief 0.5 ms depolarization, determined from pulse protocols shown in Fig. 3A. In presence of 300 mM 1-BuOH or 30 mM 1-HeOH τIGdeac at −120 mV were 0.48 ± 0.08 ms (n = 4) and 0.53 ± 0.10 ms (n = 4) respectively (Fig. 4D,E), which are indeed similar to the value in control conditions.

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