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Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide.

Lewis AH, Raman IM - J. Gen. Physiol. (2013)

Bottom Line: Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; "CW" channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement.The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor.ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdepartmental Biological Sciences Program and 2 Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the "β4 peptide"). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; "CW" channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

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Time course of recovery from inactivation and from block by the β4 peptide in WT channels. (A) Voltage protocol (top) and representative cells showing recovery from inactivation and block after conditioning for 25 ms at 0 mV in the absence (middle) and presence (bottom) of the β4 peptide. Black, control; green, with 500 nM ATX. (B) Fractional recovery at 0 mV as a function of time at −110 mV. Currents were normalized to the response to the conditioning pulse for each trace, and fractional recovery was calculated as described in Materials and methods. Black, WT; green, WT plus ATX; solid lines, double-exponential fits to the mean data. (C) As in B, with β4 peptide in the intracellular solution. (D) Comparison of recovery in solutions ±β4 peptide. (E) Comparison of recovery in ATX ±β4 peptide. Within-cell comparisons ±ATX; WT, n = 8; WT plus β4, n = 7. (F and G) As in D and E, but with conditioning for 10 ms at +60 mV; without peptide, n = 4; with peptide, n = 5.
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fig7: Time course of recovery from inactivation and from block by the β4 peptide in WT channels. (A) Voltage protocol (top) and representative cells showing recovery from inactivation and block after conditioning for 25 ms at 0 mV in the absence (middle) and presence (bottom) of the β4 peptide. Black, control; green, with 500 nM ATX. (B) Fractional recovery at 0 mV as a function of time at −110 mV. Currents were normalized to the response to the conditioning pulse for each trace, and fractional recovery was calculated as described in Materials and methods. Black, WT; green, WT plus ATX; solid lines, double-exponential fits to the mean data. (C) As in B, with β4 peptide in the intracellular solution. (D) Comparison of recovery in solutions ±β4 peptide. (E) Comparison of recovery in ATX ±β4 peptide. Within-cell comparisons ±ATX; WT, n = 8; WT plus β4, n = 7. (F and G) As in D and E, but with conditioning for 10 ms at +60 mV; without peptide, n = 4; with peptide, n = 5.

Mentions: For peptide-free WT cells, recovery was rapid: after a 10-ms recovery interval, 86.1 ± 1.8% of the inactivated current was available (n = 8; Fig. 7, A–E). Recovery followed a double exponential, with a τfast of 2.7 ± 0.1 ms, accounting for 89 ± 2% recovery, and a τslow of 149 ± 21 ms (Table 3). These measurements are consistent with previous data demonstrating that most NaV1.4 channels undergo fast inactivation on this time frame, with a small additional component of slow inactivation (Featherstone et al., 1996; Webb et al., 2009).


Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide.

Lewis AH, Raman IM - J. Gen. Physiol. (2013)

Time course of recovery from inactivation and from block by the β4 peptide in WT channels. (A) Voltage protocol (top) and representative cells showing recovery from inactivation and block after conditioning for 25 ms at 0 mV in the absence (middle) and presence (bottom) of the β4 peptide. Black, control; green, with 500 nM ATX. (B) Fractional recovery at 0 mV as a function of time at −110 mV. Currents were normalized to the response to the conditioning pulse for each trace, and fractional recovery was calculated as described in Materials and methods. Black, WT; green, WT plus ATX; solid lines, double-exponential fits to the mean data. (C) As in B, with β4 peptide in the intracellular solution. (D) Comparison of recovery in solutions ±β4 peptide. (E) Comparison of recovery in ATX ±β4 peptide. Within-cell comparisons ±ATX; WT, n = 8; WT plus β4, n = 7. (F and G) As in D and E, but with conditioning for 10 ms at +60 mV; without peptide, n = 4; with peptide, n = 5.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig7: Time course of recovery from inactivation and from block by the β4 peptide in WT channels. (A) Voltage protocol (top) and representative cells showing recovery from inactivation and block after conditioning for 25 ms at 0 mV in the absence (middle) and presence (bottom) of the β4 peptide. Black, control; green, with 500 nM ATX. (B) Fractional recovery at 0 mV as a function of time at −110 mV. Currents were normalized to the response to the conditioning pulse for each trace, and fractional recovery was calculated as described in Materials and methods. Black, WT; green, WT plus ATX; solid lines, double-exponential fits to the mean data. (C) As in B, with β4 peptide in the intracellular solution. (D) Comparison of recovery in solutions ±β4 peptide. (E) Comparison of recovery in ATX ±β4 peptide. Within-cell comparisons ±ATX; WT, n = 8; WT plus β4, n = 7. (F and G) As in D and E, but with conditioning for 10 ms at +60 mV; without peptide, n = 4; with peptide, n = 5.
Mentions: For peptide-free WT cells, recovery was rapid: after a 10-ms recovery interval, 86.1 ± 1.8% of the inactivated current was available (n = 8; Fig. 7, A–E). Recovery followed a double exponential, with a τfast of 2.7 ± 0.1 ms, accounting for 89 ± 2% recovery, and a τslow of 149 ± 21 ms (Table 3). These measurements are consistent with previous data demonstrating that most NaV1.4 channels undergo fast inactivation on this time frame, with a small additional component of slow inactivation (Featherstone et al., 1996; Webb et al., 2009).

Bottom Line: Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; "CW" channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement.The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor.ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdepartmental Biological Sciences Program and 2 Department of Neurobiology, Northwestern University, Evanston, IL 60208, USA.

ABSTRACT
Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the "β4 peptide"). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; "CW" channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

Show MeSH
Related in: MedlinePlus