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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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Tail currents and β4 peptide–induced resurgent Na currents in WT channels. (A) Voltage protocol and representative transient and tail and/or resurgent currents in WT channels without and with β4 peptide. Black, control; red, ATX. (B) Tail and resurgent currents in control solutions, as in A, expanded. (C) Tail and resurgent currents in ATX, as in A, expanded. (D) Mean peak resurgent current amplitude versus voltage in WT (black) and ATX-modified WT (green) channels. Currents normalized to peak transient current at 0 mV in each cell. Dashed lines, corrected for incomplete activation of transient current at 0 mV. (E) Mean time to peak resurgent current versus voltage. (F) Mean decay time constant (τ) of resurgent current, fit with a single exponential. Within-cell comparisons ±ATX; −β4, n = 6; +β4, n = 5.
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fig4: Tail currents and β4 peptide–induced resurgent Na currents in WT channels. (A) Voltage protocol and representative transient and tail and/or resurgent currents in WT channels without and with β4 peptide. Black, control; red, ATX. (B) Tail and resurgent currents in control solutions, as in A, expanded. (C) Tail and resurgent currents in ATX, as in A, expanded. (D) Mean peak resurgent current amplitude versus voltage in WT (black) and ATX-modified WT (green) channels. Currents normalized to peak transient current at 0 mV in each cell. Dashed lines, corrected for incomplete activation of transient current at 0 mV. (E) Mean time to peak resurgent current versus voltage. (F) Mean decay time constant (τ) of resurgent current, fit with a single exponential. Within-cell comparisons ±ATX; −β4, n = 6; +β4, n = 5.

Mentions: To further assess both accessibility and stability of block, we measured the effects of ATX exposure and the CW mutation on β4 peptide–induced resurgent currents. The potential was first stepped to 0 mV to provide a reference measurement of the transient current in each cell (Fig. 4 A, left). After recovery, the voltage was stepped beyond the Na+ reversal potential to +60 mV for 10 ms to maximize open-channel block if the β4 peptide was present (Aman and Raman, 2010). The membrane was then repolarized to a series of negative potentials (Fig. 4, A, right, and B) either to evoke tail currents (without β4 peptide) or to displace the blocker and produce resurgent current (with β4 peptide). Without peptide, WT NaV1.4 inactivated completely during the 10-ms conditioning pulse. Because no tail current could be clearly resolved, we quantified the data by averaging the current over the first 100 µs of repolarization. This current at −30 mV was 0.91 ± 0.40% of the transient current at 0 mV (Fig. 4, A, black, and B, top traces). With the β4 peptide, only tiny resurgent currents were elicited upon repolarization (Fig. 4, A, black, and B, bottom traces); the resurgent current at −30 mV was 5.2 ± 0.8% of the transient current measured at 0 mV, further demonstrating that the β4 peptide competes poorly with fast inactivation in NaV1.4. In addition, the resurgent current rose in 1.16 ± 0.15 ms and had a decay τ of 4.55 ± 0.16 ms (Fig. 4, D–F; n = 5). These time courses are two- to threefold faster than β4 peptide unblock in hippocampal neurons (Lewis and Raman, 2011). The rapid rise may result from a lower affinity blockade of expressed NaV1.4 than of native neuronal channels, and the brief decay is likely to reflect the onset of inactivation after unbinding of the blocker (Raman and Bean, 2001; Lewis and Raman, 2011), which is particularly rapid in expressed NaV1.4 channels.


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)

Tail currents and β4 peptide–induced resurgent Na currents in WT channels. (A) Voltage protocol and representative transient and tail and/or resurgent currents in WT channels without and with β4 peptide. Black, control; red, ATX. (B) Tail and resurgent currents in control solutions, as in A, expanded. (C) Tail and resurgent currents in ATX, as in A, expanded. (D) Mean peak resurgent current amplitude versus voltage in WT (black) and ATX-modified WT (green) channels. Currents normalized to peak transient current at 0 mV in each cell. Dashed lines, corrected for incomplete activation of transient current at 0 mV. (E) Mean time to peak resurgent current versus voltage. (F) Mean decay time constant (τ) of resurgent current, fit with a single exponential. Within-cell comparisons ±ATX; −β4, n = 6; +β4, n = 5.
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Related In: Results  -  Collection

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fig4: Tail currents and β4 peptide–induced resurgent Na currents in WT channels. (A) Voltage protocol and representative transient and tail and/or resurgent currents in WT channels without and with β4 peptide. Black, control; red, ATX. (B) Tail and resurgent currents in control solutions, as in A, expanded. (C) Tail and resurgent currents in ATX, as in A, expanded. (D) Mean peak resurgent current amplitude versus voltage in WT (black) and ATX-modified WT (green) channels. Currents normalized to peak transient current at 0 mV in each cell. Dashed lines, corrected for incomplete activation of transient current at 0 mV. (E) Mean time to peak resurgent current versus voltage. (F) Mean decay time constant (τ) of resurgent current, fit with a single exponential. Within-cell comparisons ±ATX; −β4, n = 6; +β4, n = 5.
Mentions: To further assess both accessibility and stability of block, we measured the effects of ATX exposure and the CW mutation on β4 peptide–induced resurgent currents. The potential was first stepped to 0 mV to provide a reference measurement of the transient current in each cell (Fig. 4 A, left). After recovery, the voltage was stepped beyond the Na+ reversal potential to +60 mV for 10 ms to maximize open-channel block if the β4 peptide was present (Aman and Raman, 2010). The membrane was then repolarized to a series of negative potentials (Fig. 4, A, right, and B) either to evoke tail currents (without β4 peptide) or to displace the blocker and produce resurgent current (with β4 peptide). Without peptide, WT NaV1.4 inactivated completely during the 10-ms conditioning pulse. Because no tail current could be clearly resolved, we quantified the data by averaging the current over the first 100 µs of repolarization. This current at −30 mV was 0.91 ± 0.40% of the transient current at 0 mV (Fig. 4, A, black, and B, top traces). With the β4 peptide, only tiny resurgent currents were elicited upon repolarization (Fig. 4, A, black, and B, bottom traces); the resurgent current at −30 mV was 5.2 ± 0.8% of the transient current measured at 0 mV, further demonstrating that the β4 peptide competes poorly with fast inactivation in NaV1.4. In addition, the resurgent current rose in 1.16 ± 0.15 ms and had a decay τ of 4.55 ± 0.16 ms (Fig. 4, D–F; n = 5). These time courses are two- to threefold faster than β4 peptide unblock in hippocampal neurons (Lewis and Raman, 2011). The rapid rise may result from a lower affinity blockade of expressed NaV1.4 than of native neuronal channels, and the brief decay is likely to reflect the onset of inactivation after unbinding of the blocker (Raman and Bean, 2001; Lewis and Raman, 2011), which is particularly rapid in expressed NaV1.4 channels.

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