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Slow inactivation does not affect movement of the fast inactivation gate in voltage-gated Na+ channels.

Vedantham V, Cannon SC - J. Gen. Physiol. (1998)

Bottom Line: In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle.We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations.These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Division of Medical Sciences, Harvard Medical School, Cambridge, Massachusetts 02138, USA.

ABSTRACT
Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (microl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (hinfinity) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

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Mutation F1304C destabilizes fast inactivation. (A)  Na+ currents were elicited from excised inside-out macropatches  by depolarization to −20 mV from a holding potential of −120  mV. Averages of two current traces each for WT and mutant  F1304C are shown normalized to peak amplitude and superimposed. Mutant channels display a slowed current decay and a  greater fraction of persistent current. (B) Voltage dependence of  steady state fast inactivation, h∞•(V), in response to a 200-ms  prepulse, is right shifted for F1304C (V1/2 = −81.6 ± 1.3 mV,  slope = 5.5 ± 0.5, n = 9), compared with WT (V1/2 = −98.3 ± 1.9,  slope = 5.7 ± 0.3, n = 12). G(V), computed as peak INa/(V − Erev),  is similar for WT (n = 12) and F1304C (n = 9). These data are  consistent with a destabilization of fast inactivation caused by substitution of cysteine for phenylalanine at site 1304.
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Figure 1: Mutation F1304C destabilizes fast inactivation. (A) Na+ currents were elicited from excised inside-out macropatches by depolarization to −20 mV from a holding potential of −120 mV. Averages of two current traces each for WT and mutant F1304C are shown normalized to peak amplitude and superimposed. Mutant channels display a slowed current decay and a greater fraction of persistent current. (B) Voltage dependence of steady state fast inactivation, h∞•(V), in response to a 200-ms prepulse, is right shifted for F1304C (V1/2 = −81.6 ± 1.3 mV, slope = 5.5 ± 0.5, n = 9), compared with WT (V1/2 = −98.3 ± 1.9, slope = 5.7 ± 0.3, n = 12). G(V), computed as peak INa/(V − Erev), is similar for WT (n = 12) and F1304C (n = 9). These data are consistent with a destabilization of fast inactivation caused by substitution of cysteine for phenylalanine at site 1304.

Mentions: Fig. 1 A shows macroscopic current traces in response to a step depolarization from −120 to −20 mV taken from patches expressing wild-type or F1304C channels. The mutant displays a slowed rate of macroscopic current decay (τh) and a greater level of persistent current (measured as the average current between 40 and 42 ms after depolarization divided by the peak current): 9.1 ± 1.2% (n = 10) for F1304C, compared with 0.3 ± 0.07% (n = 6) for WT. This persistent current eventually decays over hundreds of milliseconds, probably because of slow inactivation.


Slow inactivation does not affect movement of the fast inactivation gate in voltage-gated Na+ channels.

Vedantham V, Cannon SC - J. Gen. Physiol. (1998)

Mutation F1304C destabilizes fast inactivation. (A)  Na+ currents were elicited from excised inside-out macropatches  by depolarization to −20 mV from a holding potential of −120  mV. Averages of two current traces each for WT and mutant  F1304C are shown normalized to peak amplitude and superimposed. Mutant channels display a slowed current decay and a  greater fraction of persistent current. (B) Voltage dependence of  steady state fast inactivation, h∞•(V), in response to a 200-ms  prepulse, is right shifted for F1304C (V1/2 = −81.6 ± 1.3 mV,  slope = 5.5 ± 0.5, n = 9), compared with WT (V1/2 = −98.3 ± 1.9,  slope = 5.7 ± 0.3, n = 12). G(V), computed as peak INa/(V − Erev),  is similar for WT (n = 12) and F1304C (n = 9). These data are  consistent with a destabilization of fast inactivation caused by substitution of cysteine for phenylalanine at site 1304.
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Related In: Results  -  Collection

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Figure 1: Mutation F1304C destabilizes fast inactivation. (A) Na+ currents were elicited from excised inside-out macropatches by depolarization to −20 mV from a holding potential of −120 mV. Averages of two current traces each for WT and mutant F1304C are shown normalized to peak amplitude and superimposed. Mutant channels display a slowed current decay and a greater fraction of persistent current. (B) Voltage dependence of steady state fast inactivation, h∞•(V), in response to a 200-ms prepulse, is right shifted for F1304C (V1/2 = −81.6 ± 1.3 mV, slope = 5.5 ± 0.5, n = 9), compared with WT (V1/2 = −98.3 ± 1.9, slope = 5.7 ± 0.3, n = 12). G(V), computed as peak INa/(V − Erev), is similar for WT (n = 12) and F1304C (n = 9). These data are consistent with a destabilization of fast inactivation caused by substitution of cysteine for phenylalanine at site 1304.
Mentions: Fig. 1 A shows macroscopic current traces in response to a step depolarization from −120 to −20 mV taken from patches expressing wild-type or F1304C channels. The mutant displays a slowed rate of macroscopic current decay (τh) and a greater level of persistent current (measured as the average current between 40 and 42 ms after depolarization divided by the peak current): 9.1 ± 1.2% (n = 10) for F1304C, compared with 0.3 ± 0.07% (n = 6) for WT. This persistent current eventually decays over hundreds of milliseconds, probably because of slow inactivation.

Bottom Line: In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle.We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations.These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

View Article: PubMed Central - PubMed

Affiliation: Program in Neuroscience, Division of Medical Sciences, Harvard Medical School, Cambridge, Massachusetts 02138, USA.

ABSTRACT
Voltage-gated Na+ channels exhibit two forms of inactivation, one form (fast inactivation) takes effect on the order of milliseconds and the other (slow inactivation) on the order of seconds to minutes. While previous studies have suggested that fast and slow inactivation are structurally independent gating processes, little is known about the relationship between the two. In this study, we probed this relationship by examining the effects of slow inactivation on a conformational marker for fast inactivation, the accessibility of a site on the Na+ channel III-IV linker that is believed to form a part of the fast inactivation particle. When cysteine was substituted for phenylalanine at position 1304 in the rat skeletal muscle sodium channel (microl), application of [2-(trimethylammonium)ethyl]methanethiosulfonate (MTS-ET) to the cytoplasmic face of inside-out patches from Xenopus oocytes injected with F1304C RNA dramatically disrupted fast inactivation and displayed voltage-dependent reaction kinetics that closely paralleled the steady state availability (hinfinity) curve. Based on this observation, the accessibility of cys1304 was used as a conformational marker to probe the position of the fast inactivation gate during the development of and the recovery from slow inactivation. We found that burial of cys1304 is not altered by the onset of slow inactivation, and that recovery of accessibility of cys1304 is not slowed after long (2-10 s) depolarizations. These results suggest that (a) fast and slow inactivation are structurally distinct processes that are not tightly coupled, (b) fast and slow inactivation are not mutually exclusive processes (i.e., sodium channels may be fast- and slow-inactivated simultaneously), and (c) after long depolarizations, recovery from fast inactivation precedes recovery from slow inactivation.

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Related in: MedlinePlus