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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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Accessibility of site 1304 recovers rapidly for fast- and  slow-inactivated channels. (A) Two protocols were used to measure accessibility of site 1304 in response to variable length conditioning pulses to 0 mV from a holding potential of −120 mV. In  the experiment shown on top, the MTS-ET exposure was placed  near the end of the conditioning pulse to verify that site 1304 remains buried during long depolarizations and to determine how  quickly it becomes buried for short depolarizations. Depolarization was maintained throughout the exposure and shortly after.  (Tpre = 0.007, 0.2, 2.0, or 10.0 s; MTS-ET exposure was 20 or 50  ms; [MTS-ET] = 4 or 8 μM; n = 3 for all). In the bottom protocol, the exposure was placed 5 ms after repolarization: 4 μM, 50  ms (average n = 5 per Tcond, except Tcond = 10 s) or 8 μM, 20  ms (average n = 8 per Tcond, all points at least n = 4). The total  time between the end of the conditioning pulse and the beginning  of the exposure includes a short delay due to lag of the switching  apparatus, which we measured as described above (Fig. 2) to be 7.1  ± 0.1 ms, giving a preexposure recovery time of 12.1 ± 0.1 ms.  Therefore, for the 20-ms case, exposure occurred from 12.1 to 32.1  ms after repolarization (depicted as the shaded area in the graph  in Fig. 5). For both protocols, enough time after the conditioning  pulse was allowed for complete recovery (from 5 s for Tcond =  0.02 s to 45 s for Tcond = 10 s) before the 45-ms test pulse was administered. (B) Reaction rates were determined as described  above (Fig. 2) for the two protocols described in A. Dotted lines indicate Rmax (0.932 μmol−1 s−1) and Rmin (0.208 μmol−1 s−1). The  reaction rates for the first protocol, in which the exposures occurred during the conditioning pulse (○) were slow, consistent  with site 1304 burial. For the experiments in which the exposure  occurred shortly after the end of the conditioning pulse (•), the  measured rate was consistent with nearly complete accessibility  (experiments without any conditioning pulse are shown for comparison as Tcond = 0). This result suggests that the fast inactivation gate recovers rapidly despite the fact that >90% of the channels remain slow inactivated.
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Figure 6: Accessibility of site 1304 recovers rapidly for fast- and slow-inactivated channels. (A) Two protocols were used to measure accessibility of site 1304 in response to variable length conditioning pulses to 0 mV from a holding potential of −120 mV. In the experiment shown on top, the MTS-ET exposure was placed near the end of the conditioning pulse to verify that site 1304 remains buried during long depolarizations and to determine how quickly it becomes buried for short depolarizations. Depolarization was maintained throughout the exposure and shortly after. (Tpre = 0.007, 0.2, 2.0, or 10.0 s; MTS-ET exposure was 20 or 50 ms; [MTS-ET] = 4 or 8 μM; n = 3 for all). In the bottom protocol, the exposure was placed 5 ms after repolarization: 4 μM, 50 ms (average n = 5 per Tcond, except Tcond = 10 s) or 8 μM, 20 ms (average n = 8 per Tcond, all points at least n = 4). The total time between the end of the conditioning pulse and the beginning of the exposure includes a short delay due to lag of the switching apparatus, which we measured as described above (Fig. 2) to be 7.1 ± 0.1 ms, giving a preexposure recovery time of 12.1 ± 0.1 ms. Therefore, for the 20-ms case, exposure occurred from 12.1 to 32.1 ms after repolarization (depicted as the shaded area in the graph in Fig. 5). For both protocols, enough time after the conditioning pulse was allowed for complete recovery (from 5 s for Tcond = 0.02 s to 45 s for Tcond = 10 s) before the 45-ms test pulse was administered. (B) Reaction rates were determined as described above (Fig. 2) for the two protocols described in A. Dotted lines indicate Rmax (0.932 μmol−1 s−1) and Rmin (0.208 μmol−1 s−1). The reaction rates for the first protocol, in which the exposures occurred during the conditioning pulse (○) were slow, consistent with site 1304 burial. For the experiments in which the exposure occurred shortly after the end of the conditioning pulse (•), the measured rate was consistent with nearly complete accessibility (experiments without any conditioning pulse are shown for comparison as Tcond = 0). This result suggests that the fast inactivation gate recovers rapidly despite the fact that >90% of the channels remain slow inactivated.

Mentions: The rationale for our next experiment was to determine the conformational state of the fast inactivation gate at the tail end of varying length conditioning pulses to 0 mV. The protocol used is shown in Fig. 6 A (top). As in previous experiments, 4 μM MTS-ET was applied for a fixed duration (either 50 or 150 ms) with test pulses between each exposure. Before each exposure, there was a prepulse to 0 mV of either 0.007, 0.20, 3.0, or 10.0 s, with depolarization maintained during the exposure and shortly after. Reaction rates were determined as described above and are shown in Fig. 6 B (○). The reaction rates for each prepulse used were similar to each other and close to Rmin, demonstrating that cys1304 is buried within 7 ms of depolarization and remains buried despite the onset 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)

Accessibility of site 1304 recovers rapidly for fast- and  slow-inactivated channels. (A) Two protocols were used to measure accessibility of site 1304 in response to variable length conditioning pulses to 0 mV from a holding potential of −120 mV. In  the experiment shown on top, the MTS-ET exposure was placed  near the end of the conditioning pulse to verify that site 1304 remains buried during long depolarizations and to determine how  quickly it becomes buried for short depolarizations. Depolarization was maintained throughout the exposure and shortly after.  (Tpre = 0.007, 0.2, 2.0, or 10.0 s; MTS-ET exposure was 20 or 50  ms; [MTS-ET] = 4 or 8 μM; n = 3 for all). In the bottom protocol, the exposure was placed 5 ms after repolarization: 4 μM, 50  ms (average n = 5 per Tcond, except Tcond = 10 s) or 8 μM, 20  ms (average n = 8 per Tcond, all points at least n = 4). The total  time between the end of the conditioning pulse and the beginning  of the exposure includes a short delay due to lag of the switching  apparatus, which we measured as described above (Fig. 2) to be 7.1  ± 0.1 ms, giving a preexposure recovery time of 12.1 ± 0.1 ms.  Therefore, for the 20-ms case, exposure occurred from 12.1 to 32.1  ms after repolarization (depicted as the shaded area in the graph  in Fig. 5). For both protocols, enough time after the conditioning  pulse was allowed for complete recovery (from 5 s for Tcond =  0.02 s to 45 s for Tcond = 10 s) before the 45-ms test pulse was administered. (B) Reaction rates were determined as described  above (Fig. 2) for the two protocols described in A. Dotted lines indicate Rmax (0.932 μmol−1 s−1) and Rmin (0.208 μmol−1 s−1). The  reaction rates for the first protocol, in which the exposures occurred during the conditioning pulse (○) were slow, consistent  with site 1304 burial. For the experiments in which the exposure  occurred shortly after the end of the conditioning pulse (•), the  measured rate was consistent with nearly complete accessibility  (experiments without any conditioning pulse are shown for comparison as Tcond = 0). This result suggests that the fast inactivation gate recovers rapidly despite the fact that >90% of the channels remain slow inactivated.
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Figure 6: Accessibility of site 1304 recovers rapidly for fast- and slow-inactivated channels. (A) Two protocols were used to measure accessibility of site 1304 in response to variable length conditioning pulses to 0 mV from a holding potential of −120 mV. In the experiment shown on top, the MTS-ET exposure was placed near the end of the conditioning pulse to verify that site 1304 remains buried during long depolarizations and to determine how quickly it becomes buried for short depolarizations. Depolarization was maintained throughout the exposure and shortly after. (Tpre = 0.007, 0.2, 2.0, or 10.0 s; MTS-ET exposure was 20 or 50 ms; [MTS-ET] = 4 or 8 μM; n = 3 for all). In the bottom protocol, the exposure was placed 5 ms after repolarization: 4 μM, 50 ms (average n = 5 per Tcond, except Tcond = 10 s) or 8 μM, 20 ms (average n = 8 per Tcond, all points at least n = 4). The total time between the end of the conditioning pulse and the beginning of the exposure includes a short delay due to lag of the switching apparatus, which we measured as described above (Fig. 2) to be 7.1 ± 0.1 ms, giving a preexposure recovery time of 12.1 ± 0.1 ms. Therefore, for the 20-ms case, exposure occurred from 12.1 to 32.1 ms after repolarization (depicted as the shaded area in the graph in Fig. 5). For both protocols, enough time after the conditioning pulse was allowed for complete recovery (from 5 s for Tcond = 0.02 s to 45 s for Tcond = 10 s) before the 45-ms test pulse was administered. (B) Reaction rates were determined as described above (Fig. 2) for the two protocols described in A. Dotted lines indicate Rmax (0.932 μmol−1 s−1) and Rmin (0.208 μmol−1 s−1). The reaction rates for the first protocol, in which the exposures occurred during the conditioning pulse (○) were slow, consistent with site 1304 burial. For the experiments in which the exposure occurred shortly after the end of the conditioning pulse (•), the measured rate was consistent with nearly complete accessibility (experiments without any conditioning pulse are shown for comparison as Tcond = 0). This result suggests that the fast inactivation gate recovers rapidly despite the fact that >90% of the channels remain slow inactivated.
Mentions: The rationale for our next experiment was to determine the conformational state of the fast inactivation gate at the tail end of varying length conditioning pulses to 0 mV. The protocol used is shown in Fig. 6 A (top). As in previous experiments, 4 μM MTS-ET was applied for a fixed duration (either 50 or 150 ms) with test pulses between each exposure. Before each exposure, there was a prepulse to 0 mV of either 0.007, 0.20, 3.0, or 10.0 s, with depolarization maintained during the exposure and shortly after. Reaction rates were determined as described above and are shown in Fig. 6 B (○). The reaction rates for each prepulse used were similar to each other and close to Rmin, demonstrating that cys1304 is buried within 7 ms of depolarization and remains buried despite the onset 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.

Show MeSH
Related in: MedlinePlus