Struyk AF and Cannon SC (2002), Slow inactivation does not block the aqueous ac...

XB-ART-6460

J Gen Physiol 2002 Oct 01;1204:509-16. doi: 10.1085/jgp.20028672.

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Slow inactivation does not block the aqueous accessibility to the outer pore of voltage-gated Na channels.

Struyk AF , Cannon SC .

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Slow inactivation of voltage-gated Na channels is kinetically and structurally distinct from fast inactivation. Whereas structures that participate in fast inactivation are well described and include the cytoplasmic III-IV linker, the nature and location of the slow inactivation gating mechanism remains poorly understood. Several lines of evidence suggest that the pore regions (P-regions) are important contributors to slow inactivation gating. This has led to the proposal that a collapse of the pore impedes Na current during slow inactivation. We sought to determine whether such a slow inactivation-coupled conformational change could be detected in the outer pore. To accomplish this, we used a rapid perfusion technique to measure reaction rates between cysteine-substituted side chains lining the aqueous pore and the charged sulfhydryl-modifying reagent MTS-ET. A pattern of incrementally slower reaction rates was observed at substituted sites at increasing depth in the pore. We found no state-dependent change in modification rates of P-region residues located in all four domains, and thus no change in aqueous accessibility, between slow- and nonslow-inactivated states. In domains I and IV, it was possible to measure modification rates at residues adjacent to the narrow DEKA selectivity filter (Y401C and G1530C), and yet no change was observed in accessibility in either slow- or nonslow-inactivated states. We interpret these results as evidence that the outer mouth of the Na pore remains open while the channel is slow inactivated.

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Species referenced: Xenopus
Genes referenced: btnl2 tbx2

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	Figure 1. . Sites of informative cysteine substitution mutations in the outer pore. The SS2 segments are shown schematically in order of relative position from the DEKA Na ion selectivity region. Residues lettered in black represent sites at which cysteine substitution yielded informative data in state-dependent accessibility assays described below. Sites lettered in black outline denote locations at which we were not able to measure a modification rate of substituted cysteine residues. Residues in light gray were not tested, either because they were uninformative since they had no measurable effect when exposed to MTS-ET (D400, W756, K1237, and A1529), they did not produce a detectable INa (G1238), or the predicted modification rate was too slow for our assay (W1239).
	Figure 2. . Introduction of cysteine missense mutations into informative P-region sites has little or no effect on the recovery kinetics of fast- or slow-inactivation. All measurements were made in excised outside-out macropatches held under perfusion with control bath solution. Conditioning pulses to −10 mV of 40 ms or 3 s duration were used to induce fast and slow inactivation, respectively, (A, inset). (A) Recovery from fast inactivation at −120 mV was unchanged between cysteine substitution mutants and wild-type. (B) Recovery from slow inactivation at −120 mV was not affected by introduction of cysteine residues at most informative sites. For E758C mutant channels, a modest acceleration in the time course of recovery was observed.
	Figure 3. . The kinetics of entry to slow inactivation at −10 mV. The paired pulse protocol used to measure entry to slow inactivation is shown schematically at the top of the figure. The symbols used for each mutant are identical to those listed in the inset of Fig. 2 A. The majority of the mutations had no significant effect on the time course of entry to slow inactivation. E758C showed a modest 2.5-fold slowing in the time course of entry, and a modest decrease in the maximal extent of slow inactivation (S∞). The shaded areas represent the timing and duration of the MTS-ET exposures designed to assay cysteine availability for predominantly fast- and slow-inactivated conditions.
	Figure 4. . Measurement of state-dependent modification rate. (A) Na currents elicited by the assay pulses during serial exposures to MTS-ET are shown on a condensed time scale. The top number of the time scale represents the interval elapsed between each assay pulse, and the bottom number represents the time scale relative to individual assay pulses. In this example, accessibility of the E758C residue is being tested by serial 200 ms exposures to 2 mM MTS-ET early during the conditioning pulse (minimally slow inactivated). (B) Peak INa values from A are plotted against the cumulative time of exposure to MTS-ET, and fit with a single exponential decay. The apparent modification rate (RApparent) is then computed from the time constant.
	Figure 5. . No state-dependent change of cysteine modification rate was seen in the six mutants studied. (A) Schematic representation of the pulsed MTS-ET application protocols. Modification rates for fast-inactivated channels were assayed by exposing outside-out patches to MTS-ET for 200 ms, beginning 20 ms into the 300 ms conditioning pulse (top protocol, open circles). To measure the modification rate of slow-inactivated channels, patches were exposed to MTS-ET beginning 10.5 s after the onset of a 12 s conditioning pulse (bottom protocol, filled circles). All conditioning pulses were to −10 mV, and a gap of 10 s at −120 mV to allow for full recovery from inactivation was inserted before a test pulse to −20 mV to measure remaining peak INa. This cycle was repeated until maximal modification in each patch had taken place. (B) Comparison of modification rates of cysteines in each of the four domains measured under conditions in which channels were predominantly fast- or slow-inactivated. The mean and SEM of these rates are plotted by domain location for each mutant, with rates for the two states of the channel connected by a line. The numerical values are listed in Table II.
	Figure 6. . Absence of detectable modification of I757C by MTS-ET. Na currents elicited after successive exposures to 4 mM MTS-ET are shown on a compressed time scale. MTS-ET was applied for 200 ms, beginning 20 ms after the onset of a conditioning pulse to −10 mV (fast-inactivation protocol). No reduction in peak INa was detectable, even after a cumulative exposure time of 8 s. Onset of each trace is offset by the time interval between each trial (scale bar, top). Time scale during a trace is indicated by the number below the scale bar.

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