Xiong W et al. (2003), Molecular motions of the outer ring of charge o...

XB-ART-4867

J Gen Physiol 2003 Sep 01;1223:323-32. doi: 10.1085/jgp.200308881.

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Molecular motions of the outer ring of charge of the sodium channel: do they couple to slow inactivation?

Xiong W , Li RA , Tian Y , Tomaselli GF .

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In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation is poorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactivation of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ring of charge in inactivation gating by paired cysteine mutagenesis in the rat skeletal muscle Na channel (rNav1.4). The outer charged ring residues were substituted with cysteine, paired with cysteine mutants at other positions in the external pore, and coexpressed with rat brain beta1 in Xenopus oocytes. Dithiolthreitol (DTT) markedly increased the current in E403C+E758C double mutant, indicating the spontaneous formation of a disulfide bond and proximity of the alpha carbons of these residues of no more than 7 A. The redox catalyst Cu(II) (1,10-phenanthroline)3 (Cu(phe)3) reduced the peak current of double mutants (E403C+E758C, E403C+D1241C, E403C+D1532C, and D1241C+D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancy of slow inactivation states completely prevented Cu(phe)3 modification of outer charged ring paired mutants E403C+E758C, E403C+D1241C, and E403C+D1532C. In contrast, voltage protocols that favored slow inactivation did not prevent Cu(phe)3 modification of other double mutants such as E403C+W756C, E403C+W1239C, and E403C+W1531C. Our data suggest that slow inactivation of the Na channel is associated with a structural rearrangement of the outer ring of charge.

???displayArticle.pubmedLink??? 12913092
???displayArticle.pmcLink??? PMC2234489
???displayArticle.link??? J Gen Physiol
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Species referenced: Xenopus laevis
Genes referenced: scn4a

???displayArticle.disOnts??? epilepsy
???displayArticle.omims??? HYPERKALEMIC PERIODIC PARALYSIS; HYPP [+]

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	Figure 1. . Schematic depiction of the Na channel α subunit. It consists of four domains, each of which has six transmembrane segments. The S4 segments represent at least part of the voltage sensor. The segments between S5 and S6 (P-segments) line the outer pore. The outer ring of negative charge (E403, E758, D1241, and D1532) is illustrated in filled circles. The open circles represent the putative selectivity filter residues (D400, E755, K1237, and A1529). The domain III-IV linker underlies fast inactivation. The 13 double cysteine mutants studied are shown below the schematic. E403C, E758C, and D1241C (enclosed in the boxes) were paired with other cysteine mutants in different domains of the outer pore.
	Figure 2. . Evidence for disulfide bonding of residues in the outer ring of charge. (A) Representative traces show that current through E403C+E758C mutant Na channels increased more than twofold in magnitude in the presence of 1mM DTT. (B) The current-voltage relationship shows a similar increase in peak current amplitude of the double mutant by exposure to DTT over the entire voltage range (P < 0.01, n = 8). Peak currents measured at each voltage step were normalized to the maximum current observed in each cell, averaged and plotted as a function of voltage. (C) A plot of the time course of the decrease in peak current amplitude upon exposure to 100 μM Cu(phe)3. The effects of Cu(phe)3 were not reversed during washout. The inset shows currents recorded at time (a) and (b). (D) A plot of the peak Na current amplitude during application of Cu(phe)3, washout, and subsequent treatment with 1 mM DTT.
	Figure 3. . Rate-dependent modification by Cu(phe)3 in paired mutant channels. (A) Rate-dependent modification by Cu(phe)3 of E403C+E758C mutant channels. Increasing stimulation frequency (from 0.033 Hz to 1 Hz) enhanced the rate of current reduction of E403C+E758C, suggesting rate-dependent modification of Cu(phe)3 in E403C+E758C mutant channels. The inset shows representative currents at 2 min (a) and 12.5 min at stimulation frequencies of 1 Hz (b) and 0.033 Hz (c) after the application of Cu(phe)3. (B) In contrast, current reduction was rapid and not dependent on stimulation frequency in the E403C+W1239C mutant channels. (C) Plots of the rates of reduction in peak current amplitude at different stimulation frequencies in the presence of Cu(phe)3 (n = 2–7). Single exponential fits of the form y = Ae(−t/τ) to plots like those shown in A and B were used to determine the time constants, τ. A is the amplitude. The top panel shows the time constants of current decay of mutant channels that were significantly increased at slower stimulation frequencies. The bottom panel is a plot of the time constants of double mutant channels that did not exhibit stimulation frequency dependent rates of modification. (P < 0.05, *P < 0.01).
	Figure 4. . Slow inactivation prevented the modification of E403C+ E758C, but not E403C+W1531C mutant channels by Cu(phe)3. (A). Slow inactivation was induced by holding the cells at −20 mV for 15 min with brief repolarizations to −100 mV for 10 ms to elicit currents after recovery from fast inactivation. The mean data shows that channels entered slow-inactivated states at approximately the same rate regardless the presence (filled circles) or absence (open circles) of Cu(phe)3. (B) Upon washout, Na currents nearly completely recovered from slow inactivation induced by the voltage protocol used in A independent of prior exposure to Cu(phe)3 (P > 0.05, n = 6). The inset shows representative currents before (a) and 60 s after initiation of recovery (b) with (filled circle) or without (open circle) prior exposure to Cu(phe)3. (C) A complementary voltage protocol was employed to induce slow inactivation before application of Cu(phe)3. Channels were held at −20 mV and pulsed for 50 ms to 0 mV at 1 Hz for 30 min. After 15 min of pulsing, Cu(phe)3 (100 μM) was applied for 15 min before washout. The oocytes were voltage clamped at −20 mV during the 4 min washout, then they were stimulated at 1 Hz from a holding potential of −100 to −20 mV to elicit Na currents. The Na currents completely recovered from slow inactivation compared with control (P > 0.05) (B). (D) After washout of Cu(phe)3, Na currents in E403C+W1531C mutant channels did not significantly recover from slow inactivation induced by the voltage protocol used in A (P < 0.05, n = 3). E403C+W1239C mutant channels were also subjected to modification by Cu(phe)3 and recovered incompletely (P < 0.05, n = 3).
	Figure 5. . A bar plot of the percent recovery from slow inactivation elicited by the voltage protocol in Fig. 4 A. The percent recoveries of E403C+E758C, E403C+K1240C, E403C+D1241C, and E403C+D1532C channels were not significantly changed in the presence of Cu(phe)3 (P > 0.05, n = 3–6). Significant Cu(phe)3-induced modification of the other paired mutants occurred during induction of slow inactivation (P < 0.05, n = 3–4).
	Figure 6. . A schematic model showing how gating influences the rate and extent of disulfide bond formation of double mutants in the external pore of the Na channel. In the case of E403C+E758C, enhanced rates of current reduction with higher stimulation frequencies suggest that the activated or fast inactivated states are modified more readily than the rested state. In contrast, double mutant channels such as E403C+W1531C (Fig. 3 C) are rapidly modified at both slow and fast stimulation rates, suggesting that the rested state may be accessible to modification by Cu(phe)3. Slow inactivation protects E403C+E758C channels (and E403C+D1241C and E403C+D1532C in the outer ring of charge) but not E403C+W1531C from Cu(phe)3-induced modification. The results are consistent with a change in the conformation of the outer ring of charge during slow inactivation of Na channels.

References [+] :

Alekov, Enhanced inactivation and acceleration of activation of the sodium channel associated with epilepsy in man. 2001, Pubmed