Title Molecular motions of the outer ring of charge of the sodiumchannel: Do they couple to slow inactivation?

Author(s) Xiong, W; Li, RA; Tian, Y; Tomaselli, GF

Citation Journal Of General Physiology, 2003, v. 122 n. 3, p. 323-332

Issued Date 2003

URL http://hdl.handle.net/10722/91680

Rights

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J. Gen. Physiol.

© The Rockefeller University Press

•

0022-1295/2003/09/323/10 $8.00Volume 122 September 2003 323–332http://www.jgp.org/cgi/doi/10.1085/jgp.200308881

Molecular Motions of the Outer Ring of Charge of the Sodium Channel: Do They Couple to Slow Inactivation?

Wei Xiong, Ronald A. Li, Yanli Tian,

and

Gordon F. Tomaselli

Molecular and Cellular Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21205

abstract

In contrast to fast inactivation, the molecular basis of sodium (Na) channel slow inactivation ispoorly understood. It has been suggested that structural rearrangements in the outer pore mediate slow inactiva-tion of Na channels similar to C-type inactivation in potassium (K) channels. We probed the role of the outer ringof 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 inthe external pore, and coexpressed with rat brain

�

1

in

Xenopus

oocytes. Dithiolthreitol (DTT) markedly increasedthe current in E403C

�

E758C double mutant, indicating the spontaneous formation of a disulfide bond and prox-imity of the

�

carbons of these residues of no more than 7 Å. 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, andD1241C

�

D1532C) at a rate proportional to the stimulation frequency. Voltage protocols that favored occupancyof slow inactivation states completely prevented Cu(phe)

3

modification of outer charged ring paired mutantsE403C

�

E758C, E403C

�

D1241C, and E403C

�

D1532C. In contrast, voltage protocols that favored slow inactiva-tion 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 rear-rangement of the outer ring of charge.

key words:

rNav1.4 channel • cysteine mutagenesis • disulfide bond • electrophysiology

I N T R O D U C T I O N

The voltage-gated Na channel mediates the rapid up-stroke of action potential and is responsible for excita-tion and conduction in nerve and muscle. Upon depo-larization, the channel activates a process that involvesoutward movement of all four charged S4 segments(Stuhmer et al., 1989; Hirschberg et al., 1995; Yang andHorn, 1995; Yang et al., 1996; Kontis et al., 1997). Nachannel opening is transient, with inactivation rapidlyensuing. Na channels undergo several different kineti-cally distinct forms of inactivation. The time constantsof recovery from inactivated states range from millisec-onds (fast inactivation) to hundreds of milliseconds(intermediate inactivation) and seconds (slow inactiva-tion) (Simoncini and Stuhmer, 1987; Ruff et al., 1988;Nuss et al., 1996; Kambouris et al., 1998).

In

Shaker

K channels, N-type inactivation is mediatedby an intracellular region at the NH

2

terminus acting as“ball” occluding the pore (Hoshi et al., 1990). Theanalogous region in the Na channel is the cytoplasmicIII-IV linker, which serves as a tethered pore blockerbinding to an inactivation gate receptor in the intracel-lular mouth of the channel (Armstrong, 1981; Stuhmer

et al., 1989; Vassilev et al., 1989; Patton et al., 1992;West et al., 1992). Unlike fast inactivation, the molecu-lar basis of slow inactivation in Na channels is poorlyunderstood. However, slow inactivation plays a criticalrole in membrane excitability and firing properties bygoverning the availability of Na channels in neuronsand muscle (Ruff et al., 1988; Fleidervish et al., 1996).The physiological significance of slow inactivation inNa channels has been established by studies demon-strating that abnormalities of slow inactivation are asso-ciated with a variety of human diseases, including hy-perkalemic periodic paralysis (Cummins and Sigworth,1996; Hayward et al., 1999; Bendahhou et al., 2002),Brugada syndrome (Veldkamp et al., 2000), and epi-lepsy (Alekov et al., 2001; Spampanato et al., 2001).

In

Shaker

K channels, C-type inactivation, which canbe slow, is a mechanism of inactivation that involvesa conformational rearrangement of the outer pore.C-type inactivation persists when N-type inactivation is de-leted (Hoshi et al., 1991), is inhibited by extracellulartetraethylammonium (Choi et al., 1991) and is sensitiveto extracellular [K

�

] and mutations in the outer pore(Lopez-Barneo et al., 1993).

The outer ring of charge (E403, E758, D1241, andD1532 in domains I-IV, respectively) is located three

Address correspondence to Gordon F. Tomaselli, Department ofMedicine, Johns Hopkins University School of Medicine, 720 Rut-land Ave./Ross 844, Baltimore, MD 21205. Fax: (410) 955-7953;email: gtomasel@jhmi.edu

Abbreviations used in this paper:

DTT, dithiolthreitol; MTS, methane-thiosulfonate.

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Outer Ring of Charge and Na Channel Slow Inactivation

to four residues external to the putative selectivity fil-ter (D400, E755, K1237, and A1529) of the Na chan-nel (Heinemann et al., 1992). Whether the conservedouter ring of charge in the external pore of the Nachannel is involved in slow inactivation is not well de-fined, although charge neutralization of one residue(E403) in this ring markedly delays recovery fromslow forms of inactivation (Zhang et al., 2003). We hy-pothesized that slow inactivation in the Na channel isassociated with a structural rearrangement of thisouter ring of charge. This rearrangement changes therelationship of side chains of residues that comprisethe outer pore of Na channels, including those in theouter ring of charge. In the present study, we intro-duced pairs of cysteines into the outer ring of charge(EEDD) and other pore residues in each of the do-mains of rNav1.4 (Fig. 1). Paired-cysteine substitu-tions in the outer pore of Na channels can form disul-fide bonds both spontaneously and in the presence ofa redox catalyst (Benitah et al., 1996, 1997, 1999). Thecombination of paired-cysteine mutagenesis and disul-fide trapping was employed to explore the changes inspatial relationships of residues in the outer pore thatmay participate in slow inactivation. We determinedthe conformation-dependent rate of disulfide bondformation in the paired-cysteine mutants and investi-gated the effects of the induction of slow inactivationon the extent of disulfide bond formation. Our results

suggest that motion in the outer ring of charge isclosely associated with slow inactivation gating of theNa channel.

M A T E R I A L S A N D M E T H O D S

Mutagenesis and Channel Expression

A 1.9Kb BamHI-SphI or 2.5kb SphI-KpnI fragment of the

�

1

skeletal muscle Na channel (rNav1.4 channel) cDNA (Trimmeret al., 1989) was subcloned into pGEM-11Zf

�

and pGEM-7Zf

�

(Promega), respectively. The 1.9- and 2.5-kb cassettes were em-ployed for oligonucleotide-directed mutagenesis in domains Ior II–IV, respectively (Kunkel, 1985). Mutations were confirmedby sequencing mutagenic cassettes, which were cloned backedinto pSP64T (Krieg and Melton, 1984). Complementary RNAwas prepared via in vitro transcription using SP6 RNA polymerase.The

�

subunit was coexpressed with the rat brain

�

1

subunit (1:1weight ratio) (Isom et al., 1992) in

Xenopus

laevis

oocytes as de-scribed previously (Tomaselli et al., 1995; Perez-Garcia et al.,1996; Benitah et al., 1997). Oocytes were stored in the followingsolution (in mM): NaCl 96, KCl 2, MgCl

2

1, CaCl

2

1.8, HEPES 5,Na Pyruvate 5, theophylline 0.5, supplemented with penicillin100 U/ml and streptomycin 100

�

g/ml (pH 7.6 with NaOH).

Electrophysiology and Data Analysis

Macroscopic Na currents were recorded 24–48 h after injectionof cRNA using a two-microelectrode voltage-clamp (OC-725B;Warner Instrument Corp.) in frog Ringer’s solution containing(in mM): NaCl 96, KCl 2, MgCl

2

1, HEPES 5 (pH 7.6 withNaOH). All experiments were performed at room temperature.The stock solution of the redox catalyst Cu (II) (1,10-phenan-

Figure 1. Schematic depiction of theNa channel � subunit. It consists of fourdomains, each of which has six trans-membrane segments. The S4 segmentsrepresent at least part of the voltagesensor. The segments between S5 andS6 (P-segments) line the outer pore.The outer ring of negative charge(E403, E758, D1241, and D1532) is il-lustrated in filled circles. The open cir-cles represent the putative selectivity fil-ter residues (D400, E755, K1237, andA1529). The domain III-IV linker un-derlies fast inactivation. The 13 doublecysteine mutants studied are shown be-low the schematic. E403C, E758C, andD1241C (enclosed in the boxes) werepaired with other cysteine mutants indifferent domains of the outer pore.

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throline) (Cu(phe)

3

) was a mixture of 150 mM Cu(II)SO

4

and500 mM 1,10-phenanthroline in a 4:1 water/ethanol solution(Careaga and Falke, 1992) and was diluted to its final concentra-tion just before use. Dithiothreitol (DTT) was dissolved in extra-cellular solution just before use. Acquisition and analysis ofwhole-cell currents was performed with custom-written software.Current-voltage relationships were fitted to a transform of theBoltzmann distribution:

where

V

rev

is the reversal potential,

G

max

the maximum normal-ized conductance,

V

1/2

the potential of half activation, and

k

theslope factor. Average data are expressed as mean

�

SEM. Statisti-cal significance was evaluated by ANOVA or Student’s

t

test (Ori-gin; MicroCal) with P

�

0.05 representing significance.

R E S U L T S

Disulfide Bonding of Paired Cysteine Substitution Mutants

Fig. 1 shows a schematic depiction of rNav1.4 with theresidues of the selectivity filter and the outer ring ofcharge highlighted. Amino acids in the outer ring ofcharge (E403, E758, D1241, and D1532) in the ascend-ing limb of the P-segment (SS2 region) were substi-tuted singly with cysteine and paired with other cys-teine substitutions in the external pore. 13 pairedcysteine substitution mutants (Fig. 1, bottom) werestudied and exhibited distinct phenotypes. For exam-

I Imax⁄ V Vrev–( )Gmax 1 V1 2⁄ V–( )exp+ k⁄[ ],⁄=

ple, Fig. 2 A shows the Na currents elicited by a voltagestep from

�

100 to

�

20 mV through E403C

�

E758Cmutant channels. The magnitude of the Na current wasmarkedly increased after a 1–1.5 min exposure to thereducing agent DTT (1 mM) (Fig. 2 A). The mean datashows that the peak Na current at

�

20 mV more thandoubled in the presence of DTT and the increase wasobserved over a wide range of voltages (

n

8, P

�

0.01) (Fig. 2 B). The enhancement of Na currentsupon exposure to DTT suggests that a disulfide bondforms spontaneously between the cysteines at the 403and 758 positions, cross-linking the pore-lining (P) seg-ments of domains I and II, and partially occluding thepore. DTT did not increase the magnitude of the basalcurrent of any of the other paired mutants examined inthis study.

The redox catalyst Cu(phe)

3

facilitates disulfidebond formation in paired cysteine mutant channels,but at concentrations of 100

�

M or less has no effect oneither the wild-type Na channel or single-cysteine poremutant channels (Benitah et al., 1997, 1999). Disulfidebond formation was also induced by Cu(phe)

3

inE403C

�

E758C mutant channels. The modification byCu(phe)

3

was irreversible during washout (Fig. 2 C).However, the application of DTT, at least partially, re-stored the Na current through E403C

�

E758C mutantchannels (Fig. 2 D).

Figure 2. Evidence for disulfidebonding of residues in the outer ring ofcharge. (A) Representative traces showthat current through E403C�E758Cmutant Na channels increased morethan twofold in magnitude in the pres-ence of 1mM DTT. (B) The current-voltage relationship shows a similar in-crease in peak current amplitude of thedouble mutant by exposure to DTTover the entire voltage range (P � 0.01,n 8). Peak currents measured at eachvoltage step were normalized to themaximum current observed in eachcell, averaged and plotted as a functionof voltage. (C) A plot of the time courseof the decrease in peak current am-plitude upon exposure to 100 �MCu(phe)3. The effects of Cu(phe)3 werenot reversed during washout. The insetshows currents recorded at time (a) and(b). (D) A plot of the peak Na cur-rent amplitude during application ofCu(phe)3, washout, and subsequenttreatment with 1 mM DTT.

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Outer Ring of Charge and Na Channel Slow Inactivation

Conformation-dependent Rate of CatalyzedDisulfide Bond Formation

We used different voltage protocols to examine theconformation-dependent rate of disulfide bond forma-tion of the paired cysteine mutants. The rates of modi-fication of paired mutants were determined using de-polarizing test pulses to

�

20 mV for 50 ms from a hold-ing potential of

�

100 mV at stimulation frequencies of1 Hz or 0.033 Hz in the presence of Cu(phe)

3

. Usingthis protocol at 1 Hz, voltages that favored the activatedand fast inactivated states (

�

20 mV) occupied 4.76%duty cycle, compared with only 0.17% of the duty cycleat 0.033 Hz. At test pulse durations of 50 ms very littleslow inactivation was recruited in the wild-type or any ofthe paired mutant channels. The rates of disulfidebond formation were indexed by the peak current re-duction curves and the time constants were deter-mined by single exponential fits to the plots of currentreduction (Fig. 3, A and B). A plot of the time con-stants of the modification rates demonstrates that cys-teine substitutions of the outer ring of charge residuesexhibit specific patterns of frequency-dependent di-sulfide bond formation when combined with otherP-segment cysteine substitutions (Fig. 3 C). Paired cys-teine substitutions of outer charged ring residues indomains I and II (E403C

�

E758C), I and III (E403C

�

D1241C), I and IV (E403C

�

D1532C), and III and IV(D1241C

�

D1532C) exhibited significantly faster ratesof catalyzed disulfide bond formation at 1 Hz com-pared with 0.033 Hz stimulation frequencies (Fig. 3, Aand C). Similarly, the other paired outer charged ringmutant (II

�

IV E758C

�

D1532C) exhibited a morerapid rate of modification with 1 Hz stimulation fre-quency compared with 0.033Hz, but this difference didnot reach statistical significance (Fig. 3 C). The pairingof charge ring mutants with other cysteine mutations inthe P-segments, but not in the outer charged ring, gen-erally produced double mutants with rapid Cu(phe)

3

-induced disulfide bond formation insensitive to thestimulation frequency (Fig. 3, B and C). The excep-tions were when E403C was combined with M1240C (in

F

igure

3. Rate-dependent modification by Cu(phe)

3

in pairedmutant channels. (A) Rate-dependent modification by Cu(phe)

3

of E403C

�

E758C mutant channels. Increasing stimulation fre-quency (from 0.033 Hz to 1 Hz) enhanced the rate of current re-

duction of E403C

�

E758C, suggesting rate-dependent modifica-tion of Cu(phe)

3

in E403C

�

E758C mutant channels. The insetshows representative currents at 2 min (a) and 12.5 min at stimula-tion frequencies of 1 Hz (b) and 0.033 Hz (c) after the applicationof Cu(phe)

3

. (B) In contrast, current reduction was rapid and notdependent on stimulation frequency in the E403C

�

W1239C mu-tant channels. (C) Plots of the rates of reduction in peak currentamplitude at different stimulation frequencies in the presence ofCu(phe)

3

(

n

2–7). Single exponential fits of the form y

Ae

(

�

t

/

)

to plots like those shown in A and B were used to determine thetime constants,

.

A

is the amplitude. The top panel shows the timeconstants of current decay of mutant channels that were signifi-cantly increased at slower stimulation frequencies. The bottompanel is a plot of the time constants of double mutant channelsthat did not exhibit stimulation frequency dependent rates ofmodification. (*P

�

0.05, **P

�

0.01).

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Cu(phe)3. Fig. 4 B shows that the recovery of the Nacurrent was �90% complete by 1 min of washout in-dependent of the presence of Cu(phe)3. Similarly,E403C�E758C mutant channels exhibited completerecovery from slow inactivation, which was induced byholding the channels at �20 mV and pulsing to 0 mVat a frequency of 1 Hz before and during exposure toCu(phe)3 (Fig. 4 C). Slow inactivated E403C�E758Cmutant channels recovered with a similar time courseindependent of prior exposure to Cu(phe)3 (P �0.05) (Fig. 4, B and C). These results demonstratethat slow inactivation in E403C�E758C mutant chan-nels can completely prevent the formation of a disul-fide bond between the cysteines at the 403 and 758positions in the presence of Cu(phe)3. In contrast,there was no significant recovery of the Na currentthrough E403C�W1531C mutant channels in thepresence of Cu(phe)3 (Fig. 4 D).

The induction of slow inactivation prevented cata-lyzed disulfide bond formation in specific patterns withpaired mutants in the outer pore of the channel. Whenthe domain I outer-charged ring mutant E403C wascombined with a mutation in any one of the charge res-idue positions in the other domains (domain II E758C,domain III D1241C, and domain IV D1532C), disul-fide bond formation was prevented by voltage pro-tocols that populated slow inactivated states (Fig. 5).In contrast, the pairing of E403C with other cys-teine mutations in the P-segments generated doublemutants (e.g., E403C�W756C, E403C�W1239C, andE403C�W1531C) that were not protected from modifi-cation by Cu(phe)3 during slow inactivation. Inter-estingly, slow inactivation in other paired mutantsin the outer ring (III�IV D1241C�D1532C, II�IVE758C�D1532C) provided less protection from Cu(phe)3-induced disulfide bond formation comparedwith the mutants containing E403C (Fig. 5). However,slow inactivation does not appear to render the E403Cside chain completely inaccessible, since it remainsable to form disulfide bonds with other cysteine substi-tutions in the pore (W1531C and possibly W1239C andW756C).

Interestingly, E403C�K1237C Na currents were alsoprotected from Cu(phe)3-induced disulfide bond for-mation (83%) by slow inactivation. These results, alongwith the findings that charge-altering mutations at resi-due K1237 favor slow forms of inactivation (Todt et al.,1999), suggest a role for K1237 in slow inactivation.With the exception of E403C�K1237C mutant, doublemutants that have more protection (i.e., 60% or more)from catalyzed modification in slow inactivated states(Fig. 5) also exhibit significant differences in the modi-fication rate at different stimulation frequencies fromthe �100 mV holding potential (Fig. 3). These findingssuggest that in these mutant channels that exhibit ahigh degree of flexibility of P-segments during gating,

T A B L E I

Extent of Current Recovery

Mutant Extent of current recovery

Protocol A (1 Hz)

Cu(phe)3

Protocol B(0.033 Hz)Cu(phe)3

Protocol C(slow inact)

Control

Protocol C(slow inact)Cu(phe)3

% % % %

E403C�E758C 0.16 � 0.03 0.14 � 0.02 96.55 � 0.55 88.64 � 5.09

E403C�D1241C 0.18 � 0.04 0.16 � 0.02 93.14 � 3.34 81.01 � 6.26

E403C�D1532C 0.15 � 0.02 0.18 � 0.04 97.28 � 2.72 94.43 � 2.27

E403C�M1240C 0.14 � 0.03 0.12 � 0.03 93.88 � 4.30 84.95 � 8.54

E403C�K1237C 0.12 � 0.03 0.15 � 0.03 97.94 � 2.73 83.21 � 3.31

D1241C�D1532C 0.15 � 0.02 0.13 � 0.02 97.33 � 1.67 60.69 � 1.38

E758C�K1237C 0.14 � 0.02 0.13 � 0.03 98.02 � 0.91 57.75 � 9.76

E403C�G1530C 0.16 � 0.02 0.19 � 0.04 95.13 � 1.15 47.22 � 1.56

E403C�W756C 0.13 � 0.02 0.15 � 0.03 96.02 � 2.80 40.04 � 3.85

E403C�W1239C 0.19 � 0.04 0.16 � 0.02 96.13 � 3.87 37.52 � 4.85

E758C�D1532C 0.17 � 0.03 0.15 � 0.02 94.02 � 2.80 33.02 � 3.15

E403C�W1531C 0.15 � 0.02 0.17 � 0.02 97.91 � 1.10 10.16 � 6.40

E758C�W1531C 0.12 � 0.03 0.15 � 0.03 98.13 � 0.76 1.76 � 1.41

(Protocols A and B) Depolarizing test pulses to �20 mV for 50 ms from aholding potential of �100 mV at stimulation frequencies of 1 Hz or 0.033Hz, respectively (n 2–7 cells). (Protocol C) Slow inactivation wasinduced by voltage clamping cells at �20 mV and then repolarizing to�100 mV for 10 ms every 5 s (Fig. 4 A). Recovery at �100 mV was measuredby voltage steps to �20 mV for 50 ms at 1 Hz after washout of Cu(phe)3

(n 3–6 cells).

domain III) and G1530C (in domain IV) (Fig. 3 C). Inall the paired mutant channels examined, the modifi-cation by Cu(phe)3 was irreversible in the absence ofa reducing agent (Table I), consistent with disulfidebond formation (Benitah et al., 1997, 1999).

Slow Inactivation and Cu(phe)3-inducedDisulfide Bond Formation

We used two different protocols to induce slow inacti-vation in the presence and absence of Cu(phe)3 to in-vestigate the effect of conformational changes of theouter pore associated with slow inactivation on the ex-tent of disulfide bond formation. Slowly recovering in-activated states were populated by holding the cells at�20 mV and then repolarizing to �100 mV for 10 msevery 5 s. The brief repolarization permitted recoveryfrom fast inactivation and monitoring of the residualpeak current amplitude. Cu(phe)3 was washed out for4 min before returning the channels to a holding po-tential of �100 mV. The recovery component afterwashout represented channels that had slow inacti-vated but had not undergone catalyzed disulfide bondformation. Fig. 4 A shows that the rate of developmentof slow inactivation of E403C�E758C mutant chan-nels was not significantly different in the presence andabsence of Cu(phe)3. The reduction of current waslargely attributed to cumulative slow inactivation,rather than induced disulfide bond formation by

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328 Outer Ring of Charge and Na Channel Slow Inactivation

neither closed state nor slow inactivated states favor cat-alyzed disulfide bond formation (Fig. 6).

In summary, the side chains of amino acid residues inthe outer ring of charge of the voltage-gated Na chan-nel (especially other outer ring mutants paired withE403C) undergo unique motions in slow inactivationthat prevents Cu(phe)3-induced modification (Fig. 5).

D I S C U S S I O N

Slow inactivation is a collection of distinct gating pro-cesses. However, the molecular determinants of slow in-activation remain unknown. Site-directed mutagenesissuggests that residues in the S4 (Mitrovic et al., 2000),S4-S5 linkers (Bendahhou et al., 2002), S5 (Bendahhouet al., 1999), and S6 segments (Hayward et al., 1997;Wang and Wang, 1997) may play a role in slow inactiva-tion. Moreover, several lines of evidence have sup-ported a role for the outer pore in slow inactivation.First, mutations in the P-segments disrupt slow inactiva-tion in Na channels (Tomaselli et al., 1995; Balser et al.,1996; Kambouris et al., 1998; Todt et al., 1999; Ong etal., 2000; Hilber et al., 2001, 2002; Vilin et al., 2001;

Zhang et al., 2003). Second, alkali metal cations ap-plied to the external surface inhibit slow inactivation(Townsend and Horn, 1997), similar to observationsfor C-type inactivation in Shaker K channels (Pardo etal., 1992; Lopez-Barneo et al., 1993; Baukrowitz andYellen, 1995). Third, residues in the outer pore of Nachannels may underlie isoform-specific differences inslow inactivation (Vilin et al., 2001).

In the K channel, C-type inactivation is clearly associ-ated with constriction of the outer mouth of the pore(Liu et al., 1996). In the Na channel, whether a similarconstriction of the outer pore occurs is not certain.Townsend and Horn showed that increasing the extra-cellular Na� concentration inhibits slow inactivation,suggesting that binding of Na� to a site near the outermouth of the pore inhibits closing of the slow inactiva-tion gate (Townsend and Horn, 1997). On the otherhand, gating- and conformation-independent modifi-cation of cysteine mutants in the outer pore by meth-anethiosulfonate (MTS) reagents argues that the outerpore remains largely unobstructed when slow inacti-vated (Struyk and Cannon, 2002). However, in the lat-ter study, only a limited number of cysteine mutations

Figure 4. Slow inactivation pre-vented the modification of E403C�E758C, but not E403C�W1531C mu-tant channels by Cu(phe)3. (A). Slowinactivation was induced by holding thecells at �20 mV for 15 min with brief re-polarizations to �100 mV for 10 ms toelicit currents after recovery from fastinactivation. The mean data shows thatchannels entered slow-inactivated statesat approximately the same rate regard-less the presence (filled circles) or ab-sence (open circles) of Cu(phe)3. (B)Upon washout, Na currents nearly com-pletely recovered from slow inactivationinduced by the voltage protocol used inA independent of prior exposure toCu(phe)3 (P � 0.05, n 6). The insetshows representative currents before(a) and 60 s after initiation of recovery(b) with (filled circle) or without (opencircle) prior exposure to Cu(phe)3. (C)A complementary voltage protocol wasemployed to induce slow inactivationbefore application of Cu(phe)3. Chan-nels were held at �20 mV and pulsedfor 50 ms to 0 mV at 1 Hz for 30 min.After 15 min of pulsing, Cu(phe)3 (100�M) was applied for 15 min beforewashout. The oocytes were voltageclamped at �20 mV during the 4 minwashout, then they were stimulated at 1 Hz from a holding potential of �100 to �20 mV to elicit Na currents. The Na currents completelyrecovered from slow inactivation compared with control (P � 0.05) (B). (D) After washout of Cu(phe)3, Na currents in E403C�W1531Cmutant 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).

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329 Xiong et al.

were informative and most of these were superficial inthe pore (Struyk and Cannon, 2002), thus a dynamicconstriction of the deeper reaches of the pore duringslow inactivation cannot be excluded. Indeed, we ob-served reduced accessibility to the side chain of F1236Cto (2-aminoethyl)methanethiosulfonate (MTSEA) withvoltage protocols favoring slow inactivation (Ong et al.,2000). Therefore, a structural rearrangement of theouter pore is likely during slow inactivation of Na chan-nels, although it is uncertain whether such a conforma-tion change completely prevents ion flux.

The P-segments are asymmetrically organized in theNa channel and each segment in domains I–IV makes aunique contribution to the formation of the pore (Chi-amvimonvat et al., 1996). Unexpected flexibility ofP-segments of Na channels allows disulfide formationbetween the side chains of paired engineered cysteinesin P-segments of different domains (Benitah et al., 1997;Tsushima et al., 1997). The unique ability of cysteinemutants in domains I and II to form spontaneous disul-fide bonds suggests that these two domains are in closeproximity (Benitah et al., 1997). The spontaneous orcatalyzed disulfide bonding of E403C and E758C in thisstudy is consistent with this close proximity of the do-mains I and II P-segments and a separation of no morethan 7 Å between the � carbons.

The outer charged ring appears to exhibit a high de-gree of flexibility during channel gating, consistentwith previous studies (Benitah et al., 1996, 1997, 1999;Tsushima et al., 1997). Paired cysteine mutants in theouter ring exhibited conformationally sensitive rates ofmodification in the presence of a redox catalyst (Fig. 3C). At the faster stimulation frequencies from a hold-ing voltage of �100 mV, the channels spend a greaterproportion of time in the activated and fast inactivatedstates compared with the closed state. The reactivethiol side chains in paired mutant channels that exhib-ited faster rates of modification at 1 Hz compared with0.033 Hz stimulation frequencies may come into closeproximity during activation or fast inactivation. Nota-bly, even those mutant channels, which did not exhibitstimulation frequency–dependent differences in therate of disulfide bond formation, were rapidly modifiedby Cu(phe)3 (Fig. 3 C). This is consistent with preferen-tial modification of closed channels or nearly equalrates of modification of closed and activated/fast inacti-vated channels.

Figure 5. A bar plot of the percent recovery from slow inactiva-tion elicited by the voltage protocol in Fig. 4 A. The percent recov-eries of E403C�E758C, E403C�K1240C, E403C�D1241C, andE403C�D1532C channels were not significantly changed in thepresence of Cu(phe)3 (P � 0.05, n 3–6). Significant Cu(phe)3-induced modification of the other paired mutants occurred dur-ing induction of slow inactivation (P � 0.05, n 3–4).

Figure 6. A schematic model showinghow gating influences the rate and ex-tent of disulfide bond formation ofdouble mutants in the external poreof the Na channel. In the case ofE403C�E758C, enhanced rates of cur-rent reduction with higher stimulationfrequencies suggest that the activatedor fast inactivated states are modifiedmore readily than the rested state. Incontrast, double mutant channels suchas E403C�W1531C (Fig. 3 C) are rap-idly modified at both slow and fast stim-ulation rates, suggesting that the restedstate may be accessible to modifica-tion by Cu(phe)3. Slow inactivation pro-tects E403C�E758C channels (andE403C�D1241C and E403C�D1532Cin 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 ofcharge during slow inactivation of Na channels.

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W756C was previously shown to be insensitive tobulky charged MTS reagents (Perez-Garcia et al.,1996), but in this study E403C forms a disulfide bondwith W756C in the presence of Cu(phe)3. This appar-ent discrepancy is not well understood. MTS agentsmay modify W756C without a significant effect on thecurrent amplitude. Alternatively, the combination ofboth mutations may alter the pore structure so that theside chain of W756C is accessible for disulfide bond for-mation.

Residues in the outer ring of charge are also involvedin the conformational changes in the channel poreassociated with slow inactivation. For example, Nacurrents through E403C�E758C mutant channelsthat were slow inactivated before the application ofCu(phe)3 completely recovered (Fig. 4 C), suggestingthat slow inactivation protects the channels from modi-fication by the redox catalyst. The structural rearrange-ment in the outer pore during slow inactivation ap-pears to alter the spatial relationships of the thiol sidechains of E403C (domain I) and E758C (domain II)(Fig. 6). Similar protection was afforded to E403C�D1241C (domain III) and E403C�D1532C (domainIV) channels. An alternative explanation is the accessi-bility of the redox catalyst to the outer ring of chargechanged with slow inactivation. It is possible that the re-dox catalyst was excluded from the pore; however, thiswould have to be a selective exclusion because somedouble mutant channels involving residues deep in thepore (e.g., E403C�W756C, E403C�W1531C, E758C�W1531C, Fig. 5) were modified by Cu(phe)3. Fur-thermore, the same paired cysteine mutants in theouter ring of charge underwent catalyzed disulfidebond formation by Cu(phe)3 when cells were pulsedfrom a holding potential of �100 mV (Fig. 3). In anycase, slow inactivation appears to involve movementsof the P-segments and changes in the spatial relation-ships of the side chains of the outer charged ring resi-dues.

In addition to residues in the external charge ring,two other cysteine mutants when combined with E403Cprovided nearly complete protection from disulfidebond formation when the channels were slow inacti-vated. Na currents through E403C�K1237C mutantchannels exhibited comparable recovery from slow in-activation independent of prior exposure to Cu(phe)3,suggesting a role for K1237 in slow forms of inactiva-tion. These data support the study by Todt et al. (1999)that charge-altering mutations at position 1237 favoroccupancy of the ultra-slow inactivation state, similar toC-type inactivation in shaker K channels. Slow inactiva-tion also protected E403C�M1240C from oxidation;however, M1240C undergoes extraordinarily slow mod-ification by MTS agents that is independent of thechannel conformation (Struyk and Cannon, 2002). We

cannot exclude this as the cause of the redox insensitiv-ity of this paired mutant channel.

The varying susceptibility of different outer ring dou-ble mutants further highlights the asymmetry of the Nachannel pore (Chiamvimonvat et al., 1996; Perez-Gar-cia et al., 1996). The S4 segment, half of a “voltage-sen-sor paddle” (Jiang et al., 2003), may also play a signifi-cant role in slow inactivation of the Na channel (Kontisand Goldin, 1997; Mitrovic et al., 2000). It has beenproposed that if slow inactivation depends on the mo-bility of S4 segments, S4 immobilization by fast inactiva-tion may inhibit slow inactivation (Featherstone et al.,1996; Richmond et al., 1998). Indeed, Cha et al. (1999)have shown that S4 voltage sensors in domains III andIV, but not I and II, are immobilized by Na channel fastinactivation. Furthermore, the largest effects on Nachannel slow inactivation result from mutations of S4in domains I and II (Kontis and Goldin, 1997; see alsoMitrovic et al., 2000). Thus, it is likely that the S4 seg-ments in domains I and II make significantly differentcontributions to slow inactivation compared with do-mains III and IV. However, whether the effects of eachS4 on slow inactivation transfer to its outer pore seg-ments remains unknown.

In summary, our findings demonstrate that the roleof the outer ring of charge is not limited to conven-tional notion that it increases the conductance by at-tracting cations into the channel entryway via its elec-trostatic action (Bell and Miller, 1984; Green et al.,1987). The molecular motions of the outer ring ofcharge (especially E403) suggest an unexpected role inslow inactivation of the Na channel.

We thank Debbie DiSilvestre and Ailsa Mendez-Fitzwilliam fortechnical assistance.

This work was supported by the National Institutes of Healthgrants R01 HL50411 (to G.F. Tomaselli). W. Xiong was supportedby a fellowship grant from American Heart Association(0225589U). R.A. Li was a recipient of a Research Career Devel-opment Award from the Cardiac Arrhythmias Research and Edu-cation Foundation, Inc.

Olaf S. Andersen served as editor.

Submitted: 30 May 2003Accepted: 17 July 2003

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