local anesthetic compounds , such as lidocaine , suppress the ionic current through na channels . by attenuating central and peripheral neuronal excitability , these compounds enjoy widespread use in the treatment of epilepsy and the relief of pain . in addition , local anesthetic compounds block na channels in skeletal and cardiac muscle , and are used to treat neuromuscular diseases and cardiac arrhythmias . an essential characteristic of the local anesthetic action is use dependence , which is the sustained loss of excitability lasting many hundreds of milliseconds that is induced only when the drug - exposed channel is depolarized ( courtney 1975 ) . although studies have identified amino acid residues in the cytoplasmic end of the pore that , when mutagenized , attenuate the action of local anesthetic compounds ( ragsdale et al . 1994 ; nau et al . 1999 ) , the structural basis of the sustained , depolarization - dependent loss of excitability associated with use dependence is not understood . when briefly depolarized , na channels inactivate rapidly within a few milliseconds ( fast inactivation ) , a process mediated by residues situated near the cytoplasmic face of the channel ( west et al . these nonconducting states have diverse lifetimes ranging from tens of milliseconds ( kambouris et al . 1999 ) to many seconds ( cummins and sigworth 1996 ; featherstone et al . 1996 ; hayward et al . 1997 ) , and are distinct from fast inactivation that typically recovers within 10 ms between stimuli . importantly , slow inactivation influences cellular excitability , particularly in the pathophysiological conditions associated with prolonged membrane depolarization such as epilepsy ( fleidervish et al . 1995 ) , arrhythmias ( veldkamp et al . 2000 ) , and neuromuscular diseases ( cannon 1996 ) . amino acid residues from the p - segments ( s5-s6 linkers ) , donated from each of the four homologous domains , line the extracellular aspect of the na channel pore ( see fig . 1 ) . although the structural basis of slow inactivation gating is not defined , site - directed mutations and chimeric studies suggest a role for the p - segments ( balser et al . 1996a ; benitah et al . 1999 ; todt et al . 1999 ; vilin et al . 1999 ) . in addition , p - segment mutations and extracellular cation substitutions that reduce slow inactivation ( townsend and horn 1997 ) seem to inhibit use - dependent lidocaine action ( zilberter et al . 2000 ) , suggesting a potential mechanistic linkage between these phenomena . investigating the structural basis of slow inactivation is hampered by its electrical silence , and requires a means to detect relatively slow changes in the conformational architecture of a nonconducting channel . cysteine substitution of a p - segment residue in domain iii ( see fig . 1 , f1236c ) yields a na channel reactive with the positively charged methanethiosulfonate ( mts ) reagent mts - ethylammonium ( mtsea ) applied from outside the cell , yet the residue is entirely resistant to mtsea applied from inside the cell ( yamagishi et al . adjacent to the domain iii lysine that contributes to the na channel selectivity filter ( see fig . 1 , k1237 ; heinemann et al . 1992 ) , this cysteine can not be modified by larger ( mts - ethyltrimethylammonium [ mtset ] ) or negatively charged ( mts - ethylsulfonate , mtses ) reagents ( yamagishi et al . we postulated that sulfhydryl modification of a residue harboring such restrictive access might be sensitive to the voltage - gated conformational state of the ion channel . the results show that modification of f1236c is greatly enhanced by brief depolarizations , but is inhibited by more sustained depolarizations that induce slow inactivation . moreover , lidocaine inhibited depolarization - induced mtsea modification during sustained , but not brief , depolarizations . we propose that na channel slow inactivation and use - dependent lidocaine action are linked to a common structural rearrangement involving the outer pore . for heterologous expression , the wild - type rat skeletal muscle nachannel ( 1 )  subunit was subcloned into the hindiii - xbai site of the vector gfp - irs for bicistronic expression of the channel protein and gfp reporter as previously described ( johns et al . the rat skeletal muscle mutant na channel f1236c was prepared ( yamagishi et al . 1997 ) using a pcr - based method ( quickchange site - directed mutagenesis kit ; stratagene ) . in brief , sense and antisense oligonucleotides ( 3034 mer ) containing the desired mutation were used as primers for amplification with pfu dna polymerase . the pcr product was treated with dpni to select for the mutant plasmid and transformed into escherichia coli for subsequent isolation and purification . wild - type and mutant na channels  subunits were transiently transfected into hek 293 ( human embryonic kidney cell line ) cells using lipofectamine ( gibco brl ) , and were cultured in mem medium supplemented with 10% fetal bovine serum and 1% pen - strep in a 5% co2 incubator at 37c for 13 d. in all cases , the hek cells were cotransfected with the na channel 1 subunit ( provided by dr . alfred george , vanderbilt university ) . whole - cell na currents ( ina ) were recorded ( axopatch 200b ; axon instruments ) using electrodes with resistances of 13 m when filled with a pipet solution containing ( in mm ) : 140 naf , 10 nacl , 5 egta , 10 hepes , ph 7.40 . replacing the intracellular k with na eliminated the time - dependent k currents in our hek cell recordings . current magnitudes were 12 na , and 85% of the series resistance was compensated , yielding a maximum voltage error of 1 mv . the bath solution contained ( in mm ) : 150 nacl , 4.5 kcl , 1.5 cacl2 , 1 mgcl2 , 10 hepes ( titrated to ph 7.40 with naoh ) . mtsea , mtses , and mtset ( toronto research chemicals ) were kept at 4c as high concentration stock solutions and were diluted to 25100  in the appropriate bath solution immediately before use . the disulfide reducing agents dithiothreitol ( dtt ) and glutathione were dissolved directly in the extracellular solution at a concentration of 5 mm ( titrated to ph 7.4 with naoh ) . lidocaine hcl ( sigma - aldrich ) or qx-314 ( almone labs ) were diluted from stock solutions to the bath concentrations indicated in the text . cells were dialyzed for a 15-min equilibration period before recording data . to avoid junction potentials with solution changes , inactivation gating kinetics and use - dependent block were assessed using the voltage - clamp protocols described in the text and figure legends . whole - cell currents were sampled at 20 khz ( digidata 1200 a / d converter ; axon instruments ) and low pass  filtered at 5 khz . the results are expressed as mean  sem , and statistical comparisons were made using one - way anova ( microcal origin ) with p < 0.05 indicating significance . multiexponential functions were fitted to the data using nonlinear least - squares methods ( origin ) . for heterologous expression , the wild - type rat skeletal muscle nachannel ( 1 )  subunit was subcloned into the hindiii - xbai site of the vector gfp - irs for bicistronic expression of the channel protein and gfp reporter as previously described ( johns et al . the rat skeletal muscle mutant na channel f1236c was prepared ( yamagishi et al . 1997 ) using a pcr - based method ( quickchange site - directed mutagenesis kit ; stratagene ) . in brief , sense and antisense oligonucleotides ( 3034 mer ) containing the desired mutation were used as primers for amplification with pfu dna polymerase . the pcr product was treated with dpni to select for the mutant plasmid and transformed into escherichia coli for subsequent isolation and purification . wild - type and mutant na channels  subunits were transiently transfected into hek 293 ( human embryonic kidney cell line ) cells using lipofectamine ( gibco brl ) , and were cultured in mem medium supplemented with 10% fetal bovine serum and 1% pen - strep in a 5% co2 incubator at 37c for 13 d. in all cases , the hek cells were cotransfected with the na channel 1 subunit ( provided by dr . alfred george , vanderbilt university ) . whole - cell na currents ( ina ) were recorded ( axopatch 200b ; axon instruments ) using electrodes with resistances of 13 m when filled with a pipet solution containing ( in mm ) : 140 naf , 10 nacl , 5 egta , 10 hepes , ph 7.40 . replacing the intracellular k with na eliminated the time - dependent k currents in our hek cell recordings . current magnitudes were 12 na , and 85% of the series resistance was compensated , yielding a maximum voltage error of 1 mv . the bath solution contained ( in mm ) : 150 nacl , 4.5 kcl , 1.5 cacl2 , 1 mgcl2 , 10 hepes ( titrated to ph 7.40 with naoh ) . mtsea , mtses , and mtset ( toronto research chemicals ) were kept at 4c as high concentration stock solutions and were diluted to 25100  in the appropriate bath solution immediately before use . the disulfide reducing agents dithiothreitol ( dtt ) and glutathione were dissolved directly in the extracellular solution at a concentration of 5 mm ( titrated to ph 7.4 with naoh ) . lidocaine hcl ( sigma - aldrich ) or qx-314 ( almone labs ) were diluted from stock solutions to the bath concentrations indicated in the text . cells were dialyzed for a 15-min equilibration period before recording data . to avoid junction potentials with solution changes , inactivation gating kinetics and use - dependent block were assessed using the voltage - clamp protocols described in the text and figure legends . whole - cell currents were sampled at 20 khz ( digidata 1200 a / d converter ; axon instruments ) and low pass  filtered at 5 khz . the results are expressed as mean  sem , and statistical comparisons were made using one - way anova ( microcal origin ) with p < 0.05 indicating significance . multiexponential functions were fitted to the data using nonlinear least - squares methods ( origin ) . 1 ) cysteine side chain to sulfhydryl modification using 100 m mtsea . a 3-min exposure to mtsea during hyperpolarization ( 100 mv ; fig . 2 a , protocol i ) reduced f1236c peak ina by 31  3% ( after mtsea washout ; summary data fig . this exceeded wild - type modification ( 13  3% , p < 0.05 ; fig . b ) , but was less than that previously seen with f1236c using a much higher concentration of mtsea ( 49  15% , 2.5 mm ; yamagishi et al . however , modification was substantially increased by clamping to 20 mv during mtsea exposure for f1236c ( 73  5% , p < 0.0001 ; fig . identical experiments using the larger , positively charged reagent mtset , or the negatively charged analogue mtses , produced reactivity indistinguishable from the wild type , even with sustained ( protocol ii ) depolarization ( fig . b indicates that after 3 min of depolarization - induced mtsea modification , the fractional reduction in peak ina ( relative to pre - mtsea ) after an additional 20 min of dtt exposure is reduced to 49  5% ( p < 0.005 versus the 73% pre - dtt value ) , indicating the ina reduction associated with mtsea exposure results from formation of a reducible disulfide bond . the prolonged time ( 20 min ) required for only partial dtt reversal suggests the reducing agent accesses the disulfide bond with some difficulty , perhaps through a hydrophobic pathway . we could not achieve reversal using glutathione , a larger , more hydrophilic , and generally less reactive reducing agent ( fig . 2 ) suggest that although depolarization increases the accessibility of the f1236c side chain ( allowing enhanced modification by mtsea ) , the residue still lies at a relatively inaccessible position in the outer pore , limiting the effects of the larger sulfhydryl modification compounds or reducing agents . the rate of f1236c ina decay during the depolarizing pulse did not differ from wild type ( fig . 2 a ) , suggesting the mutant had no marked effect on the fast inactivation gating process . to examine the kinetics of slow inactivation , wild - type and f1236c peak ina were measured after prepulses of incremental duration ( fig . each pair of depolarizations was separated by a brief , 20-ms hyperpolarization that allowed channels to recover fully from fast , but not slow , inactivation . wild - type and f1236c slow inactivation were described by biexponential functions with similar time constants ( parameters given in legend ) , corresponding to intermediate ( i m ,   30 ms ) and slower ( is ,   2 s ) kinetic components of slow inactivation described in previous studies of 1 expressed in xenopus oocytes ( kambouris et al . 1999 ) . however , the rate of slow inactivation increased in wild - type channels as the membrane potential increased from 20 to + 20 mv ( fig . 3 a ) because of an increase in the amplitude of the i m component ( from 0.2 to 8% ) and a shorter is time constant ( 2 , from 2,519 to 1,255 ms ; see fig . 3 legend ) . conversely , the rate of development of slow inactivation in f1236c was similar at 20 mv and + 20 mv ( fig hence , consistent with previous evidence that p - segment substitutions alter slow inactivation gating processes in na channels ( balser et al . 1996a ; todt et al . 1999 ) , the f1236c mutation did modify ( increase ) the propensity of the na channels to slow inactivate . a meaningful analysis of the depolarization - dependent rate of mtsea modification of the f1236c side chain must recognize the distinctive kinetic features of slow inactivation , including the biexponential characteristics noted in fig . 3 . therefore , we used parameters derived from the kinetic analysis of f1236c slow inactivation ( 20 mv ; fig . 3 legend ) to select depolarization pulse widths that would maximize the intermediate and slow kinetic components , i m ( 100 ms ;  3  m ) and is ( 6 s ;  3  s ) . in addition , a brief , 5-ms pulse width was used to examine depolarization - induced mtsea modification in the complete absence of slow inactivation ( fig . notably , while the pulse widths are selected to bias the channel toward distinct kinetic components of slow inactivation , caution is needed when relating these components to occupancy of individual gated states . the inactivated state dwell - times depend on many factors , including the detailed connectivity of the kinetic states involved . 4 ( a and b ) shows the time - dependent reduction of ina due to mtsea modification during trains of either 5- or 100-ms pulses ( 20 mv ) . in all experiments , mtsea was allowed to equilibrate in the bath for at least 1 min before application of the first depolarization pulse . for these studies , 2 ) to minimize tonic , depolarization - independent modification ; as such , the reduction in ina during the first pulse after mtsea addition was consistently 5% ( fig . 4 , currents are plotted from every hundredth pulse ( a ) or every fifth pulse ( b ) , and the currents shown in the two panels are aligned to reflect matching cumulative depolarization time . a comparison of a and b in fig . 4 reveals that extending the depolarization duration from 5 to 100 ms markedly reduced the rate of modification . the time - dependent reduction in ina , due to mtsea modification for a number of cells , as a function of matching ( cumulative ) depolarization time , allowing direct visual comparison of the mtsea modification rates for the three pulse widths ( 5 ms , 100 ms , and 6 s ) . the repolarization intervals between pulses ( 200 ms , 4 s , and 240 s , respectively ) were sufficiently long to prevent cumulative reduction in the current because of slow inactivation , and also were chosen such that the cumulative depolarization time increased as a function of total experimental time at the same rate for all three pulse widths . at the 6-s pulse width , the total ( 18 s ) depolarization period was generated by only three pulses ( fig . in contrast to the 100-ms pulse train , channels transiently occupy the i m component , but spend a much higher percentage of their total depolarized time in the is kinetic component . nonetheless , the rate of depolarization - dependent mtsea modification is still reduced compared with the brief , 5-s depolarizations ( fig . 4 c ) , indicating that pulses recruiting is ( like i m ) 4 c were fitted to an exponential function ( solid line ) to determine reaction rates ( kon ; fig . 4 d , see legend ) for each depolarization pulse width ; increasing the pulse width to either 100 ms or 6 s significantly reduced kon . fig . 4 d also indicates that the effects of pulse width ( 5 and 100 ms ) were insensitive to changing the depolarization voltage from 20 to + 20 mv , which is consistent with the similar rate of slow inactivation for f1236c ( in contrast to wild type ) at these two membrane potentials ( fig . the rate of mtsea modification changes with the depolarization pulse width ; the extent of modification also appears to change . incomplete elimination of the current could partly result from residual current flow through mtsea - modified channels . even under conditions where slow inactivation is prevented ( i.e. , brief 5-ms pulses ) , the rate of depolarization - dependent modification is still 100-fold slower than in previous studies examining mts modification of freely accessible cysteinyl groups ( i.e. , iii 1996 ; vedantham and cannon 1998 ) , which is consistent with restricted access of mtsea to the 1236c intrapore cysteinyl . given the even slower rate of mtsea modification at the longer pulse widths , we may speculate that a slow , competing side reaction may gradually oxidize the 1236 cysteine ( while the channel remains na permeable ) , as proposed in previous studies of intrapore cysteine disulfide formation ( benitah et al . moreover , the modification rate , with long pulses at a low mtsea concentration ( 25 m , fig . 4 c ) , is quite slow ( relative to the practical limitations on experimental time ) , making it difficult to determine precisely when modification saturates . hence , the modification rates for 100-ms and 6-s pulses may be even slower than estimated from the fitting procedure . we next considered whether accessibility of f1236c to sulfhydryl modification was modified by use - dependent lidocaine block . since the f1236c mutation alone modifies slow inactivation under control conditions ( fig . 3 ) , we first established whether lidocaine would induce significant depolarization - dependent ina suppression in this mutant . 5 a ) reduced ina availability mainly by increasing the amplitude of an intermediate (  = 68 ms ) kinetic component ( see fig . hence , the mutant channel retains use - dependent lidocaine sensitivity typical for skeletal muscle na channels expressed in hek cells ( wang et al . 1996 ) . to determine whether the effects of lidocaine on use - dependent loss of ina availability could be linked to a structural change in the outer pore , we examined voltage - dependent mtsea modification during lidocaine exposure . b shows the rate of mtsea modification induced by 100- ms depolarizing pulses to 20 mv during mtsea exposure , alone and with lidocaine superfusion . for these experiments , we raised the mtsea concentration from 25 to 50 m to allow significant modification of the cysteinyl side chain using a 100-ms pulse width in lidocaine - free conditions ( note , for comparison , only minimal modification using a 100-ms pulse width in fig . notably , in lidocaine - free solutions , the rate of mtsea modification ( kon , 10 ms ) using 100-ms pulses ( fig . 5 b , 4.4  0.6 ) was slower than with 5-ms pulses ( fig . 5 c , 9.6  1.4 , p = 0.004 vs. 100-ms pulses ) even though the mtsea concentration was raised in fig 4 , indicating a slower rate of depolarization - dependent modification when the pulse duration is lengthened . the data also reveal a marked reduction in mtsea modification with lidocaine exposure ; in fig . 5 b ( 100-ms pulse width ) , kon in lidocaine was as decreased to 0.7  0.3 ( p = 0.0002 ) . in contrast , lidocaine exposure had no effect on the rate of mtsea modification when a brief pulse width was used ( fig . hence , the protection from sulfhydryl modification afforded by lidocaine is use - dependent and requires a sustained depolarization . this pulse width sensitivity suggests the observed lidocaine effect on mtsea modification was not a nonspecific , gating - unrelated antagonism between the local anesthetic the mts reagent . based on evidence that enzymes ( cahalan 1978 ; yeh 1978 ) and mutations ( bennett et al . 1995 ; balser et al . 1996b ) that remove fast inactivation also attenuate use - dependent suppression of ina by lidocaine and other na channel blockers , a high affinity interaction between lidocaine and the fast - inactivated state has been a candidate structural motif for the sustained inactivation induced by these compounds . however , a recent study suggests that amino acid residues critically involved in fast inactivation ( west et al . 1992 ) are not trapped in the fast - inactivated conformational state by lidocaine ( vedantham and cannon 1999 ) , raising the possibility that this conformational state may not form a high affinity drug receptor as previously proposed . p - segment mutations that reduce an intermediate kinetic component of slow inactivation ( termed i m ) also attenuate use - dependent lidocaine block ( kambouris et al . 1998 ) , as do cation substitutions that inhibit this component of slow inactivation ( zilberter et al . 1991 although these and earlier studies ( khodorov et al . 1976 ) postulated a linkage between slow inactivation gating and a use - dependent local anesthetic block , unambiguous interpretation of these data has not been possible because the interventions themselves ( mutations and cation substitutions ) could alter drug binding through gating - independent mechanisms ( hille 1992 ) . here , we show that slow inactivation alters the accessibility of an outer pore cysteine to sulfhydryl modification ( fig . 3 and fig . 4 ) . moreover , lidocaine attenuates mtsea modification of the same cysteine when exposed to sustained ( 100 ms ) depolarizations ( fig . 5 b ) , but not brief , 5-ms depolarizations ( fig . 5 c ) , suggesting the lidocaine effect to suppress mtsea modification is specifically linked to gated state(s ) associated with sustained depolarization . the findings suggest that the use - dependent lidocaine block and slow inactivation share a common , outer pore structural rearrangement that reduces mts accessibility . these results do not resolve whether the impeded sulfhydryl modification rate during slow inactivation results from movement of the domain iii p - segment to a less accessible position , or rather from movement of other pore structures into positions that somehow protect the f1236c side chain from mtsea . nonetheless , a conceptual model based upon recent studies of the na channel pore demonstrating exceptional mobility of the p - segments ( benitah et al . 6 . whereas the 1236 cysteine side chain is largely inaccessible in the hyperpolarized channel ( fig . 6 , left ) , upon depolarization , we postulate that the p - segments assume positions that increase mtsea accessibility of the cysteine residue . as the depolarization is prolonged , the accessibility of the 1236c residue is once again compromised ( fig . 6 , right , kb < ka ) , which is consistent with our results ( fig . na channel gating during sustained depolarization ( i.e. , slow inactivation ) might involve a constriction of the outer pore , analogous to that envisioned for c - type inactivation of potassium channels ( baukrowitz and yellen 1996 ; liu et al . our results show that pulse widths sufficient to recruit either the intermediate ( i m ) or slower ( is ) inactivated state kinetic components ( fig . 3 ) are both effective in reducing depolarization - dependent modification ( fig . 4c and fig . d ) , which is consistent with studies linking the p - segments to slow - inactivated states spanning a wide kinetic range ( kambouris et al . 1998 ; todt et al . 1999 ; vilin et al . 1999 ) . nonetheless , the marked reduction in mtsea modification at the 100-ms pulse width is substantial , given the relatively small amplitude of intermediate ( i m ) inactivation developing by 100 ms ( fig . we speculate that a gating motion involving the pore may develop with intermediate kinetics closely related to i m ( recognizing 5-ms pulses do not inhibit modification , whereas 100-ms pulses do ) , which reduces 1236c accessibility but does not suppress na permeation ( or else , recovers more rapidly than 20 ms upon hyperpolarization and , therefore , is not visible in fig . the 1236 cysteine side chain is accessible only to modification from outside the cell ( yamagishi et al . 1997 ) , while lidocaine binds to a site in the aqueous pore on the cytoplasmic side ( ragsdale et al . 1 ) of the skeletal muscle na channel act as electrostatic barriers , preventing extracellular access and escape of the local anesthetic ( sunami et al . although mutations in the na channel domain iv - s6 segment may create an extracellular access pathway for charged lidocaine derivatives ( ragsdale et al . 1995 ) , we find that the p - segment f1236c mutation does not make the skeletal muscle na channel sensitive to extracellular qx-314 ( 1 mm , n = 4 , data not shown ) . hence , it is unlikely that lidocaine directly inhibits extracellular mtsea modification of the cysteine residue . at the same time , electrostatic interactions between residue k1237 in domain iii ( fig . 1 ) and lidocaine suggest that drug receptor in domain iv , s6 lies near the p - segment selectivity filter residues ( sunami et al . 6 , right ) we postulate that the structural rearrangements associated with slow inactivation may move the na channel p - segments into positions that stabilize the interaction between lidocaine and the pore .
voltage - gated sodium ( na+ ) channels are a fundamental target for modulating excitability in neuronal and muscle cells . when depolarized 
 , na+ channels may gradually enter long - lived , slow - inactivated conformational states , causing a cumulative loss of function . 
 although the structural motifs that underlie transient , depolarization - induced na+ channel conformational states are increasingly recognized , the conformational changes responsible for more sustained forms of inactivation are unresolved . 
 recent studies have shown that slow inactivation components exhibiting a range of kinetic behavior ( from tens of milliseconds to seconds ) are modified by mutations in the outer pore p - segments . 
 we examined the state - dependent accessibility of an engineered cysteine in the domain iii , p - segment ( f1236c ; rat skeletal muscle ) to methanethiosulfonate - ethylammonium ( mtsea ) using whole - cell current recordings in hek 293 cells . 
 f1236c was reactive with mtsea applied from outside , but not inside the cell , and modification was markedly increased by depolarization . 
 depolarized f1236c channels exhibited both intermediate ( i m ;   30 ms ) and slower ( is ;   2 s ) kinetic components of slow inactivation . trains of brief , 5-ms depolarizations , which did not induce slow inactivation , produced more rapid modification than did longer ( 100 ms or 6 s ) pulse widths , suggesting both the i m and is kinetic components inhibit depolarization - induced mtsea accessibility of the cysteine side chain . 
 lidocaine inhibited the depolarization - dependent sulfhydryl modification induced by sustained ( 100 ms ) depolarizations , but not by brief ( 5 ms ) depolarizations . 
 we conclude that competing forces influence the depolarization - dependent modification of the cysteine side chain : conformational changes associated with brief periods of depolarization enhance accessibility , whereas slow inactivation tends to inhibit the side chain accessibility . 
 the findings suggest that slow na+ channel inactivation and use - dependent lidocaine action are linked to a structural rearrangement in the outer pore .