structural determinants of drugs acting on the nav1.8 channel

Structural Determinants of Drugs Acting on theNav1.8 Channel*

Received for publication, September 30, 2008, and in revised form, February 17, 2009 Published, JBC Papers in Press, February 19, 2009, DOI 10.1074/jbc.M807569200

Liam E. Browne‡, Frank E. Blaney§, Shahnaz P. Yusaf¶, Jeff J. Clare�, and Dennis Wray‡1

From the ‡Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom, §Computational and StructuralChemistry and ¶Biological Reagents and Assay Development, GlaxoSmithKline, Harlow CM19 5AW, United Kingdom, and the �IonChannel Group, Millipore, Cambridge CB5 8PB, United Kingdom

The aim of this work is to study the role of pore residues ondrug binding in the NaV1.8 channel. Alanine mutations weremade in the S6 segments, chosen on the basis of their roles inother NaV subtypes; whole cell patch clamp recordings weremade from mammalian ND7/23 cells. Mutations of some resi-dues caused shifts in voltage dependence of activation and inac-tivation, and gave faster time course of inactivation, indicatingthat the residues mutated play important roles in both activa-tion and inactivation in the NaV1.8 channel. The resting andinactivated state affinities of tetracaine for the channel werereduced by mutations I381A, F1710A, and Y1717A (for the lat-ter only inactivated state affinity was measured), and by muta-tion F1710A for the NaV1.8-selective compound A-803467,showing the involvement of these residues for each compound,respectively. For both compounds,mutation L1410A caused theunexpected appearance of a complete resting block even atextremely low concentrations. Resting block of native channelsby compound A-803467 could be partially removed (“disinhibi-tion”) by repetitive stimulation or by a test pulse after recoveryfrom inactivation; the magnitude of the latter effect wasincreased for all the mutants studied. Tetracaine did not showthis effect for native channels, but disinhibitionwas seen partic-ularly formutants L1410A and F1710A. The data suggest differ-ing, but partially overlapping, areas of binding of A-803467 andtetracaine. Docking of the ligands into a three-dimensionalmodel of the NaV1.8 channel gave interesting insight as to howthe ligands may interact with pore residues.

Voltage-gated Na� channels are essential for the initiationand propagation of action potentials in excitable cells and arethe molecular targets for local anesthetics and other com-pounds (1, 2). Themajor structural component of voltage-gatedNa� channels is a large (230–270kDa) �-subunit, which isalone sufficient to form a functional Na� conducting channel.This subunit contains four homologous domains (I–IV), eachcontaining six membrane-spanning segments (S1–S6) (3). Inresponse to membrane depolarization, an outward movementof the positively charged S4 segments induces the conforma-tional changes in the pore leading to the conducting activatedstate (4). The channels then enter inactivated stateswithin a fewmilliseconds of channel opening.

To date, nine distinct�-subunit subtypes (NaV1.1 toNaV1.9)have been identified that differ in their primary structure, ionicpermeation, tissue distribution, functional properties, andpharmacology (5). The NaV1.8 channel is responsible for theslowly-inactivating tetrodotoxin-insensitive Na� current ofsmall diameter neurons of dorsal root ganglion cells (6–8), andis a promising target for the development of anti-nociceptivedrugs (9). The NaV1.8 channel plays a clear role in pain signal-ing following noxious mechanical and cold stimulation, andparticularly in inflammation-induced thermal and mechanicalhyperalgesia (10–14), although the role of this channel in neu-ropathic pain is less certain (10, 15–18). The NaV1.7 channelalso plays a role in pain signaling; indeed lack of function of theNaV1.7 channel in congenital disorders leads to insensitivity tocertain types of pain (19).Local anesthetics and other chemically related agents bind

selectively to the open or inactivated state of sodium channels,leading to use-dependent block during periods of repetitive fir-ing or sustained depolarization (20). Using site-directedmutagenesis, the molecular determinants for drug block inNaV1.2 to NaV1.5 sodium channels were identified as a numberof key pore-lining amino acid residues of the IS6, IIIS6, andIVS6 segments (21, 22). These S6 residues correspond toNaV1.8 amino acids Ile381, Asn390 in domain I, Leu1410, Asn1411,Val1414 in domain III, and Ile1706, Phe1710, Tyr1717 in domain IV(Fig. 1). Thesemutations also produced appreciable shifts in thevoltage dependence of activation and inactivation, supportingthe role of S6 segment amino acid residues in the gating mech-anisms. Thus, the transmembrane S6 segments appear to beimportant for both the functional properties and drug bindingin a number of NaV subtypes.

The NaV1.8 channel shows both slower kinetics and moredepolarized voltage-dependent activation and inactivationthan other NaV subtypes (6–8), although the voltage depend-ence of inactivation of humanNaV1.8 occurs at less depolarizedpotentials (23). Furthermore, despite highly homologous S6segments between NaV1.8 and the other subtypes, differencesin drug action from the other subtypes have been observed. Forexample, compared with tetrodotoxin-sensitive channels, use-dependent block by lidocaine is more pronounced for NaV1.8(24, 25) and remarkably, inactivated state block by compoundA-803467 is more than 100-fold more selective for NaV1.8 (9).The latter compound also showed greater inactivated stateblock of the human NaV1.8 channel than the rat channel (9).These properties have led to the speculation that compoundA-803467 may not bind to the usual local anesthetic binding

* This work was supported by the Biotechnology and Biological SciencesResearch Council and GlaxoSmithKline.

1 To whom correspondence should be addressed. Tel.: 44-113-3434320;E-mail: [email protected].

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 16, pp. 10523–10536, April 17, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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site (26). Furthermore, compound A-803467 showed anunusual removal of resting block (“disinhibition”) for thehuman NaV1.8 channel during repetitive stimulation.2Whereas the role of the S6 transmembrane segments in volt-

age-gated Na� channel functional properties and drug bindingis well established for other subtypes, and the key residues havebeen identified (see above), the corresponding residues for theNaV1.8 subtype are yet to be investigated. Here we have usedwhole-cell patch clamp to study the functional properties ofmutantNaV1.8 channels containing alanine substitutions at thecorresponding key positions in the S6 segments. The effect ofthese mutations on the action of compound A-803467 (Fig. 1)on theNaV1.8 channelwas investigated, and comparedwith theactions of tetracaine (Fig. 1), to further understand the action ofthis compound, which selectively acts on this promising thera-peutic target for anti-nociceptive drugs. In this study for thefirst time the drug binding sites of the NaV1.8 channel havebeen addressed, and furthermore, we have investigated thehuman channel in an appropriatemammalian host backgroundmore like the native situation, rather than in oocytes.

EXPERIMENTAL PROCEDURES

Mutagenesis of Human Nav1.8Channels—The human NaV1.8�-subunit (Swiss-Prot accessionQ9Y5Y9, polymorph 1073V, Ref. 7)in the pFastBacMam1 vector (27)was used in this study. Mutantchannels I381A, N390A, L1410A,V1414A, I1706A, F1710A, andY1717A were generated using theQuikChange XL Mutagenesis Kit(Stratagene) together with appro-priatemutagenic primers. All muta-tions were validated by restrictionmapping and sequencing of theentire channel cDNA.ND7/23 cells (ECACC, Salisbury,

UK) were cultured in Dulbecco’smodified Eagle’s medium supple-mentedwith 2mM L-glutamine, 10%heat-inactivated fetal bovine serum,and 1� non-essential amino acids.Cells were seeded at 60% confluenceand co-transfected with 3.0 �g ofwild type or mutant NaV1.8 �-sub-unit cDNAand 0.3�g of EBO-pCD-Leu2 cDNA (for CD8 marker) orpEGFP-N1 cDNA (for fluorescencemarker) using Lipofectamine 2000(Invitrogen). Transfection-positivecells were identified 2–4 days aftertransfection with Immunobeads(anti-CD8 Dynabeads; Invitrogen)or green fluorescence.Electrophysiological Recording

and Data Analysis—Whole-cell patch clamp recordings wereperformed at room temperature (20–22 °C) using patchpipettes pulled from thin-walled borosilicate glass capillariesand coated with Sigmacote (Sigma). Pipettes (tip resistances1.5–2.5 M�) were filled with solution containing (mM): CsF,120; HEPES, 10; EGTA, 10; and NaCl, 15 adjusted to pH 7.2with CsOH. Cells were continuously perfused with an externalsolution containing (mM): NaCl, 140; HEPES, 5; MgCl2, 1.3;CaCl2, 1; glucose, 11; and KCl, 4.7, adjusted to pH 7.4 withNaOH.Tetrodotoxin (200 nM,Tocris) was used to block endog-enous Na� channel currents.

Cells were allowed to equilibrate for 10min in the whole-cellconfiguration before currents were acquired using an Axo-patch-1C or Axopatch 200B amplifier (Molecular Devices) fil-tered at 5 kHz, and sampled at 10 kHz using pClamp8 orpClamp9 software (Clampex; Molecular Devices). The onlineP/4 subtraction procedure was used to subtract linear leak andcapacitance currents where appropriate. Clampfit versions 9 or10 (Molecular Devices) and Origin version 5.0 (MicroCal Inc.)were used for offline data analysis.Conductance-voltage curves were determined from the peak

sodium current (INa), using the equation:GNa � INa/(V � ENa),2 Browne, L. E., Clare, J. J., and Wray, D. (2009) Neuropharmacology

10.1016/j.neuropharm.2009.01.018.

FIGURE 1. A, the structures of the compounds tetracaine and A-803467. B, alignments of the S6 segments ofdifferent human NaV subtypes. The figure also summarizes results from this study. The residues indicated areshown in this study to be important for the affinity of tetracaine (T) and A-803467 (A) using mutagenesis, ortetracaine (t) and A-803467 (a) using computational modeling. The residues indicated (�) did not appear to beimportant for the binding of tetracaine or A-803467 by mutagenesis or by computational modeling.

Sites of Drug Action on NaV1.8

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where V is the membrane potential and ENa is the reversalpotential obtained from the I–V curve for each cell. Thesecurves were fit with the Boltzmann equation: G � Gmax/(1 �exp((V1⁄2 � V)/k)), whereGmax is maximum conductance, V1⁄2 isthe potential for half-maximal activation, and k is the slopefactor. Voltage-dependence of inactivation curves were fit withthe Boltzmann equation: I � A �(B � A)/(1 � exp((V � V1⁄2)/k)), where B is the maximum current, A is the amplitude of anon-inactivating component, V1⁄2 is the potential for half-max-imal inactivation.Inactivation time constants were obtained by fitting the

decay phase of individual currents with double exponential fits.Recovery from inactivation was studied in twin pulse experi-ments, and the time course of recovery was fit with doubleexponentials; in the presence of some drugs used, a third expo-nential was required.All test compounds were prepared in dimethyl sulfoxide and

diluted to the desired concentration in the external solutiongiving a final concentration of�1%dimethyl sulfoxide. Controltest currents were recorded before the application of com-pounds. Compounds were applied during a 3–4-min incuba-tion period after which currents were recorded in the continualpresence of test compound. The dissociation constants for rest-ing channels (Kr) and inactivated channels (Ki) were deter-mined using themodel of Kuo andBean (28) as described by Liuet al. (29) and Yarov-Yarovoy et al. (30). The resting state drugdissociation constant was calculated using a test pulse beforeand after drug application from a resting potential of�120mV,at which channels are in the resting state. The dissociation con-stants were determined usingKr � [D]/((1/IDr)� 1), whereD isthe concentration of test compound and IDr is the peak testcurrent amplitude in the presence of drug expressed as a frac-tion of the peak test current amplitude in the absence of drug.The protocol for obtaining inactivated state dissociation con-stants is shown in Fig. 5C, where a 4-s depolarizing pulse wasused to inactivate currents, and a test pulse before and afterdrug application was used to measure the extent of inactivatedcurrent block. The inactivated state dissociation constantswerecalculated using Ki � [D](1 � h)/((1/IDi) � 1), where D is theconcentration of test compound, h is the fraction of non-inac-tivated sodium current (in the absence of the compound) fol-lowing the 4-s depolarization, and IDi is the peak test currentamplitude in the presence of drug expressed as a fraction of thepeak test current amplitude in the absence of drug (Fig. 5C).The voltage of the 4-s depolarizing pulse was chosen to alwaysgive approximately the same (60–80%) inactivation in wildtype and mutant channels; the measurement of the Ki valueshould therefore only represent the effect of the mutation ondrug binding rather than any effect of the mutation on inacti-vation itself. Indeed, in previous work the effects of mutationson inactivation and on the Ki value were not found to correlate(29, 30). Statistics are presented as mean � S.E., and the Stu-dent’s t test was used to test significance.Computational Modeling of the Human Nav1.8 Channel and

Docking of Drugs—Previous models of sodium channelsdescribed in the literature have been based on bacterial potas-sium channel structures.More recently, however, a structure ofthe rat KV1.2 potassium channel has been solved (31). It was

considered that this would be closer in structure to humanNaV1.8, and our model was therefore based on its homologywith this structure (PDB code 2A79). Our model consisted ofthe S5 helices, P-loop, and S6 helices (along with the shortlinker between the P-loop and the S6 helix in the first domain).Whereas sequence alignments between the rat KV1.2 channeland NaV1.8 domains were unsuccessful; Kyte-Doolittlehydropathy plots (32) were used to identify the S5-P-loop-S6region of each domain. Alignment of the loops was made so asto form a good filter geometry with the DEKA residues. Align-ment of the S6 regions was straightforward and was based onthe conserved glycine (serine in domain IV). The alignment ofthe S5 helices was not obvious so use was made of the conser-vation moment method using the HELANAL program (33).The starting structure was constructed manually using thehomology tools within the Quanta program (WavefunctionInc.). This was subsequently refined by energy minimizationwith CHARMm (34). Initially the backbone was held fixed butthis was subsequently relaxed and the helices were maintainedby using distance (“nuclear Overhauser effect”) constraintsbetween the backbone amide bonds. 500 steps of SteepestDescent followed by 5000 steps of Adopted Basis Newton-Rhaphson were used for the minimization phases. The Karplusrotamer library (35) was used to set the side chain dihedralangles at standard values. The two ligands were constructedwith the Spartan program using HF-3–21G* charges (accelrys.com). They were docked manually into the protein in multipleposes, ensuring each time that initial side chain dihedral angleswere set at the rotamer library values. Each posewasminimizedas above and ranked according to interaction energy.

RESULTS

Effects of S6 Segment Mutations on the Functional Propertiesof the NaV1.8 Channel—Mutations were chosen at positions inthe S6 segments of domains I, III, and IV of theNaV1.8 channel,corresponding to the positions found in otherNaV channel sub-types where a number of drugs have been found to act (21, 22).Example current traces for each human NaV1.8 channel muta-tion are shown in Fig. 2. The voltage dependence of activation(Fig. 3A) was measured at a holding potential of �120 mV.Conductance-voltage relationships for some of the mutantNaV1.8 channels showed shifts in the curves as compared withwild type channels (Fig. 3A); a 9-mV shift to more positivepotentials was observed for mutations N390A and V1414A(Fig. 3B), and negative shifts of�6mVwere observed formuta-tions I381A and F1710A. The V1⁄2 for activation was unaffectedby mutations L1410A, I1706A, and Y1717A. The k values werenot affected by most of the mutations, although there weresmall but significant reductions for two of themutants (Fig. 3B).To study the effects of the mutations on the voltage depend-

ence of inactivation (Fig. 3C), a 4-s prepulse to various poten-tials was used to inactivate channels followed by a test pulse to0 mV (holding potential �120 mV). The steady-state inactiva-tion curves show that all the mutations except F1710A showedshifts tomore negative potentials (Fig. 3,C andD). The k valueswere not significantly affected as compared with wild type(10.5 � 0.4 mV, n � 84), except for mutations L1410A(decrease in k to 6.4 � 0.4 mV, n � 6) and Y1717A (increase in




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k to 15.6� 1.2mV, n� 14). Forwild type channels, inactivationwas not complete even at very positive inactivating potentials(A/B parameter in Fig. 3D, see “Experimental Procedures”);mutations I1706A, F1710A, andY1717A (all located in the IVS6segment) showed even less complete inactivation at positivepotentials.During depolarizing pulses, inactivation kinetics were faster

than wild type for the mutations. An example current trace forV1414A, and the voltage dependence of the inactivation timeconstant are shown in Fig. 3E for all the mutants. It can be seenthat the time constants were generally reduced for themutants,particularly at the �10 mV test potential (Fig. 3F). Thus themutations studied here enter inactivated states from openstates faster than the wild type NaV1.8 channel.Binding Sites for Tetracaine and A-803467 at Inactivated

Wild Type Channels—Tetracaine and A-803467 have beenshown to bind preferentially to inactivated rather than restingwild type NaV1.8 channels (9, 36). Drugs that preferentially acton the inactivated state rather than on the resting state show ahyperpolarizing shift in the steady-state inactivation curve (�V)without altering the slope (k) (37). Themagnitude of the shift in

the inactivation curvemay be used to determine whether tetra-caine and A-803467 act on the same binding site. Inactivationcurves were obtained in the presence and absence of 10 �Mtetracaine (Fig. 4A), 150 nM A-803467 (Fig. 4B). The valuesobtained for these shifts agree with the predictions calculatedfrom a model with single binding sites (37) (Fig. 4D). For bothdrugs applied together (at concentrations of 5 �M tetracaineand 75 nM A-803467, Fig. 4C), the shift clearly agrees with thepredicted value for overlapping binding sites, rather than sepa-rate binding sites for each drug, again using the models in Ref.37 (Fig. 4D). Details of the formula used in these models aregiven in Fig. 4, legend.Affinity of Tetracaine and A-803467 for Resting and Inacti-

vated Mutant NaV1.8 Channels—To determine the site ofaction for tetracaine andA-803467 on theNaV1.8 channel, herewe have studied the effects of S6 mutations on both the restingand inactivated state affinities.In the resting state (holding potential �120 mV), the extent

of block of a test pulse current by the compounds was used tomeasure the affinity of compounds for the resting state (moreprecisely, dissociation constant, Kr, see “Experimental Proce-dures”). For tetracaine, the resting state affinity was signifi-cantly reduced (i.e. dissociation constants were increased) formutations I381A andF1710Aas comparedwithwild type chan-nels (Fig. 5A). For compound A-803467, an appreciabledecrease in the resting state affinity was only observed formutation F1710A (Fig. 5B). Thus residues Ile381 and Phe1710 areinvolved in the resting state binding of tetracaine, whereas res-idue Phe1710 is involved in resting state binding of compoundA-803467.To calculate the inactivated state affinity of test compounds

formutantNaV1.8 channels, a twin pulse protocol (Fig. 5C) wasused before and after application of tetracaine or A-803467.Before compound application, the fractional amount of non-inactivated current during the test pulse after a 4-s depolariza-tion (h, see “Experimental Procedures”) was obtained from theratio of test to control pulse peak amplitude. After compoundapplication, the fraction of test current not blocked by the com-pounds (IDi, see “Experimental Procedures”) wasmeasured andthe inactivated state dissociation constant,Ki, calculated from hand IDi. Example currents are shown in Fig. 5D. MutationsI381A, F1710A, andY1717A showedmarked decreases in affin-ity for tetracaine (Fig. 5E). For compound A-803467, onlymutation F1710A caused a decrease in the inactivated stateaffinity (Fig. 5F). Thus residue Phe1710 is important in deter-mining both resting and inactivated state affinities for both tet-racaine and A-803467, whereas residues Ile381 and Tyr1717 arealso important for tetracaine binding. The effects of test com-pounds on mutant L1410A channels are considered separatelyin the next section.Tetracaine and A-803467 Drug Block of NaV1.8 Mutation

L1410A—Data for the affinity of mutant L1410A channelscould not readily be obtained using the above protocols becauseeven concentrations 1000 times smaller than used in the previ-ous section still gave almost complete resting block (Fig. 6A)(indicating very high resting state affinities of 10 nM for tet-racaine, and 100 pM for A-803467), and also because ofunusual behaviors under repetitive stimulation (Fig. 6B). As can

FIGURE 2. Example current traces for human NaV1.8 channels. Currenttraces are shown for ND7/23 cells transfected with wild type or mutanthNaV1.8 channel cDNA in the presence of 200 nM tetrodotoxin. Currents wereelicited for voltage steps to �100 to �60 mV (in 10-mV increments) from aholding potential of �120 mV. The peak current amplitudes were �79 � 7pA/pF (n � 79) for wild type NaV1.8, �53 � 5 pA/pF (n � 50) for mutant I381A,�112 � 21 pA/pF (n � 52) for mutant N390A, �32 � 5 pA/pF (n � 31) formutant L1410A, �64 � 6 pA/pF (n � 58) for mutant V1414A, �35 � 4 pA/pF(n � 38) for mutant I1706A, �69 � 7 pA/pF (n � 45) for mutant F1710A, and�40 � 3 pA/pF (n � 54) for mutant Y1717A.




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be seen in the latter figure, in the presence of tetracaine orA-803467, during repetitive stimulation at 10 Hz, currents sur-prisingly increased from almost zero to values comparable with

currents before test compound, as if stimulation removed theunusually high affinity resting block of the compounds(disinhibition).

FIGURE 3. Effects of S6 mutations on Na� channel activation and inactivation. A, conductance-voltage curves are shown for wild type NaV1.8 (f, n � 79),and mutations I381A (F, n � 50), N390A (Œ, n � 52), L1410A (�, n � 31), V1414A (�, n � 58), I1706A (E, n � 38), F1710A (�, n � 45), and Y1717A (ƒ, n � 54).Curves were fit with the Boltzmann equation and normalized to maximum conductance. The pulse protocol is shown in the inset. B, bar diagrams show thevoltage for half-maximal activation (V1⁄2) and the slope factor (k) for wild type and mutant channels (same experiments as in A). C, curves are shown for thevoltage dependence of inactivation for wild type NaV1.8 (f, n � 84), and mutations I381A (F, n � 13), N390A (Œ, n � 14), L1410A (�, n � 6), V1414A (�, n �25), I1706A (E, n � 12), F1710A (�, n � 18), and Y1717A (ƒ, n � 14). The curves were fit with the Boltzmann equation and normalized to the maximum current.The pulse protocol is shown in the inset. D, bar diagrams (same experiments as C) showing the voltage for half-maximal inactivation (V1⁄2), and the amplitudeof the non-inactivated component normalized to the maximum current (A/B, see “Experimental Procedures”). *, p 0.05. E, the inset shows example currentsfor wild type and mutant (V1414A) NaV1.8 channels elicited by a voltage step to 0 mV from a holding potential of �120 mV. Current traces were normalized andsuperimposed. The time constant, �, obtained from the inactivation time course is shown for wild type NaV1.8 (f, n � 56), and mutations I381A (F, n � 35),N390A (Œ, n � 34), L1410A (�, n � 32), V1414A (�, n � 36), I1706A (E, n � 32), F1710A (�, n � 37), and Y1717A (ƒ, n � 38), for the test potentials shown. F, bardiagrams are shown for the mean values of the time constant of inactivation, �, for each mutant at �10 mV test potential (same experiments as in E). *, p 0.05.




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To determine whether sustained depolarization would showa similar effect, a 600-ms conditioning depolarizing pulse wasfirst applied, followed 100 ms later (to allow for recovery frominactivation) by a test pulse (Fig. 6C). The 600-ms pulse is com-parable with the 60 10-ms pulses used during repetitive stimu-lation. Although current during the conditioning pulse wasalmost completely blocked by tetracaine or A-803467, thetest current was comparable with that in the absence of thecompounds (Fig. 6D). Thus it appears that depolarizationremoves the high-affinity resting block of the compounds,underlying the process of disinhibition.The Effect of A-803467 and Tetracaine on the Recovery from

Inactivation of S6 Segment Mutations—The effects ofA-803467 and tetracaine on the recovery from inactivation (inthe continual presence of the compound) were investigatedusing the protocol shown in the inset in Fig. 7C. Following theapplication of A-803467, the time course for recovery involvednot simply the removal of inactivation, but also an additional

component with increased current above resting control values(disinhibition), apparently due to partial removal of the restingblock by stimulation in the presence of the compound. Thiseffect, although small for wild type, can be seen from examplecurrent traces (Fig. 7A). The effect can also be seen for meancurrent values in Fig. 7C, where all currents have been normal-ized to control pulse amplitude in the absence of the com-pound. Recovery from disinhibition followed a very slow timecourse (1.7 � 0.2 s, Fig. 7F).

For themutants in the presence of compoundA-803467 (100nM), this disinhibitory component was larger than for wild type.For instance, example current traces for V1414A clearly showthe effect (Fig. 7B), as do the mean current values for V1414Aand L1410A (Fig. 7,D andE). For the lattermutant (with almostzero current in the resting state in the presence of the com-pound at 100 pM), marked disinhibitory current was seen, cor-responding to extensive removal of resting block during stim-ulation (Fig. 7E). Data for all themutants are summarized in Fig.

FIGURE 4. Binding sites for tetracaine and A-803467 on the wild type NaV1.8 channel. The figure shows steady-state inactivation curves for (A) tetracaine(10 �M, E, n � 4), (B) A-803467 (150 nM, E, n � 4), and (C) tetracaine (5 �M) plus A-803467 (75 nM) (E, n � 8). Control curves in the absence of drug are shown(F, paired values in each case). The protocol used was as in Fig. 3C, and Boltzmann curves fit as before. The shifts in the inactivation curves (�V1⁄2) weredetermined and are shown in D. Predicted values of the shifts are shown for separate binding sites and for overlapping binding sites using the model of Kuo(37). Briefly, in this model, in the presence of a single drug of concentration ([D]), and affinity Ki, the shift is given by �V � k(ln(1 � ([D]/Ki))). For both drugsapplied together, if the two drugs act on an overlapping site, the shift of the inactivation curve is given by �V � k(ln(1 � ([D1]/Ki1) � ([D2]/Ki2))), where [D1] and[D2] are the concentrations of each drug with the respective Ki values Ki1 and Ki2. In contrast, if the two drugs act on separate sites, then the shift in theinactivation curve is given by �V � k(ln(1 � ([D1]/Ki1) � ([D2]/Ki2) � ([D1]/Ki1)([D2]/Ki2))). For the predictions in D, values of Ki are as indicated in Fig. 5, andvalues of k for each drug application were obtained from the above inactivation curves, taking mean values in each case.




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7F, where it can be seen that the disinhibitory component(expressed as a fraction of the current blocked at rest by thecompound) is greater for all the mutants considered than for

wild type. For mutant Y1717A (notshown in the figure), currents weretoo small to be measured in thepresence of 100 nM A-803467; how-ever, even at 10 nM, disinhibitionwas much greater than for wild typeat 10 nM. All mutants (except forI1706A) showed a similar time con-stant for the recovery of this disin-hibitory component (Fig. 7F).For tetracaine (10 �M), by con-

trast, the wild type channel showedno component of disinhibition, ascan been seen from the example ofcurrent traces (Fig. 8A), and the cur-rents during recovery shown in Fig.8C. On the other hand, mutantL1410A showed a striking disinhibi-tory component in the presence oftetracaine (Fig. 8D). For mutantF1710A, a clear disinhibitory com-ponent was observed in the pres-ence of tetracaine (Fig. 8, B and E);this component was relatively largewhen expressed as a fraction of theresting block (Fig. 8F). For the othermutants, disinhibitory componentswere observed in detailed fits to thetime course but their amplitudesrelative to extent of resting blockwere small (Fig. 8F). Time constantsof recovery were in the range 0.8 to1.8 s (Fig. 8F). Taken together thedata show that for tetracaine, muta-tions L1410A and F1710A clearlyinduced the disinhibitory com-ponent, whereas for compoundA-803467, all the mutations consid-ered led to more pronounced disin-hibitory components. The timecourse of recovery of this compo-nent appears to be simply a reflec-tion of the time course of reinstate-ment of the resting block. It is alsonoteworthy that the extent of disin-hibition (Figs. 7F and 8F) for eachmutant did not correlate with theeffects of the mutations themselveson inactivation (Fig. 3), suggestingthat disinhibition is not the result ofaltered channel function per se.Molecular Modeling—A model

was constructed for the S6 and Ploop regions of the NaV1.8 channel,using the alignment shown in Fig. 9

(see “Experimental Procedures”) with the rat KV1.2 channel inthe open state. The model suggests that, unlike the other S6residues mutated in this study, Asn390 and Val1414 do not face

FIGURE 5. Dissociation constants for resting and inactivated states of mutant NaV1.8 channels. A, bardiagrams are shown representing mutant NaV1.8 channel resting state dissociation constants (Kr) for tetra-caine. Values were calculated for tetracaine at 10 �M for wild type NaV1.8 (n � 6) and mutations I381A (n � 7),N390A (n � 8), V1414A (n � 5), I1706A (n � 6), and F1710A (n � 4). B, bar diagrams are shown representingresting state dissociation constants (Kr) for compound A-803467. Values were calculated for A-803467 at 100nM for wild type NaV1.8 (n � 5) and mutations I381A (n � 4), N390A (n � 9), V1414A (n � 6), I1706A (n � 2), andF1710A (n � 7). It was not possible to determine the resting state dissociation constant for mutation Y1717Aunder the conditions used here, because a large proportion (23%) of channels are inactivated at a holdingpotential of �120 mV as a consequence of the strong negative shift in the inactivation curve. C, the figureshows the twin pulse protocol used to determine the inactivated state affinities (Ki). This was used before andafter test compound application; the depicted 4-s depolarizing pulse was to potentials such that 60 – 80%inactivation was observed. In the case of mutant Y1717A, the resting level of inactivation at �120 mV (asabove) was taken into account in obtaining the parameter h. D, the figure shows example current traceselicited by the pulse protocol in C, where currents in bold are before test compound application and fine tracesare after test compound application; the currents larger in magnitude correspond to the control pulses andcurrents smaller in magnitude correspond to the test pulse following a 4-s depolarization. E, bar diagrams areshown representing mutant NaV1.8 channel-inactivated state dissociation constants (Ki) for tetracaine. Valueswere calculated for tetracaine at 1–10 �M for wild type NaV1.8 (n � 7) and mutations I381A (n � 6), N390A (n �6), V1414A (n � 6), I1706A (n � 5), F1710A (n � 7), and Y1717A (n � 7). F, bar diagrams are shown representingmutant NaV1.8 channel-inactivated state dissociation constants (Ki) for compound A-803467. Values werecalculated for A-803467 at 10 –100 nM for wild type NaV1.8 (n � 7) and mutations I381A (n � 6), N390A (n � 7),V1414A (n � 5), I1706A (n � 6), F1710A (n � 7), and Y1717A (n � 7). *, p 0.05.




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into the pore but rather form interhelical interactions withadjacent helices. Thus Asn390 in domain I forms hydrogenbonds with Asn1724 in S6 of domain IV and Thr250 in S5 ofdomain I, whereas Val1414 in domain III makes hydrophobiccontacts with Leu889 and Phe893 in S6 of domain II (Fig. 10, Aand B). These residues are all located in the cytoplasmic side ofthe transmembrane bundle.Using the model, docking studies were performed with tet-

racaine and compoundA-803467 (Fig. 10,C–F). The lack of anyacidic residues in the S6 helices suggested that binding of theprotonated nitrogen of tetracaine and the amide NH ofA-803467 might occur with the aspartate, Asp356 in the P loopof domain I (i.e. the Asp of the DEKA motif). This allowed theligands to adopt poses where all the observed mutation resultscould be explained. Residue Phe1710 forms�-� stackingwith anaromatic ring in both ligands, whereas Leu1410 forms goodhydrophobic interactions with both drugs, consistent with theexperimental finding that its mutation altered the drug action.However, compound A-803467 extends further into the P loopof the channel where the twomethoxy groups of the compoundcan form hydrogen bonds with Thr354 and Ser1660 in P loops.These hydrogen bonds effectively lock A-803467 in this posi-tion so that the substituted ring cannot form a favorable hydro-phobic interaction with Ile381 in S6. The dimethyl amino groupof tetracaine, however, is able to interact favorably with Ile381.The tyrosine residue, Tyr1717 in S6, can form a good hydrogenbond to the ester carbonyl of tetracaine, but it is relatively farfrom the biaryl rings of A-803467, so cannot interact favorablywith it. The experimental results for affinities of the com-pounds found in our mutational study support the model.

DISCUSSION

Functional Properties of NaV1.8 S6 Mutants—Mutations inAsn390 and Val1414 gave positive shifts in steady-state activa-tion, suggesting that the corresponding native residues stabilizeopen states relative to closed states. This seems to be consistentwith our molecular model in the open state, because these two

residues and those that interactwiththem are all located in the cytoplas-mic side of the transmembrane bun-dle, and interactions are probablymore likely to be found in the openstate, so that these residues mayindeed stabilize open states. At res-idues corresponding to these posi-tions in rNaV1.2 and rNaV1.4, simi-lar positive shifts were observed inprevious work (30, 38, 39).Other mutations in NaV1.8 gave

negative shifts in activation (I381AandF1710A), indicating relative sta-bilization of corresponding nativeclosed states. However, for residuescorresponding to these, negativeshifts were not observed for muta-tions in the rNaV1.2 channel (30,40). Furthermore, in contrast to ourresults showing lack of shift for

mutations L1410A and I1706A inNaV1.8, homologous rNaV1.2mutations showed positive shifts (41). Thus, the S6 segmentresidues play an important role in voltage dependence of acti-vation but this role in activation appears to be different for theNaV1.8 channel from other subtypes.

For the voltage dependence of steady-state inactivation inNaV1.8, all mutations studied here (except F1710A) causedstrong negative shifts. One possibility might be that shifts ininactivation curves might simply be the result of shifts in theactivation curves. However, because NaV1.8 activation showedboth negative and positive shifts depending on the mutationstudied, this suggests that inactivation gating is not simplylinked to activation, as already noted for NaV1.4 (42). Thesecurves represent mainly inactivation from closed states (41).Thus, for NaV1.8, closed-state inactivation is less favored forthe native channel than for the mutants. We also showed thatthe time course of inactivation of NaV1.8 was faster for all themutants considered here. Because this represents open-stateinactivation (41), the data show that open-state inactivation isalso less favorable for the native channel than for the mutants.Themutational data shows that all the S6 residues studied hereare involved in inactivation processes in the native channel.As for activation, the effects of mutations on inactivation

appear to be subtype-specific. For closed-state inactivation,shifts for N390A, V1414A, and I381A in NaV1.8 were in theopposite direction to those for corresponding mutations inNaV1.2; there was no shift for F1710A in NaV1.8, whereas cor-respondingmutations inNaV1.2 andNaV1.5 gave positive shifts(30, 40, 41, 43). For open state inactivation (observed from thetime course of decay of currents), all the mutations in NaV1.8caused a faster time course, whereas the corresponding muta-tions in NaV1.2 did not affect it (nor did mutations in NaV1.4corresponding to I1706A and Y1717A in NaV1.8). Also themutation corresponding to F1710A in NaV1.8 was slower forNaV1.4 (30, 40–42). The underlying reason at the molecularlevel for these differences is not known, but may be related to

FIGURE 6. Disinhibition of resting block for mutation L1410A. A, the figure shows example L1410A mutantcurrents in the absence and presence of very low concentrations (indicated) of tetracaine and A-803467. Thecurrent traces were elicited by a test pulse to 0 mV from a holding potential of �120 mV. B, currents, Inorm, formutant L1410A NaV1.8 channels are shown during a 10-Hz train of pulses (10-ms duration to 0 mV from aholding potential of �120 mV), plotted against pulse number and normalized to the first pulse of the untreatedcell. The currents are shown before the application of tetracaine (f, n � 6) or A-803467 (F, n � 8) and aftertetracaine (10 nM, �) or A-803467 (100 pM, E) in paired cells. C, as shown in the protocol, current amplitude wasmeasured at a test pulse following a 600-ms depolarizing pulse to 0 mV and a 100-ms recovery period. D, thebar diagrams show the mean currents using the protocol in C, before (filled bars) and after (unfilled bars) theapplication of tetracaine (10 nM, n � 5) or compound A-803467 (100 pM, n � 6) in paired cells.




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FIGURE 7. The effect of A-803467 on the recovery from inactivation. Example NaV1.8 channel currents are shown for wild type (A) and V1414A mutant (B) in thepresence or absence of A-803467 (100 nM). Currents were elicited by an initial control pulse (to 0 mV), followed by test pulses (to 0 mV) at the indicated times duringrecovery (protocol shown in the inset of C). The graphs show the test pulse amplitude normalized to control pulse during the recovery from inactivation for wild type(C), and example mutations, V1414A (D) and L1410A (E), using the protocol shown in the inset. Time courses of recovery from inactivation are shown before (f) andafter (F) the application of A-803467 (100 nM, except 100 pM for L1410A) in paired cells. F, bar diagrams are shown for the amplitude (Idis, normalized to extent of restingcurrent block) and time course (�) of the slowest component of the three-exponential fit to the time course of recovery of inactivation for wild type (n�5), and mutantsI381A (n � 7), N390A (n � 7), L1410A (n � 5), V1414A (n � 6), I1706A (n � 2), and F1710A (n � 7). The dotted line in C–E represents the level of resting block.




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FIGURE 8. The effect of tetracaine on the recovery from inactivation. Example NaV1.8 channel currents are shown for wild type (A) and F1710A mutant (B) in thepresence or absence of tetracaine (10 �M). Currents were elicited by an initial control pulse (to 0 mV), followed by test pulses (to 0 mV) at the indicated times duringrecovery (protocol shown in the inset of C). The graphs show the test pulse amplitude normalized to control pulse during the recovery from inactivation for wild type(C), and example mutations, L1410A (D) and F1710A (E), using the protocol shown in the inset. Time courses of recovery from inactivation are shown before (f) andafter (F) the application of tetracaine (10 �M, except 10 nM for L1410A) in paired cells. F, bar diagrams are shown for the amplitude (Idis, which is the disinihibitorycomponent I3 expressed as a fraction of resting current block) and time course (�) of the slowest component of the three-exponential fit to the time course of recoveryof inactivation for mutants N390A (n � 4), L1410A (n � 7), V1414A (n � 3), I1706A (n � 3), F1710A (n � 4), and Y1717A (n � 6), whereas wild type (n � 6) and mutantI381A (n � 6) did not show disinhibition. The dotted line in C–E represents the level of resting block.




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the property of slower inactivation observed in native NaV1.8than in other NaV subtypes.For the IVS6 segment mutations I1706A, F1710A, and

Y1717A, but not for the othermutations considered here, therewas incomplete inactivation, even at very positive potentials. Asimilar phenomenon of incomplete inactivation has also beenreported for NaV1.2 for the mutations homologous to F1710Aand Y1717A in NaV1.8, and for NaV1.4 mutation homologoustoN390A inNaV1.8 (40, 44). Thus, as for the other subtypes, forNaV1.8 these residues play an important role in inactivation.Mutation Y1717A gave the largest effect in NaV1.8; as thismutation is located at the intracellularmouth of the porewherethe inactivation domain III–IV linker acts, it may be that thisresidue is somehow involved in the receptor site for fast inacti-vation (40).Effects of S6 SegmentMutations onDrugAction on theNaV1.8

Channel—Tetracaine and A-803467 have been previouslyshown to preferentially bind to inactivated rather than restingwild type NaV1.8 channels (9, 36). Here we have analyzed theactions of tetracaine and compound A-803467 on the humanchannel using an appropriate mammalian expression system.Similar magnitude shifts in the steady-state inactivation curvewere observed following the application of either tetracaine (10�M) or compound A-803467 (150 nM) separately, or followingthe application of both drugs together (at half the above con-centrations). This finding indicates that one drug precludes thebinding of the other. The observed shifts were all of a similarmagnitude to the value predicted by amodel with a single bind-ing site, rather than amodel with separate binding sites for eachcompound. Thus, tetracaine and A-803467 appear to bind tooverlapping, or partially overlapping, binding sites on theNaV1.8 channel.To determine more precisely the site of action of tetracaine

and A-803467 on the NaV1.8 channel, residues were chosen formutation in the S6 regions guided by their role in drug bindingfor other NaV subtypes (21, 22). We have shown that residuesIle381, Leu1410, Phe1710, and Tyr1717 are involved for tetracaineaffinity, but only residues Leu1410 and Phe1710 for A-803467.The roles of these residues in binding of the respective com-pounds was fully supported in our docking study using the

molecular model for NaV1.8 (Fig. 10, C–F). In addition, themodel implicates binding of other residues in the pore region ofthe channel. The S6 residues implicated in binding toNaV1.8 bymolecular modeling and by our mutational studies are summa-rized in Fig. 1B.The increased resting affinity observed with the L1410A

mutant is difficult to explain in our molecular model; one pos-sibility would be that the compounds are being trapped in thebound state, although it is difficult to understand how the ala-nine mutation would enhance trapping. As the mutation didnot show unusual functional properties in the absence of drugs(see above), it is unlikely that themutation causes severe distor-tion of the molecular structure.In previous studies with a range of drugs and NaV subtypes,

the residue corresponding to Phe1710 in NaV1.8 has generallybeen found to be most important in determining inactivatedstate affinity (36). We have indeed found this residue to beimportant in the present study for NaV1.8, both for binding oftetracaine and for A-803467. For tetracaine the F1710A muta-tion reduced the inactivated state affinity far more than theresting affinity, and so this residue has a key role in contributingto the preferential inactivated state block of tetracaine. A sim-ilar result for tetracainewas found forNaV1.3 channels (36). Forcompound A-803467, no previous mutagenesis work has beencarried out, and as mentioned in the Introduction, it was spec-ulated that this drug does not bind to the local anesthetic bind-ing site (26). However, our results for mutation of the localanesthetic binding site Phe1710 indeed further suggests bindingof this compound to at least part of the established local anes-thetic binding site. Although this residue is indeed importantfor binding of compoundA-803467, by contrast with tetracaineother residues not mutated here must also be important forbinding. The reason for this is that mutation of this residue inNaV1.8 affected both resting and inactivated state affinities forA-803467 by similar proportional amounts, and because affin-ity of this compound for inactivated native channels is muchgreater than for resting native channels, other residues mustalso be involved. Our modeling leads us to suggest that otherresidues in S6 regions of all four domains and the P-loop mayalso be important for A-803467 binding (Fig. 10, C–F), and itwould be interesting to investigate the role of these residues infuture mutational studies.Although the residues considered here have been shown to

be involved for a range of NaV subtypes and compounds, tetra-caine has so far only been examined for NaV1.3 where residuescorresponding to Phe1710 (as above) and Tyr1717 are involved,although the interaction with the latter residue was suggestedto be indirect (36). For NaV1.2 channels, the local anestheticetidocaine also has important interactions with these residues(45). The key role for residue Phe1710 in drug binding has alsobeen generally shown forNaV1.3,NaV1.4, andNaV1.5 (22). Thisresidue is proposed to directly bind to local anesthetics by acation-� interaction (46), although our model for NaV1.8 sug-gests a �-� stacking interaction. Overall it is surprising thatcompound A-803467 is selective for NaV1.8, whereas thesequence alignments with other human NaV subtypes (Fig. 1B)show that the residues that we have implicated in drug binding(whether by the mutational study or by modeling) are almost

FIGURE 9. Sequence alignments used in the model. The S5 helices, P-loops,and S6 helices are aligned with the rat KV1.2 channel (31). The DEKA motif inthe filter and the glycines in S6 are shown shaded.




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completely identical between NaV subtypes. Of the residuesmutated here, only the Val1414 residue is different in other sub-types; the isoleucine present in other subtypes at this positionmay contribute to subtype differences, although it is remote tothe ligand binding site. However, the other regions of the chan-nel may indirectly involve drug binding; indeed slow inactiva-tion processes and their conformational implications for thewhole channel have been suggested to play a role in selectivityof compounds (24). Another possible explanation of thereported selectivity may lie in the presence of a double glycinemotif in the S6 helix of domain III. The glycine at 1406 is aserine in other NaV channels. This double glycine greatlyincreases the flexibility in the helix and indeed a briefmoleculardynamics simulation of 200 ps3 showed that Leu1410 movedcloser to the chlorophenyl ring of A-803467, thus further stabi-lizing the interaction. A more extensive mutational study ofother residues coupled with more extensive molecular dynam-ics simulations in other NaV channel models would be requiredin the future to establish the reason for the selectivity ofA-803467 for the NaV1.8 channel.For native NaV1.8 channels, in the continual presence of

compound A-803467, depolarization and subsequent repolar-ization leads not only to inactivation and recovery but also topartial removal of resting block by the compound. The lattermechanism involves a component with an increase in current(disinhibition), an entirely distinct process from removal ofinactivation during recovery. All themutations considered hereincreased this disinhibitory current for A-803467. Anextremely interesting example of this was for mutant L1410A,where resting block by the compoundwas almost complete. Forthis mutant, stimulation in the presence of the compound gavean appreciable current corresponding tomarked removal of theresting block. This phenomenon was observed whether stimu-lation was applied repetitively (Fig. 6B), or after long (600 ms,Fig. 6D) or short (50 ms, Fig. 7E) depolarizations. For the othermutations considered here, whereas resting blockwas generallynot substantially affected, all mutations showed greater disin-hibition than for the wild type channel. Thus all the residuesmutated here appear to be involved in this disinhibitory effectof A-803467.Tetracaine did not show the disinhibitory effect for native

NaV1.8 channels. However, mutations caused the appearanceof disinhibitory components after stimulation in the presenceof tetracaine. As for compound A-803467, tetracaine almost

completely blocked resting channels with high affinity formutant L1410A, and yet stimulation in the presence of tetra-caine again showed a disinhibitory component, whether forrepetitive stimulation or for long or short pulses (Figs. 6 and 8).Of the remaining mutants considered here, clear disinhibitorycurrents were seen for F1710A, although smaller effects wereseen for most of the other mutants. Taken together, the datasuggest that for tetracaine, residues Leu1410 and Phe1710 con-tribute to the mechanism underlying the main disinhibitorycomponent on the NaV1.8 channel.The phenomenon of disinhibition is not easy to understand

by the molecular model. One may speculate that disinhibitionmay be the result of weakened ligand binding to the pore region(and the DEKA filter) caused by channel opening and the com-pound moving away from the pore loop region, so allowingincreased channel currents. With tetracaine, the salt bridgewith Asp356 of the DEKA filter is stronger than the hydrogenbonds formed with A-803467, and so less disinhibition wouldoccur for tetracaine. The hydrogen bonds from the P-loop res-idues Thr354 and Ser1660 toA-803467 hold the ligand to the sideof the P loop and would then perhaps allow partial flow of ionsthrough the channel, perhaps allowing disinhibition for thenative channel in the case of A-803467, although not fortetracaine.Although it seems reasonable to interpret the disinhibitory

component as due to removal of resting block by stimulation inthe presence of the compounds, another possible mechanismmay be the induction of a new single channel state with a higherconductance. Thus it would be interesting to test this in thefuture using single channel recordings. Either way, duringrecovery fromdisinhibition there is reinstatement of the restingequilibrium state in the presence of the drug. Although thedetailedmechanism for this process is not understood, becausethe time course of removal of the disinhibitory component isvery slow (order of seconds), that would imply that the processdoes not involve simply unbinding and rebinding of the drug(which would bemuch quicker). Onemay perhaps hypothesizethat depolarization in the presence of the drug induces a newtype of state with slow recovery back to equilibrium.In summary, our mutational study has indicated that various

residues in the S6 region play important roles in the activationand inactivation properties of the NaV1.8 channel. We haveidentified residues in S6 involved in the resting and inactivatingstate block by tetracaine and A-803467. We also identified res-idues involved in the phenomenon of removal of resting block(disinhibition) by stimulation in the presence of these com-3 F. E. Blaney, unpublished data.

FIGURE 10. Modeling of the pore region of NaV1.8. A, local environment around Asn390, with hydrogen bonds between this residue and Asn1724 on S6 ofdomain IV and Thr250 on S5 of domain I. B, local environment around Val1414, with hydrophobic interactions with Leu889 and Phe893 on S6 of domain II. Theseinteractions occur at the cytoplasmic side of the bundle in the open state model. C, the figure shows docking of tetracaine to IIS6, IVS6, and P-loops, with �-�stacking Phe1710 (in IVS6) and Tyr1717 (in IVS6), whereas the latter residue also forms a hydrogen bond with the ester carbonyl group of tetracaine. Hydrophobicinteractions are also observed between tetracaine and Leu882 (in IIS6) and Leu1711 (IVS6). D, docking is shown for tetracaine to IS6, IIIS6, and P-loops, with theprotonated amino group of the compound forming a salt bridge with Asp356 (in the P-loop), whereas the amino-methyl groups have hydrophobic interactionswith Ile381 (in IS6) and Phe382 (in IS6). The butyl group of tetracaine also forms hydrophobic interactions with Leu1410 (in IIIS6) and Phe1413 (in IIIS6). E, dockingof A-803467 is shown to IIS6, IVS6, and P-loops, with P-loop residues Thr354 and Ser1660 hydrogen bonding to the two methoxy groups of the compound. Thereis �-� stacking between the ligand and Phe1710 (in IVS6) and additional hydrophobic interaction with Leu882 (in IIS6) and Val1714 (in IVS6). F, the figure showsdocking of A-803467 to IS6, IIIS6, and P-loops, with primary interaction with Asp356 (in the P-loop) and Leu1410 (in IIIS6) in the proximity of the chlorophenyl ringof the compound. There is also a hydrogen bond between the amide carbonyl group of the compound and Ser385 (in IS6). Residues Asn390 (in IS6) and Val1414

(in IIIS6) do not interact with the ligand. The figures in C–F show P-loops for domains I and IV and S6 helices for all four domains. The �-carbon ribbons arecolor-coded as follows: gray, pore loops; blue, IS6; yellow, IIS6; orange, IIIS6; red, IVS6. The underlined residues were mutated.




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pounds. The data suggest differing but partially overlappingareas of binding for tetracaine and compound A-803467.

Acknowledgments—We thank Dr. Andrew Powell and Tim Dale forcontinuing expert technical assistance and advice. We are grateful toDr. Lin-Hua Jiang for help with initial experiments in his laboratoryand for comments on the manuscript.

REFERENCES1. Catterall, W. A., Goldin, A. L., andWaxman, S. G. (2005) Pharmacol. Rev.

57, 397–4092. Hille, B. (2001) Ionic Channels of Excitable Membranes, Sinauer, Sunder-

land, MA3. Catterall, W. A. (2000) Neuron 26, 13–254. Yang, N., George, A. L., Jr., and Horn, R. (1996) Neuron 16, 113–1225. Goldin, A. L., Barchi, R. L., Caldwell, J. H., Hofmann, F., Howe, J. R.,

Hunter, J. C., Kallen, R. G.,Mandel, G.,Meisler,M. H., Netter, Y. B., Noda,M., Tamkun, M. M., Waxman, S. G., Wood, J. N., and Catterall, W. A.(2000) Neuron 28, 365–368

6. Akopian, A. N., Sivilotti, L., and Wood, J. N. (1996) Nature 379, 257–2627. Rabert, D. K., Koch, B. D., Ilnicka, M., Obernolte, R. A., Naylor, S. L.,

Herman, R. C., Eglen, R. M., Hunter, J. C., and Sangameswaran, L. (1998)Pain 78, 107–114

8. Sangameswaran, L., Delgado, S. G., Fish, L.M., Koch, B. D., Jakeman, L. B.,Stewart, G. R., Sze, P., Hunter, J. C., Eglen, R.M., andHerman, R. C. (1996)J. Biol. Chem. 271, 5953–5956

9. Jarvis, M. F., Honore, P., Shieh, C. C., Chapman,M., Joshi, S., Zhang, X. F.,Kort, M., Carroll, W., Marron, B., Atkinson, R., Thomas, J., Liu, D., Kram-bis, M., Liu, Y., McGaraughty, S., Chu, K., Roeloffs, R., Zhong, C., Mikusa,J. P., Hernandez, G., Gauvin, D.,Wade, C., Zhu, C., Pai,M., Scanio,M., Shi,L., Drizin, I., Gregg, R., Matulenko, M., Hakeem, A., Gross, M., Johnson,M., Marsh, K., Wagoner, P. K., Sullivan, J. P., Faltynek, C. R., and Krafte,D. S. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 8520–8525

10. Abrahamsen, B., Zhao, J., Asante, C. O., Cendan, C. M., Marsh, S., Mar-tinez-Barbera, J. P., Nassar, M. A., Dickenson, A. H., and Wood, J. N.(2008) Science 321, 702–705

11. Akopian, A. N., Souslova, V., England, S., Okuse, K., Ogata, N., Ure, J.,Smith, A., Kerr, B. J., McMahon, S. B., Boyce, S., Hill, R., Stanfa, L. C.,Dickenson, A. H., and Wood, J. N. (1999) Nat. Neurosci. 2, 541–548

12. Kerr, B. J., Souslova, V., McMahon, S. B., and Wood, J. N. (2001) Neuro-report 12, 3077–3080

13. Laird, J. M., Souslova, V., Wood, J. N., and Cervero, F. (2002) J. Neurosci.22, 8352–8356

14. Nassar, M. A., Levato, A., Stirling, L. C., andWood, J. N. (2005)Mol. Pain1, 24

15. Joshi, S. K., Mikusa, J. P., Hernandez, G., Baker, S., Shieh, C. C., Neelands,T., Zhang, X. F., Niforatos, W., Kage, K., Han, P., Krafte, D., Faltynek, C.,Sullivan, J. P., Jarvis, M. F., and Honore, P. (2006) Pain 123, 75–82

16. Lai, J., Gold, M. S., Kim, C. S., Bian, D., Ossipov, M. H., Hunter, J. C., andPorreca, F. (2002) Pain 95, 143–152

17. Porreca, F., Lai, J., Bian, D., Wegert, S., Ossipov, M. H., Eglen, R. M.,

Kassotakis, L., Novakovic, S., Rabert, D. K., Sangameswaran, L., andHunter, J. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7640–7644

18. Dong, X. W., Goregoaker, S., Engler, H., Zhou, X., Mark, L., Crona, J.,Terry, R., Hunter, J., and Priestley, T. (2007) Neuroscience 146, 812–821

19. Cox, J. J., Reimann, F., Nicholas, A. K., Thornton, G., Roberts, E., Springell,K., Karbani, G., Jafri, H., Mannan, J., Raashid, Y., Al-Gazali, L., Hamamy,H., Valente, E. M., Gorman, S., Williams, R., McHale, D. P., Wood, J. N.,Gribble, F. M., and Woods, C. G. (2006) Nature 444, 894–898

20. Hille, B. (1977) J. Gen. Physiol. 69, 497–51521. Catterall, W. A. (2002) Novartis Found. Symp. 241, 206–218; Discussion

218–23222. Nau, C., and Wang, G. K. (2004) J. Membr. Biol. 201, 1–823. Akiba, I., Seki, T., Mori, M., Iizuka,M., Nishimura, S., Sasaki, S., Imoto, K.,

and Barsoumian, E. L. (2003) Receptors Channels 9, 291–29924. Leffler, A., Reiprich, A.,Mohapatra, D. P., andNau, C. (2007) J. Pharmacol.

Exp. Ther. 320, 354–36425. Roy, M. L., and Narahashi, T. (1992) J. Neurosci. 12, 2104–211126. Cummins, T. R., and Rush, A. M. (2007) Exp. Rev. Neurother. 7,

1597–161227. Condreay, J. P., Witherspoon, S. M., Clay, W. C., and Kost, T. A. (1999)

Proc. Natl. Acad. Sci. U. S. A. 96, 127–13228. Kuo, C. C., and Bean, B. P. (1994)Mol. Pharmacol. 46, 716–72529. Liu, G., Yarov-Yarovoy, V., Nobbs, M., Clare, J. J., Scheuer, T., and Catter-

all, W. A. (2003) Neuropharmacology 44, 413–42230. Yarov-Yarovoy, V., McPhee, J. C., Idsvoog, D., Pate, C., Scheuer, T., and

Catterall, W. A. (2002) J. Biol. Chem. 277, 35393–3540131. Long, S. B., Campbell, E. B., and Mackinnon, R. (2005) Science 309,

897–90332. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105–13233. Blaney, F. E., and Tennant, M. (1996) inMembrane ProteinModels (Find-

lay, J. B. C., ed) pp. 161–176, Bios Scientific Publishers Ltd., Oxford34. Brooks, B. R., Bruccoleri, R. E., Olafson, B. D., States, D. J., Swaminathan,

S., and Karplus, M. (1983) J. Comput. Chem. 4, 187–21735. Dunbrack, R. L., Jr., and Karplus, M. (1993) J. Mol. Biol. 230, 543–57436. Li, H. L., Galue, A., Meadows, L., and Ragsdale, D. S. (1999)Mol. Pharma-

col. 55, 134–14137. Kuo, C. C. (1998)Mol. Pharmacol. 54, 712–72138. Nau, C., Wang, S. Y., Strichartz, G. R., andWang, G. K. (1999)Mol. Phar-

macol. 56, 404–41339. Wang, S. Y., and Wang, G. K. (1997) Biophys. J. 72, 1633–164040. McPhee, J. C., Ragsdale, D. S., Scheuer, T., and Catterall, W. A. (1995)

J. Biol. Chem. 270, 12025–1203441. Yarov-Yarovoy, V., Brown, J., Sharp, E. M., Clare, J. J., Scheuer, T., and

Catterall, W. A. (2001) J. Biol. Chem. 276, 20–2742. Kondratiev, A., and Tomaselli, G. F. (2003)Mol. Pharmacol. 64, 741–75243. Carboni, M., Zhang, Z. S., Neplioueva, V., Starmer, C. F., and Grant, A. O.

(2005) J. Membr. Biol. 207, 107–11744. Nau, C., Wang, S. Y., and Wang, G. K. (2003) Mol. Pharmacol. 63,

1398–140645. Ragsdale, D. S.,McPhee, J. C., Scheuer, T., andCatterall,W.A. (1996)Proc.

Natl. Acad. Sci. U. S. A. 93, 9270–927546. Ahern, C. A., Eastwood, A. L., Dougherty, D. A., and Horn, R. (2008) Circ.

Res. 102, 86–94




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Liam E. Browne, Frank E. Blaney, Shahnaz P. Yusaf, Jeff J. Clare and Dennis Wray1.8 ChannelvStructural Determinants of Drugs Acting on the Na

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