Molecular Interactions of the Gating Modifier Toxin ProTx-IIwith Nav1.5IMPLIED EXISTENCE OF A NOVEL TOXIN BINDING SITE COUPLED TO ACTIVATION*□S

Received for publication, November 9, 2006, and in revised form, February 15, 2007 Published, JBC Papers in Press, March 5, 2007, DOI 10.1074/jbc.M610462200

Jaime J. Smith‡, Theodore R. Cummins§, Sujith Alphy‡, and Kenneth M. Blumenthal‡1

From the ‡Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York,Buffalo, New York 14214 and §Department of Pharmacology and Toxicology, Indiana University School of Medicine,Indianapolis, Indiana 46202

Voltage-gated Na� channels are critical components in thegeneration of action potentials in excitable cells, but despitenumerous structure-function studies on these proteins, theirgatingmechanism remains unclear. Peptide toxins oftenmodifychannel gating, thereby providing a great deal of informationabout these channels. ProTx-II is a 30-amino acid peptide toxinfrom the venom of the tarantula, Thrixopelma pruriens, thatconforms to the inhibitory cystine knot motif and which modi-fies activation kinetics of Nav and Cav, but not Kv, channels.ProTx-II inhibits current by shifting the voltage dependence ofactivation tomore depolarized potentials and, therefore, differsfrom the classic site 4 toxins that shift voltage dependence ofactivation in the opposite direction. Despite this difference infunctional effects, ProTx-II has been proposed to bind to neu-rotoxin site 4 because it modifies activation. Here, we investi-gate the bioactive surface of ProTx-II by alanine-scanning thetoxin and analyzing the interactions of each mutant with thecardiac isoform, Nav1.5. The active face of the toxin is largelycomposed of hydrophobic and cationic residues, joining a grow-ing group of predominantly Kv channel gating modifier toxinsthat are thought to interact with the lipid environment. In addi-tion, we performed extensive mutagenesis of Nav1.5 to locatethe receptor site with which ProTx-II interacts. Our data estab-lish that, contrary to prior assumptions, ProTx-II does not bindto the previously characterized neurotoxin site 4, thusmaking ita novel probe of activation gating inNav channels with potentialto shed new light on this process.

Voltage-gated cation channels are integral membrane pro-teins that play a critical role in electrical signaling by controllingthe flow of Na�, K�, and Ca2� across the plasma membrane inresponse to changes in voltage. Nav channel � subunits arecomposed of four homologous domains, DI-DIV, each havingsix transmembrane segments, S1-S6. The first four segments ofeach domain comprise the voltage sensor of these proteins,

whereas S5 and S6 form the central ion conducting pore (forreview, see Ref. 1). The homologous Kv channels are tetramersof four identical subunits, each similar to aNav channel domain.

Site-directed fluorescent labeling has shown that Navdomains I and IImovewith activation and are unaffected by fastinactivation gating, whereas domains III and IV exhibit kineticcomponents associated with deactivation and fast inactivation(2, 3). DIV-S4 has a unique role in gating in that its charges onlymove during inactivation (4, 5), and there is evidence for strongcooperativity among Nav channel domains throughout the gat-ing process. In contrast, subunit coupling is only seen late in Kvchannel gating transitions (6–8). It has been suggested that thismajor difference between Nav and Kv channel kinetics under-lies the basis of fast electrical transmission, i.e. domain cooper-ativity is necessary for the rapid upstroke of an action potential(6). The functional differences among Nav channel domainsraise the possibility that distinct domain structures might existas well.Given the many distinctions between Nav and Kv channels,

detailed studies of Nav channel gating mechanisms are essen-tial. Studies using neurotoxins that bindNav channels with highaffinity to alter conductance or gating properties have beenextremely useful for this purpose. Polypeptide toxins derivedfrom the venom of spiders, sea anemones, scorpions, and snailsinteract with voltage sensors to modify activation or inactiva-tion and have been tremendously useful probes of gatingmech-anisms. These gating modifier toxins bind to sites 3 and 4,respectively. Site 3 has been localized to the extracellular S3/S4linker of domain IV (9, 10), whereas residues in domain II S3/S4make a major contribution to site 4 (11, 12).Site 3 toxins, such as those from sea anemone venom, delay

channel inactivation upon binding,most likely by inhibiting thenormal outward movement of gating charges in DIV, resultingin the inability of the inactivation particle to mobilize (5). Incontrast, site 4 toxins enhance channel activation and shift thevoltage dependence of activation to more hyperpolarizedpotentials via a voltage sensor-trapping mechanism (11, 13). Itis thought that the open probability of the channel increasesbecause of the toxin locking the channel in its activated confor-mation after a depolarizing pre-pulse. ProTx-II is a 30-aminoacid peptide toxin purified from the venom of the tarantula,Thrixopelma pruriens, that modifies activation of both Nav andCav, but not Kv, channel isoforms by inhibiting peak currentand shifting the voltage dependence of activation tomore depo-

* The costs of publication of this article were defrayed in part by the paymentof page charges. This article must therefore be hereby marked “advertise-ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1.

1 To whom all correspondence and reprint requests should be addressed:Dept. of Biochemistry, School of Medicine and Biomedical Sciences, StateUniversity of New York, 3435 Main St., Buffalo, NY 14214. Tel.: 716-829-2727; Fax: 716-829-2725; E-mail: kblumen@buffalo.edu.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 17, pp. 12687–12697, April 27, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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larized potentials (14, 15). ProTx-II conforms to the inhibitorycystine knot (ICK)2 motif, a common structural fold amongspider toxins targeting ion channels (see The KNOTTIN data-base online). ICK peptides are defined by a 1-4, 2-5, 3-6 cystineconnectivity and often have limited regular secondary struc-ture. Based on its ability to modify activation, but not inactiva-tion kinetics, it has been suggested that ProTx-II binds to site 4,but no direct evidence exists to validate this claim (14–17).The Nav channel isoform-specific actions of other tarantula

venom ICK peptide toxins have been characterized. These tox-ins modify activation by inhibiting sodium current and causinga depolarizing shift in gating (16). Like ProTx-II, their receptorsites have not been identified. However, detailed studies on theKv channel ICK toxins hanatoxin and SGTx have shown thatthey inhibit potassium current by shifting channel opening tomore depolarized potentials via an interaction with a receptorsite on the C-terminal end of S3 near the extracellular surface(18, 19). The similar functional effects of ProTx-II and these Kvchannel toxins on their targets raise the possibility that theirreceptor sites are similar as well, although Nav channel asym-metrywill likely introduce an additional level of complexity intobinding site identification.Because ProTx-II modifies activation in a manner distinct

from the previously characterized site 4 toxins, it is importantto understand the basis for its activity. In the present study weinvestigate the bioactive surface of ProTx-II by alanine-scan-ning the peptide and analyzing the interactions of the mutantswith the cardiac isoform, Nav1.5, by whole-cell voltage clamp.In addition, we carried out extensive mutagenesis of Nav1.5 toascertain whether ProTx-II, like other toxins that modify Navchannel activation, interacts with receptor site 4. Our resultsindicate that the active face of ProTx-II consists ofmany hydro-phobic as well as cationic residues that likely interact with areceptor site on Nav channels that is separate and distinct fromsite 4.

EXPERIMENTAL PROCEDURES

Molecular Biology

ProTx-II—The ProTx-II coding sequence and upstreamenterokinase site were amplified from a previously constructedexpression vector in our laboratory (15) using standard PCRprocedures and cut with EcoRI/HindIII using sites introducedin the primers. The cleaved product was then subcloned into anoctahistidine version of the pMALc2x vector (New EnglandBiolabs) between EcoRI and HindIII using standard molecularbiology protocols. As described previously, two additionalN-terminal amino acids derived from a StuI restriction siteremain in the ProTx-II coding sequence (15). Site-directedmutagenesis was performed using the QuikChange method(Stratagene, La Jolla, CA) to create all recombinant toxinmutants described in this paper, and all constructswere verifiedby sequencing.

Molecular Biology

NaV1.5—The pBluescript plasmid containing the SCN5Agene encoding humanNav1.5� subunitwas used for all channelmutant constructs.We initially sought to swap the extracellularS3/S4 linkers in domains II and IV to assess the functionaleffects of toxins that are known to bind these regions. Toreplace DIV S3/S4 with its DII counterpart, five residues(SPTLF) were deleted in DIV S3/S4 using a loop out PCR pro-cedure to obtain a DIV S3/S4 linker that matched the length ofDII S3/S4. The remaining linker residues were thenmutated tomatch the sequence of DII- S3/S4 using the QuikChangemethod.We named this construct II:II. To create the constructin which the DIV S3/S4 linker replaced the corresponding DIIlinker, splicing by overlap extension (20) was used to insert fiveresidues (SPTLF) downstream of the DII S3/S4 linker to matchthe length of DIV S3/S4. This was followed by mutation of theeight upstream residues in DII S3/S4 to match the completesequence to that of DIV S3/S4. We called this construct IV:IV.The swapped construct was created by digesting the IV:IV con-structwithAgeI/NheI to remove the newly created S3/S4 linkerregion in DII and subcloning that region into the II:II constructdigested with AgeI/NheI. This new construct became IV:II.Sites in the remaining extracellular linker regions were tar-

geted bymutating residues inNav1.5 that differ fromNav1.7, anisoform for which ProTx-II has a 100-fold higher affinity (14,21). 1–7 amino acidmutations weremade in a single primer setto account for any binding determinant in that particular linkerusing an inverse PCR protocol. Transmembrane segmentmutations were made as single amino acid replacements.

Production of Recombinant Toxins

Wild-type and mutant toxins were expressed as fusion pro-teins containingmaltose-binding protein upstream of the toxinin Escherichia coli BL21 (DE3) as described (15). After lysis in aFrench press, the supernatants obtainedwere purified onNi2�-nitrilotriacetic acid resin and reduced with 10 mM dithiothrei-tol for 1 h at 37 °C. After diluting the proteins to 0.2mg/ml, theywere dialyzed against 2.5 mM GSH, 50 mM Tris, 100 mM NaCl,pH 8.3. After dialysis, the proteins were oxidized by dropwiseaddition ofGSSG to a final concentration of 0.5mMand allowedto incubate for 72 h. Fusion proteins were then dialyzed against50 mM NH4HCO3 and cleaved overnight at room temperaturewith enterokinase (Novagen/EMD Biosciences). Toxins werepurified via RP-HPLC as described (15). Molecular weights ofpurified toxinswere confirmed byMALDI-TOFmass spectros-copy analysis on a Bruker Biflex IV spectrometer. Because offolding difficulties encountered with a subset of mutants, somepositions were mutated to an amino acid with a larger sidechain to facilitate proper packing. These include K4Q, R13Q,W24L, K26Q, and K27Q.

Folding of Synthetic Toxins

In addition to recombinant mutant toxins, some syntheticmutants were studied, including Y1A, S11A, K14A, E17A,L23A, K28A, L29A, and W30A (GenScript Corp., Piscataway,NJ). Lyophilized peptides were resuspended to a peptide con-centration of 5 mg/ml in nitrogen saturated 8 M urea, 50 mMTris, 50 mM NH4HCO3, 120 mM GSH, pH 7.8. The peptides

2 The abbreviations used are: ICK, inhibitory cystine knot; HPLC, high perform-ance liquid chromatography; RP, reverse phase; MALDI-TOF, matrix-as-sisted laser desorption ionization time-of-flight; HEK cells, human embry-onic kidney cells; SGTx, toxin 1 from the spider Scodra griseipes.

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were then diluted to a concentration of 0.5 mg/ml and a GSHconcentration of 12 mM. A final dilution brought the peptideconcentration to 0.125 mg/ml and urea to 2 M, whereas GSHremained at 12 mM. GSSG was then added dropwise to a finalconcentration of 1.2 mM and allowed to incubate at 4 °C for48 h. Samples were taken at various stages throughout the fold-ing reactions for RP-HPLC analysis. MALDI-TOF analysis ofsamples confirmed that the peptides were oxidized. To purifyfolded peptides, we used cation exchange chromatography(HiTrap SP FF,GEHealthcare) followed byRP-HPLC.Wewereunable to produce significant amounts of the G18A mutant,presumably because of its inability to fold.

Cell Culture and Electrophysiology for Wild-type and MutantProTx-II Studies

All cell culture reagents were purchased from Invitrogen.Standard whole-cell voltage-clamp recordings weremade fromall cells. To analyze the effects of wild-type and mutantProTx-II on the human cardiac Nav channel, a stable cell lineexpressing Nav1.5 was constructed in HEK 293 cells as previ-ously described (22). To verify previously reported affinity datafor ProTx-II on the peripheral nerve Nav channel, a stable HEK293 cell line expressing human Nav1.7 was utilized (23). Tostudy the effects of ProTx-II on neuronal Nav channels, themurine neuroblastoma cell line, N1E-115 was obtained fromATCC (Manassas, VA). Because these cells predominantlyexpress Nav1.2, we ascribe ProTx-II modification to this iso-form (24). Cells were maintained in Dulbecco’s modifiedEagle’smedium supplementedwith 10% fetal bovine serum and1% penicillin/streptomycin at 37 °C in a 5% CO2 atmosphere.200 �g/ml G418 was used for selection of HEK 293 cellsexpressing Nav1.5 or Nav1.7 channels. Toxin affinities forNav1.5 and Nav1.2 channels were measured after introductionby gravity perfusion into a 300-�l bath chamber at a flow rate of�3ml/min. For Nav1.7 measurements, toxin was diluted into a250-�l recording chamber and mixed by repeatedly pipetting25�l over�5 s to achieve the specified concentration. All toxinsolutions contained 1 mg/ml bovine serum albumin to preventadsorption to tubing. Single cell recordings were made at roomtemperature using an Axopatch 200B amplifier with a Digidata1322A analogue to digital converter and pCLAMP software(Axon Instruments). Pipettes were pulled from borosilicateglass (World Precision Instruments) and fire-polished to a finalresistance of 1–3 megaohms when filled with recording solu-tion. Solutions used for sodium current measurementsthrough Nav1.5 channels contained the following: bath solu-tion, 10 mM NaCl, 130 mM CsCl, 2 mM CaCl2, 10 mM HEPES,pH 7.4 with CsOH; pipette solution, 95 mM CsF, 30 mM CsCl,5 mMNaCl, 10 mM EGTA, 10 mMHEPES, pH 7.0 with CsOH.Solutions used for Nav1.7 channels contained the following:bath solution, 140 mM NaCl, 3 mM KCl, 1 mM MgCl2, 1 mMCaCl2, 10 mM HEPES, pH 7.3; pipette solution, 140 mM CsF,1 mM EGTA, 1 mM MgCl2, 10 mM NaCl, 10 mM HEPES, pH7.3. Solutions used for Nav1.2 channels contained: bath solu-tion, 70 mM NaCl, 70 mM CsCl, 2 mM CaCl2, 10 mM HEPES,pH 7.4; pipette solution, 10 mM NaCl, 90 mM CsF, 30 mMCsCl, 10 mM EGTA, 10 mM HEPES, pH 7.0. Recordings wereinitiated 5–8 min after patch rupture.

To measure time courses of modification and dissociation,cells were held hyperpolarized at �130 mV then stepped toeither�30mV (Nav1.5 channels) or�10mV (Nav1.2 channels)for 10 ms at a frequency of 1 Hz as either toxin-containing ortoxin-free solution was introduced into the bath. To generatecurrent-voltage (I-V) relationships, cells were stepped in 5-mVincrements from �80 to �20mV (Nav1.5 channels), from �80to�70mV (Nav1.7 channels), or from�50 to�50mV (Nav1.2channels) from a holding potential of �120 or �130 mV for 30ms (Nav1.5 and 1.2) or 50 ms (Nav1.7), and the resulting peakcurrentswere plotted against voltage. Current-voltage relation-ships were obtained just before toxin application and aftersteady-state inhibition was achieved. Only cells having a�4.2-mV slope were included. Currents were capacity-cor-rected using MatLab 6.5 (The MathWorks, Inc., Natick, MA).Only cells with a leak resistance of �750 megaohms wereincluded in analyses. Toxin test concentrations ranged from250 to 20 �M and were determined empirically for each toxinmutant.Conductance-voltage (g-V) relationships quantitating the

voltage dependence of activation were obtained from peak cur-rent-voltage (I-V) relationships according to g � INa/V � Vr,where INa is the peak Na� current at test potential V, and Vrrepresents reversal potential. To assess the voltage dependenceof inactivation, a 2-step protocol was used in which cells werestepped in 5-mV increments from�130 to�30mV for 300msfollowed by a step to the test potential �30 mV for 20 ms toevaluate channel availability. Normalized activation andinactivation curves were fit to a Boltzmann function y � 1/[1 � e(V � V0.5)/k], where y is normalized gNa or INa, V is mem-brane potential, V0.5 is the midpoint of activation or inactiva-tion, and k is the slope factor. Data are depicted as �S.E.The half-blocking concentration (IC50) for ProTx-II on

Nav1.7 was calculated based on the single-site Langmuir inhi-bition isotherm using the following function: (Itoxin/I0) �[toxin]/(1 � Itoxin/I0), where I0 and Itoxin are the peak sodiumcurrentsmeasuredwith a test pulse to�30mVbefore and afterapplication of toxin, respectively, and[toxin] is the concentra-tion of toxin.

Cell Culture and Electrophysiology for Mutant NaV1.5 Studies

Mutant channel DNA was transiently transfected into HEK293 cells followed bywhole-cell voltage clamp analysis to assessmutant channel function and interaction with wild-typeProTx-II. Cells weremaintained inDulbecco’smodified Eagle’smedium supplemented with 10% fetal bovine serum and 1%penicillin/streptomycin. Lipofectamine reagent in combina-tion with PLUS reagent (Invitrogen) was used for transienttransfections, and after 24–36 h of incubation cells weretrypsinized and moved to coverslips for analysis. Because oflower expression levels of mutant channels, [Na�] wasincreased in the bath solution to either 70mM (mediumNa�) or140mM (high Na�), and [Cs�] was adjusted accordingly. Othercomponents were kept constant as described above. Corre-sponding pipette solutions contained either 35 mM NaCl, 65mM CsF (medium Na) or 70 mM NaCl, 30 mM CsF (high Na).The voltage dependence of activation and inactivation of linkerswap mutants was analyzed and compared with wild-type

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Nav1.5 to ensure normal channel function using protocolsdescribed above. A wild-type toxin concentration of 1 �M wasused to assess modification of mutant channels. We looked fora loss of inhibition during simple step protocols from �130 to�30 mV that would indicate the receptor site on the channelhad been disrupted.

Kinetic Calculations

Peak currents were plotted versus time from the point oftoxin wash-in or wash-out. The data were fit to a first orderexponential decay equation usingOrigin 6.1 software (MicrocalSoftware Inc., Northampton, MA). We calculated the kineticconstants for channel modification (kmod) and toxin dissocia-tion (koff) using the inverse of � of the fit for toxin wash-in andwash-out, respectively. To determine the rate of toxin associa-tion, the following equationwas used: kon � kmod � koff/[toxin].The dissociation constant was determined using koff/kon � KD.Data are reported as �S.E.

Molecular Modeling

An energy-minimized molecular model of ProTx-II was cre-ated using the Protein Data Bank coordinates for HpTx-2, anICK motif peptide targeting Kv4 channels (PDB code 1emx;Refs. 25 and 26). Conversion to the ProTx-II sequencewas donein the Biopolymer module of InsightII, and the resulting modelwas then subjected to energy minimization (initially using asteepest descents protocol followed by at least 2500 cycles ofconjugate gradients) in Discover to remove steric clashes. Afterminimization, the total energy of themodel structurewas�300kcal/mol, and it retained the backbone structure typical of theICK motif.

RESULTS

Functional Characterization ofWild-type andMutant Formsof ProTx-II—The first aim of the present study was to charac-terize the effects of ProTx-II mutants on Nav1.5 to isolate thepharmacophore of the toxinmolecule. In addition, we hoped toidentify the channel receptor site and ultimately establish amechanism of action for this novel acting peptide toxin. Weproduced wild-type ProTx-II and several mutated formseither recombinantly or synthetically and purified them tohomogeneity using RP-HPLC. To characterize the effects ofwild-type or mutated ProTx-II on Nav1.5, we used a whole-cell voltage clamp on HEK 293 cells stably expressing thischannel. We evaluated the extent of channel modification bydepolarizing the cell membrane to �30 mV as toxin-freesolution was replaced with toxin-containing solution. At 1�M ProTx-II, we observed rapid and near complete inhibitionof sodium current (� � 2.5 s) (Fig. 1A). This inhibition wascompletely reversible upon toxin wash-out (� � 40 s) (Fig. 1B).To verify that toxin binding is concentration-dependent, weexamined channelmodification over the range of 250–1000 nMProTx-II. Modification decreased accordingly at both concen-trations (�250 nM � 9.3 � 0.23 S, n � 3; �500 nM � 5.48 � 0.17 S,n� 3). In contrast, dissociation remained a zero-order reactionas expected (�250 nM � 53.3� 3.2 S, n� 3; �500 nM � 41.7� 2.2 S,n � 3). ProTx-II also shifts the voltage dependence of gating tomore depolarized potentials, indicating that the toxin does not

inhibit through a pore-blocking mechanism but, rather, inter-feres with the energetics of gating (15). Analysis of ProTx-II onsteady-state activation and inactivation kinetics revealed thatthe midpoint of the activation curve shifted by 23 mV in thedepolarizing direction, whereas inactivation remained unaf-fected by the toxin (Fig. 2, A and B). This mode of channelmodification is similar to that of other ICK toxins that targetNav and Kv channels but very different from site 4 toxins tar-geting Nav channels. Our kinetic analysis yielded an equilib-rium dissociation constant for ProTx-II of 93 nM, similar to thevalue obtained using natural material (14).To identify side chains of ProTx-II that play key functional

roles, we introduced point mutations at every position outsideof the cystine framework. To overcome folding difficultiesencountered with some mutants, it was necessary to introduceamino acids other than alanine at some positions. We consider

FIGURE 1. Kinetics of ProTx-II modification and dissociation. HEK 293 cellsstably expressing Nav1.5 were stepped to �30 mV from a holding potential of�130 mV for a duration of 10 ms at a frequency of 1Hz. A, the addition of 1 �M

ProTx-II, indicated by the arrow, results in almost complete inhibition of Na�

current (� � 2.5 s). The first and last current traces from toxin wash-in areshown in the inset. B, ProTx-II inhibition is completely reversible upon toxinwash-out (� � 40 s); same representative cell as shown in panel A. Data arenormalized to the peak current value. (n � 12 cells).

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a mutant properly folded based on several criteria. First, anHPLC retention time similar to wild type as well as a distribu-tion as a dominant homogenous chromatographic form sug-gests a correct folding pattern. Second, a molecular weightidentical to the calculatedmass of oxidized toxin as analyzed byMALDI-TOF indicates that all disulfide bonds have formed.Finally, a circular dichroism spectrum that overlays that ofwild-type toxin is characteristic of a normal fold. Fig. 3 com-pares the far UV CD spectra of wild-type ProTx-II to those ofthe homologous ICK motif-gating modifiers HpTx-2 andGsMTx-4. The CD spectra of all ProTx-II mutants we exam-ined overlay with that obtained from wild-type toxin and aresimilar to those of HpTx2 and GsMTx4. G18A was the only

mutant toxin that we were unable to produce in significantyield, perhaps indicating a unique requirement for flexibility atthis position during folding. All other mutants met the abovecriteria.After purification of each mutant toxin, we characterized its

interaction with Nav1.5 as described for wild-type ProTx-IIusing an empirically determined test concentration. Mutanttoxins were tested at concentrations at or above theirKD exceptin cases where insufficient availability of toxin made this pro-hibitive. Onset kinetics for a subset of mutants are comparedwith that of wild-type ProTx-II in Fig. 4. We define a residue asessential if its mutation results in a loss of affinity of at least

FIGURE 2. ProTx-II modifies the steady-state voltage dependence of acti-vation, but not inactivation, of NaV1.5 channels. A, ProTx-II shifts thesteady-state activation curve 23 mV in the depolarizing direction. Cells (n �10) stably expressing Nav1.5 were stepped in 5-mV increments from �80 to�20 mV from a holding potential of �130 mV for 30 ms. For control cells (E),the midpoint of activation Va � �42.5 � 0.57mV, and the slope factor k �5.91 � 0.22 mV; for cells treated with 1 �M ProTx-II (F), midpoint of activationVa � �19.6 � 0.5 mV, and the slope factor k � 7.48 � 0.32 mV. B, ProTx-II hasno effect on steady-state inactivation. Cells (n � 8) were stepped in 5-mVincrements from �130 mV to �30 mV for 300 ms followed by a test pulse to�30 mV for 20 ms. For control cells (E), the midpoint of inactivation Vh ��79.2 � 0.7 mV, and the slope factor k � 4.26 � 0.20 mV; for cells treated with1 �M ProTx-II (F), the midpoint of inactivation Vh � �79.5 � 0.7 mV, and theslope factor k � 3.91 � 0.44 mV. Normalized conductances (activation) andcurrents (inactivation) were generated for both data sets, fit to a Boltzmannfunction, and are shown as data � S.E.

FIGURE 3. Circular dichroism spectra of ICK motif toxins. Spectra ofProTx-II (‚), HpTx-2 (E), and GsMTx-4 (�) were recorded at peptide concen-trations of 0.15 mg/ml in a Jasco-720 spectropolarimeter at a scan rate of 20nm/min in 5 mM sodium phosphate buffer, pH 6.9. All spectra shown repre-sent the average of four complete sweeps and were smoothed using theJasco data analysis package. The CD spectra of all ProTx-II mutants reported inTable 1 overlay that of the wild-type toxin.

FIGURE 4. Modification of NaV1.5 currents by ProTx-II mutants. Onsetkinetics for toxins with mutations to hydrophobic or cationic residues aredramatically decreased as compared with wild-type ProTx-II. Cells stablyexpressing Nav1.5 were stepped to �30 mV from a holding potential of �130mV for 10 ms every 1 s until modification was complete. Data shown are fromrepresentative cells (n � 3, except for K27Q, where insufficient toxin wasavailable), and currents are normalized to peak current values. Results shownare representative of the three general phenotypic classes observed; that is,minimal loss of affinity, 10 –20-fold loss of affinity, and �20-fold loss of affin-ity. Toxin concentrations used were wild type, D10A, and E12A, 1 �M; W7A,W30A, and K27Q, 5 �M; M6A, 20 �M.

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10-fold. As shown in Table 1, the affinities of 11mutants do notdiffer substantially from that of wild-type toxin, suggesting thatthese residues do not play a significant role in toxin-channelinteractions. Most of these sites were polar to alanine substitu-tions. In contrast, 10 mutant toxins show losses of affinity from10- to 125-fold. W5A and K26Q are inactive at concentrationsup to 40 �M, indicating either a severely reduced channel affin-ity or a folding impairment. Interestingly, most of the essentialresidues are hydrophobic. Analysis of the ICK toxin SGTx,which targets Kv channels, has identified a similar number ofhydrophobic residues that are important for channel modifica-tion (19). It has been suggested that hydrophobic amino acidsmay contribute to interactions with the surrounding lipidmembrane, but this has yet to be directly examined. It is possi-ble that these residues contribute to direct protein-proteininteractions within the toxin-channel complex as well.In addition to hydrophobic residues, a subset of the seven

cationic residues, including Lys-27, Arg-13, and Arg-22, wereidentified as critical for toxin activity. Neutralization of the ani-onic residues, Asp-10, Glu-12, andGlu-17, resulted in no loss ofaffinity, indicating that positively charged channel residues aremost likely not involved in direct binding interactions.Increases inKD can be dominated by either an increased rate

of dissociation or a decreased rate of association. For example,our data show that the loss of affinity observed for M6A,W7A,L29A, and W30A is wholly ascribable to decreased rates ofassociation. Taken together with our functional analysis thatshows hydrophobic residues are important binding determi-nants, this may have implications for how the toxin accesses apotentially membrane-restricted receptor site. If these residueswere involved in initial interactions with lipid membranebefore the toxin molecule reaches its channel receptor site, wemight expect that mutational effects would be restricted to kon.

Analysis of Mutant Nav1.5 Interactions with Wild-typeProTx-II—To identify the channel receptor site responsible forinteractingwith ProTx-II, we employed amutagenesis strategy.Nav channel site 4 has been suggested as the receptor site forProTx-II and similar NaV channel toxins (14–17). Because themajor site 4 epitope within the domain II S3/S4 linker was firstidentified (11, 12), additional components have been detectedin other extracellular linker regions including DIII SS2-S6 (27).However, because the II S3/S4 sitemakes the largest single con-tribution to affinity for site 4 scorpion toxins, we chose to targetthis region first.We createdmutant Nav1.5 constructs in whichthe DII and DIV S3/S4 linkers were swapped. This approachyielded a IV:II mutant in which the sequence of DIV S3/S4replaced the DII S3/S4 linker and vice versa. Using this nomen-clature, the wild-type construct would be referred to as II:IVand the swapped construct as IV:II. As described under “Exper-imental Procedures,” obligatory intermediates in this strategywere the II:II and IV:IV channels. The availability of IV:IVallowedus to directly test the interaction of ProTx-IIwith site 4.Cestele et al. (11) have shown that the site 4 scorpion toxin

CssIV from the venom of Centruroides suffusus suffusus shiftsthe voltage dependence of NaV1.2 activation in a hyperpolariz-ing direction, thereby enhancing activation when currents areanalyzed after a prepulse to �50 mV. In contrast, NaV1.5 acti-vation is shifted in the opposite direction and inhibited slightly(11). To ascertain whether ProTx-II also behaves as a site 4toxin, we first examined its effects on current-voltage relation-ships in NaV1.2 and 1.5. As shown in Fig. 5A, application ofProTx-II (1 �M) to HEK 293 cells expressing human NaV1.5results in a rightward shift in the I-V curve and an �70% inhi-bition of maximum current. This is qualitatively similar to theeffects of CssIV, but the effects of ProTx-II on both inhibitionand I-V shift are much larger. We next analyzed its effects on

TABLE 1Interactions between ProTx-II mutants and Nav1.5Kinetic analysis of 23 ProTx-II mutants compared to wild-type toxin identifies 10 residues likely to be involved in channel modification. For five of these, the loss of affinityis dominated by kon, whereas only one is dominated by koff. The remaining residues that are essential for channelmodification exhibit amixed effect on both rates.W5A andK26Q were inactive up to 40 �M. N/D, not determined.

Toxin kmod koff kon KDmut/KDwt Test concentrations�1 s�1 105 M�1 s�1 �M

Wild type (n � 12) 0.347 � 0.043 0.025 � 0.002 3.20 � 0.42 1 1Y1A (n � 8) 0.400 � 0.030 0.100 � 0.005 3.01 � 0.31 4.1 1Q3A (n � 8) 0.441 � 0.040 0.062 � 0.007 3.79 � 0.35 1.8 1K4Q (n � 6) 1.190 � 0.052 0.250 � 0.023 4.70 � 0.35 5.9 2W5A N/D N/D N/D N/D N/DM6A (n � 3) 0.300 � 0.090 0.070 � 0.01 0.10 � 0.03 76.3 20W7A (n � 5) 0.140 � 0.010 0.031 � 0.005 0.21 � 0.02 16.4 5T8A (n � 7) 0.245 � 0.033 0.051 � 0.004 1.90 � 0.31 3.1 1D10A (n � 6) 0.645 � 0.070 0.089 � 0.008 5.50 � 0.74 2.1 1S11A (n � 5) 0.758 � 0.101 0.028 � 0.005 5.70 � 1.23 0.6 1.3E12A (n � 6) 0.502 � 0.078 0.091 � 0.007 4.10 � 0.78 3.0 1R13Q (n � 7) 0.660 � 0.062 0.160 � 0.010 0.99 � 0.12 20.1 5K14A (n � 6) 0.835 � 0.096 0.086 � 0.007 7.48 � 0.93 1.3 1E17A (n � 8) 0.317 � 0.053 0.023 � 0.002 4.70 � 0.83 0.6 0.625M19A (n � 8) 0.686 � 0.097 0.145 � 0.019 0.54 � 0.088 34.8 10V20A (n � 4) 0.522 � 0.160 0.178 � 0.014 0.34 � 0.14 76.6 10R22A (n � 6) 0.580 � 0.084 0.315 � 0.030 0.52 � 0.13 68.1 5L23A (n � 6) 0.648 � 0.071 0.084 � 0.003 5.78 � 0.74 1.7 1W24L (n � 6) 1.240 � 0.206 0.196 � 0.033 2.08 � 0.36 10.8 5K26Q N/D N/D N/D N/D N/DK27Q (n � 2) 0.117 � 0.022 0.080 � 0.02 0.07 � 0.01 126.3 5K28A (n � 7) 0.540 � 0.105 0.110 � 0.012 3.32 � 0.53 4.0 1.3L29A (n � 6) 0.151 � 0.017 0.037 � 0.004 0.22 � 0.03 18.9 5W30A (n � 6) 0.067 � 0.003 0.019 � 0.002 0.09 � 0.007 21.9 5

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NaV1.2 currents using the protocol described under “Experi-mental Procedures.” This protocol does not include any pre-pulse step analogous to that used by Cestele et al. (11). Asshown in Fig. 5B, under these conditions ProTx-II causes adepolarizing shift in the I-V relationship and inhibits current toan extent comparable with that seenwithNaV1.5. These resultsare clearly in contrast to those observed for CssIV and are thefirst indication that ProTx-II is not a classical site 4 toxin.Although CssIV has been shown to interact with multiple

extracellular sites in NaV1.2, the most dramatic effect on itsaffinity is seen upon mutation of G845 in the domain II S3-S4linker. We, therefore, analyzed the ability of our NaV1.5 IV:IVchannels to be modified by ProTx-II and CssIV in separateexperiments. As shown in Fig. 5C, we see near inhibition ofsodium current by 1 �M ProTx-II in this construct, indicatingthat the toxin does not bind to site 4 and likely interacts with anunknown neurotoxin receptor site. To demonstrate that we didin fact obliterate site 4, we next tested the functional effect ofCssIV on IV:IV channels. At a concentration of 200 nM, CssIVhad no effect on sodium current as compared with toxin-freecontrol (Fig. 5D), although the same CssIV concentration

resulted in 65% inhibition of currentand a depolarizing shift in wild-typeNav1.5 (n� 2). This effect is consist-ent with that reported previously byCestele et al. (11). We then per-formed the same experiment usingProTx-II on the II:II channel to ruleout its interaction with site 3 andsaw the same extent of modificationas observed with wild-type Nav1.5and IV:IV. To confirm abolition ofsite 3, we tested a well characterizedsite 3 toxin, ApB, on II:II and repro-ducibly observed a 90% reduction inactivity. These results verify ourexpectation that ProTx-II also failsto bind to site 3.Because ProTx-II does not bind

to the previously characterized gat-ing modifier neurotoxin receptorsites 3 or 4, and because all gatingmodifier toxins characterized todate have been shown to bindwithin or proximal to extracellularlinkers, we next targeted theremaining extracellular linkerregions. Our mutagenesis strategyexploited the fact that ProTx-II hasbeen reported to have an �50-foldhigher affinity for the peripheralnerve sodium channel isoform,Nav1.7, over the cardiac isoform,Nav1.5 (Fig. 6; Refs. 14 and 21).Alignment of Nav1.5 and Nav1.7S1/S2 and S3/S4 linker sequencesidentified 21 residues that differbetween the two isoforms. Addi-

tionally, 5 residues in DIII SS2/S6 were targeted, based on theiridentification as determinants of site 4 toxin isoform selectivity(27). Because we had already demonstrated that DII and DIVS3/S4 linkers are not involved in ProTx-II binding, we elimi-nated them from this analysis and mutated the remaining resi-dues to alanine (Table 2). All residues targeted formutation in asingle linker were altered simultaneously rather than as singlepoint mutations to account for all possible binding contribu-tions within a single extracellular linker. We were able toexpress allmutant channels after transient transfection ofDNAinto HEK293 cells. Upon the addition of 1 �M ProTx-II, allchannel mutants tested were modified to a similar extent asobserved for wild-type channels (Fig. 7). We interpret thesedata as evidence that ProTx-II does not make any critical bind-ing interactions with extracellular linker regions of sodiumchannels.Because we have identified several hydrophobic and cationic

residues in ProTx-II as essential for modification of Nav1.5, wenext focused on potential binding sites within transmembranesegments. It has been suggested that gating modifier toxinsmight access their receptor site on their target channel after

FIGURE 5. The electrophysiologic phenotype of ProTx-II is distinct from that of the site 4 toxin, CssIV.E, control; ●, toxin. A, the effect of 1 �M ProTx-II on Nav1.5. Cells stably expressing Nav1.5 were stepped in 5-mVincrements from �80 mV to �20 mV from a holding potential of �130 mV for 30 ms. An �70% reduction inpeak current and a 20-mV depolarizing shift in the voltage dependence of activation were observed (n � 12).B, the effect of 1 �M ProTx-II on Nav1.2. N1E-115 cells expressing predominantly Nav1.2 were stepped in 5-mVincrements from �50 mV to �50 mV from a holding potential of �130 mV for 30 ms. A �80% reduction in peakcurrent and a 5-mV depolarizing shift in the voltage dependence of activation were observed (n � 5). Theexperimentally determined KD for ProTx-II on Nav1.2 was �200 nM. C, the effect of 1 �M ProTx-II on the Nav1.5IV:IV mutant channel which lacks the DII S3/S4 linker. Cells transiently expressing the IV:IV channel were sub-jected to the same step protocol as described in A. A similar extent of modification to that of wild-type Nav1.5was observed, indicating retention of the binding site (n � 7). The experimentally determined KD for ProTx-II onthe IV:IV channel was �89 nM. D, the effect of 200 nM CssIV on the Nav1.5 mutant IV:IV channel. Cells transientlyexpressing the IV:IV channel were subjected to the same step protocol as described in A and C. CssIV failed tomodify IV:IV due to ablation of the binding site (n � 3). Data are �S.E.

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diffusing through the lipid membrane to reach a membrane-restricted site (28–30).We, therefore, created single site chargeneutralization mutations of several anionic transmembraneresidues in Nav1.5 and analyzed their ability to be modified byProTx-II (Table 2 and supplemental Table 1). All mutant chan-nels displayed near complete inhibition of current upon theaddition of toxin, indicating that ProTx-II does not make crit-ical binding interactions with any of the targeted residues intransmembrane segments.

DISCUSSION

The molecular mechanisms fundamental to channel modifi-cation by sites 3- and 4-gating modifier toxins have been thefocus of several in depth studies, and the results have shed lighton the roles of individual domains in voltage sensing (5, 11, 13).However, much less is currently known about the mechanismof action of ICKpeptides onNav channels.We, therefore, inves-tigated the interaction of the ICKpeptide toxin ProTx-II and 23mutant forms with the human cardiac Na� channel isoform,Nav1.5. ProTx-II modifies Nav and Cav channels in a mannerdistinct from previously characterized Nav channel gatingmodifier toxins, and its channel receptor site is unknown.Our results identify all functionally essential residues on the

toxin surface, thereby establishing the bioactive face of themol-ecule. Of the 24 non-cysteine residues, we were able to produceand characterize 23 mutants (Table 1), all of which have CD

spectra identical to that of the wild-type toxin. Interestingly,whereas these spectra closely resemble those of homologousgating modifier toxins of the ICK family, there are also cleardifferences among them, and all are dramatically different froma fourth homolog, SgTx-1 (19). In addition, we note thatalthough these spectra are atypical for native proteins, all ofthese toxins are in fact fully active. It is, therefore, likely thatthese polypeptides as a group have very little regular secondarystructure, perhaps contributing to their ability to target distinctchannels and channel isoforms with high affinity.Eleven ProTx-II mutants exhibit activity similar to that of

wild-type toxin, and most of the residues having severelydecreased channel affinities are hydrophobic. The N-terminalmutations M6A and W7A in addition to the C-terminalmutantsW24L, L29A, andW30A exhibitKD values 10–76-foldhigher than wild-type toxin, clearly demonstrating that thesepositions contribute strongly to binding affinity. Trp-5 maycontribute as well since we observed no channel inhibition byW5A up to a concentration of 40 �M, and because the corre-sponding position has been implicated as a binding determi-nant in the homologous Kv channel ICK toxin, SGTx (19). Asseen in Fig. 8, these residues contribute to a hydrophobic pro-trusion on the surface of the ProTx-II. A similar cluster of func-

FIGURE 6. NaV1.7 channels are more sensitive to ProTx-II than NaV1.5channels. Representative Nav1.7 currents recorded from a HEK 293 cell areshown under control conditions (A) and after application of 100 nM ProTx-II(B). The cell stably expressing Nav1.7 was stepped in 5-mV increments from�80 mV to �70 mV from a holding potential of �120 mV for 50 ms. ProTx-IIinhibition was allowed to reach steady state before recording the tracesshown in B. C, the current-voltage relationship from this cell is shown undercontrol (E) conditions and after exposure to toxin (F). D, the dose-responserelationship for ProTx-II inhibition of human Nav1.7 (F; n � 14) and humanNav1.5 (E; n � 9) channels stably expressed in HEK 293 cells. Current ampli-tudes were obtained before toxin application with 50-ms test pulses to �30mV from a �120-mV holding potential. The solid curves show the single-siteLangmuir inhibition isotherm fits to the data.

TABLE 2Mutational analysis of Nav1.5

To determine the ProTx-II receptor site, a total of 76 residues were mutated intransmembrane or extracellular linker regions of Nav1.5 either singly or as part of alarger substitution. Themajority of linker residues were selected formutation basedupon sequence conservation between Nav1.5 and Nav1.7, and detailed analysis ofthese channels is depicted in Fig. 7. Acidic transmembrane residues were targetedbased on (a) the importance of several cationic residues for ProTx-II affinity, (b) theimportance of several hydrophobic residues for ProTx-II affinity in conjunctionwith the ability of ProTx-II to bind phospholipids, both, raising the possibility of amembrane-restricted binding site, and (c) the importance of transmembrane resi-dues for binding of the ICK toxin, hanatoxin, to Kv2.1 (34). These data are depictedin supplemental Table I. The extent ofmodification of eachmutant channel by 1�MProTx-II was similar to that observed for wild-type Nav1.5, i.e., 65–85% inhibition(n � 2–3 per mutant). Mutations in bold font were made as part of a large substi-tution in that particular linker. All others were created as single mutations.

Mutation Location Mutation LocationH151A DI S1/S2 E1202Q DIII S1D152A D1 S1/S2 E1207Q DIII S1P153A DI S1/S2 E1224A DIII S1/S2P155A DI S1/S2 D1225A DIII S1/S2W156A DI S1/S2 L1228A DIII S1/S2E161Q DI S2 E1229A DIII S1/S2E171Q DI S2 E1230A DIII S1/S2D197N DI S3 R1231A DIII S1/S2E208Q DI S3 E1239Q DIII S2D211A DI S3/S4 D1242N DIII S2C373A DI SS2/S6 E1252Q DIII S2R376A DI SS2/S6 D1274N DIII S3D716N DII S1 D1279N DIII S3D720N DII S1 F1292A DIII S3/S4E737A DII S1/S2 E1294A DIII S3/S4Y739A DII S1/S2 M1295A DIII S3/S4N740A DII S1/S2 R1431A DIII SS2/S6S743A DII S1/S2 G1432A DIII SS2/S6E744A DII S1/S2 Y1433A DIII SS2/S6E746A DII S1/S2 E1434A DIII SS2/S6E747A DII S1/S2 E1435A DIII SS2/S6E763Q DII S2 T1548A DIV S1/S2D785N DII S3 D1550A DIV S1/S2E795A DII S3 P1553A DIV S1/S2L796A DII S3 E1554A DIV S1/S2L798A DII S3 K1555A DIV S1/S2Arg-800—Leu-807a DII: S3/S4 N1557A DIV S1/S2F810A DII S4 D-(1609–1621)a DIV S3/S4L812A DII S4

a Linker swap mutants.

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tionally important hydrophobic residues has been shown toform a protrusive patch on the active face of SGTx (19). Thiscommon feature points toward a dominant role of hydrophobicresidues in ICK toxin activity irrespective of the target channel.Furthermore, our data highlight the importance of Met-19 andVal-20 for toxin activity with affinities of their alanine replace-ments decreased �35 and 77-fold, respectively. The impor-tance of these positions in channel modification is unique toProTx-II since Ala-20 is a nonessential residue in SGTx, andLeu-19 is a folding determinant (19).In addition to illustrating the importance of hydrophobic

amino acids in ProTx-II, we also demonstrate that the bioactiveface is amphipathic in nature. The mutations R13Q, R22A, andK27Q cause dramatic increases in KD, suggestive of a role inelectrostatic interactions with the channel or phospholipidhead groups. Although K26Q also lacks activity up to a 40 �Mtest concentration, this is more likely because of a folding aber-ration since its HPLC profile displays a heterogeneous popula-tion of peptide forms. In contrast, W5A behaves chromato-graphically as a well folded species and is similarly devoid ofactivity. Because mutation of the anionic residues Asp-10, Glu-12, and Glu-17 resulted in minor changes in affinity, we inter-pret this to mean that cationic channel residues do not directlyinfluence toxin binding.

Wang et al. (19) also found basicresidues in SGTx that are critical forinhibition of Kv2.1, whereas muta-tion of acidic residues in factincreased the affinity of that toxinfor its target. In the SGTx solutionstructure, the basic residues form aring around the hydrophobic patch,presumably creating the active face(31). Because the solution structurefor ProTx-II is unknown, we choseto construct a molecular modelbased upon the known solutionstructure of a similar ICK toxin thattargets Kv4 channels, HpTx-2. Ourmodel clearly shows the existence ofa hydrophobic cluster of residues,all of which we found to be essentialbinding determinants (Fig. 8) Resi-dues we identified as nonessentialcluster to the opposite face of themolecule. Similar molecular phar-macophores for toxins that modifyeither Nav or Kv channels is of par-ticular interest because it couldpoint to a similar mechanism ofaction for these peptides on theirrespective targets.In a previous study we found that

ProTx-II has the ability to bind toliposomes, raising the possibilitythat the toxin might insert intomembranes as part of its mode ofaction (15). Together with our

kinetic analysis of mutant toxins showing a critical role ofhydrophobic residues in channel modification, these resultscould suggest a membrane-restricted channel receptor site fortoxins that modify activation gating. While the functional roleof lipid binding requires further study, it is clearly a propertyrestricted to toxins affecting activation, but not inactivation,kinetics. This result was not particularly unexpected becausesite 3 is known to be extracellular; thus, membrane partitioningwould confer no advantage to toxins affecting inactivationkinetics.Cohen et al. (17) recently reported that site 4 is not “dipped”

in the lipid bilayer and concluded that the lipid binding activityexhibited by site 4 toxins has no functional relevance. Althoughit is possible that this is true for the �-toxins with which theyconducted their studies, it still remains to be seen if the essentialhydrophobic patch on ProTx-II is primarily important for pro-tein-protein interactions or for facilitating entry into thebilayer.Elucidation of the channel receptor site with which ProTx-II

interacts is necessary to further characterize the molecularmechanism through which this toxin works. We previouslyinferred that ProTx-II was a site 4 toxin based on its ability tomodifyNav channel activation and because awell characterizedsite 4 toxin, CssIV, also exhibits the ability to bind to liposomes.

FIGURE 7. ProTx-II does not bind to extracellular linkers of NaV1.5. Top, transmembrane architecture of theNav channel � subunit is provided as a reference to channel mutations indicated in B as well as the remainingmutations listed in Table 2. Bottom, mutant Nav1.5 channels containing alanine replacements for all extracel-lular linker residues which differ between Nav1.5 and Nav1.7 were transiently expressed in HEK 293 cells. Datashown are scaled currents from representative cells (n � 3) stepped in 5-mV increments from �80 mV to �20mV from a holding potential of �130 mV. Inhibition by 1 �M ProTx-II (F) ranged from 65 to 85% for all mutantchannels as compared with toxin-free control (E). Amino acid linker sequences are shown above each tracewith the mutated residues depicted in large bold font. DII S3/S4 and DIV S3/S4 linker mutants are not repre-sented because they were analyzed separately in the linker swap experiments.

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Having already demonstrated that the effects of ProTx-II andCssIV on Nav1.2 differ when analyzed using a simple step pro-tocol (Fig. 5B and Ref. 11), we also examined its effect with avoltage-sensor trapping protocol. Intriguingly, whenwe use thevoltage-sensor trapping protocol developed by Cestele et al.(Ref. 11 and data not shown) to evaluate whether toxin-modi-fied channels activate at more negative potentials than is typi-cally observed, no ProTx-II-induced current is observed. Theseresults confirm the mechanistic differences between ProTx-IIand CssIV. To locate residues necessary for ProTx-II binding,we have carried out extensive channel mutagenesis. The resultsof linker swap experiments established that ProTx-II does notinteract with either sites 3 or 4 in theDIV andDII S3/S4 linkers,respectively. These results were of particular interest given thatall Nav channel gating modifier peptide toxins characterized todate bind to these extracellular linkers (9–11). Furthermore,linker mutagenesis targeting sites at which Nav1.5 and Nav1.7differ clearly indicate the unlikelihood of the ProTx-II receptorsite existing in these regions (Fig. 7).Because ProTx-II displays a bioactive surface containing

hydrophobic and cationic residues and can bind phospholipids,we considered that the toxin molecule might interact first withthe lipid bilayer before accessing a target wholly or partiallywithin the membrane. To evaluate potential interactions withcationic toxin residues, we created 16 individual mutations intransmembrane segments, each neutralizing an acidic residuepredicted to reside in the outer leaflet of DI, DII, or DIII (Table2 and supplemental Table 1). DIVwas excluded from this studybecause a great deal of evidence supports its role in inactivation,rather than activation, gating (2, 5). None of the mutations weanalyzed caused any reduction in channel modification by

ProTx-II. We have also observed that after pretreatment ofTTX-sensitive C373Y Nav1.5 channels with sub-saturatingProTx-II, the addition of 20 nM tetrodotoxin completely blockscurrent, strongly suggesting that ProTx-II does not bind in thepore vestibule (data not shown and Ref. 32). It remains possiblethat basic toxin residues interact with charged phospholipidhead groups or that hydrophobic toxin residues make contactwith hydrophobic channel residues, and a very recent molecu-lar dynamics analysis suggests that the former scenario appliesin the case of SGTx (33). However, a full analysis of potentialinteractions of hydrophobic toxin residues with hydrophobicsites in the transmembrane regions of the channel is beyond thescope of this study.Although ProTx-II has no affinity for Kv channels, it behaves

in a manner similar to Kv channel ICK toxins and also displaysa similar active face. Glu-795, Leu-796, and Leu-798 in theC-terminal end of Nav1.5 DII S3 correspond to residues in theS3b region of Kv2.1 that render that channel sensitive to hana-toxin (17). Nonetheless, mutagenesis at these sites in Nav1.5fails to disrupt channel modification, suggesting that ProTx-IIdoes not interact with the previously identified gating modifier“hot spot” on Nav, Cav, and Kv channels (34). It has been shownkinetically that Kv channel ICK toxins have multiple receptorsites arising from their tetrameric channel structure (35). How-ever, there is no precedent for Nav channel gating modifiertoxins binding to multiple sites with high affinity, presumablybecause Nav channel domains do not have identical amino acidsequences. Although mutations in several extracellular linkershave been shown to decrease site 4 toxin (CssIV) binding affin-ity, most of the binding energy is associatedwith residues inDIIS3/S4, a finding we were able to confirm (11). Moreover, a sin-gle mutation in DIV S3/S4 renders Nav1.5 and 1.2 almost com-pletely insensitive to the site 3 toxins, ApB and LqqV (9, 10).These results give credence to the notion that ProTx-II inter-acts with only a single channel site.In this study we havemapped the results from our toxin scan

onto a structural model of ProTx-II to establish its bioactivesurface, thus providing insight into its mechanism of action. Inaddition, we demonstrated that ProTx-II does not interact withpreviously characterized gating modifier peptide toxin sites.Likewise, it is highly unlikely that ProTx-II makes any directbinding interactions with other extracellular linker regions onNav1.5. These results establish the novelty of this toxin as agatingmodifier whosemechanism of inhibition differs dramat-ically from those of previously characterized Nav channel tox-ins. This uniqueness demonstrates its potential as an extremelyuseful probe of gatingmechanisms inNav as well as Cav channels.Given the intriguing, if elusive mechanism of action associatedwith this toxin, future studies aimed at identifying the channelreceptor site are imperative and are currently under way.

Acknowledgments—We are very grateful to Dr. James Garrity for cre-ating several mutant toxin contructs, Drs. Gerardo Corzo andLourival Possani for the generous gift of CssIV, Drs. Michael Moralesand Philip Gottlieb for gifts of HpTx and GsMtx4, respectively,Dr. DorothyHanck for cells expressing theC373Ymutant ofNav1.5, andDrs. Morales and Hanck for valuable comments on the manuscript.

FIGURE 8. The bioactive surface of ProTx-II. A molecular model ofProTx-II was created by in silico mutagenesis from the Protein Data Bankcoordinates for HpTx-2, a homologous ICK motif peptide targeting Kv4.2(36). The model was subjected to energy minimization to remove stericclashes. Essential residues, shown in cyan, are defined by a �10-fold lossof affinity for Nav1.5 upon mutation and are all either hydrophobic orcationic in nature. Nonessential residues are shown in magenta and seg-regate to the opposite faces of the molecule than essential residues.

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

57, 397–4092. Cha, A., Ruben, P. C., George, A. L., Fujimoto, E., and Bezanilla, F. (1999)

Neuron 22, 73–873. Chanda, B., and Bezanilla, F. (2002) J. Gen. Physiol. 120, 629–6454. Chahine, M., George, A. L., Zhou, M., Ji, S., Sun, W., Barchi, R. L., and

Horn, R. (1994) Neuron 12, 281–2945. Sheets, M. F., Kyle, J. W., Kallen, R. G., and Hanck, D. A. (1999) Biophys. J.

77, 747–7576. Chanda, B., Asamoah, O. K., and Bezanilla, F. (2004) J. Gen. Physiol. 123,

217–2307. Zagotta, W. N., Hoshi, T., and Aldrich, R. W. (1994) J. Gen. Physiol. 103,

321–3628. Schoppa, N. E., and Sigworth, F. J. (1998) J. Gen. Physiol. 111, 313–3429. Rogers, J. C., Qu, Y., Tanada, T.N., Scheuer, T., andCatterall,W .A. (1996)

J. Biol. Chem. 271, 15950–1596210. Benzinger, G. R., Kyle, J. W., Blumenthal, K. M., and Hanck, D. A. (1998)

J. Biol. Chem. 273, 80–8411. Cestele, S., Qu, Y., Rogers, J. C., Rochat, H., Scheuer, T., and Catterall,

W. A. (1998) Neuron 21, 919–93112. Cestele, S., Scheuer, T., Mantegazza, M., Rochat, H., and Catterall, W. A.

(2001) J. Gen. Physiol. 118, 291–30113. Cestele, S., Yarov-Yarovoy, V., Qu, Y., Sampieri, F., Scheuer, T., and Cat-

terall, W. A. (2006) J. Biol. Chem. 281, 21332–2134414. Middleton, R. E.,Warren, V.A., Kraus, R. L., Hwang, J. C., Liu, C. J., Dai, G.,

Brochu, R. M., Kohler, M. G., Gao, Y. D., Garsky, V. M., Bogusky, M. J.,Mehl, J. T., Cohen, C. J., and Smith, M. M. (2002) Biochemistry 41,14734–14747

15. Smith, J. J., Alphy, S., Seibert, A. L., and Blumenthal, K. M. (2005) J. Biol.Chem. 280, 11127–11133

16. Bosmans, F., Rash, L., Zhu, S., Diochot, S., Lazdunski,M., Escoubas, P., andTytgat, J. (2006)Mol. Pharmacol. 69, 419–429

17. Cohen, L., Gilles, N., Karbat, I., Ilan, N., Gordon, D., and Gurevitz, M.(2006) J. Biol. Chem. 281, 20673–20679

18. Swartz, K. J., and MacKinnon, R. (1997) Neuron 18, 675–682

19. Wang, J. M., Roh, S. H., Kim, S., Lee, C. W., Kim, J. I., and Swartz, K. J.(2004) J. Gen. Physiol. 123, 455–467

20. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K., and Pease, L. R. (1989)Gene (Amst.) 77, 61–68

21. Kraus, R. L., Warren, V. A., Smith, M. M., Middleton, R. E., Blumenthal,K. M., and Cohen, C. J. (2002) Biophysical Society 46th Annual Meeting,February 23–27, San Francisco, Abstract 85c

22. Seibert, A., Liu, J. R., Hanck, D. A., and Blumenthal, K.M. (2003)Biochem-istry 42, 14515–14521

23. Cummins, T. R., Howe, J. R., and Waxman, S. G. (1998) J. Neurosci. 18,9607–9619

24. Hirsch, J. K., and Quandt, F. N. (1996) Brain Res. 706, 343–34625. Brahmajothi, M. V., Campbell, D. L., Rasmusson, R. L., Morales, M. J.,

Trimmer, J. S., Nerbonne, J. M., and Strauss, H. L. (1999) J. Gen. Physiol.113, 581–600

26. Bernard, C., Legros, C., Ferrat, G., Bischoff, U., Marquardt, A., Pongs, O.,Francisco, and Darbon, H. (2000) Protein Sci. 9, 2059–2067

27. Leipold, E., Hansel, A., Borges, A., and Heinemann, S. H. (2006) Mol.Pharmacol. 70, 340–347

28. Lee, S. Y., and MacKinnon, R. (2004) Nature 430, 232–23529. Jung,H. J., Lee, J. Y., Kim, S. H., Eu, Y., Shin, S. Y.,Milescu,M., Swartz, K. J.,

and Kim, J. I. (2005) Biochemistry 44, 6015–602330. Bemporad, D., Wee, C. L., Sands, Z., Grottesi, A., and Sansom, M. S. P.

(2006) Biochemistry 45, 11844–1185531. Lee, C. W., Kim, S., Roh, S. H., Endoh, H., Kodera, Y., Maeda, T., Kohno,

T., Wang, J. M., Swartz, K. J., and Kim, J. I. (2004) Biochemistry 43,890–897

32. Satin, J., Kyle, J. W., Chen, M., Bell, P., Cribbs, L. L., Fozzard, H. A., andRogart, R. B. (1992) Science 256, 1202–1205

33. Wee, C. L., Bemporad, D., Sands, Z., Gavaghan, D., and Sansom, M. S. P.(2006) Biophys. J. 92, 7–9

34. Winterfield, J. R., and Swartz, K. J. (2000) J. Gen. Physiol. 116, 637–64435. Swartz, K. J., and MacKinnon, R. (1997) Neuron 18, 665–67336. Priest, B. T., Blumenthal, K. M., Smith, J. J., Warren, V. A., and Smith,

M. M. (2007) Toxicon 49, 194–201

Novel Neurotoxin Receptor Site Coupled to NaV Activation

APRIL 27, 2007 • VOLUME 282 • NUMBER 17 JOURNAL OF BIOLOGICAL CHEMISTRY 12697

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Jaime J. Smith, Theodore R. Cummins, Sujith Alphy and Kenneth M. BlumenthalACTIVATION

IMPLIED EXISTENCE OF A NOVEL TOXIN BINDING SITE COUPLED TO 1.5:vMolecular Interactions of the Gating Modifier Toxin ProTx-II with Na

doi: 10.1074/jbc.M610462200 originally published online March 5, 20072007, 282:12687-12697.J. Biol. Chem. 

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