Design of a Specific Activator for Skeletal Muscle Sodium

Design of a Specific Activator for Skeletal Muscle SodiumChannels Uncovers Channel Architecture*SReceived for publication, June 6, 2007, and in revised form, August 6, 2007 Published, JBC Papers in Press, August 8, 2007, DOI 10.1074/jbc.M704651200

Lior Cohen, Nitza Ilan, Maya Gur, Walter Stuhmer, Dalia Gordon1, and Michael Gurevitz2

From the Department of Plant Sciences, George S. Wise Faculty of Life Sciences, Tel-Aviv University, Ramat-Aviv,69978 Tel-Aviv, Israel and Department of Molecular Biology and Neuronal Signaling, Max Planck Institute of ExperimentalMedicine, D-37075 Gottingen, Germany

Gating modifiers of voltage-gated sodium channels (Navs)are important tools in neuroscience research and may havetherapeutic potential in medicinal disorders. Analysis of thebioactive surface of the scorpion -toxin Css4 (from Centru-roides suffusus suffusus) toward rat brain (rNav1.2a) and skel-etal muscle (rNav1.4) channels using binding studies revealedcommonality but also substantial differences, which wereused to design a specific activator, Css4F14A/E15A/E28R, ofrNav1.4 expressed in Xenopus oocytes. The therapeuticpotential of Css4F14A/E15A/E28R was tested using an rNav1.4mutant carrying the same mutation present in the geneticdisorder hypokalemic periodic paralysis. The activatorrestored the impaired gating properties of the mutant chan-nel expressed in oocytes, thus offering a tentative new meansfor treatment of neuromuscular disorders with reduced mus-cle excitability. Mutant double cycle analysis employing toxinresidues involved in the construction of Css4F14A/E15A/E28R

and residues whose equivalents in the rat brain channelrNav1.2a were shown to affect Css4 binding revealed signifi-cant coupling energy (>1.3 kcal/mol) between F14A andE592A at Domain-2/voltage sensor segments 12 (D2/S1-S2),R27Q and E1251N at D3/SS2-S6, and E28R with both E650Aat D2/S3-S4 and E1251N at D3/SS2-S6. These results showthat despite the differences in interactions with the rat brainand skeletal muscle Navs, Css4 recognizes a similar region onboth channel subtypes. Moreover, our data indicate that theS3-S4 loop of the voltage sensor module in Domain-2 is in veryclose proximity to the SS2-S6 segment of the pore module ofDomain-3 in rNav1.4. This is the first experimental evidencethat the inter-domain spatial organization of mammalian Navsresembles that of voltage-gated potassium channels.

Among the superfamily of voltage-gated ion channels,sodium channels (Navs)3 play a central role in the generation ofaction potentials in excitable cells and are the target of a largevariety of neurotoxins, drugs, and insecticides (1, 2). Althoughthe structure of homo-tetrameric voltage-gated potassiumchannels (Kvs) was recently resolved (3, 4), the structure ofNavshas not yet been determined.Eukaryotic Navs are constituted from one large protein that

forms four homologous, non-identical domains. As for Kvs,each domain of theNav comprises two functionalmodules. Thepore module consists of transmembrane segments S5 and S6connected by a membrane-associated re-entrant loop (SS1-SS2). The voltage-sensing module, which consists of segmentsS1-S4, undergoes a conformational alteration in response tochanges in the membrane potential, enabling channel activa-tion (1, 5). Estimation of the distances between transmembranesegments of the homo-tetrameric bacterial Nav, NaChBac, sug-gested an intra-subunit organization similar to that of Kvs in alipid bilayer (6), but the inter-domain organization of the poreand voltage-sensing modules in eukaryotic Navs have not beendetermined.A variety of mammalian Navs, encoded by at least nine

genes, have been described (1, 7). Expression of these chan-nels varies greatly across tissues and developmental stages(1). Nav1.11.3 and Nav1.6 are expressed in the central nerv-ous system, Nav1.6 and Nav1.7 are expressed in the periph-eral nervous system, Nav1.8 and Nav1.9 are expressed in sen-sory neurons, and Nav1.4 and Nav1.5 are expressed inskeletal and cardiac muscle, respectively. Many genetic dis-orders leading to abnormal function of these channels havebeen described in humans (e.g. Refs. 812). For example,mutations in the SCN4A gene, encoding for the human skel-etal muscle channel Nav1.4, lead to various types of periodicparalysis, paramyotonia congenital, and Nav myotonias(reviewed in Ref. 8). Several mutations rendering hypokale-mic periodic paralysis (hypoPP) (8, 1315) were identified inthe voltage sensor segment S4 of Domain 2 (D2/S4) of thehuman skeletal muscle sodium channel, hNav1.4, causing apositive shift in the voltage dependence of channel activationand gating pore currents at resting membrane potential (1316). Action potentials evoked from human hypoPP musclesamples are sluggish and smaller than those obtained in

* This work was supported by German-Israeli Foundation for ScientificResearch and Development Grant G-770-242.1/2002 (to D. G. and W. S.), byUnited States-Israel Binational Agricultural Research and DevelopmentGrant IS-3480-03 (to M. G. and D. G.), by Israeli Science Foundation Grants733/01 (to M. G.) and 1008/05 (to D. G.), and by National Institutes of HealthGrant 1 U01 NS058039-01 (to M. G.). The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked advertisement 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 Figs. S1 and S2 and Table S1.

1 To whom correspondence may be addressed. Tel.: 972-3-6409844; Fax: 972-3-6406100; E-mail: [email protected].

2 To whom correspondence may be addressed. E-mail: [email protected].

3 The abbreviations used are: Nav, voltage-gated sodium channel; Css4, Cen-truroides suffusus suffusus -toxin 4; hypoPP, hypokalemic periodicparalysis.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 40, pp. 29424 29430, October 5, 2007 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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healthy muscle tissue (15). Gating modifiers that facilitatechannel activation could potentially restore the properties ofdefective Navs, but to be therapeutically applicable they haveto be highly specific for hNav1.4.

Scorpion -toxins (e.g. the anti-mammalian Css2 and Css4from Centruroides suffusus suffusus) are short polypeptidesreticulated by four disulfide bonds that target Navs andmod-ulate their gating properties (17). They are typified by theshift they induce in the voltage dependence of channel acti-vation to more negative membrane potentials upon bindingto receptor site 4 (17), shown to be associated with Domain 2of the Nav (1821). Four conserved residues in D2/S1-S2 andS3-S4 have been implicated in the interaction of the -toxinCss4 with mammalian Navs (19, 20), and two residues inD3/SS2-S6 have recently been linked to the preference of the-toxin Tz1, from the scorpion Tityus zulianus, for the mus-cle channel rNav1.4 over the brain channel rNav1.2a (22).These data, and the fact that Css4 is more potent at hNav1.2athan hNav1.4 (23), have suggested that the set of residuescomprising receptor site 4 on both channels are notidentical.As the binding of -toxins is independent of membrane

potential and because a depolarizing prepulse is required toobserve their effect, it was suggested that prebound -toxinstrap the D2/S4 segment in its outward activated position,leading to enhanced channel activation upon subsequentdepolarizations (19). Despite the accumulated knowledgeabout their mode of interaction with the Nav, complete elu-cidation of neurotoxin receptor site 4 and the extent of itsconservation in various channel subtypes remained to bedescribed.Toxins with high preference for Nav subtypes are useful for

identifying distinct channels and studying their mechanism ofaction and may also be instrumental in development of newdrugs for treating neurological and muscular disorders. More-over, as the receptor binding sites for toxins incorporate resi-dues of different, not necessarily adjacent, extracellular loops(1, 20, 22), by identifying interacting residues of toxins andchannels and by relying on the known toxin structures we mayelucidate the spatial organization of the channel extracellularface.Mutagenic dissection of scorpion -toxins that affect both

mammalian (Css4) and insect (the excitatory toxin Bj-xtrITfrom Buthotus judaicus and the depressant toxin LqhIT2 fromLeiurus quinquestriatus hebraeus) Navs revealed a spatiallysimilar cluster of bioactive residues (2426). Moreover, wehave suggested that a spatially conserved Glu residue in -tox-ins (position 28 in Css4) forms a hot spot for interaction withreceptor site 4 (25).Here we show profound variations in the bioactive surface of

Css4 toward the rat muscle and brain Navs, which we used toconstruct aCss4 triplemutant that exclusively binds and affectsrNav1.4. Thismutant toxin can restoremost of the gating prop-erties of a rNav1.4 mutant designed according to a mutationfound in hypoPP patients. We also identified Css4-rNav1.4 res-idue pairs that exhibit coupling energies indicating close prox-imity of specific extracellular channel loops. These data enabledthe first estimation of the distance between extracellular loops

of the gating and pore modules of two adjacent Nav domainsand provide evidence that the inter-domain architecture ofNavs resembles that of Kvs.

EXPERIMENTAL PROCEDURES

Mutagenesis of Css4PCR-driven mutagenesis, expressionin Escherichia coli, in vitro folding, and purification of Css4derivatives have been described in detail elsewhere (25).Expression of Sodium Channels in OocytesThe pNa200

vectors bearing the genes encoding rNav1.2a, rNav1.3, andrNav1.6 were a gift fromDr. A. Goldin (University of California,Irvine). The pAlter vectors bearing the genes encoding rNav1.4and hNav1.5 were a gift from Dr. R. G. Kallen (University ofPennsylvania, Philadelphia, PA). These genes and the auxiliaryh1 were transcribed in vitro using T7 RNA polymerase andthe mMESSAGE mMACHINETM system (Ambion, Austin,TX) (27, 28) and injected into Xenopus laevis oocytes as previ-ously described (21).Site-directed Mutagenesis of rNav1.4pAlter containing the

entire rat skeletal muscle sodium channel -subunit was usedfor oligonucleotide-based mutagenesis. The PCR-amplifiedfragment containing the mutations E592A, H599Q/D601S/N602S, E650A, L653A, Q657E, G658N, and R666G wascleaved by BsiwI and BssHII and inserted into the corre-sponding sites of the vector. E1251N and H1257K werecleaved by EcoNI and inserted into the corresponding sitesof the vector, which was further used for transcription afterDNA sequence verification.Two Electrode Voltage Clamp Recording and Data Analysis

Currents were measured 12 days after injection using a twoelectrode voltage clamp and a Gene Clamp 500 amplifier(Axon Instruments, Union City, CA). Data were sampled at10 kHz and filtered at 5 kHz. Toxins were diluted with bathsolution and applied directly to the bath to achieve thedesired final concentration. Data acquisition was controlledby a Macintosh PPC 7100/80 computer equipped with anITC-16 analog/digital converter (Instrutech Corp., PortWashington, NY), using Synapse (Synergistic Systems). Thebath solution contained 96 mM NaCl, 2 mM KCl, 1 mMMgCl2, 2 mMCaCl2, and 5mMHEPES, pH 7.85. Oocytes werewashed with bath solution flowing from a BPS-8 perfusionsystem (ALA Scientific Instruments, Westbury, NY) with apositive pressure of 4 psi.Capacitance transients and leak currents were removed by

subtracting a scaled control trace using a P/6 protocol (29).For the GV analysis, mean conductance (G) was calculatedfrom the peak current/voltage relationship using the equa-tion G I/(V Vrev), where I is the peak current, V is themembrane potential, and Vrev is the reversal potential. Thenormalized conductance/voltage relationship was fit witheither a one- or two-component Boltzmann distributionaccording to Equation 1G/Gmax (1 A)/(1 exp[(V112 V)/k1]) A/(1

exp[(V212 V)/k2])where V112 and V212 are the respective membrane potentials

for two populations of channels for which the mean conduct-ance is half maximal, k1 and k2 are their respective slopes, andAdefines the proportion of the second population (amplitude)

Nav1.4 Architecture Uncovered by a Specific Activator

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with respect to the total. For fits inwhich only one population ofchannels was apparent, A was set to zero. The voltage depend-ence of steady-state fast inactivation was described using a sin-gle Boltzmann distribution as shown in Equation 2I/Imax 0 1/(1 exp[(V V12)/k])where I is the peak current measured during the test depo-

larization step, Imax is the current without a preceding condi-tioning step, V is the membrane potential of the conditioningstep, V12 is themembrane potential at which half-maximal inac-tivation is achieved, k is the slope factor, 0 is the remainingnormalized peak current at highly depolarizing conditioningpotentials, and1 is the normalized amplitude (30). Formutantdouble cycle analysis, the dose response for Css4 effects onrNav1.4 was determined based on the current induced by thetoxin following a test pulse to a voltage of35mV from theV0.5of activation determined for each channel mutant (Table 1).The free energy change in toxin binding to the wild-type/mu-tant channel pair (G) was calculated as the difference of theaverage RTln (EC50) for the wild type and mutant, where R isthe gas constant and T stands for temperature in 0K. G wastaken as the difference of the Gs for Css4 and the toxinmutant: G (GWT, native Gmutant, native) (GWT, mutant Gmutant, mutant), where the first and second subscript posi-tions refer to the channel and the toxin, respectively. RT 0.59 kcal/mol.Binding ExperimentsSkeletalmusclemembranes and brain

synaptosomes were prepared from adult albino Wistar rats(300 g, laboratory-bred) as previously described (31, 32). Therats were sacrificed according to the rules of the Animal CareUnit at Tel Aviv University (permit number L-03-54) followingNational Institutes of Health guidelines. Radio-iodination ofCss4, purification of monoiodotoxin, and binding assays wereperformed as previously described (25). Nonspecific toxinbindingwas determined in the presence of 1Munlabeled toxinand consisted typically of 1530% of total binding. Each exper-iment was performed in duplicate and repeated at least threetimes as indicated (n). Data are presented as the mean S.D.for independent experiments.

RESULTS

Comparison of Css4 Interactions with Rat Skeletal Muscleand Brain Navs and Construction of a Specific Modifier forNav1.4To clarify the difference in Css4 potency at Nav1.2aversus Nav1.4 we sought for the toxin bioactive surfacetoward the skeletal muscle Nav and compared it with thereported surface toward brain Navs (25). We have previouslyshown that substitution of Tyr-4, Thr-10, Phe-14, Leu-19,Tyr-24, Arg-27, Glu-28, Gln-32, Tyr-40, Tyr-42, Phe-44,Trp-47, Trp-58, and Lys-63 for Ala considerably affectedCss4 binding affinity for rat brain synaptosomes (supple-mental Table S1 and Ref. 25). Most of these substitutionsalso decreased the binding affinity for rat muscle mem-branes, except for those at Thr-10, Phe-14, Glu-28, Gln-32,and Lys-63 (supplemental Table S1 and Fig. 1). These resultsraised a subset of residues that are involved in the surface ofinteraction of the toxin with both rat brain and skeletal mus-cle Navs and highlighted a few residues whose roles differ.This difference is notable in light of the CD alteration ren-

dered by T10A, F14A, and K63A and the fact that Glu-28 andGln-32 have been assigned to the hot spot in the surface ofinteraction of Css4 with the rat brain channel (25). Thus,receptor site-4, which pertains to the interface between Css4and the Nav, differs in mammalian brain versusmuscle Navs,especially in respect to Phe-14 and Glu-28 (supplementalTable S1), which laid the groundwork for construction of amutant toxin specific for Nav1.4. Because the affinity ofmutant E15A for both channel types exceeded 10-fold that ofthe unmodified toxin (supplemental Table 1 and Fig. 1), weconstructed Css4 mutants bearing various combinations ofsubstitutions F14A and E28R in the background of E15A.The most significant among these constructs was the triplemutant Css4F14A/E15A/E28R, whose affinity for the brain chan-nel dropped 254-fold whereas its affinity for the skeletalmuscle channel was similar to that of Css4E15A (supplemen-tal Table S1), thus providing a highly selective ligand formuscle channels.We then analyzed the effects of these mutants on a variety of

mammalian Navs co-expressed in Xenopus oocytes with the 1subunit, using Css4E15A as the first point of reference. Css4E15Awas active at rNav1.2a and rNav1.4 but had no effect at rNav1.3,hNav1.5, and rNav1.6 even at a concentration of 5 M (Table 1and supplemental Fig. S1). Hence, the preference of Css4E15Afor themammalianNav subtypeswas similar to that of Css4 (19,23, 33), yet the potency of Css4E15A increased10-fold (EC500.46 for rNav1.2a and 0.88 M for rNav1.4) compared with theunmodified toxin (25). Css4E15A induced a25-mV shift in thethreshold of activation for both rNav1.2a and rNav1.4 (at 0.5and 1 M, respectively) (Fig. 2 and supplemental Fig. S1). Theconductance-voltage relation (G/V) curves for rNav1.2a andrNav1.4 in the presence of Css4E15A exhibit two componentswith a minor negative shift in the voltage for half activation(V0.5) of the entire channel population and a stronger shift inthe V0.5 of a fraction of the channel population (Fig. 2 andTable1, values in parentheses). The effects of Css4E15A on channelactivation resembled those of Css4 in the requirement for adepolarizing prepulse of similar duration and the rates of asso-

FIGURE 1. Competition of wild-type and mutant Css4 toxins on binding torat brain and skeletal muscle membrane preparations. Membranes wereincubated with 0.1 nM 125I-His-Css4 and increasing concentrations of the indi-cated mutants at 22 C for 60 min. Nonspecific binding, determined in thepresence of 1 M His-Css4, was subtracted. The Ki values (in nM, n 3) for ratbrain synaptosomes and rat skeletal muscle membranes, respectively, wereCss4, 0.98 0.1, 3.9 1.17; Css4E15A, 0.07 0.01, 0.3 0.1; Css4F14A, 141 18,3.7 0.4; Css4R27A, 31 6.3, 56.5 10.5; Css4E28A, 635 98, 6.2 2.3. Thecurves are from representative experiments.




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ciation and dissociation at rNav1.2a and rNav1.4 (supplementalFig. S1) (19, 20, 23).Css4F14A/E15A, Css4E15A/E28R, and Css4F14A/E15A/E28R had no

effect on the voltage dependence of activation of the neuronalchannels rNav1.2a, rNav1.3, and rNav1.6 and the cardiac chan-nel hNav1.5. However, they shifted the voltage dependence ofrNav1.4 activation equally as well as Css4E15A, demonstratingcomplete specificity for the skeletal muscle channel (Table 1and Fig. 2).Effect of Css4F14A/E15A/E28R on the Gating Properties of

rNav1.4R666G, an Equivalent of the Genetic DisorderhNav1.4R672GAn R672G mutation in D2/S4 of the humanNav1.4 was identified in the SCN4A gene of patients withhypoPP (8) and shown to generate an 8-mV rightward shift in the

voltage dependence of activation and a 5-mV leftward shift in thesteady-state fast inactivation (13). Because Css4F14A/E15A/E28Rinduced a hyperpolarizing shift in the voltage dependence ofrNav1.4 activation (Fig. 2), we examined its effects on an iden-tical mutation constructed in the equivalent position of the ratNav1.4 (rNav1.4R666G). We found that the voltage dependenceof channel activation indeed was right-shifted by 8 mV (V0.5 17 1.3 mV) relative to the unmodified channel (V0.5 24.9 0.3 mV, Fig. 3A), as well as its steady-state fast inacti-vation that was left-shifted by 5 mV (V0.5 55.5 0.7 mV)relative to the unmodified channel (V0.5 49 0.6 mV, Fig.

FIGURE 2. Conductance-voltage relations for rNav1.2a and rNav1.4 in thepresence of various Css4 mutants. A, rNav1.4. B, rNav1.2a. Concentrations ofthe mutant toxins and activation parameters (V0.5) are shown in Table 1. Thecurrent-voltage relations were determined as described in supplemental Fig.S1 using the voltage protocol with a depolarizing prepulse.

FIGURE 3. Characterization of rNav1.4R666G and Css4F14A/E15A/E28R effects

on channel gating properties. A, conductance-voltage relations of theunmodified rNav1.4 (V0.5 24.9 0.3 mV) and of rNav1.4

R666G in theabsence (V0.5 17 1.3 mV) and presence of 1 M Css4

F14A/E15A/E28R (V0.5 25 0.7 and 34.2 1.3 mV for 13% of the channel population). The current-voltage relations were determined as described in Fig. 2. B, steady-state inactiva-tion of rNav1.4 fits a Boltzmann function (see Experimental Procedures, andEquation 2) with V0.5 49 0.6 and V0.5 55.5 0.7 mV for rNav1.4

R666G

and V0.5 50.4 0.7 mV for rNav1.4R666G in the presence of 1 M

Css4F14A/E15A/E28R. Steady-state fast inactivation was determined using a50-ms prepulse to 60 mV followed by a hyperpolarizing pulse to 100 mVand a series of 50-ms prepulses from 90 to 20 mV in 5-mV incrementsbefore the test pulse of 20 mV (see inset).

TABLE 1Effects of Css4 mutants on the activation of various Navs and rNav1.4 mutantsThe G/V relations in the presence of Css4 mutants exhibit two components: a minor negative shift in the V0.5 of activation of the entire channel population (uppernumber), and a stronger shift in the V0.5 (lower number) of a fraction (in parentheses) of the channel population. Data represent the mean S.E. of at least sixindependent experiments. The EC50 of Css4E15A was 0.46 and 0.88 M for rNav1.2a and rNav1.4, respectively. Current-voltage relations were determined as describedin Fig. 2. ND, not determined.

Css4Channel

ControlV0.5 mV

E15A E15A/F14A E15A/R27Q E15A/E28R F14A/E15A/E28RM V0.5 mV M V0.5 mV M V0.5 mV M V0.5 mV M V0.5 mV

rNav1.2a 24.9 0.4 0.5 29.6 0.5 10 25.3 0.3 10 25.1 0.2 10 26.1 0.3 10 25.2 1.242 3 (39%)

rNav1.3 19.1 0.5 5 18.1 0.6 ND ND 10 19.4 0.7 10 18.8 1.4hNav1.5 35.1 0.3 5 35 0.5 ND ND 10 35 0.4 10 35.1 0.4rNav1.6 27.7 0.5 5 25.8 1.6 ND ND 10 26.3 0.4 10 26.7 0.9rNav1.4 24.9 0.3 1 31 0.2 1 31.4 0.4 25 29.2 0.4 1 31 0.2 1 31.6 0.3

36.1 2.1 (21%) 40.1 2.5 (22%) 34.5 1.5 (12%) 36.3 1.6 (21%) 39.5 2.5 (24%)E592A 15.1 0.6 1 16.3 0.2 20 16.2 0.4 10 14.9 0.4 1 16.7 0.3 10 15.1 0.3

31 2.7 (25%) 30 2.1 (25%) 35.7 2.6 (28%)H599Q/D601S/N602S

25 0.2 1 25.3 0.2 1 25.3 0.2 10 24.4 0.7 1 25 0.3 1 24.3 1.2

33.8 3.9 (19%) 34.2 2.2 (20%) 34.6 1.7 (21%) 33.2 2.4 (21%)E650A 20 0.4 1 22.3 0.3 1 22.5 0.5 10 -20 0.3 30 21.3 0.3 10 20.2 0.6

42.7 2.1 (30%) 43.2 1.7 (28%) 37.7 2.1 (16%)L653A 22.3 0.2 10 23.6 0.1 10 23.6 0.1 10 23.3 0.7 10 23.6 0.1 10 23.8 0.4Q657E 25 0.2 1 26.4 0.1 1 26.1 0.3 10 25.2 0.8 1 26.4 0.9 1 26.3 1.3

45 2 (18%) 41 3.1 (19%) 46 1.6 (20%) 43 2.1 (18%)G658N 26.8 0.2 10 26.8 0.3 10 26.8 0.3 10 26.6 0.5 10 26.7 0.3 10 26.8 0.3E1251N 27.2 0.5 2 29.5 0.5 1 30 0.7 2 29.7 0.5 25 30.2 0.6 10 26.8 0.8

52 2.3 (27%) 56 5.2 (26%) 52.3 3.1 (30%) 54 3.2 (25%)H1257K 29.7 0.4 5 31.7 0.9 5 31.5 0.2 10 31.1 0.2 5 32.7 0.3 10 31.7 0.5

39.3 2.7 (17%) 38.7 2.1 (16%) 38.5 2.8 (14%) 39.1 1.8 (16%)




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3B). In a concentration of 1 M, Css4F14A/E15A/E28R shifted thevoltage dependence of activation of rNav1.4R666G to V0.5 25 0.7 mV, and less than 15% of the mutant channel pop-ulation was activated at more negativemembrane potentials (Fig.3A). In addition, the steady-state fast inactivation of rNav1.4R666G

was right-shifted, providing a V0.5 50.4 0.7 mV (Fig. 3B). Theseeffects byCss4F14A/E15A/E28R demon-strated its ability to restoremost ofthe altered gating properties ofrNav1.4R666G, which under theinfluence of this specific modula-tor performed much like theunmodified rNav1.4 under controlconditions.Aside from the therapeutic

potential arising from the specificityof Css4F14A/E15A/E28R for rNav1.4,our results have raised the questionofwhetherCss4 recognizes a similarregion in rNav1.2a and rNav1.4.Therefore, we examined the effectsof mutations in both the toxin andthe channel on their interaction.Css4E15A Effects on rNav1.4

MutantsBased on recent reportsabout substitutions introduced torNav1.2a (E779Q in D2/S1-S2;E837Q, L840C, and G845N inD2/S3-S4) and rNav1.4 (G658N inD2/S3-S4; E1251N and H1257Kin D3/SS2-S6) that reduced theeffects of the -toxins Css4 (19, 20)and Tz1 (22), we constructedrNav1.4 mutants E592A in D2/S1-S2; E650A, L653A, and G658N inD2/S3-S4; and E1251NandH1257Kin D3/SS2-S6 (see Fig. 4A forsequence alignment). In addition,four residues that differ betweenrNav1.2a and rNav1.4 at D2/S1-S2and S3-S4 were substituted atrNav1.4 with their rNav1.2a equiva-lents (H599Q/D601S/N602S andQ657E). Analysis of the eight channelmutants in the presence of Css4E15Arevealed a similar negative shift in the

G/V relations for rNav1.4E592A, rNav1.4H599Q/D601S/N602S,rNav1.4E650A, and rNav1.4Q657E and the unmodified channel. Incontrast, channel mutations L653A and G658N abolished theCss4E15A effect as indicated by the unaffected G/V relationsmeasured with up to 10 M toxin (Table 1 and Fig. 4). The G/Vrelations of rNav1.4E1251N and rNav1.4H1257K were affected byCss4E15A, but with lower potency (EC50 1.91 and 5.3 M,respectively) (Tables 1 and 2 and Fig. 4B). These results suggestthat Leu-653 and Gly-658, and to a lesser extent Glu-1251 andHis-1257, are involved in the interaction of Css4E15A withrNav1.4.Mutant Double Cycle Analysis of Css4 against rNav1.4The

high activity of Css4F14A/E15A, Css4E15A/E28R, and especiallyCss4F14A/E15A/E28R at the skeletal muscle channel and the lack ofeffect at the brain channel indicate that Phe-14 and Glu-28 aretwo major points of difference in the interface between Css4

FIGURE 4. Effects of Css4E15A on rNav1.4 mutants. A, alignment of D2/S1-S2, D2/S3-S4, and D3/SS2-S6 Nav regionsof a number of Navs (Swissprot accession numbers are P15390 for rNav1.4, P04775 for rNav1.2a, P04774 forrNav1.1, P08104 for rNav1.3, Q14524 for hNav1.5, CAA70364 for rNav1.6, O08562 for rNav1.7). Numbers insuperscript provide the position of the indicated residues in the channel sequence. B, differences in conduc-tance-voltage relations of rNav1.4 mutants in the presence of Css4

E15A. Open circles designate control andclosed circles the results obtained at various concentrations of Css4E15A (see Table 1). The current-voltagerelations were determined as described in Fig. 2.

TABLE 2EC50 for activation of mutant rNav1.4 channels by Css4 mutantsEC50 values (M) were determined as described in supplemental Fig. S2. The datarepresent the mean S.E. for the independent experiments (number in parenthe-ses). ND, not determined.

rNav1.4mutant

EC50Css4E15A Css4F14A/E15A Css4E15A/R27Q Css4E15A/E28R

M M M M

Wild-type 0.88 0.12 (6) 0.93 0.15 (4) 26.2 2.9 (4) 0.98 0.03 (4)E592A 1.11 0.3 (5) 19.2 2.1 (4) ND NDE650A 1.26 0.17 (4) ND ND 29.8 3.1 (3)E1251N 1.91 0.17 (4) ND 2.31 0.3 (4) 23.9 2.7 (3)




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and the two types of Navs. Based on the general conservation ofmammalian Navs and the elucidation of a subset of commonresidues involved in the interaction of Css4 with the two chan-nel types, we compared the effects of Css4E15A, Css4F14A/E15A,Css4E15A/E28R, and Css4F14A/E15A/E28R on the activation prop-erties of the eight rNav1.4 mutants. In addition, we included inthis comparative analysis Css4E15A/R27Q because of the proxim-ity of Arg-27 to Glu-28 and the similar effect of its substitutionon both rNav1.2a and rNav1.4 (supplemental Table S1 andTable 1) (25). Where a significant change in activity wasobtained (Table 1), the EC50 values were determined, enablingmutant double cycle analysis (Table 2 and supplemental Fig.S2). The EC50 of Css4F14A/E15A at rNav1.4E592A was 20-foldhigher than its EC50 at the unmodified channel (19.2 2.1versus 0.93 0.15 M; Table 2), indicating a coupling energy of1.65 kcal/mol between F14A of the toxin and E592A of thechannel. Css4E15A/R27Q hardly affected rNav1.4 (EC50 26.2 2.9 M), indicating the importance of Arg-27 for Css4 interac-tion with its rNav1.4 receptor site (Table 2). Of the eight chan-nel mutations, E1251N significantly increased the ability ofCss4E15A/R27Q to produce a negative shift in the conductance-voltage relations of the channel (Tables 1 and 2). Indeed, thepotency of Css4E15A/R27Q at rNav1.4E1251N was very similar tothat of Css4E15A (Table 2). This indicates that R27Q has a sig-nificant positive coupling energy with E1251N (G 1.89kcal/mol). Most intriguing were the effects of Css4E15A/E28R onthe channelmutants.Whereas the EC50 ofCss4E15A/E28R for theunmodified channelwas very similar to that ofCss4E15A (0.980.03 M), it increased prominently for rNav1.4E650A andrNav1.4E1251N (29.8 3.1 and 23.9 2.7 M, respectively)(Tables 1 and 2 and Fig. 4B). Hence, a single Css4 residue (Glu-28) exhibits negative coupling energywith two channel residues(G 1.8 and 1.41 kcal/mol for E650A and E1251N,respectively) positioned on two distinct extracellular loops inDomains 2 and 3 (D2/S3-S4 and D3/SS2-S6).

DISCUSSION

The design of Css4F14A/E15A/E28R,a specific activator of rNav1.4, wasbased on systematic analysis of thebinding of a large collection of Css4mutants to Navs in rat brain and ratskeletal muscle membrane prepara-tions and the finding that substitu-tion of Phe-14 and Glu-28 for Alamarkedly decreased activity at thebrain channel without affectingactivity at the muscle channel.Despite these differences, the sub-stitution of a number of other Css4residues had a similar impact on thetwo channel subtypes, suggestingthat the toxin recognizes a similarregion on both channels. The sim-ilar/non-similar face of toxin inter-action with these channels raisedtwo experimental avenues thatcould exploit Css4F14A/E15A/E28R.The first was to assess the therapeu-

tic potential of such a specific Nav activator, and the secondwasto examine its interaction with the skeletal muscle channel.The Putative Therapeutic Potential of Css4F14A/E15A/E28R in

Neuromuscular DisordersOver thirty mutations in SCN4A,the gene encoding the skeletal muscle Nav, have been linked toneuromuscular disorders such as hypo- and hyperkalemicperiodic paralyses, paramyotonia congenital, Nav myotonias,and congenital myasthenic syndrome (8). Three of these muta-tions were localized to D2/S4 of hNav1.4 (R669H, R672G/H/S,and R675G/Q/W) (reviewed in Ref. 8), and one was localized toD3/S4 of hNav1.4 (R1132Q) (14). Skeletal muscle fibers from apatient heterozygous for R672G displayed depolarization andweakness in low potassium extracellular solution (15). Both theincreased inactivation and the impaired voltage dependence ofactivation caused by the R672Gmutationmay contribute to thereduction of Nav performance and reduced membrane excit-ability (14). Sternberg et al. (34) reported a very severe hypoPPphenotype in a family carrying the R672G mutation where thefrequency and severity of attacks increased in response to treat-ment with acetazolamide. The ability of Css4F14A/E15A/E28R torestore the gating properties of rNav1.4R666G (Fig. 3), whichmimicked the hypoPP mutation hNav1.4R672G, demonstrates aputative therapeuticpotentialwhenseekinga remedy to thedefec-tiveSCN4Ageneproduct.The specificityofCss4F14A/E15A/E28R forthe skeletal muscle Nav suggests that it merits investigation as apossible treatment for hypoPP and perhaps other neuromuscu-lar disorders with symptoms of reduced muscle excitability.Derivation of Channel Architecture from Css4-rNav1.4

Mutant Double Cycle AnalysisThe interaction of Css4 andrNav1.4 was examined by mutant double cycle analysis, focus-ing on residues employed in the design of the selective activatorand on rNav1.4 channel residues whose equivalents were eitherproposed to be involved in the interaction ofCss4with rNav1.2a(20) or whose substitution was shown to affect the interaction

FIGURE 5. Schematic presentation of the inter-domain arrangement of the voltage-sensing and poremodules in rNav1.4. A, schematic Css4 structural model (in ribbon) (23) based on the known structure of the-toxin Cn2 (90% similarity in sequence) (35) (PDB accession 1cn2) highlighting the three residues shownhere to have coupling energies with channel residues derived from three distinct extracellular loops ofDomains 2 (yellow) and 3 (blue). B, external view of the proposed inter-domain arrangement of the transmem-brane segments (S1-S6) in the mammalian Nav based on panel A and following the proposed similarity inintra-domain arrangement between NaChBac and Kv1.2 (3, 6). The dashed line illustrates a Css4 projection in itsputative bound form to demonstrate the size relations of the toxin and the channel.




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of the toxin Tz1 with rNav1.4 (22). Four conserved residues inthe external loops of rNav1.2a have been implicated in the inter-action with Css4: Glu-779 in D2/S1-S2 and Glu837, Leu-840,and Gly-845 in D2/S3-S4 (Fig. 4A) (20). The decrease in theability of Css4E15A to modulate channel activation followingsubstitutions of L653A andG658N in rNav1.4 (equivalent posi-tions of Leu-840 andGly-845 in rNav1.2a) (Table 1 and Fig. 4A)suggests that these residues belong to a conserved region ofreceptor site 4. However, the lack of effect of substitutionsE592A and E650A in rNav1.4 (equivalent positions in rNav1.2aare Glu-779 and Glu-837) on Css4E15A action indicates thatdespite their conservation in all mammalian Navs these resi-dues do not belong to the common receptor for scorpion-tox-ins on Navs (Table 1 and Fig. 4A).Overall, the substantial variations in the receptor site for

Css4 in rNav1.2a and rNav1.4 are consistent with the results ofbinding assays of Css4 mutants on rat brain and muscle Navsand are further demonstrated by the difference in the face ofinteraction between the two channels and the triple mutantCss4F14A/E15A/E28R (supplemental Table S1). In light of the gen-eral conservation ofmammalianNavs, the high specificity of thetriple Css4 mutant for Nav1.4 suggests that the toxin residuesPhe-14 and Glu-28 encounter a different face upon binding torNav1.2a versus rNav1.4. This prompted us to examine bymutant cycle analysis Css4-rNav1.4 interacting pairs. Wefocused on the toxin residues Phe-14 and Arg-28, whose sub-stitution abolished the activity toward rNav1.2a, but notrNav1.4, and Arg-27, whose substitution affected both channeltypes (Tables 1 and 2 and Fig. 2). In the channel we selectedthose residues whose substitution was shown to influence theeffect of scorpion -toxins (20, 22). The significant couplingenergies obtained between F14A (toxin) and E592A atD2/S1-S2 (channel), aswell as E28R (toxin) and the two channelresidues E650A at D2/S3-S4 and E1251N at D3/SS2-S6 (Table2), along with the three-dimensional model of Css4 (Fig. 5) (25,35) enabled to estimate the distances between the toxin channelinteracting pairs. As the distance between C of Phe-14 and Cof Glu-28 is 68 , Glu-592 of D2/S1-S2 is likely to reside 10 from Glu-650 of D2/S3-S4. Because E28R of the toxin dem-onstrated a negative energy of interaction with both E650A ofD2/S3-S4 and E1251N of D3/SS2-S6, we conclude that the twochannel residues are very close to one another (Fig. 5). Thisconclusion is further corroborated by the high positive cou-pling energy between R27Q of the toxin and E1251N of thechannel (Tables 1 and 2). Based on these data and in the absenceof a three-dimensional structure of the Nav, we suggest thatloop S3-S4 of the voltage sensor module in Domain 2 is in veryclose proximitywith loop SS2-S6 of the poremodule inDomain 3.Although substantiation of this suggestion requires resolution ofthe channel three-dimensional structure, the proposed architec-ture resembles that reported for Kvs, where the voltage-sensingmodule of eachdomain is in close proximity to the poremodule ofthe adjacent domain, in a clockwise orientation (Fig. 5) (3).

AcknowledgmentWe thank Prof. F. Frolow, Tel Aviv University, forhelp with the illustration of the toxin-channel interaction.

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