Reconstitution of Neurotoxin-Modulated Ion Transport

Proc. Nati. Acad. Sci. USA Vol. 81, pp. 1239-1243, February 1984 Neurobiology

Reconstitution of neurotoxin-modulated ion transport by the voltage-regulated sodium channel isolated from the electroplax of Electrophorus electricus (action potential/ion flux/veratnidine/tetrodotoxin/local anesthetics) ROBERT L. ROSENBERG, SALLY A. TOMIKO, AND WILLIAM S. AGNEW Department of Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510 Communicated by Joseph F. Hoffman, October 18, 1983

ABSTRACT The functional reconstitution of the voltage- STX have been used to monitor the purification of the regulated Na channel purified from the electroplax of Elec- TTX/STX-binding components from eel electroplax (4, 5), trophorus electricus is described. Reconstitution was achieved rat muscle sarcolemma (6), and rat brain synaptosomes (7). by removing detergent with Bio-Beads SM-2 followed by The reported peptide compositions of the proteins from freeze-thaw-sonication in the presence of added liposomes. these tissues are somewhat different. Whereas the TTX- This preparation displayed heat-stable binding of 3H-labeled binding activity of the eel electroplax copurifies to homoge- tetrftlotoxin (TTX) (Kd = 33 nM). 22Na' influx was stimulated neity with a single, heavily glycosylated peptide of Mr 2- to 5-fold by alkaloid neurotoxins and blocked by TTX. Ve- 260,000-300,000 (5), additional peptides of Mr 37,000 and ratridine activated Na' influx with a KY2 of 18 IAM, and this 39,000 are present in preparations from brain (8), and pep- activation was blocked by TTX precisely in parallel with spe- tides of Mr 45,000, 38,000, and 39,000 appear in preparations CifiC [3HITTX binding. Batrachotoxin stimulated 22Na' flux from muscle (9). Because TTX and STX binding identifies more effectively than did veratridine. No effect of the peptide only a part of the Na channel, these differences may reflect anemone toxin II was found. Insertion of the Na channel into losses of important peptide subunits during isolation, incom- membranes resulted in 60-70% of the TTX-binding sites fac- plete purification, proteolytic degradation, or actual differ- ing the vesicle exterior. Thus, external TTX partially inhibited ences in the proteins from different tissues or species. flux, whereas blockade was complete when TTX was also To determine whether the isolated proteins are functional- equilibrated with the vesicle interior. The lipid-soluble local ly intact, the TTX/STX-binding components of muscle (10) anesthetics tetracaine and dibucaine inhibited flux completely. and brain (11, 12) were previously reconstituted into phos- QX-222, a charged derivative of lidocaine, blocked only a frac- pholipid vesicles and alkaloid-stimulated, TTX-blocked tion of the channels, apparently those oriented inside-out. Pu- 22Na+ influx was demonstrated. In this communication we rified samples were predominantly composed of the Mr report the incorporation of the Na channel purified from 260,000-300,000 glycopeptide but contained variable quanti- electroplax into lipid vesicles. Alkaloid toxins, local anes- ties of smaller peptides. Veratridine-dependent flux and pep- thetics, and TTX all modified Na+ influx in a manner con- tide compositions were determined on fractions across a gel sistent with their known pharmacology. The large glycopep- filtration column profile. Stimulated flux codistributed only tide accounted for -80% of the protein present in the prepa- with the large peptide. rations tested. Furthermore, evidence is presented that the smaller peptides present probably are not required for func- The early depolarizing currents of the propagated action po- tional reconstitution. tential are mediated by a voltage-regulated sodium channel. The channel is an integral membrane protein that forms an MATERIALS AND METHODS aqueous pathway through which sodium ions can pass in re- Materials. Citrate-free TTX was the kind gift of Y. Kishi, sponse to electrochemical driving forces. The conductance Harvard University, and it was tritiated by the Wilzbach state of the channel is regulated by time- and voltage-depen- procedure and purified as described (4). The toxin was of dent gating mechanisms. Because the regenerative depolar- specific activity 59.1 Ci/mol (1 Ci = 37 GBq) and =65% ra- izations in many nerve, muscle, and related types of excit- diochemical purity as determined by frog sciatic nerve bioas- able cells are controlled by this protein, its structure and say. Egg phosphatidylcholine (Sigma type IX-E) ran as a sin- mechanisms for transport and regulation are of considerable gle spot in a standard thin-layer chromatography solvent sys- interest. tem. Veratridine was from Aldrich. BTX was the generous Among a variety of pharmacological agents that are func- gift of J. Daly (National Institutes of Health). QX-222 from tional probes of the Na channel, four classes of compounds Astra Pharmaceutical (Worcester, MA) was the gift of R. Al- have been especially useful: (i) tetrodotoxin (TTX) and saxi- drich. Dibucaine was from ICN Pharmaceuticals. Samples of toxin (STX) bind with high affinity to an external site to anemone toxin II (ATX II) were gifts of M. Lazdunski and block ion conductance; (ii) lipid-soluble alkaloid toxins, in- L. Beress. Carrier-free 22NaCl was from Amersham. Bio- cluding veratridine and batrachotoxin (BTX), alter gating Beads SM-2 from Bio-Rad were washed according to mechanisms to cause persistent channel activation; (iii) local Holloway (13). All other reagents were from Sigma. anesthetics, including derivatives of procaine and lidocaine, Purification of the TTX-Binding Protein. The purification interact with an internal site to block conductance; and (iv) procedures were essentially as described earlier (4, 5), with peptide toxins isolated from species of scorpion and sea the following modifications: Protease inhibitors (50 ,uM o- anemone perturb gating so as to enhance the effects of the phenanthroline, 50 ,uM L-1-tosylamido-2-phenylethyl chloro- alkaloid toxins (for reviews see refs. 1-3). Tritiated TTX and methyl ketone, 100 ,uM phenylmethanesulfonyl fluoride, 1.0 mM EGTA, and pepstatin A at 0.1 ,ug/ml) were added during The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: FTS, freeze-thaw-sonication; TTX, tetrodotoxin; in accordance with 18 U.S.C. §1734 solely to indicate this fact. STX, saxitoxin; BTX, batrachotoxin; ATX II, anemone toxin II.

1239 Downloaded by guest on October 2, 2021 1240 Neurobiology: Rosenberg et al. Proc. NatL Acad Sci. USA 81 (1984)

solubilization of membrane suspensions and were present somes. Thermal stability is one criterion for measuring inser- throughout purification. Protein was eluted stepwise from tion of the protein into artificial liposomes. Unlike toxin- the DEAE-Sephadex A-25 in a column maintained at 00C. binding sites in native membranes, the detergent-solubilized An Amicon XM-50 ultrafiltration membrane was used for protein is quite unstable (4, 19), and binding activity is rapid- pressure concentration prior to Sepharose 6B chromatogra- ly lost at a temperature of 370C, as shown in Fig. 1. When the phy. TTX was not added for stabilization. The Sepharose 6B detergent is adsorbed from the solution by Bio-Beads treat- column and the collected fractions were maintained at 00C. ment, about 50% of the binding activity and total protein is Protein concentrations were determined by the method of lost. The remaining TTX-binding sites, however, are rela- Peterson (14) or by the fluorescamine method (15). [3H]TTX tively stable to elevated temperature (Fig. 1). This material is binding was measured by the Sephadex G-50 assay (4). Sodi- associated only with lipid present in buffers during purifica- um dodecyl sulfate/polyacrylamide gels were run according tion (4, 5). However, no greater heat stability was obtained to the method of Laemmli (16). after lipid supplementation followed by an FTS cycle (Fig. Reconstitution Protocols. The sodium channel was incor- 1). In experiments in which the protein was supplemented porated into phospholipid vesicles as follows: Detergent was with a lipid/detergent mixture before detergent removal, the removed by the addition of Bio-Beads SM-2 (0.3 g/ml) to reconstituted material was completely stable to a 30-min in- purified preparations (containing 100-150 ,ug of protein per cubation at 37TC (Fig. 1). By this criterion, therefore, the ml in 0.1% Lubrol, 0.183 mg of phosphatidylcholine per ml, latter method, similar to the methods of Weigele and Barchi 100 mM sodium phosphate, and protease inhibitors, pH 7.4) (10) and Talvenheimo et al. (11), would seem to be optimal. followed by agitation at 40C for 5 hr (10, 11, 13). This prepa- However, large and more reproducible flux signals (general- ration was then fused with added liposomes in a freeze- ly 1000-3500 nmol/mg of protein per min) were consistently thaw-sonication (FTS) cycle as described by Kasahara and observed with protein reconstituted with an FTS cycle, sig- Hinkle (17): Phosphatidylcholine (40 mg/ml) dried from a nals completely reproducible for up to 4 days after Bio- chloroform solution was sonicated to opalescence in sodium Beads treatment. Thus, FTS preparations were used for the phosphate buffer (90 mM, pH 7.4) in a bath type sonicator studies described here. (Laboratory Supplies, Hicksville, NY). Lipid sonicate was 22Na' Influx Mediated by the Reconstituted Na Channel. added to the Bio-Beads-treated samples to produce a mix- Veratridine-stimulated 22Na' influx was measured to assess ture of 75-100 ,ug of protein per ml and 10 mg of lipid per ml the Na transport function of the reconstituted TTX binding and aliquots were frozen rapidly in dry ice/acetone, thawed component. As illustrated in Fig. 2A, vesicles preincubated slowly at room temperature for approximately 5 min, and for 5 min at 30TC exhibited some basal 22Na' influx. When sonicated for 10 sec at full power. veratridine was present at 100 uM, however, 22Na' influx The assay of alkaloid-dependent 22Na' influx was a modi- was stimulated by 2- to 3-fold (Fig. 2A). Such stimulation by fication of the method of Epstein and Racker (18). Small col- veratridine was observed in 20 experiments. No stimulated umns were packed with Dowex 50X8-100 resin prepared in flux was observed in pure phosphatidylcholine vesicles re- the Tris form as described (18) and were stored in the cold constituted without protein. Addition of 1 ,uM TTX at 0C 15 for a short time before use. Vesicles were preincubated with min prior to adding veratridine resulted in partial inhibition or without TTX for 15 min at 0°C and were incubated in ve- of flux. This inhibition was 60% in the example shown (Fig. ratridine or the veratridine vehicle for 5 min at 30°C. 22Na' 2A) and was between 45% and 80% in all experiments con- influx was initiated by a 1:4 dilution of the prepared sample ducted. This may be explained by the vectorial orientation of into isotonic Tris sulfate (170 mM, pH 7.4) containing the the channel in the membrane and is consistent with studies same concentrations of toxins and approximately 10 ,uCi of of [3H]TTX binding to reconstituted preparations before and 22Na+ per ml. Flux was measured at 30°C. At the appropri- after FTS. In both cases only 60-70% of the sites were ac- ate times, 0.2 ml of the flux mixture was applied to the cold cessible to external TTX. In keeping with this, when TTX Dowex column and was eluted with 2 ml of ice-cold isotonic was added before FTS, allowing access to inside-out as well sucrose solution (220 mM sucrose, 1 mg of bovine serum as outside-out oriented channels, inhibition of stimulated albumin per ml). Internalized 22Na+ was quantified by liquid flux was always complete (Fig. 2B). scintillation counting. Veratridine was prepared as a 2 mM The dependence of 22Na' influx on veratridine concentra- suspension in Tris sulfate (170 mM, pH 7.4) by brief sonica- tion was determined in duplicate assays in three experiments tion. BTX stock solutions (500 ,uM) were in ethanol. Vesicle total internal space was determined from the amount of 22Na+ internalized after incubations of more than 24 hr. >; lOoo 0 u~~~~ RESULTS n u x 75 Purification and Reconstitution of the TTX-Binding Compo- 0 E nent. The purification of the TTX-binding protein was a cecon modification of methods previously reported (4, 5). Although .0 50 past studies employed TTX to stabilize the protein, it was not used here because of difficulties in removing the toxin X 25 before reconstitution. This reduced the yield of TTX-binding H- activity from the Sepharose 6B column to -50% of normal, 0 10 20 30 with no effect on protein recovery or peptide composition. Time at 370C, min The problem with stability was partially offset by maintain- ing the column at 0°C, but specific activities of preparations FIG. 1. Thermal stability of TTX-binding sites before and after used in these studies were 800-1000 pmol of [3H].TTX bound reconstitution. Samples were tested for stability by incubation for per mg of protein, indicating the preparations were 25-30% the indicated times at 370C, followed by assay for TTX binding. active. These samples were relatively pure on the basis of Samples tested: before reconstitution (0), after treatment with Bio- peptide composition, as indicated below. Beads in the presence (z) and absence (A) of additional lipid (phos- phatidylcholine at 6.0 mg/ml in 1.5% Lubrol PX), and after treat- The methods tested for reconstitution were based on stud- ment with Bio-Beads followed by FTS in the presence of phosphati- ies by Kasahara and Hinkle (17) on glucose transport sys- dylcholine vesicles (10 mg/ml) (0). Before assay, reconstituted tems and by Weigele and Barchi (10) and Talvenheimo et al. preparations were solubilized at 0C with sufficient Lubrol PX to (11) on Na channel proteins from sarcolemma and synapto- give a maximal signal (0, 1% Lubrol; o and A, 0.25% Lubrol). Downloaded by guest on October 2, 2021 Neurobiology: Rosenberg et aL Proc. NatL. Acad Sci. USA 81 (1984) 1241

25- A B 100- Ha,K.' 04)- Ver. - ~~~~~Ver '4-oon 0 75 /- ' 15- * Ver+eTTX >e = = E 50 r- 1+ +~~*,;//- ^ r ; Ver + TTX + x Z Control Ver+T 5 - Control Z E 25sl I I~~~~~~~~ 20 40 60 20 40 60 / L Is/ Time, sec 0 A20 40 60 80 100 200 Veratridine, FIG. 2. Veratridine-stimulated 22Na' influx into vesicles con- ALM taining the reconstituted Na channel. (A) Vesicles prepared by FTS FIG. 3. ConcentrationHAdependence/AAAof/AAactivationO offlux by verat- (containing 50-75 Mg of protein per ml) were incubated for 15 min at ridine. Vesicles containing the reconstituted Na channel were incu- 00C in the presence (A) or absence (e, o) of 1 MM TTX. Then 0.1 mM bated for 5 min at 30'C in the presence of veratridine at the concen- veratridine (Ver) was added (a, A), or the veratridine vehicle (170 trations shown. 22Na' influx was measured in a 20-sec incubation. mM Tris sulfate, pH 7.4) was added to the control preparation (o). Each point is the result of six individual determinations except at Samples were incubated for 5 min at 30'C and 22Na' influx was veratridine concentrations of 20 uM (n = 5) and 200 MiM (n = 2). measured. (B) Vesicles containing the reconstituted Na channel Bars indicate SEM. Maximal specific influx of these preparations were prepared and incubated as in A, except that 1 MM TTX was was 1.2-2.1 Mmol of Na' per mg of protein per min. added prior to FTS. In these experiments 10% of the internal space was equivalent to an uptake of 0.98 Mmol of Na' per mg of protein. vation (2, 21). As indicated in Fig. 5, BTX stimulated 22Na' Measured at 10 sec, veratridine-stimulated flux was 3.5 Amol of Na' influx. After 45-min incubations with this toxin at 30'C, up to per mg of protein per min (A) and 3.2 Amol of Na' per mg of protein 5-fold increases in flux were observed. As with veratridine, per min (B). BTX-stimulated flux was inhibited partially by TTX added after FTS (Fig. SA) and fully by TTX added before FTS (Fig. employing two preparations (Fig. 3). Veratridine stimulation SB). was saturable, with maximal and half-maximal flux being Peptide neurotoxins isolated from the scorpion Leiurus elicited by 100 MM and 18 MM veratridine, respectively. quinquestriatus (ScTX) and the anemone Anemonia sulcata The interaction of TTX with the reconstituted Na channel (ATX II) bind to an external site on the channel and enhance was examined in more detail. Fig. 4A illustrates a Langmuir the chronic activation of the channel by alkaloid neurotoxins binding isotherm from a reconstituted preparation. The equi- (2, 22). Therefore the effect of ATX 11 (10 MM) on flux stimu- librium dissociation constant (Kd) was 33 nM. Fig. 4B illus- lated by a submaximal concentration of veratridine (5 MM) trates the inhibition of veratridine-stimulated 22Na' influx as was examined. The response to added ATX II alone or ATX a function of the total TTX concentration added after FTS. II plus veratridine was identical to controls with no additions The stimulated flux was inhibited up to 80%, with 45 nM or the veratridine alone, respectively (data not shown). TTX causing half-maximal inhibition. In Fig. 4C, the percent The effects of local anesthetics are summarized in Table 1. of maximal TTX binding and percent of maximal inhibition In these experiments the anesthetics or TTX were added af- are plotted as a function of total TTX concentration, to illus- ter FTS. After incubation with the inhibitor and veratridine, trate the close correlation between binding and blockade. 22Na' influx at 60 sec was measured. TTX (1 MM) inhibited BTX is a more effective alkaloid believed to interact with stimulated flux by 59%, whereas the membrane-permeant the same binding site as veratridine, resulting in chronic acti- anesthetics tetracaine and dibucaine (1, 23) caused complete

I-I 0 360 - 100 - C 1-

+ E< = 4c6 E CZ 50 i H 0 0 B6 r. la 2 0 .00 0 1 0 100 206 440 50 10( 0 50 1001000 TTXfree, nM TTXtotal, nM TTXtotal, nM

FIG. 4. The interaction of TTX with the reconstituted Na channel. (A) Binding of [3H]TTX. Vesicles containing the reconstituted Na channel (no FTS cycle) were incubated in [3H]TTX for at least 20 min at 0C and bound [3H]TTX was determined by the Sephadex G-50 assay (4). [3H]TTX binding was measured in the absence of any detergent. Nonspecific [3H]TTX binding, determined in the presence of 1 AM unlabeled TTX, was less than 10% of the total signal and was subtracted from the data reported. The curve is drawn according to the equation [TTXbound] = Bmax4ITTXtotal - TTXbund]/([TTXtotal - TTXbound] + Kd), using the results of a linear regression fit of the data plotted in Inset according to Scatchard (20). Data points are single determinations. Bmax = 71 pmol/ml, Kd = 33 nM. (B) TTX inhibition of veratridine- stimulated 22Na+ influx. Vesicles containing the reconstituted Na channel were incubated for 15 min at 0C in TTX at the concentration shown. Veratridine (0.1 mM) was added and the vesicles were incubated for 5 min at 30C. 22Na+ influx was determined in a 60-sec incubation. Control 22Na+ influx ih the absence of any toxin was subtracted from the data. Data shown are the triplicate determinations of 22Na+ influx from a single preparation of reconstituted sodium channels. At 60 sec, maximum veratridine activated influx in this preparation was 0.63 Mmol of Na' per mg of protein. (C) [3H]TTX binding and TTX blockade as a function of total TTX concentration. Data from A are shown as the percent of maximal specific binding versus total [3H]TTX concentration (e), and data from B and a different TTX-inhibition experiment from another preparation were averaged and plotted as percent of maximal TTX blockade of 22Na' influx (o). The data shown are from six individual determinations of influx except at 50 nM TTX (n = 5) and 100 nM TTX (n = 4). Half-maximal effect for both binding and blockade is 45 nM TTX. Downloaded by guest on October 2, 2021 1242 Neurobiology: Rosenberg et al. Proc. NatL Acad Sci. USA 81 (1984)

Table 1. Inhibition of veratridine-stimulated Na' transport by 36 A B T4-V BTX / BTX TTX and local anesthetics 0I% 27 Veratridine- *BTX+TTX stimulated C' Na' influx, Inhibition, .= 18 V ~~~~~~BTX+TTX Exp. Inhibitor Amol/mg per min % (n) + 4 Vehicle Vehicle z 9 1 None 0.83 0 TTX (1 /iM) 0.34 59 ± 2.7 (3) 0 10 20 30 40 10 20 30 40 Tetracaine -0.05 105 ± 2.6 (3) Time, sec (0.1 mM) Dibucaine -0.22 127 ± 0.3 (3) FIG. 5. BTX activation of 22Na' influx by the reconstituted Na (0.1 mM) channel. (A) Vesicles containing the protein were incubated in 5 kuM BTX (m), 5 MxM BTX plus 1 MLM TTX (A), or 1% ethanol (z), for 45 2 None 2.54 0 min at 30'C and 22Na' influx was measured (n = 1). (B) Same as A, TTX (1 uM) 1.03 60 ± 1 (2) except TTX was added to 1 MLM before FTS (A), and all samples were then incubated in BTX or ethanol as in A and assayed for QX-222 (3 mM) 0.72 72 ± 3 (2) 22Na' influx. At 10 and 40 sec, BTX and vehicle n = 2, otherwise n TTX (1 iM) + -0.16 106 ± 3 (2) = 1. The 10% internal space for these preparations represents influx QX-222 (3 mM) of 1.4 Amol of Na' per mg of protein. BTX-stimulated flux at 10 sec FTS vesicles were incubated in the presence of the inhibitors is 14.4 tkmol of Na' per mg of protein per min (A) and 11.4 tkmol of shown for 15 min at 0C and then incubated in 0.1 mM veratridine Na' per mg of protein per min (B). for 5 min at 30'C. 22Na+ influx was measured at 60 sec. Veratridine- stimulated influx is calculated from the difference between 22Na+ inhibition at 0.1 mM. The quaternary derivative of lidocaine, uptake measured in the presence and absence of veratridine. Per- QX-222, is charged and cannot readily cross membranes (1, cent inhibition is shown ± SEM (n = 3) or + range (n = 2). 23). When added to the vesicles, 3 mM QX-222 inhibited only 72% of the stimulated flux. This could be explained if tuted samples of highest specific activity fractions pooled the anesthetic blocked transport only by inside-out directed from the Sepharose 6B column (4). Fig. 6 shows sodium do- channels. In keeping with this, QX-222 and TTX together decyl sulfate/polyacrylamide gels of such material. The sam- blocked 100% of the stimulated flux. ples were not completely homogeneous, containing smaller Peptide Composition of the Purified Reconstituted Na Chan- peptides in addition to the -300,000-dalton species, though nel. The flux studies were generally performed on reconsti- none in the 18,000- to 40,000-dalton range. The large peptide A 13

Al. x ()

00- --

- 2()(

-93 06-

L4 \ --66 45

I (10 I 1(1(1 K, 2)6

45

- D-F 1)1- 28 S-no36( .A(41) 4AA4 6n-s") 44l4(1 44 -6 40 I ;()o1

FIG. 6. Correlation of veratridine-stimulated 22Na' influx with the distribution of peptides present in the purified preparation. Individual and pooled fractions from the Sepharose 6B column purification either were incorporated into phosphatidylcholine vesicles by Bio-Beads treatment followed by FTS or were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (16). Veratridine-stimulated 22Na' influx of each sample was determined by using 0.1 mM veratridine and an influx time of 20 sec. In each panel, the percent stimulation of 22Na' influx of the veratridine-treated vesicles over control vesicles is plotted against fraction number. Polyacrylamide gels run on individual and pooled fractions were stained with Coomassie brilliant blue and subjected to scanning densitometry. Representative gels (A = 6% acrylamide; B = 5-12% linear acrylamide gradient to resolve the low molecular weight region) are shown on the right. Peptides Of Mr 270,000-300,000, 95,000, 53,000, 49,000, 47,000, and 42,000 were identified and the area under each peak was determined. No additional peptides in the molecular weight range of 40,000-18,000 were observed on the gradient gels. In each graph the relative area of an individual peptide peak is plotted against the fraction number. Note the different scales for the Mr 270,000-300,000 and 95,000 peptides. DF, dye front. Downloaded by guest on October 2, 2021 Neurobiology: Rosenberg et aL Proc. NatL Acad. Sci. USA 81 (1984) 1243

accounted for more than 80% of the total protein present in ed together with TTX, which blocks outside-out channels, most samples, as estimated from the areas under densitome- inhibition was complete. That both toxins each blocked 60- ter scans of sodium dodecyl sulfate gels. 72% may indicate a finite membrane permeability to QX-222. To determine whether neurotoxin-activated ion transport Attempts to demonstrate flux activation by the peptide is mediated exclusively by the large glycopeptide, complete- toxin ATX II were unsuccessful. However, this toxin ap- ly homogeneous preparations will have to be reconstituted. pears to act at the same site as the peptide toxin from the As a step in this direction, however, reconstitution was used scorpion Leiurus (22), and recent reports with the synapto- as a semiquantitative assay for function in the following ex- somal protein indicate a requirement for an unidentified lipid periment. Fractions across the Sepharose 6B column profile factor for the binding of that toxin (12). were reconstituted for comparison with the distribution of An important consideration for elucidation of transport peptides measured from densitometer scans of stained sodi- and regulatory mechanisms is the peptide composition of the um dodecyl sulfate/polyacrylamide gels. In Fig. 6 the per- molecule. Common to all three Na channel proteins that cent of veratridine-stimulated 22Na' influx and the relative have been isolated is a large glycopeptide, of Mr 260,000- amount of each peptide from 300,000 to 18,000 daltons are 300,000 for electroplax and synaptosomal proteins and plotted versus fraction number. It was evident that only the 130,000-230,000 in sarcolemma. Whether smaller peptides distribution of the -300,000 dalton peptide correlated with found in the synaptosomal (8) and sarcolemmal (9) proteins veratridine-stimulated ion transport. The slight offset of the are essential for channel function is at present uncertain. peak of flux activity relative to the large peptide may reflect Smaller subunits have not been found in the purest prepara- the quantitative limitations of reconstitution; smaller pep- tions of the electroplax protein. Although samples reconsti- tides do not appear to be involved because their levels tuted here were only -80% homogeneous, the distribution of change negligibly in the range of the flux activity peak. the remaining smaller peptides was not correlated with flux activity and [3H]TTX binding, consistent with the proposal DISCUSSION that the large glycopeptide alone may bind the neurotoxins In the present studies the purified TTX-binding protein from and local anesthetics and mediate ion transport. eel electroplax has been incorporated into membrane vesi- cles. Na channel-specific ion transport was demonstrated, as The authors thank Drs. C. F. Stevens and R. L. Huganir for stim- well as the presence of several sites of drug action character- ulating and helpful discussions. This work was supported by Nation- istic of the physiologically defined Na channel. The reconsti- al Institute of Neurological and Communicative Disorders and tution procedure involved treatment of the purified protein Stroke Grant NS17928, and by a grant to W.S.A. from the Multiple Sclerosis Society. R.L.R. was supported by National Institutes of with Bio-Beads SM-2, followed by addition of sonicated lip- Health Predoctoral Training Grant GM07527, and S.A.T., by Na- osomes and fusion by FTS. The first step appears to reform tional Institutes of Health Postdoctoral Training Grant NS07102. the protein into vesicles with lipid contained in the purifica- tion buffer, as evidenced by improved thermal stability and 1. Cahalan, M. (1980) in The Cell Surface and Neuronal Func- the fact that only 60-70% of the TTX binding sites are acces- tion, eds. Cotman, C. W., Poste, G. & Nicholson, G. L. (Else- sible to [3H]TTX. The FTS cycle produces largely unilamel- vier/North-Holland, New York), pp. 1-47. lar vesicles (for otherwise less than 60-70% of the TTX bind- 2. Catterall, W. A. (1980) Annu. Rev. Pharmacol. Toxicol. 20, ing sites would be exposed), incorporating the protein into 15-43. vesicles with sufficient internal volume to permit flux mea- 3. Agnew, W. S. (1984) Annu. Rev. Physiol. 46, 517-530. 4. Agnew, W. S., Levinson, S. R., Brabson, J. S. & Raftery, surements. M. A. (1978) Proc. Nati. Acad. Sci. USA 75, 2606-2610. Both BTX and veratridine were active in this preparation. 5. Miller, J. A., Agnew, W. S. & Levinson, S. R. (1983) Bio- The KY, (concentration for half-maximal effect) for veratri- chemistry 22, 462-470. dine of 18 p.M is comparable to values reported with the pro- 6. Barchi, R. L., Cohen, S. A. & Murphy, L. E. (1980) Proc. teins from synaptosomes (11 p.M) (11) and sarcolemma (35 Natl. Acad. Sci. USA 77, 1306-1310. p.M) (10), and within the dose-response range observed with 7. Hartshorne, R. P. & Catterall, W. A. (1981) Proc. Natl. Acad. intact eel electroplax (10-30 A.M) (24). Stimulation offlux by Sci. USA 78, 4620-4624. BTX (up to 5-fold) was consistently greater than with vera- 8. Hartshorne, R. P., Messner, D. J., Coppersmith, J. C. & Cat- tridine. terall, W. A. (1982) J. Biol. Chem. 257, 13888-13891. 9. Barchi, R. L. (1983) J. Neurochem. 40, 1377-1385. The use of [3H]TTX permitted comparisons of toxin bind- 10. Weigele, J. B. & Barchi, R. L. (1982) Proc. Natl. Acad. Sci. ing with flux inhibition. The Kd of 33 nM was slightly higher USA 79, 3651-3655. than the Kd of 7 nM reported for solubilized, unpurified, pro- 11. Talvenheimo, J. A., Tamkun, M. M. & Catterall, W. A. (1982) tein (4). Nevertheless, the concentration dependence of J. Biol. Chem. 257, 11868-11871. binding was closely correlated with that of flux inhibition 12. Tamkun, M. M., Talvenheimo, J. A. & Catterall, W. A. (1983) (Fig. 4C), indicating that TTX blockade of 22Na' influx can Biophys. J. 41, 142a (abstr.). be quantitatively attributed to binding of the toxin to its site 13. Holloway, P. W. (1973) Anal. Biochem. 53, 304-308. on the channel. This correlation extends to observations re- 14. Peterson, G. L. (1977) Anal. Biochem. 83, 346-356. to orientation of the channel. After 15. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leim- lating vectorial FTS, only gruber, W. & Weigele, M. (1972) Science 178, 871-872. 60-70% of the total binding sites were accessible to 16. Laemmli, U. K. (1970) Nature (London) 227, 680-685. [3H]TTX, consistent with the incomplete block offlux (Figs. 17. Kasahara, M. & Hinkle, P. C. (1977) J. Biol. Chem. 252, 7384- 2A, 4B, and 5A). The lipid-soluble alkaloids can presumably 7390. activate channels of either orientation, while TTX should 18. Epstein, M. & Racker, E. (1978) J. Biol. Chem. 253, 6660- block only outside-out channels. Consistent with this inter- 6662. pretation, TTX added before the disruptive FTS completely 19. Agnew, W. S. & Raftery, M. A. (1979) Biochemistry 10, 1912- inhibited all stimulated flux. 1919. Local anesthetics bind to an internal site on the Na chan- 20. Scatchard, G. (1949) Ann. N. Y. Acad. Sci. 51, 660-672. 21. Huang, L.-Y. M., Moran, N. & Ehrenstein, G. (1982) Proc. nel (1, 23). The presence of this site was demonstrated by Natl. Acad. Sci. USA 79, 2082-2085. inhibition of flux by tetracaine, dibucaine, and QX-222. Tet- 22. Catterall, W. A. & Beress, L. (1978) J. Biol. Chem. 253, 7393- racaine and dibucaine are membrane permeant and caused 7396. complete block of specific flux. The impermeant derivative, 23. Hille, B. (1977) J. Gen. Physiol. 69, 497-515. QX-222, inhibited -72% of stimulated flux, presumably act- 24. Bartels, E. & Rosenberry, T. L. (1973) Biochim. Biophys. ing on inside-out directed channels. When QX-222 was add- Acta 298, 973-985. 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