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Reconstitution of Neurotoxin-Modulated Ion Transport

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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.
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