pharmacological and biochemical properties of

0041-0101/91 53.00 + .00 1990 Pergamon Press plc

Tosicon Vol . 29. No. l, pp. 571, 1991 . Printed N Great Britain.

Tj

PHARMACOLOGICAL AND BIOCHEMICAL PROPERTIES OF SAXIPHILIN, A SOLUBLE SAXITOXIN-BINDING PROTEIN FROM THE BULLFROG (RANA CATESBEIANA) JANKE MAHAR, 1 GERGELY L. LLJK .ACS,1'* YI LI,1 SHERWOOD HALLZ EDWARD MOCZYDLOWSKII t

arid

'Department of Pharmacology and Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06510, and 2U.S. Food and Drug Administration, 200 C Street, SW, Washington, DC 20204, U.S.A . (Acceptedfor publication 21 June 1990)

G. L. Lurcncs, Y. LI, S. HALL and E. MOCZYDLOWSKI . Pharmacological and biochemical properties of saxiphilin, a soluble saxitoxin-binding protein from the bullfrog (Rang catesbeiana) . Toxicon 29, 53-71, 1991 .-Supernatant fractions of various tissues and plasma from the North American bullfrog, Rana catesbeiana, specifically bind saxitoxin with high affinity . Binding of [3H]saxitoxin to bullfrog plasma follows single-site behavior with an equilibrium dissociation constant of Kd = 0.16±0.03 nM at 0° C and a maximum binding capacity of 380t 60 pmole/ml plasma . Highaffinity binding of [3H]saxitoxin is chemically specific since it is unaffected by tetrodotoxin and a variety of cationic peptides, amino acids and drugs. The structure-activity dependence of binding to this site was investigated with eight different natural and synthetic derivatives of saxitoxin. Substitution of the carbamoyl side chain or the C-12 ß-hydroxyl group of saxitoxin with a hydrogen atom had little effect on binding affinity, but addition of a hydroxyl group at the N-1 position decreased the binding affinity from 430- to 710-fold in three different molecular pairs. High performance size exclusion chromatography of supernatant from bullfrog skeletal muscle showed that the ['H]saxitoxin-binding component migrates with an apparent molecular weight of Mr = 74,000 f 8000 or a Stokes radius of 35 t 2A. The ['H]saxitoxin-binding protein in skeletal muscle extract or plasma is retained on a cation-exchange column at pH 6.0, suggesting that the protein contains a region of exposed basic residues . Column isoelectric focusing of a sample from plasma indicated that the protein has a basic isoelectric point near pH = 10.7. These pharmacological and biochemical properties imply that this soluble saxitoxin-binding activity is associated with a novel protein named saxiphilin that is structurally distinct from known subtypes of functional sodium channel proteins which are the biological target of saxitoxin's paralyzing effect . J. MAHAR,

'Present address: Division of Cell Biology, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario MSG 1X8, Canada . tAuthor to whom correspondence should be addressed. 53

54

J . MAHAR er at. INTRODUCTION

SnxrroxtN (STX), a small heterocyclic molecule (mol. wt = 301 .3) containing two guanidinium groups, is a potent blocker of voltage-dependent sodium channels that provide the basis for nerve and muscle excitability . As such, STX and its derivatives are responsible for occasional human cases of paralytic shellfish poisoning (ICno, 1966; Rrrct-uE and ROGART, 1977; SCHANTZ, 1986) . This poisoning is generally due to the consumption of bivalve shellfish, such as clams and mussels, that have accumulated the saxitoxin-type toxins from marine dinoflagellates- various species of the genus Alexandrium (most previously assigned to the genera Gonyaulax or Protogonyaulax), Pyrodinium bahamense, and Gymnodinium catenatum (St-ntenzu, 1986; HALL et al., 1990). In freshwater environments, STX has been found in association with some blooms of the cyanobacterium, Aphanizomenon Jlos-aquae (IxAWn et al., 1982) . The toxigenic strains of this blue-green alga produce both STX and neosaxitoxin (IKAWA et al., 1982; SHIMIZU et al., 1984; Mnt-n~toon and CARhnct-tAEt., 1986), the latter derivative containing an N-1 hydroxyl group in place of a hydrogen atom (SHIMIZU et al., 1978) . Tetrodotoxin (TT'X), a different small molecule (mol. wt = 319 .28) with one guanidinium group, is likewise a potent blocker of voltage-dependent sodium channels. TTX has been found in a variety of sea animals such as annelids, starfish, molluscs, crabs, octopuses and fish, and in land animals such as certain salamanders and frogs (FUIIRMAN, 1986; YnsuMOTO et al., 1986) . In some cases, both STX and TTX are found within the same animal (YASUMOTO et al., 1986) . Although it was previously thought that TTX was a metabolic product of the animals that contain it, recent evidence suggests that TTX is produced by certain bacteria (YASUMOTO et al., 1986; Nocuct-n et al., 1986; Yo~rsu et al., 1987) . How STX- and TTX-containing animals avoid the lethal effects of these neurotoxins is not completely understood. While some of these animals have been shown to exhibit toxin-insensitive action potentials (Kno and FUI-IRMAN, 1967; TWAROG et al., 1972; Ktnotcoxo et al., 1974; DAIGO et al., 1988), the molecular mechanisms that underly this insensitivity are largely unknown . STX and TTX bind to a common site on sodium channels and are widely used ligands in biochemical studies of this particular class of voltage-dependent ion channels (RITCHIE and ROGART, 1977). The sodium channel binding site for these two toxins is associated with a 26()-280,000 mol . wt integral membrane glycoprotein that has only been solubilized in an active micellar form with the use of detergents and supplementary phospholipids required to stabilize the toxin-binding activity (Acxt:w and Rnr-rExY, 1979; MILLER et al., 1983; BARCHt, 1983; HARTSHORNE and CATTERALL, 1984) . In 1982, DOYLE and coworkers reported an apparent exception to this rule for frog heart extracts, where high-affinity ['H]STX binding activity was found in the high-speed supernatant of heart homogenates without the use of detergents (DOYLE et al., 1982) . Independently, our laboratory observed a similar phenomenon in an attempt to investigate the possible distribution of sodium channel subtypes in frog muscle (MOCZYDLOWSKt et al., 1988b). We resolved a distinct class ofhigh-affinity (Kd = 0.1 nM) binding sites for [3H]STX that were insensitive to TTX and present in two forms. One form appeared to be in tight association with muscle membranes and the other form was abundantly present in the high-speed supernatant of homogenized muscle. This soluble ['H]STX binding activity is pharmacologically distinct from functional sodium channels in muscle by the absence of binding competition with a high concentration of TTX (100 pM) and by the absence of binding competition with monovalent cations of alkali metals (MOCZYDLOWSKI et al., 1988b). These observations raise a number of questions about the biochemical origin and

Saziphilin, a Saxitoxin-binding Protein from the Bullfrog

55

significance of this unusual STX-binding activity. Is the "soluble" binding activity due to a cytoplasmic protein or is it released from cell membranes in the process of homogenization? Does this activity correspond to an unusual form of functional sodium channels, an evolutionarily-related protein or a completely novel STX-binding protein? What is the physiological significance of this protein? To address these questions, we have begun a biochemical characterization of the soluble STX-binding activity in the North American bullfrog, Rana catesbeiana. In this paper, we describe the tissue distribution and structureactivity requirements for STX binding to this soluble site. We also report the apparent molecular size and iscelectric point of the protein associated with this activity . Since our results suggest that this protein does not function as a sodium channel, we have named it saxiphilin after its specificity and high affinity for saxitoxin. MATERIALS AND METHODS Materials [3H]STX obtained from Amersham-Searle (Arlington Height, IL, U.S.A .) was repurified and standardized as described previously (MoczYnLOwsxt et al., 19886) . The final specific activity of f H]STX was 25 .5 Ci/mmole with a radiochemical purity of 86% . Unlabeled STX and TTX were purchased from Calbiochem Corp . (San Diego, CA, U.S .A .) and stock solutions of these toxins were prepared in 1 mM citrate buffer, pH 5.0. The peptide toxins p~onotoxin GIIIA and GIIIB were kindly provided by Dr BALDOSff?RO Ouveae (Department of Biology, University of Utah, U.S .A .). Leupeptin and pepstatin were purchased from Bcehringer-Mannheim Biochemicals (Indianapolis, IN, U.S .A.) . Phenyhnethylsulfonyl fluoride, choline chloride (3x crystallized), EDTA (ethylenediaminetetraacetic acid), Hepes (N-2-hydrozyethylpiperazine-N=2~thanesulfonic acid), Mes (2(N-morpholino~thanesulfonic acid), Mops (3-(N-morpholino)propanesulfonic acid), Tris, tricaine methanesulfonate, heparin and other amino acids, peptides and drugs mentioned in Results were purchased from Sigma Chemical Co . (St Louis, MO, U.S.A.). Proteins used for mol. wt standards were purchased from PharmaciaLKB (Piscataway, NJ, U.S .A.) and Sigma Chemical Co. (St Louis, MO, U.S .A.) . Saxitoxirt derivatives The naturally occurring saxitoxin derivatives used in the present study, B1, C1, NEO, B2 (Fig . 2), were isolated from cultures of Alexarrdrium sp . as previously described (S . H~u.t-, 1982, Ph .D. thesis, University of Alaska, Fairbanks, AK). a-Saxitoxinol (a-STX-OH) was prepared by reduction of STX with sodium borohydride and chromatographic separation of the reaction products (Roca:as and RerroPOR'.r, 1980; SHt~uzu et al., 1981 ; KoexN et al., 1981). Decarbamoyl saxitoxin (dcSTX) was prepared by acid hydrolysis of saxitozin (KOEttx et al., 1981). Decarbamoyl neosaxitoxin (dcNEO) was similarly prepared by hydrolysis of NEO (aqueous 6 M HCI, 100°C, 30 min, sealed under nitrogen), although the yield was poor compared to the hydrolysis of STX, consistent with other experience with the N-1-hydroxyl saxitozin derivatives. Acetyldecarbamoylsaxitoxin (AodcSTJ~ was prepared by peracetylation of dcSTX (acetic anhydride/pyridine, room temperature) followed by selective hydrolysis in dilute aqueous acetic acid. The synthetic derivatives were characterized by conventional 'H-NMR and "C-NMR spectra. Concentrations of stock solutions of the synthetic derivative were confirmed to within f l0% by quantitative 'H-NMR experiments using internal standards calibrated with known concentrations of saxitoxin dihydrochloride . Details of the preparation and characterization of the synthetic derivatives will be published separately. Equilibrium (3FIJSTX binding assays The standard assay for [3H]STX binding was carried out in the presence of 5 to l0 nM f H]STX, 20 mM Mops-NaOH, pH 7.4, 0.2 M choline chloride, 0.1 mM EDTA and various bullfrog protein samples in a final volume of 0.25 ml (Moczmt.owsxr et al., 1986, 19886) . Binding was allowed to equilibrate at 0°C for 30 to 60 min before removing the free toxin at 4°C by passing duplicate 100 kl aliquots of the reaction mixture through small cation-exchange columns followed by 0.5 ml of 20 mM Tris-HCI, pH 7.2 wash . The columns contained I ml of the microporous resin, AG SOW-X2 (Tris+ form, 100-200 mesh, Bio-Rad, Richmond, CA, U.S.A.) . Specific binding is defined as the difference of binding measured in the absence and presence of 10 pM STX. For quantitative determination of binding activity in a given sample, bound [;H]STX was not allowed to exceed 20% of the total [3H]STX in the assay. Under these conditions, binding activity was a linear function of sample protein. Tritium was measured by liquid scintillation counting at 40% efliciency using 6 ml Ecoscint (National Diagnostics). Duplicate determinations of bound [3H]STX did not differ by more than 10%. Equilibrium binding parameters such as the dissociation constant (~, maximal binding capacity (B°) and Hill coefficient (n) were evaluated by fitting [3H]STX saturation or competition data to a one-site model using

56

J. MAHAR et al.

EBDA and LIGAND programs from Elsevier-Biosoft (Cambridge, U .K .) . Kd values of various unlabeled STX derivatives were determined with the LIGAND program by analysis of competition displacement titrations of ['H]STX using a non-linear least squares fit of the titration curve to a one-site model of competition of two ligands binding to the same site. A Kd value of 0 .10 nM for ['H]STX was assumed in this analysis, as determined by titration with unlabeled STX . Bullfrog tissue preparations Specimens of adult Rang catesbeiana were purchased from Connecticut Valley Biological Supply Co. (Southhampton, MA, U.S .A .) and tadpoles of the same species were purchased from Charles D . Sullivan Co . (Nashville, TN, U .S.A .). Animals were handled according to guidelines of the Yale Animal Care and Use Committee. Adult frogs were anesthetized with 30 mg/kg of tricaine methanesulfonate administered by i .p. injection . Blood was drawn from the aortic arch into a syringe containing 0 .1 ml of 1 mg/ml heparin . Plasma was prepared by centrifuging whole blood for 30 sec at 15,000 x d . For tissue studies, selected organs were dissected from five adult bullfrogs (3 males, 2 females). Pooled organs were collected in sucrose buffer (0 .3 M sucrose, 10 mM Mops-NaOH, pH 7 .4, 3 mM NaN 3, 5 mM EDTA) at 4°C, weighed and minced in 3-8 ml sucrose buffer/g tissue. Protease inhibitors were added (4 pM pepstatin, 4 pM leupeptin, 200 pM phenylmethysulfonyl ftouride) and tissues were homogenized at 8000 rpm for 1 min with a Tissumizer (Tekmar, Cincinnati, OH, U.S .A .) and then for 30 sec with a Potter-Elvehjem tissue grinder . Supernatant was obtained by two consecutive centifugations, first at 12,000 x g for 10 min and then at 100,000 x g for 30 min . A similar procedure was used for preparation of whole animal supernatant extract from young tadpoles, except that the tadpoles were first anesthetized by hypothermia on ice and killed by rapid decapitation before homogenization in a food blender at high speed . For large scale preparation of skeletal muscle supernatant, leg muscle (440 g) was dissected from large bullfrogs . The muscle was minced and 440 ml sucrose buffer with protease inhibitors was added . The mixture was homogenized twice for 30 sec in a food blender at high speed . The homogenate was centrifuged at 2,000 x g for 10 min and the supernatant was saved. The pellets were rehomogenized with 440 ml of sucrose buffer and centrifuged as before. The combined supernatant was passed through cheesecloth and centrifuged at 120,000 xg for 1 hr . Supernatant preparations were stored in aliquots at -80°C. ['H]STX binding activity of frozen samples did not vary appreciably over six months . Nigh performaxce size exclusion chromatography The sieving column for these experiments was an Ultropac TSK G3000SW (7 .5 x 600 mm) with a TSK SW precolumn (7 .5 x 75 mm) purchased from Pharmacia-LKB (Piscataway, NJ, U .S .A .) . The column was equilibrated and run at a constant flow rate of 1 .0 ml/min at 22°C using a Waters 600 pump and delivery system (Millipore Corp ., Millford, MA, U .S.A .). The running buffer was 20 mM Hepes-NaOH, pH 6 .8, 0 .5 M ammonium acetate . The elution position of molecular weight standards was monitored by absorbance at 280 nm and ['H]STX binding activity was assayed in small aliquots taken from 0.5 or I .0 ml fractions. Following the gel permeation theory of HrM~r. and $QUIRE (1981), the apparent molecular weight of the ['H]STX-binding component was determined from a linear calibration curve of a volume function, F vs the cube root of molecular weight, MW~/'. F is given by the following equation: F °(y4'-Vôl')l(~l'-Vôl°) (I) where V is the elution volume of a standard protein or unknown, V is the void volume determined with calf thymus DNA and Y, is the total column volume determined with NaN, or uridine . The hydrodynamic Stokes radius, R~, was determined from a similar calibration curve of R~ vs the inverse of the error function complement, erfc - ' (l-F~) according to the method of ActcERS (1967) . Cation-exchange chromatography A sample of skeletal muscle supernatant (16 ml, IS mg/ml) prepared as described above was thawed and adjusted to pH 6 .0 with acetic acid . The sample was gently mixed in a flask for 30 min at 4°C with 40 ml of S-Sepharose Fast Flow chromatography media (Pharmacia-LKB, Piscataway, NJ, U .S .A .) that was previously equilibrated with buffer A (10 mM Mes-NaOH, 5 mM EDTA, pH 6 .0, 1 pM leupeptin, I pM pepstatin) containing 25 mM sodium acetate. The mixture was poured into a column at 4°C . After settling, the column was eluted with 120 ml of 0.l M sodium acetate in buffer A followed by a linear gradient of 100 ml 0.1 M sodium acetate/l00 ml 1 .0 M sodium acetate in buffer A . The flow rate was maintained at 2 ml/min with a peristaltic pump . The batch loading procedure described above was necessary to prevent clogging and flow rate distubance that occurred with direct application of this sample to columns of S-Sepharose. For skeletal muscle supernatant, an empirically determined ratio of 5 pmoles of ['H]STX binding activity per ml bed volume of S-Sepharose was used to estimate the amount of sample loaded in this procedure. A similar procedure was used to chromatograph samples of bullfrog plasma except that this sample was diluted 10-fold with equilibration buffer and applied directly to a column of S-Sepharose . For plasma, a sample ratio of 20 pmoles of ['H]STX binding activity per ml bed volume of S-Sepharose was used .

Saxiphilin, a Saxitoxin-binding Protein from the Bullfrog

57

Isoelectric focusing

Samples of ['H]STX binding activity that were partially purified by S-Sepharose chromatography were analyzed by iscelectric focusing according to the method of VESTERBERG (1971) in a 110 ml preparative column using a 0~0% sucrose gradient with 2% ampholines (Phatmalyte 3-10, Pharmacia-LKB, Piscataway, NJ, U.S.A .). The sample was first dialyzed overnight against 2 liters of 25 mM sodium acetate. The sample was added to the light solution before gradient formation and focused for 40 hr at a constant power of 15 W. The jacketed column was maintained at 4°C with a circulating bath. Column fractions (I ml) were collected for pH measurement and assayed for [3H]STX binding after adjustment to pH 7 with acetic acid or Tris base . Protein measurements

Protein samples were assayed according to the method of PETERSON (1977) using bovine serum albumin as a standard. RF4ULTS

Distribution of soluble (3HJSTX binding activity in bullfrog tissues

Previous work established the presence of an apparently soluble form of [ 3 H]STX binding activity in homogenates from frog heart (DOYLE et al., 1982; TANAxA et al., 1984) and skeletal muscle (MOCZYDLOWSKI et al., 1988b). Ifthis activity were due to a precursor or cleavage product of a conventional sodium channel protein, one would expect it to be distributed primarily in excitable tissues such as heart, skeletal muscle and brain where voltage-activated sodium channels function in the generation of action potentials . Table 1 lists the results of screening supernatant extracts of various tissues of adult bullfrogs for ['H]STX binding activity. Unexpectedly, we found activity in all tissues tested, with the f

TABLE 1 . YIELD AND SPECIFIC ACTIVITY OF SOLUBLE H1STX BINDING ACTIVITY EXTRACTED FROM VARIOU3 TL4SUFS OF ADÙLT BULLF(LOOS AND WHOLE TADPOLFS

Tissue Plasma Liver Kidney Lung Heart Eggs Testis Stomach Skeletal muscle Brain Tadpoles

Yield (pmole/g tissue)

Specific activity (pmole/mg protein)

380 t ~ 60 f 23 170 (169, 172) 140 (138, 146) 140 (133, 142) 130 (119, 137) 86 (86, 87) 75 (74, 76) 36 (36, 37) 17 (17, l8) 120 t ~

14 .1 f 3.3 1 .4 f0.6 4.2 (4 .1, 4 .2) 6.2 (6 .0, 6.4) 5.2 (5 .0, 5.3) 4.3 (4 .0, 4.6) 3.4 (3 .4, 3.4) 2.0 (2 .0, 2.0) 1.1 (l .l, 1 .1) 1.2 (1 .1, 1 .2) 3.7 f 1 .1

Supernatant fractions of various tissues of adult speciments of Rana catesbeiana and whole tadpoles of the same species were prepared by

homogenization and centrifugation as described in Methods. Specific binding activity was assayed as in Fig. l using 5 nM [3H]STX and a blank containing 10 pM STX. Results are expressed as a yield or tissue content relative to the weight of the original tissue and as specific activity, based on the protein concentration of the isolated supernatant . For plasma, liver and whole tadpoles, the means and standard deviations of six individual animals are listed . For the other tissues, the means (of duplicate deteralinations in parentheses) are listed for samples prepared by pooling organs from five animals as described in Methods.

58

J . MAHAR et al.

FIG. 1 . EQUILIBRIUM BINDING OF fH]STX TO BULLFROG PLASMA . Plasma (0 .1 mg protein/ml) was incubated at 0°C for 30 min in the presence of 20 mM Mops-NaOH, pH 7 .4, 0 .2 M choline chloride, 0.1 mM EDTA, the indicated concentrations of f H]STX and either no additional toxins (~), 20 ~M TTX (Q) or 10 pM STX (/) . Data points are means of duplicate determinations of bound [~H]STX assayed as described in Methods . The inset is a Scatchard plot transformation of the data according to: [B]/[F][P] = Bo/Ka-[B]/[P]Kd, where [B] and [F'] are concentrations of bound and free [3H]STX, respectively, [P] is the protein concentration in mg/ml, Bm~ is the maximum binding capacity in units of pmole/mg protein and Kd is the equilibrium dissociation constant . The solid line in the inset is a least-squares fit of the data with Bo x = 17 .1 ±0.8 pmoles/mg and Kd = 0 .16±0 .03 nM .

highest levels observed for non-excitable tissues such as plasma, kidney, lung and eggs. Supernatant prepared from brain and skeletal muscle actually had the lowest amount of binding activity relative to other tissues. The level of activity in the plasma of adult bullfrogs was relatively constant from frog to frog, with a range of 300 to 440 pmoles ['H]STX binding sites/ml plasma as measured in a comparison of plasma samples from six animals. We also found that the soluble ['H]STX binding activity was present in early stage (limbless) tadpoles of R. catesbeiana, at levels comparable to those found in the unfertilized eggs of adult female animals. In six tadpoles weighing from 4.9 to 7.6 g, we found an average of 120 pmoles of [3H]STX binding activity per g of tissue in extracts of soluble protein prepared from the whole tadpole (Table 1) . Pharmacological spec city of the high-affinity (jHJSTX binding site in bullfrog plasma Figure 1 presents the results of a titration of a fixed amount of bullfrog plasma (0.1 mg/ml) with increasing concentrations of [3H]STX up to 8 nM in the absence or presence of a large excess of either STX or TTX. In the presence of 10 kM unlabeled STX, the measured binding activity exhibits a linear dependence on (3H]STX concentration, characteristic of non-specific binding or a background component. In the absence of unlabelled toxins or in the presence of 20 ~M TTX, a saturable component of ['H]STX binding is observed. These results indicate that high affinity binding of ['H]STX to a saturable site in bullfrog plasma is completely unaffec;`~:d by a concentration of TTX that normally displaces STX binding to sodium channels from mammalian skeletal muscle or brain (MOCZYDLOWSKI et al., 1986). Scatchard plot analysis of the data in Fig. 1 according to a single-site model of binding gave an equilibrium dissociation constant of Kd = 0.16±0.03 nM and maximum binding capacity of 17 pmoles/mg plasma protein. In


59

R3

STX dcSTX AcdcSTX ac-STX-OH B1 C1 NEO dCNEO B2

R1 CONHZ H COCH3 CONH 2 CONHS03 CONHS03 CONH z H CONHS03

R2 H H H H H H OH OH OH

R3 H H H H H OS03 H H H

R4 OH OH OH H OH OH OH OH OH

FIG. Z. STRUCTURFS OF SAXITOXIN DERIVATIVFS USED 1N THIS STUDY .

another experiment using a different bullfrog plasma sample and a different batch of ~H]STX we obtained a similar Kd value of 0.10 nM. The results of these experiments with bullfrog plasma are in close agreement with previous results for bullfrog skeletal muscle supernatant, where [3H]STX binding was also insensitive to TTX and exhibited a Ka of 0.14±0.02 nM (MOCZYDLOWSKI et al., 1988b) . To further characterize the chemical specificity of the soluble ~H]STX binding site, we considered the possibility that this activity might be due to a component that binds a variety of cationic compounds such as STXZ+ by an electrostatic mechanism. If this were the case, one would expect that binding of ['H]STX would be inhibited by various types of organic rations and polycations. To test this idea we screened a variety of such rations for the ability to inhibit specific [3H]STX binding to bullfrog plasma under the conditions of Fig. 1 in the presence of 5 nM ['H]STX. We have found no compounds other than STX and STX-derivatives that appreciably inhibit [3H]STX binding in this assay. One class of compounds which had no significant effect included the basic amino acids o-lysine, Llysine, n-arginine and L-arginine, each tested at a concentration of 10 mM . Likewise, 0.1 mM concentrations of poly-~-lysine (average M~ = 9200) or poly-i-arginine (average M, = 12,000) had no effect . Also without effect were two basic ~-conotoxin peptides, GIIIA and GIIIB (tested at 2 ~M), that compete with [3H]STX binding to sodium channels from skeletal muscles (MOCZYDLOWSKI et al., 1986). Apamin (10 pM), a basic bee peptide specific for certain CaZ +-activated potassium channels (MOCZYDLOWSKI et al., 1988a), and other small organic compounds known to block a variety of sodium and potassium channels (methylguanidine, 10 mM; tetraethylammonium, 10 mM; amiloride, 1 mM; 4-aminopyridine, 10 mM) were also ineffective. In addition to these compounds, we found that 10 mM concentrations of chloride salts of Mgt+, CaZ+, MnZ +, CoZ+ and ZnZ+ were without effect . In contrast to such negative results, Fig. 2 shows the structures of a variety of natural and synthetic derivatives of STX which are potent inhibitors in the ['H]STX-binding

J . MAHAR et a(.

-10 FIG.

3.

DISPLACEMENT

-9 COMPETITION

-8 log[toxin] !M) TITRATIONS OF DERIVATIVES.

-7

f H]STX

-6 BINDING

WITH

VARIOUS

STX

Bullfrog plasma (0 .05 mg protein/ml) was incubated at 0°C for 30 min in the presence of l0 mM Mops-NaOH, pH 7 .4, 0 .2 M choline chloride, 0.1 mM EDTA, 4.8 nM fH]STX and the indicated concentrations of the following toxins with structures listed in Fig. 2 : STX (~), BI (Q), a-STX-OH (~), AcdcSTX (p) and dcSTX (/). Data points are means of duplicate determinations of specifically bound fH]STX expressed as a fraction of a control value measured in the absence of unlabeled toxins . Specific binding is defined as that in excess of a blank measured in the presence of IO uM STX . The initial level of specific binding in the absence of unlabeled toxin ranged from 0 .8 to 1 .1 nM [IH]STX bound in various experiments . Results with toxins STX, B1, a-STX-OH and toxins STX, AcdcSTX, dcSTX are compared in panels A and B, respectively, for the sake of clarity .

assay . Figures 3 and 4 show the results of competition displacement experiments in which inhibition of [3H]STX binding by unlabelled STX and eight different STX-derivatives is plotted as a function of increasing concentrations of inhibitor. The displacement curves described by these titrations are in accord with binding competition at a single site as indicated by Hill coefficients for the various titrations which are not significantly different from 1 .0 (range = 0.85 to 1 .17; mean = 1 .01 ±0 .12 (±S.D .), n = 9 derivatives) . Figure 3 compares results with unlabeled STX and four of the derivatives in Fig. 2. that are approximately equipotent inhibitors. These equipotent derivatives include three molecules with modifications at the carbamoyl side chain of STX: a derivative, B1, with a sulfo substitution on the carbamoyl-NHz group, decarbamoyl saxitoxin (dcSTX), acetyldecarbamoylsaxitoxin (AcdcSTX) and a derivative with a hydrogen atom substitution at the


61

C-12 position, a-saxitoxinol (a-STX-OH) . Figure 4 compares results with four other derivatives that exhibit greatly reduced binding affinity (150- to 1500-fold) compared to STX: C1, a C-11 hydroxysulfate derivative of B1, and the following derivatives containing an N-1 hydroxyl group: neosaxitoxin (NEO), decarbamoylneosaxitoxin (dcNEO), and B2, an N-1 hydroxyl derivative of B1 . The derived Kd values for the various STX derivatives are summarized in Table 2. The results of Table 2 can be used to infer some of the structural requirements of the high-affinity STX binding site in bullfrog plasma. Removal of the carbamoyl side chain at the 0-18 position results in similar 2 .1 fold and 2.9 fold decreases in affinity, respectively, in the two molecular pairs, dcSTX/STX and dcNEO/NEO. A somewhat smaller effect is observed for removal of an acetyl side chain synthetically substituted at C-18 oxygen : a 1 .6-fold reduction in atiïnity for the dcSTX/AcdcSTX comparison . Similarly, the addition of a negatively charged sulfo group at the carbamoyl nitrogen has virtually no effect on the binding affinity as observed for the B1/STX and B2/NEO pairs. Taken together, these results indicate that the carbamoyl portion of the STX molecule is a relatively unimportant determinant of the high affinity binding interaction. Likewise, removal of the C-12 ß-hydroxyl group has essentially no effect as demonstrated by the nearly identical Ka values of STX and a-STX-OH . In contrast, addition of the N-1 hydroxyl group has a profound effect on the binding affinity as demonstrated by 550-, 430- and 710-fold reductions in binding affinity in the respective molecular pairs, NEO/STX, B2/B1 and dcNEO/dcSTX . Addition of an TABLE 2. COMPARL40N OF THE BINDING APFINrfY OF VARIOUS SAXITOXIN DERIVATIVPS TO BULLFROG PLASMA AND THE BLOCKING AFFINITY FOR RAT MUSCLE AND DOG HEART SODIUM CHANNELS IN PLANAR HILAYERS

Toxin

STX dcSTX AcdcSTX a-STX-0H Bl C1 NEO dcNEO B2

lib

K~(nM) Bullfrog plasma

Bullfrog plasma

0 .11 f 0.02 0 .24 t 0.03 0 .15 f 0.02 0 .092 f 0.01 0.10 f O.OI 16 _+ 4 60 t 4 170 t 30 43 t 7

1 .0 2 .2 1 .4 0.84 0.91 150 550 1500 390

(toxin)/Kd (STX) Dog heart Rat muscle Na* channel Na+ channel 1 .0 2 .3 5 .0 64 41 360 0 .27 230 43

1 .0 4 .3 8 .8 80 N .D. 31 1 .6 2200 91

The equilibrium dissociation constant, Kd, of the listed STX derivatives (structures shown in Fig . was determined by displacement competitions titrations of ['H]STX binding to bullfrog plasma at 0°C as illustrated in Figs 3 and 4. Best fit Ilb values with standard deviations (of the fit) were obtained by fitting the displacement titrations to a single-site model of binding using the LIGAND program as described in Methods. The relative affinity of bullfrog plasma for the various saxitoxins is expressed as the ratio of the listed Kd's to that of STX. The relative affinity of Na+ channels from rat skeletal muscle (TTX-sensitive) and dog heart (TTX-insensitive) is expressed as the relative Kd at 0 mV measured for block of single, batrachotoxin-modified Na+ channels in planar bilayers at 22°C . The original data for these Na+ channel experiments is taken from Guo et nl. (1987) and from similar experiments by A. RAVINDRAN and E . MOCZYDLOWSKI (unpublished results). The standard deviations of the Kd values from such Na+ channel blocking experiments ranges from 6-22% . The absolute hCd values for STX block of Na+ channels from rat muscle and dog heart are 4.4 f 0.9 nM and 100 t 7 nM, respectively (Guo et ol ., 1987) . N .D ., not determined . 2)

ti2

1 . MAHAR et at.

bg[to~xinl (MI FIG .

4.

DISPLACEMENT

COMPEPEI7TION

TITRATIONS OF DERIVATIVES .

['H]STX

BWDING

WITH

VARIOUS

STX

The protocol for this experiment was the same as that described in Fig . 3 except that the following toxins were tested : C1 (~), B2 (Q), NEO (/) and dcNEO (p). Note that the abscissa scale for these derivatives covers toxin concentrations that are two orders of magnitude lower than those used in Fig. 3 .

a-hydroxysulfate moiety at the C-11 carbon also appears to markedly affect toxin binding as noted by the 160-fold reduction in afl"tnity for the C1/B1 pair. Thus, the experiments of Figs 3 and 4 have identified two portions of the STX molecule that are relatively unimportant in the binding interaction: the carbamoyl side chain and the C-12 ß-hydroxyl group; and, two positions where the addition of polar groups results in a large decrease in afïtnity : the N-1 position and the C-11 position . These results define a unique pharmacology for the soluble bullfrog STX binding site. The above results indicate that the soluble STX binding site in bullfrog tissues has a high degree of chemical specificity for STX and its derivatives. Nevertheless, it is possible that the high level of ['H]STX binding activity in plasma may be fortuitous binding to an ordinary enzyme or plasma protein that bears no particular relevance to the action of STX as a neurotoxin . If this were the case, one might expect the acitivity to be widely distributed in the plasma of other animal species. However, our assays of rat and human plasma and similar extracts of rat liver failed to detect any significant [3H]STX binding activity in these mammalian tissues ( < 0.1 pmole/g tissue or < 0.01 pmole/mg protein) . Also, TANAKA et al. (1984) readily demonstrated such activity in soluble heart extracts of Rana pipiet:s (leopard frog) and Bufo marinas (marine toad), but did not observe the activity in extracts of rabbit, chicken and turtle heart or Electrophorus electricus (electric eel) electric organ. These results imply that the soluble ['H]STX-binding activity is only present in certain animal species and is probably not a ubiquitous plasma protein. Since the STX-binding activity appears to be associated with a novel soluble protein, we have named it saxiphilin after its specificity for saxitoxin. Biochemical properties of the soluble (jHJSTX binding activity To investigate the stability of saziphilin for eventual purification, we examined the effect of various treatments and conditions of incubation . The [3H]STX-binding activity was completely lost when a sample of skeletal muscle supernatant was boiled for 5 min. However, no loss of activity was observed for muscle supernatant incubated at 24°C for


63

3

0.8 E c

0 _N

0.4

0

3

F1G. S . APPARENT MOLECULAR WEIGHT OF [ H]STX BINDING ACTIVITY FROM BULLFROG SKELETAL MUSCLE AS DETERMINED BY HIGH PERFORMANCE SIZE EXCLUSION CHROMATOGRAPHY. (A) Calibration curve using soluble proteins of known mol. wt . The data are plotted according to

the linear relationship between the cube root of mol. wt, MW'~, and the volume function, F as derived by HIMMEL and SQUIRE (1981) . Standards: 1, ribonuclease A; 2, myoglobin; 3, chymotrypsinogen A; 4, hemoglobin 2 N; S, ovalbumin; 6, horseradish peroxidase; 7, conalbumin; 8, bovine serum albumin 1 N; 9, transferrin; 10, aldolase 2 N; 1 l, catalase 2 N; 12 glucose oxidase; 13, bovine serum albumin 2 N; l4, catalase 4 N; 15, urease 1 N; 16 apoferritin; 17 urease 2 N. The solid line is a linear regression fit to F, = m[MW]'~I+b, where m = -0.0148 and b = 1.20 with a correlation ccelficient of r = -0 .987 . The arrow shows the F~ value of the mean elution position, 18 .3 f 0.4 ml ( f S.D, n = 3) of [3 H]STX binding activity as determined in B. (B) Chromatographic profile of [~H]STX binding activity on an Ultropac TSK G3000SW column (7 .5 x 600 mm). Sample: 0.3 ml of bullfrog muscle supernatant (14.4 mgJml) prepared as described in Methods. The solid line is a tracing of relative protein concentration monitored by absorbance at 280 nm . Open circles (Q) indicate [ 3H]STX binding activity measured in 0.09 ml aliquots of various fractions assayed under standard conditions with S nM fH]STX . Filled circles (~) indicate blank cpm measured in the presence of 20 pM STX. The void volume, Vo, and column volume, V were determined with calf thymus DNA and NaN3, respectively .

3 hr in the presence of 1 mM EDTA. At 0°C, only 10% loss of activity was observed after 5 days of incubation . In another experiment, we found that incubation of a diluted sample of bullfrog plasma at 25°C in the presence of 5 mM 2-mercaptcethanol or dithiothreitol resulted in a 4055% loss of activity after 4 hr compared to an identical sample not exposed to these reducing agents . Since this experiment suggested that saxiphilin might be susceptible to disulfide reduction, reducing agents were omitted in subsequent purification experiments. To determine the relative size of the STX-binding component, we performed high performance size exclusion chromatography using a column (TSK G3000SV~ that has been shown to provide reliable mol. wt estimates of many soluble proteins in the range of

64

J. MAHAR et a1.

FIG . i) . ION-EXCHANGE CHROMATOGRAPHY OF BULLFROG MUSCLE SUPERNATANT .

A sample of bullfrog skeletal muscle supernatant was subjected to chromatography on a 40 ml column of S-Sepharose as described in Methods. After loading of sample, the column was eluted with 120 ml of 0.I M sodium acetate in buffer A (10 mM Mes-NaOH, 5 mM EDTA, pH 6 .0, 1 uM leupeptin, 1 pM pepstatin) followed by a linear gradient of 100 ml 0.1 M sodium acetate/100 ml 1 .0 M sodium acetate in buffer A. Flow rate, 2 ml/min . The fraction size was 5 ml from fraction No . 1 to No . 25 and 3 ml thereafter . Relative protein concentration of various fractions was monitored by absorbance at 280 nm (Q) and the sodium acetate gradient (---) was monitored by conductivity measurements . Aliquots (0 .045 ml) of various fractions were assayed for fH]STX binding (~) in the presence of 5 nM [3 H]STX and 100 mM Mops-NaOH, pH 7.4.

10,000 to 500,000 (TARVERS and ('xuRCx, 1985) . Since this column does appear to exclude negatively charged proteins and retard positively charged proteins at low ionic strength, we performed this experiment at an ionic strength of 0.5, where protein migration was previously observed to be independent of charge effects (TARVERS and CHIntcH, 1985) . Figure SA shows a linear calibration curve of 17 protein standards in good agreement with the results of TARVeRS and G~IUxcx (1985) for this column . Figure SB shows a typical elution profile of [3H]STX binding activity from a sample of bullfrog skeletal muscles supernatant run under identical conditions as the standard proteins. The recovery of binding activity was 72%, 100% and 51 % in three experiments . We found that the binding activity eluted as a homogeneous component with an apparent mol . wt of 74,000 f 8000 ( t S.D., n = 3). When the data were analyzed in terms of protein hydrodynamic radius according to the procedure of AcxsRS (1967), a Stokes radius of Re = 35 f 2~ was obtained . In contrast to the previous results of DOYLE et al., (1982), we did not observe any ['H]STX-binding activity in the void volume that would indicate aggregation. The apparent size of saxiphilin that we obtain by this technique is much smaller than that of sonicated phospholipid vesicles, which are the smallest known bilayer structures [R~ = 100 ~, BRUNNER et al., 1976] . This experiment therefore rules out the possibility that saxiphilin is associated with membrane vesicles . However, our results do not exclude the possibility of association with a small amount of lipid not in the form of vesicles. Since gel permeation chromatography was found to be a rather ineffective method for fractionating tissue extracts, we explored the elution behavior [3H]STX binding activity in ion~xchange chromatography. In initial attempts, we found that the soluble ~H]STX binding activity was not retained by the anion-exchange resin, DEAE-agarose, when samples of bullfrog muscle extract were applied to a column of this medium at 50 mM ionic strength and a pH of 6.2 or 7.6. However, we did find that the activity could be

Saxiphilin, a Sazitozin-binding Protein from the Bullfrog

65

14 12 10 82 a 6 4 2

10

20

30

40

50

80

70

volume (mD

80 90 100 110

h

FIG. 7. LSOELECTRIC FOCUSING OF fH]STX BINDING ACTMTY FROM BULLFROG PLASMA . A sample of partially purified fH]STX binding activity (1 .6 mg protein, specific activity = 71 pmoles/mg) prepared by S-Sepharose chromatography of bullfrog plasma was focused for 40 hr at a constant power of IS W as described in Methods. (~) pH profile of 1 ml fractions. The concentration of ['H]STX binding sites (~) present in various fractions was assayed with 5 nM fH]STX after adjustment to pH 7.4 .

fractionated on certain cation exchange media at pH 6.0. Figure 6 shows an example of such an experiment in which saxiphilin activity from a skeletal muscle extract was quantitatively retained on a column of S-Sepharose at pH 6.0 . The retained activity was eluted from this column with a sodium acetate gradient in a single peak observed at 0.45 M sodium acetate. This step could be used to obtain a 10- to 20-fold purification of ['H]STX-binding activity from crude muscle supernatant or a seven-fold purification of plasma . Although this step yielded a stable pool of activity with good recovery (6(1-90%), our attempts to concentrate such pools by various ultrafiltration methods resulted in severe losses of activity . We have found that lyophilization ca.n alternatively be used to concentrate such pools of saxiphilin activity with nearly complete recovery of activity . We also investigated the behavior of saxiphilin activity using conventional column isoelectric focusing in a sucrose gradient. Samples of plasma saxiphilin first purified sevenfold by S-Sepharose chromatography were subjected to isoelectric focusing for 40 hr at 4°C using ampholines designed to produce a continuous pH gradient in the range of pH 3-11 . The [3H]STX binding activity focused in a single sharp peak near the basic end of the pH gradient (Fig. 7). In two different experiments, peak fractions of activity were found at pH 10.7 and 10.3, indicating a basic isoelectric point for the saxiphilin protein. Unfortunately, this procedure also resulted in a 70% loss of activity, presumably due to prolonged exposure at high pH. Analysis of the most active fractions by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate (SDS-PAGE) and silver staining showed a prominent band at 88±4 kDa which exhibited a strong correlation with the relative amount of ['H]STX binding activity (not shown) . The preceding results have been recently used to develop a successful purification protocol for saxiphilin which will be published elsewhere (Lt, Y. and MOCZYDLOWSKI, E., Unpublished results). DISCUSSION

Voltage-dependent sodium channels have been previously considered to be the sole specific sites for STX in electrophysiological studies and binding studies using this toxin.

66

J. MAHAR et at.

Our experiments establish the existence of a unique STX-binding protein with properties that are markedly different from functional sodium channels by various pharmacological and biochemical criteria. Sodium channels are principally located in excitable cells such as neurons, heart and skeletal muscle fibers . Active sodium channels have also been reported in other cells without a primary role in impulse generation such as fibroblasts (GRAY et al., 1986) and glia (RrrcNm, 1987 ; YAROWSKY and ICRUEGER, 1989), but the functional significance of these observations is presently unknown. In the bullfrog, saxiphilin is present in all tissues that we examined . The highest amount of saxiphilin was found in plasma at a concentration of approximately 300140 nM when expressed in terms of STX-binding sites. The tissue studies of Table 1 do not reveal where saxiphilin is produced in the bullfrog or how it is secreted into the plasma . In any case, this ubiquitous tissue distribution is clearly unexpected for any entity related to a functional sodium channel. Pharmacological and biochemical properties ofsaxiphilin vs those of sodium channels

Table 2 presents a comparison of the relative Kd's for STX and various derivatives determined by competition with [3H]STX binding to bullfrog plasma with relative Kd's previously determined in single-channel blocking experiments with two different sodium channel subtypes in planar bilayers (Guo et al., 1987). From this comparison, several major differences in the structure-activity relationships of the soluble bullfrog STX binding site vs sodium channels can be identified. One prominent difference is the 64-fold (muscle) and 80-fold (heart) reduction in affinity due to removal of the C-12 ß-hydroxyl group in a-STX-OH for the two sodium channels vs the negligible effect of this substitution for bullfrog saxiphilin . Another major difference concerns the effect of the N-1 hydroxyl group. In the case of saxiphilin, the affinity for NEO is decreased by 550-fold relative to STX. For sodium channels, the addition of the N-1 hydroxyl group to the STX parent molecule increases the blocking affinity about four-fold for the muscle sodium channel, but has little effect on the blocking affinity for the heart subtype. Since the N-1 hydroxyl group in NEO exhibits a p~ of 6.75 (St-n~zu et al., 1978) and would be partially deprotonated at our working pH of 7 .4, these results suggest that positive charge distribution over the C-2 guanidinium/N-1 hydroxyl portion of the molecule is more critical for high affinity binding to saxiphilin than for sodium channels. However, when the N-1 hydroxyl group is added to the decarbamoylsaxitoxin molecule, a 100-fold reduction (muscle) and a 510-fold reduction (heart) in blocking affinity of sodium channels is observed for the two sodium channel subtypes for the dcNEO/dcSTX comparison. This complex, structure-dependent behaviour is not observed for the plasma binding site, since the N-1 hydroxyl group causes a large reduction in binding affinity for both STX and dcSTX parent molecules. Also, addition of a sulfo group on the carbamoyl tail of various STX derivatives generally causes a significant reduction (three-to 160-fold) in blocking affinity for sodium channels, while this sulfo substituent has a negligible effect on the binding affinity to saxiphilin . These pharmacological differences in toxin binding affinity between the STX-binding site of sodium channels and that of saxiphilin imply that structural basis for toxin binding is quite different in these two cases. At the outset of our work, it was unclear whether the STX-binding activity in supernatant fractions of frog tissues corresponded to a genuine soluble protein or a low density component associated with phospholipids as suggested by Dovt .E et al. (1982) . In our hands, saxiphilin behaves as a homogeneous component with a Stokes radius of 35 ~ and can be purified by conventional methods for soluble proteins . These results demon-

Saziphilin, a Saxitoxin-binding Protein from the Bullfrog

67

strate that saxiphilin does exist as a soluble protein. However, we previously observed similar TTX-insensitive sites for ['H]STX that were associated with fractionated muscle membranes (MoczYnt owsxl et al., 1988). It is therefore possible that saxiphilin may also exist in a membrane-bound form . Additional experiments are needed to determine whether such membrane-associated activity is due to extrinsic membrane adsorption of soluble saxiphilin or integral association of a similar protein within the phospholipid membrane. Regardless of this possibility, the soluble form of saxiphilin is inconsistent with any known behavior of functional sodium channel proteins . Purification and cloning studies have shown that the STX-binding site of functional sodium channels resides in a large glycoprotein with a protein mol. wt of 208-228,000 as calculated from protein coding sequences (None et al., 1984, 1986). The detergentsolubilized channel exhibits a Stokes radius of 95-105 /~ by gel filtration chromatography (AGNEW et al ., 1978 ; BARCHI and MuxPi-n, 1980) and migrates as a diffuse band with an M~ of ~ 300,000 by SDS-PAGE (MILLER et al., 1983 ; Bnxcfu, 1983; KxnhtBe et al., .1985) . These size characteristics are quite distinct from those of the saxiphilin protein. Ow sizeexclusion chromatography experiments (Fig . 5) and preliminary results of SDS-PAGE analysis of partially purified samples suggest that the saxiphilin protein is less than half the size of functional sodium channels. The behavior of saxiphilin in ion-exchange chromatography and iscelectric focusing is also quite different from sodium channel proteins . One might expect a protein that avidly binds a divalent cationic compound such as STXZ+ to possess negative surface charges that would attract the toxin to its binding site. However, if it exists, such a presumed local negative surface charge at the STX-binding site does not dominate the macroscopic charge behavior of saxiphilin . Saxiphilin has a net positive charge at pH 6.0 as indicated by adsorption to cation-exchange resins (Fig. 6) and an apparent iscelectric point of 10.7 (Fig. 7). Sodium channel proteins that have been purified from cell membranes have an abundance of exposed negative charges due to a content of about 100 sialic acid residues on the carbohydrate portion of the molecule (MILLER et al., 1983). This structwal feature accounts for the binding of solubilized sodium channel proteins to anion-exchange resins at neutral pH (AGNEW et al., 1978). Although the biochemical properties of saxiphilin summarized above eliminate a correspondence to any functional form of known sodium channels, it is still possible that saxiphilin could be a smaller protein with some homology to sodium channels or an altered conformation of a sodium channel fragment. To pursue an immunological approach to this question, Dr R. L. BARCHI'S laboratory (University of Pennsylvania School of Medicine, Philadelphia, PA), probed Western blots of partially purified bullfrog plasma and muscle supernatant sample (provided by us) with nine polyclonal antisera raised against synthetic oligopeptides spaced along the primary sequence of the rat skeletal muscle sodium channel. These experiments did not detect any specific immunoreactive bands in the frog samples, suggesting that saxiphilin is antigenically unrelated to rat muscle sodium channels (R. L. BARCHI, personal communication) . The only basis for presently suspecting that saxiphilin might be related to sodium channels is its pharmacological specificity for saxitoxin. Although most sodium channel currents are inhibited by nanomolar concentrations of both TTX and STX, some tissues exhibit an altered specificity. For example, complete block of sodium currents from mammalian heart requires about 30 ~M TTX (BROwx et al., 1981). Similarly, a TTX-insensitive population of sodium channels is expressed in non-innervated skeletal muscle cells of mammals (Goxol et al., 1985). Such pharmacological observations have led to the concept of multiple sodium channel subtypes (TRnrIhtER and AcxEw, 1989),

68

J . MAHAR et al.

analogous to the concept of receptor subtypes. Cloning results indicate that subtype variability arises at the genetic level in the existence of multiple sodium channel genes (NODA et al., 1986; TRIMMER et al., 1989; ROGART et al., 1989), but it is still unproven whether TTX-insensitivity is due to distinct sodium channel genes or a post-translational modification of a TTX-sensitive sodium channel . The above-mentioned cases of TTX-sensitivity are not fully analogous to the unusual specificity of bullfrog saxiphilin, which does not bind TTX at a concentration of 100 ~M (MOCZYDLOWSKI et al., 1988b). In addition to sodium channel subtypes within an organism, there is also considerable variability in STX/TTX sensitivity among animal species. For example, pufferfish and Taricha newts, whose tissues can contain large amounts of TTX, have been shown to exhibit TTX-resistant action potentials (Kno and FUHRMAN, 1967; KIDOKORO et al., 1974). Central American frogs of the genus Atelopus, have been also shown to contain TTX (KiM et al., 1975). However, the bullfrog species that we have studied is not known to contain this toxin . Although sodium currents of heart, skeletal muscle and the node of Ranvier of Rana frogs are fully blocked by 1 ~M TTX (COxxER et al., 1975; Cot.Quxoux et al., 1974; BErrorr et al., 1985), electrophysiological studies of the bullfrog sympathetic ganglion have revealed a subpopulation of sodium channels that are not blocked by l0 kM TTX but are blocked by 0.1 pM STX (JONES, 1987) . Since the toxin pharmacology of this latter current resembles the toxin specificity of saxiphilin, one may speculate that saxiphilin could be structurally related to such an unusual type of TTX-resistant sodium channel. What is the function of saxiphilin in bullfrogs? One possibility is that a high affinity STX-binding protein present at high concentration in circulating plasma could comprise a defense mechanism against saxitoxin poisoning . While this hypothesis has not yet been explicitly tested, it receives partial support from existing data in the literature. PRINZMETAL et al. (1932) working with crude extracts of "mussel poison" observed that frogs are resistant to natural sources of STX in comparison to mice. These authors noted that 15 lethal mouse doses delivered by i.p. injection were required to cause symptoms of paralysis in a large frog and they stated that "A minimum lethal dose for the frog cannot be given since the heart usually keeps beating for several days and recovery of the animal depends largely on the individual resistance, environmental conditions, etc." A later study by Kno and Fui-utMArr (1967) with pure STX confirmed the relative resistance of frogs to STX intoxication with minimal lethal STX doses of 5 f~g/kg for the mouse and 75 kg/kg for the leopard frog, Rana pipiens . In contrast to the 15-fold lower toxicity of STX in the frog, TTX exhibited nearly the same toxicity in these two animals (8 hg/kg, mouse; 12 hg/kg, frog). Furthermore, the lower toxicity of STX in the frog cannot be explained by a resistance of frog sodium channels to STX since electrophysiological experiments on isolated nerves and muscle fibers show that the Kd for TTX is 3 nM and that for STX is 1 nM for inhibition of sodium currents at 12°C (SCHWARZ et al., 1973; CAMPBELL and HILLE, 1976) . These data suggest that the presence of saxiphilin in frogs could account for their marked resistance to STX poisoning in the laboratory. However, these observations would have little biological significance unless there was evidence for possible exposure of frogs to STX-like toxins in nature. In fact, STX and certain of its derivatives do occur in the natural environment of frogs since a freshwater blue-green alga, Aphanizomenon flos-aquae, has been shown to synthesize STX and NEO (Si-uMizu et al., 1984) . The hypothesis that saxiphilin functions as a natural

Sauphilin, a Sautoxin-binding Protein from the Bullfrog

69

antidote for STX in frogs is therefore a plausible one that merits investigation. This idea does have shortcomings however, since previously studied cultures of A . flos-aquae were found to produce mostly NEO (IKAWA et al., 1982 ; SI-nhflzu et al., 1984 ; MAI-nKOOD and CARMICHAEL, 1986), which has a lower aüïnity for saxiphilin than STX. Despite this inconsistency, is it possible that other strains or species of cyanobacteria may preferentially produce STX. Further insight into the biological role of saxiphilin might also be obtained from a more complete knowledge of the species distribution of this protein . If saxiphilin were produced by a variety of species that encounter STX in the food chain, this would implicate a defensive function of the protein . Although such studies are at an early stage, we have recently found saxiphilin activity in the plasma of two species of garter snakes (Thamnophis sirtalis concinnus and Thamnophis sirtalis parietalis) and one newt (Taricha granulosa), but not in a rainbow trout (Oncorhynchus mykiss). For the positive species, the measured STX-binding activity in pmoles ['H]STX sites/ml plasma was: T.s. concinnus, 210; T.s. parietalis, 620 and T. granulosa, 8 (.I. MAHAR, D . CAMPBELL and E . MOCZYDLOWSKI, unpublished results). It should also be noted that another group investigating the resistance of crabs to STX described a high mol. wt protein (M~ = 145,000) that appears to be induced by exposure to STX (BARBER et al., 1988). However, this particular protein seems to be quite different from bullfrog saxiphilin in that it binds to an anion exchange resin at pH 5. Possible applications of saxiphilin In the area of human toxicology, diligent monitoring of commercial shellfish can virtually eliminate the threat of paralytic shellfish poisoning. However, occasional outbreaks still occur, such as 26 fatalities from contaminated clams on the Pacific coast of Guatemala in 1987 (HALL et al., 1990). A therapy based on saxiphilin could potentially be used to treat acute cases of poisoning. Also, given the remarkable specificity for STX and STX derivatives, an assay method based on saxiphilin binding could provide a rapid and sensitive method for determining the relative saxitoxin content of shellfish samples. Current methods of saxitoxin analysis are based primarily on mouse toxicity testing and high performance liquid chromatography . A saxiphilin-based assay would reduce the use of live animals and overcome the difficulty of chromatographic analysis of complex mixtures of tissue extracts. Whether or not saxiphilin itself proves effective in these roles, knowledge gained from investigation of its highly selective affinity for saxitoxin derivatives will aid in dealing with the health threat posed by these toxins . Acknowledgements-We thank Dr JoxN DALY for the gift of batrachotoun and Dr BALDOAfFdtO OLIVERA for gifts of p-conotoxin peptides . We are indebted to Dr Dox~t .D C~trHet.r- for providing samples of garter snake and trout plasma. We are grateful to Dr RO~RT L . BaRCm and members of his laboratory for screening bullfrog samples with sodium channel antibodies . This work was supported by grants from the National Institutes of Health (AR38796 and HL38156), the Searle Scholars Program/Chicago Community Trust and an Established Investigator award to E . M . from the American Heart Association . REFERENCES AcKFRS G . K . (1967) A new calibration procedure for gel filtration columns . J. blot. Chem. 242, 3237-3238 . Acxew, W. S. and R~rrRY, M . A . (1979) Solubilized tetrodotoun binding component from the electroplax of Electrophorus electricus . Stability as a function of mixed lipid~etergent micelle composition . Biochemistry 18, 1912-1919 .

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Aaxew, W. S., LEVIxsox, S. R., BRAesox, J. S. and RAFTERY, M. A. (1978) Purification of the tetrodotoxinbinding component associated with the voltage-sensitive sodium channel from Electrophorus electricus electroplax membranes. Proc . natn. Acad. Sci. U.S.A . 75, 2606-2610 . BARHER, K. G., Krrrs, D. D., TOWN3LEY, P. M. and SMITH, D. S. (1988) Appearance and partial purification of a high molecular weight protein in crabs exposed to saxitoxin . Toxicon 26, 1027-1034. BARCHI, R. L. (1983) Protein components of the purified sodium channel from rat skeletal muscle sarcolemma. J. Netrrochem . 40, 1377-1385. BARCHI, R. L. and MURPHY, L. E. (1980) Size characteristics of the solubilized sodium channel saxitoxin binding site from mammalian sarcolemma . Biochim. biophys. Acta 597, 391-398. BExorr, E., CORBII:R, A. and DuBOIS, J. M. (1985) Evidence for two transient sodium curcents in the frog node of Ranvier. J. Physiol., Lond. 361, 339-360. BROWN, A. M., LEE, K. S. and PowELL, T. (l9ß1) Sodium current in single rat heart muscle cells. J. Physiol., Lond. 318, 479-500. BRUNNPR, J., SKRABAL, P. and HAUSER, H. (1976) Single bilayer vesicles prepared without sonication : physicochemical properties. Biochim. biophys. Acta 455, 322-331. CAMPBELL, D. T. and Hale, B. (1976) Kinetic and pharmacological properties of the sodium channel of frog skeletal muscle . J. gen. Physiol. 67, 309-323. COLQUHOUN, D., RANG, H. P. and RtTCHa, J. M. (1974) The binding of tetrodotoxin and a-bungarotoxin to normal and denervated mammalin muscle. J. Physiol., Lord. 2411, 199-226. CONNER, J., BARR, L. and JAICOHSSON, E. (1975) Electrical characteristics of frog atrial trabeculae in the double sucrose gap. Biophys. J. 15, 1047-1067. DAIGO, K., NOGUCHI, T., MIWA, A., KAWAI, N. and H~smMOro, K. (1988) Resistance of nerves from certain crabs to paralytic shellfish poison and tetrodotoxin . Toxicon 26, 4ßy90. DOYLE, D. D., WoNG, M. TANAKA, J. and BARR, L. (1982) . 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