Biochemistry, Yale University, New Haven, Conn , U.S.A.

Transcription

1 J. Physiol. (1973), 235, pp With 5 text-figures Printed in Great Britain THE BINDING OF LABELLED SAXITOXIN TO THE SODIUM CHANNELS IN NERVE MEMBRANES BY R. HENDERSON,* J. M. RITCHIE AND G. R. STRICHARTZ From the Departments of Pharmacology and Molecular Biophysics and Biochemistry, Yale University, New Haven, Conn , U.S.A. (Received 18 June 1973) SUMMARY 1. Tritium labelled saxitoxin has been prepared and purified, and its binding both to intact rabbit vagus nerves and to a solubilized preparation of garfish olfactory nerve membranes has been examined. 2. In intact and solubilized nerves there is a saturable binding component of magnitude equal to that previously obtained for labelled tetrodotoxin. 3. This component of bound saxitoxin is displaced competitively by tetrodotoxin, and it is concluded that the two toxins bind to the same site. 4. The saturable saxitoxin (STX) interaction with the nerve membrane is reversible and can be described by the equation STX + R = STXX. R where R is the binding site or receptor. With the solubilized preparation of garfish nerve membranes the saxitoxin-receptor reaction rates are almost four times faster than those of tetrodotoxin. The half-life of the saxitoxin-receptor complexes is 13 see compared with 44 see for the tetrodotoxin-receptor complex. 5. A number of agents were tested for their ability to displace the labelled saxitoxin. Calcium and thallous ions each produced significant reversible reduction in binding, with apparent equilibrium dissociation constants of about mx. Toxin binding is also inhibited reversibly in acidic solutions by protons competing with toxin for a binding site with a pka of 5*6-5*9. All three ions are known to block sodium currents in myelinated nerve at similar concentrations. Our experiments indicate that they do so at the site of toxin binding. 6. Lidocaine and veratrine do not affect the binding of saxitoxin. * Present address: Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge, England.

2 784 B. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ INTRODUCTION Saxitoxin is the paralytic toxin from the dinoflagellate Gonyalaux catenella that periodically infects shellfish and causes 'red tides' (Schantz, Lynch, Vayvada, Matsumoto & Rapoport, 1966). When applied to nerve or muscle preparations in nanomolar concentrations the toxin specifically blocks the regenerative influx of sodium ions that underlies the electrical excitability of the tissue. Saxitoxin acts in a manner identical to that of tetrodotoxin, the puffer fish poison (Kao, 1966; Narahashi, Haas & Terrien, 1967; Hille, 1968a). The potent and highly specific action of these two toxins has led to an intense study of their molecular mechanism, with the hope that this would shed some light on the events occurring in the membrane during excitation (Hille, 1970; Narahashi, 1972). A number of studies of the uptake of tetrodotoxin (including [3H]- tetrodotoxin) by nerve membranes has demonstrated that there are remarkably few binding sites (Moore, Narahashi & Shaw, 1967; Keynes, Ritchie & Rojas, 1971; Colquhoun, Henderson & Ritchie, 1972; Hafemann, 1972; Henderson & Wang, 1972) and it is believed that these binding sites are the physiological sites of action of the toxin. This belief is founded to a large extent on the results of experiments with radioactively labelled tetrodotoxin that show a component of binding whose equilibrium dissociation constant and rate of binding are similar to those inferred from electrophysiological experiments (Colquhoun & Ritchie, 1972a, b; Colquhoun et al. 1972; Henderson & Wang, 1972; Schwarz, Ulbricht & Wagner, 1973). However, one criticism of any experiment using compounds labelled by the Wilzbach method is that some closely related impurity may also be produced during labelling and be undetected during purification, thereby compromising the conclusions drawn from these experimental results. The present study examines the binding of labelled saxitoxin, whose chemical structure is quite different from the puffer fish poison. The results complement those obtained with tetrodotoxin and further substantiate the hypothesis that both toxins act by binding to the same site. The results also provide several additional correlations between the pharmacology of the toxin binding and that of membrane ionic currents. supporting the belief that these sites of toxin binding are indeed the sites of the physiological action of the toxins, namely the sodium channels themselves. METHODS In the experiments with rabbit vagus nerves the procedures used by Colquhoun et al. (1972) in their experiments with radioactive tetrodotoxin were used throughout with one small modification. Preliminary experiments showed that the labelled saxitoxin was not stable when heated in aqueous solution at 600 C and ph 7 for 2 hr

3 STX BINDING TO SODIUM CHANNELS 785 (see also the section on purification). These conditions had been used previously by Colquhoun et al. (1972) to dry the nerves so that they could be weighed in order to estimate the extracellular space. In the present experiments, therefore, only the wet weight was determined directly. The extracellular space and dry weight were then obtained indirectly (see, however, p. 791) from the average values found by Colquhoun et al. (1972); this procedure seemed justifiable since the nerves were soaked for the same period (6 hr) and subjected to the same dissection procedure as in the previous experiments (Colquhoun et al. 1972). Final toxin binding was expressed in f-mole per mg dry nerve as in Colquhoun et al. (1972). Wherever possible data are given as mean values + s.e. of the means. The temperature of all experiments was about 200 C. The Locke solution for these experiments contained (mx): NaCl, 154; KCl, 5-6; CaCl2. 2-2; dextrose, 5; morpholinopropane sulphonate buffer, ph 7-2, 2-0. Solutions containing thallous ion were made by addition of thallous nitrate, a weak electrolyte which is never completely dissociated. The designated thallium ion concentrations were calculated using the stability constants for thallous nitrate in aqueous solution (Sillen & Martell, 1964). A solubilized membrane preparation of garfish olfactory nerves was obtained by incubating homogenized nerves for 4 hr in a saline solution (0-15 x-nacl; 0-01 M-Tris, ph = 73) containing 2 % Triton X- 100 detergent. The procedures that Henderson & Wang (1972) had applied to radioactive tetrodotoxin binding were used. In particular, the assay of binding used a similar column of Sephadex G25F to equilibrate the solubilized nerve with the toxin. All solutions in these particular experiments contained: NaCl, 150mM; Tris (hydroxymethyl)amino methane buffer, ph 7-2, 10 mm; Triton X-100, 2 % (v/v). For the determination of the half-life of the toxin: receptor complex a faster flow rate than in the experiments of Henderson & Wang (1972) was required because the dissociation of the toxin: receptor complex was about four times faster with saxitoxin than with tetrodotoxin. The use of a gel filtration column in the equilibration of a small molecule like saxitoxin with a large molecule or molecular aggregate such as a solubilized nerve membrane is equivalent to equilibrium dialysis; but it is much faster so that the whole experiment can be carried out within 5 min or less. For equilibrium measurements the column is first equilibrated with the labelled saxitoxin at the concentration desired, and then a small volume of the nerve membranes, dissolved in a detergent such as Triton X-100, is applied. The column is elated with the same solvent used in the equilibration. The fractions eluted from the column then contain a constant amount of radioactivity until the void volume peak containing the membrane material emerges. The void volume fractions contain an additional amount of radioactive saxitoxin corresponding to the amount of bound toxin. For a column of suitable dimensions (in these experiments 0-5 cm in diameter and 10 cm in length) the toxin has completely equilibrated with the membrane material by the time the void volume appears. By equilibrating the column with different concentrations of saxitoxin, or under different conditions, a complete binding curve can be obtained, or the effect of different conditions investigated. For kinetic measurements of the rate of release of labelled saxitoxin (e.g. Fig. 4) the column is first equilibrated with a large excess of unlabelled toxin (1000 nm). A small volume of a solution containing membrane material, to which labelled saxitoxin has been added, is then applied to the column. The column is subsequently eluted under a large hydrostatic pressure and fractions collected as usual. The shape of the elution profile can be analysed to give the amount of radioactive toxin remaining bound at different times after the start of the run. The principle of the method is that the membrane material, being excluded from the gel particles, moves about three times faster through the column than does the toxin, which is small

4 786 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ enough to permeate the relatively small holes in the gel. Clearly, any radioactive toxin that emerges in the void volume has remained bound throughout the duration of the run, which in the case of the experiment shown in Fig. 4 was 47 sec, and any radioactive toxin that emerges much later with a profile such as that obtained when free saxitoxin is applied to the column has been unbound for the duration of the run. The difference between the elution profile with the membrane fraction present and the profile when it is absent then shows toxin that was originally bound at the start of the run, some of which was released during the run and did not become rebound because of the large excess of unlabelled toxin present. The position of emergence of the toxin in the difference profile indicates the time at which the release occurred. The amount bound at different times after the start of the run can then be obtained by integrating the difference profile from the front to the back of the column (inset, Fig. 4). The off-rate constant (k_1) for the toxin :receptor interaction can thus be determined. From the value (K) for the equilibrium dissociation constant (Fig. 3), the value for the on-rate constant (k,) can be calculated: details of the procedure are described in Henderson & Wang (1972). The main difference between the earlier experiments with tetrodotoxin and the present experiments with saxitoxin is that the faster on-rate and off-rate with saxitoxin necessitated that the column be run faster. Preparation and purification of tritium-labelled 8axitoxin Saxitoxin (5 mg), generously supplied by Dr E. J. Schantz, was labelled by exposure to a tritium gas discharge (Dorfman & Wilzbach, 1959). Labile tritium was removed by repeatedly dissolving in 10-2 M acetic acid and evaporating to dryness. The above procedure was carried out by ICN Corp., Irvine, California. The resultant material contained 50 mc of radioactivity. The crude material, before purification, was subjected to high voltage paper electrophoresis at values of ph of 6-5, 8-6, and 9-1 (Fig. 1). Apart from some pigmented material that remained at the origin, the pattern of electrophoresis at ph 6-5 showed three clear peaks labelled I, II and III; these corresponded (for compounds of molecular weight about 300) to species with charges of 0, + 1 and + 2 respectively. Unlabelled saxitoxin with a charge of + 2 at ph 6-5 has the same electrophoretic mobility as that of peak III. Furthermore, only peak III changed its mobility as the ph was raised to 9. Its pka, estimated from the data in Fig. 1, was , and its charge was + 1 at high ph. The pka and charge are exactly those reported for saxitoxin (Schantz et al. 1966); and, as expected, unlabelled saxitoxin had the same mobility at ph 8-6 and 9-1 as that of peak III. The unlabelled saxitoxin was visualized by spraying with alcoholic potassium hydroxide and heating for 10 min at 900 C. The resultant product was fluorescent under ultraviolet illumination. We concluded, therefore, that peak III was labelled saxitoxin, and that peaks I and II were degradation products of the reaction. The purification, performed on 1 mg samples consisted of a single step electrophoresis at ph 6-5 in pyridine:acetic acid:water, 25:1:225. The procedure was usually repeated by cutting out the strip containing peak III and running it again under the same conditions, but this was not absolutely necessary since the improvement in purity was only slight. The radioactive peak was then eluted into a small volume of water buffered at ph 4-0 with sodium acetate, and the solution stored at 00 C. Under these conditions the saxitoxin is chemically stable for several months. However, we observed, by electrophoretic analysis and by evaporation, that an appreciable fraction of the tritium originally incorporated in the purified saxitoxin had exchanged with the solvent hydrogen over a period of several months at 0 C. The same phenomenon was observed at room temperature and ph 7. Here one half

5 STX BINDING TO SODIUM CHANNELS 787 of the radioactive label was removed from the toxin in 7 days while its biological activity was diminished by less than 10%. Similarly, at elevated temperatures (500 C) the exchange occurred in a matter of hours. These observations lead us to believe that it is mainly the ac-methylene protons of the propionyl group in saxitoxin (Wong, Oesterlin & Rapoport, 1971) that are labelled. Approximate rate data for the deuteration of this methylene group (J. Wong, personal communication) in saxitoxin are of the same order of magnitude as the rate of loss of tritium observed 111H6 I:t ~~ph 6.5 J I I I - II ph 86.1 ~*i~ { ~~ph 9-1 c 14 \ l i Distance moved (cm) Fig. 1. Distribution of radioactivity obtained on electrophoresis of the crude material tritiated by the gas discharge method at ph values of 6-5, 8*6 and 9-1. The interrupted vertical line represents the position to which an uncharged marker will migrate during the electrophoresis, which was for 60 min at 40 V/cm. For discussion of peaks, I, II and III, see text. here. This meant that a continual check of the radiochemical purity was required every few months and repurification performed when necessary. The purified material was considered homogeneous by our criteria and represented a twenty-fold radiochemical purification from the starting material. Because of the relatively clean behaviour of peak III in Fig. 1, and the sensitivity of the group whose pk. is 8-2, to

6 788 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ changes in the structure of the saxitoxin molecule (Schantz, 1960), it is highly unlikely that our purified material is anything but tritium-labelled saxitoxin. The concentration of the stock solution of radioactive saxitoxin was calibrated against a solution of known concentration using the bio-assay procedure described by Colquhoun et al. (1972). The specific activity of the final material was about 400 mclm-mole. RESULTS Binding to rabbit vagus nerve The uptake of radioactive saxitoxin by rabbit vagus nerve during a 6 hr soak is shown in Fig. 2. The continuous line is a least squares fit (weighted as in Colquhoun et al. 1972) to the experimental data points of the equation: amount bound = b [STX] + Al. [STX]/[STX] + K). This represents a saturable component with M sites and an equilibrium dissociation constant K, together with a linear non-saturable component of slope b. The number of sites obtained, 127 f-mole. mg dry-', is close to 200- E E 1 50 TTX--_-- E S50WX X Saxitoxin concentration (nm) Fig. 2. The uptake of labelled saxitoxin (STX) by rabbit desheathed vagus nerve at different external concentrations of STX. The nerves were equilibrated for 6 hr with the STX. The broken line is the asymptote of the binding curve. The total binding curve is the relation U = STX bound = 1-2[STX] + 127[STX]/(6.7 x (STX]) where U is given in f-mole. mg dry-l and [STX] is given in nm. The linear and saturable components are drawn separately. The total binding curve is a least squares fit to the points that are shown, together with the following points for uptakes (f-mole. mg dry-') at higher concentrations; 100 nm; 238, 236, 237, 241; 200 nm: 425, 427, 409, 447, 290, 534; 300 nm: 403, 403; 400 nm: 627, 623, 698, 602; 600 nm: 772, 845.

7 STX BINDING TO SODIUM CHANNELS 789 the value of 152 f-mole. mg dry-' obtained by Colquhoun et al. (1972) for the same nerve using radioactive tetrodotoxin. The dissociation constant (K) was nm. Colquhoun et al. (1972) obtained a value of 1-6 nm for the equilibrium dissociation constant of saxitoxin in experiments where the competitive inhibition of saxitoxin on tetrodotoxin uptake was studied. The experimental points in the earlier study were, however, not very precise, and the present value is certainly more reliable. This value of 6-7 nm is similar to the value of 3 nm obtained by Colquhoun et al. (1972) for tetrodotoxin. Since the potency of the two toxins in reducing the size of the compound action potential of rabbit vagus nerve is quite similar, differing by less than 20% (unpublished observations), this close agreement is what would be expected if the two toxins act with equal effectiveness at the same sites. More data on the rabbit vagus nerve, demonstrating, for example, the displacement of saxitoxin by tetrodotoxin is presented in the section describing the effects of some pharmacologically active compounds. Binding of solubilized garfish olfactory nerve membrane Binding of the labelled saxitoxin to a detergent solubilized preparation from garfish olfactory nerve membrane was carried out using the procedure of Henderson & Wang (1972). The results at equilibrium are shown in Fig. 3. Again the continuous line is a least squares fit of the data. In contrast to binding by intact nerve (Colquhoun et al. 1972) there was no need to assume that any non-specific binding occurred, and the amount bound was simply given by amount bound = M. [STX]/([STX] + K) where again M is the number of sites and K the equilibrium dissociation constant. The assumption of no non-specific binding appears to be valid since the addition of a large excess of either unlabelled saxitoxin or unlabelled tetrodotoxin resulted in the complete abolition of the binding peak. Thus, the non-specific binding to intact rabbit vagus nerves found for both saxitoxin (Fig. 2) and tetrodotoxin (Colquhoun et al. 1972) is clearly associated with the intactness of the nerve membranes. During the preparation of membrane fragments much of the phospholipid is centrifuged out as fairly large aggregates. Since such solubilization also abolishes the non-specific binding of both tetrodotoxin and saxitoxin, it is tempting to conclude that non-specific binding involves the phospholipids or the cholesterol (Villegas & Barnola 1972) of the membranes. Another possibility (that the non-specific binding in intact nerve represents uptake into the axoplasm) seems unlikely in view of the finding that tetrodotoxin

8 790 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ injected internally into squid giant axons does not seem to be able to reach the outside surface of the nerve (Narahashi, Anderson & Moore, 1966). The value for the maximum binding capacity (M) of saxitoxin was 1*4 p-mole. mg membrane protein-1, which is close to the value obtained previously for the number of binding sites for radioactive tetrodotoxin (1.1 p-mole.mg protein-'); the value for the equilibrium dissociation constant (K) of 6*3 nm is also close to the value of 7 nm obtained previously. Table 1 shows the good agreement in the data on the dissociation constants 1-1 EU0 E 0~ -Y 0 x._ x 'U 25r I- 10I 5 A( / /0 C/ / X -0 0 / V/ r. e I I I Saxitoxin concentration- (nm)' Fig. 3. The uptake of labelled saxitoxin by solubilized garfish nerve membranes. Note the double reciprocal plot. The lines are least squares fit to the points in the absence (@) and presence (4)) of 15 nm tetrodotoxin. TABIE 1. Measurements of equilibrium dissociation constants of saxitoxin (STX) and tetrodotoxin (TTX) for garfish olfactory nerve Method KTTX KSTX (nm) (nm) Reference 10 - Colquhoun et al Henderson & Wang, This study [3H]TTX binding to intact nerve [3H]TTX binding, STX competition; solubilized membranes [3H]STX binding, TTX competition; solubilized membranes [3H]TTX binding to membranes 22Na ion flux measurements in intact nerve Benzer & Raftery, R. Henderson & G. Strichartz (unpublished experiments)

9 STX BINDING TO SODIUM CHANNELS79791 for saxitoxin and tetrodotoxin for the garfish preparation available from several methods. Fig. 3 also shows the binding of radioactive saxitoxin in the presence of 15 nm tetrodotoxin. The binding of saxitoxin appears to be inhibited competitively by tetrodotoxin, with the equilibrium dissociation constant for tetrodotoxin estimated as 12 nm (see Table 1). In a previous paper (Henderson & Wang, 1972) the dissociation rate of tetrodotoxin (TTX) from its binding site was measured and it was shown that the reaction was reversible with no destruction of the toxin, the equation describing the process being TTX+R =TTX.R. In this reaction k-1 was 0-95 min-1 and K = (k-]/kl) was 6 niv. The dissociation rate constants for saxitoxin have now been measured (as described in Methods, pp ). Fig. 4 demonstrates that the release of saxitoxin closely follows an exponential time course with an offset rate constant(kc1) of 3*2 min-' (corresponding to a half-life of 13 sec). Thus, 1cj, which is given by ik_1/k, is 5 x 108 m-1 mim-1, K being taken to be 6 3 nm (Fig. 3). The equation STX + R STX -R describes the process, the reaction being reversible with no degradation of the toxin during binding and release. If the toxin had been hydrolysed, for example, almost all the toxin would have been destroyed during the 5 min incubation period before the application of the sample to the column; and as a result of binding, the two elution proifiles of Fig. 4 would have been identical. However, the expected amount of bound toxin did emerge ahead of the profile's unbound material indicating that no degradation occurred during the binding reaction. Effect of various procedures on saxitoxin binding Colquhoun et al. (1972) examined the effect of a variety of agents and procedures on tetrodotoxin binding. We have extended some of the observations (with calcium, ph, and local anaesthetics) to saxitoxin binding, and, in addition, we have examined the effect of thallous ions and of veratrine. In most of these experiments the nerves were soaked in the test solution for 6 hr and the uptake of radioactivity was determined relative to the uptake by the paired nerve from the same rabbit from normal Locke solution containing the same concentration of saxitoxin. One hour before the end of the period of exposure to saxitoxin one third of the soaking-in solution was removed and [14C]mannitol added to it. About a third of each nerve was then cut off and placed in the labelled mannitol solution for the final hour so that we could measure the extracellular space, and hence correct for the saxitoxin in this space.

10 792 1?. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ C 60.i E Z 40 C 0 o-0 20 ~40 C M 20 ~' Time after start of flow (sec) I II I Fraction number Fig. 4. Elutionprofile (M) from a Sephadex G25 column of a sample (0-25 ml.) containing 1 1-6nIm saxitoxin equilibrated with garfish nerve membrane 1 mg protein). The circles (@) show the result of a similar experiment done under identical conditions but with a sample containing only saxitoxin in solution and no nerve membranes. For further details see Methods section, and for treatment of result, see text. The inset shows the time course of the dissociation of saxitoxin from the saxitoxin-receptor complex: note the semni-logarithmnic scale. The temperature of the experiment was 20' C. Dependence on the ph. As Fig. 5 shows, the saturable component of saxitoxin binding was reduced when the bathing solution was made more acidic. Since saxitoxin is virtually fully ionized at all values of ph studied ( ) and labelled saxitoxin is stable under acidic conditions, this reduction must reflect an effect of ph on the binding site itself rather than on the toxin molecule. Binding of saxitoxin to the rabbit vagus was halved when the solution (containing 6-8 nm toxin) was at ph 5*5. If it is assumed that protons compete with toxin molecules at a common binding site, value is obtained (twelve experiments) for the inhibitory dissociation constant for protons, K. +, of tm. This value of K. + corresponds with a protonation site whose PKa is 5-85 if one proton were bound at one toxin binding site. On this basis, binding of toxin from solutions where the toxin concentration is near the K toxin' as in Fig. 5, will follow a titration curve that centres on a value log10 2, i.e. 0-3 ph unit, lower than the pka of the binding site, as in Fig. 5. The ph dependence of tetrodotoxin binding was measured in gar olfactory nerves, in the ph range , but using only 2 nm tetrodotoxin solutions. Again the specific binding was halved near ph 5-5. But with a

11 STX BINDING TO SODIUM CHANNELS79793 tetrodotoxin, calculations of K.1 + from a simple competition model (five experiments) yield a value for K], of 2-28 ± 0-82 /tzm; this would correspond with a single proton binding site whose pka is The possibility of irreversible degradation of the nerves under acidic conditions was excluded by experiments in which test nerves were exposed to low ph (4.5-5) for 6-12 hr and then subsequently tested for their ability to bind saxitoxin at ph 7. As the open circles in Fig. 5 show, prior long-term exposure of the nerves to acidic solutions had little or no effect o-0,75~~~~~~ o 0~~~~~~~~ 750 U 0 x ~ ~ External ph Fig. 5. The dependence on external ph of the saturable uptake of saxitoxin by the desheathed rabbit vagius nerve. The uptakes at the different values of ph are expressed relative to the uptakes by paired nerves from solutions of the same saxitoxin concentration but at ph 7-0. Uptake was measured after 6 hr exposure time in solutions containing STX at 6 flm (@); 7 nm (U); 8 nm~(c); and 200 nm (15) concentrations. The open circles (0) represent the uptakes at ph 7-0 by nerves that had previously been exposed for 3 hr in saxitoxin-free solutions at the ph indicated. The continuation line is a titration curve for a single protonation site with pk. = 5.5. on binding. It should be noted, however, that nerves which have been exposed for any appreciable period of time to solutions whose ph is below 6 no longer conduct impulses. There are many possible reasons for this other than damage to the sodium channels; for example, the nerves may no longer be able to generate ATP, the membrane may be leaky, or the sodium pump may be impaired. Any one, or a combination of these factors may cause the loss of electrophysiological activity but leave the toxinbinding properties intact. The non-specific binding was also affected by the ph changes. At ph 4-5, for example, the total uptake from 200 nmi saxitoxin (which is largely non-specific) was reduced to about a half. It was assumed that this point lay on a single protonation titration curve for non-specific binding 26 PH Y 235

12 794 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ so that the effect on specific binding (Fig. 5) could be calculated. This correction factor was, however, always small since the non-specific uptake from the low concentrations of saxitoxin used in Fig. 5 was small (< 6 f- mole. mg dry-'). Calcium. The replacement of all the sodium chloride of Locke solution by an isosmotic amount of calcium chloride (113 mm) had little effect on the uptake of saxitoxin in high concentrations (200 nm). However, at low concentrations of saxitoxin when the total uptake is largely determined by the saturable component the binding was markedly reduced (Table 2). TABLE 2. Inhibition by calcium of the saturable component of saxitoxin binding to rabbit vagus nerves. In a all the NaCl of Locke solution was replaced with 113 mm- CaC12. In b the NaCl was replaced by varying amounts of CaCl2 and appropriate amounts of sucrose to keep both the osmolarity and the ionic strength constant. The value 6 is the ratio of the saturable saxitoxin uptakes in the test (high calcium) and normal Locke (2.2 mm Ca) solutions, obtained after a relatively small correction had been applied for the calcium-insensitive, non-saturable sax-toxin uptake (1.2 f- mole.nm-1.mg-1). The equilibrium dissociation constant for saxitoxin binding was taken to be 6-7 nm (Fig. 2) a [Ca] [STX] Number of Kc. (mm) (nm) experiments (mm) 115* Mean +s.e b * Mean + s.e One possible explanation for this reduction in binding is that one calcium ion competes for the site where one positively charged molecule of toxin is reversibly bound. On this basis one can calculate an apparent equilibrium dissociation constant, KCa, for the inhibitory effect of calcium. The binding of any inhibitor, I, that lowers the observed toxin affinity in a reversible manner can be characterized by an equilibrium dissociation constant, KI, given in the equation: Amount bound (specific) = M[STX]/{[STX] + K( 1+ [I]/KI)} where M is the total specific binding capacity, K is the equilibrium dissociation constant for the toxin and [STX] its concentration. Such calculations from experiments at four different toxin concentrations are consistent with an

13 STX BINDING TO SODIUM CHANNELS79795 inhibitory constant of about 30 mm for calcium ions (final column, Table 2). In the experiments of Table 2 a, in which 113 mmv calcium replaced the 154 mm sodium chloride of normal Locke solution, the ionic strength of the high calcium Locke solution was greater than in normal Locke solution (0.345 m compared to m) and may have diminished the binding by some non-specific mechanism. However, the following experiments (Table 26b) done at constant ionic strength (0.165 m) strongly suggest that the effects in Table 2 a reflect an action of calcium per se rather than a change in ionic strength. When the nerves were bathed in a calcium-locke solution with the same tonicity and ionic strength as normal sodium Locke, the saxitoxin binding was still reduced, to 47% of its control value. This calcium- Locke contained 53-2 mm calcium chloride and 100 mmi sucrose to replace completely the 154 mmv sodium chloride normally present. If the calcium concentration was 314- or 10 mm (adjusted by mixing the above mentioned high calcium solution with normal Locke solution) the reduction in saxitoxin binding was less (Table 2 b). From the equation introduced above, these reductions yield an average value for the apparent Kc_ of *7 mm. The near constancy of the individual values with varying external calcium concentrations provides some support for the initial assumption that calcium ions compete with saxitoxin for the same binding sites. In nerves it has been observed that an increase of extracellular calcium raises the firing threshold and shifts the voltage dependence of ionic conductance parameters along the voltage axis (Brink, 1954; Frankenhaeuser & Hodgkin, 1957). Both of these actions of calcium can be attributed to a change of the electric potential at the outer surface of the axon membrane, a change that can be assigned almost completely to the ability of divalent ions to screen fixed charges on the membrane by accumulating in the electric double layer adjacent to the nerve (Chandier, Hodgkin & Meves, 1965; Gilbert & Ehrenstein, 1970; McLaughlln, Szabo & Eisenman, 1971). The concentration of a charged species at the membrane surface will depend on the surface potential according to Boltzmann's law(mc- Laughiin et al. 1971). Since both saxitoxin and tetrodotoxin are cations at neutral ph, a change in surface potential at the binding site should affect the local concentrations of these toxins and their apparent K's, hence affecting the amount of toxin bound, particularly at low concentrations. In other words, calcium could decrease toxin binding by screening fixed charges at or near the binding site rather than by competing with a toxin molecule for a specific binding site. One means of discriminating between the two possible modes of calcium inhibition is to compare the inhibition of saxitoxin binding by calcium with the inhibition of tetrodotoxin binding by calcium. Since saxitoxin is 26-2

14 796 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ doubly charged, it will be affected more by the same surface potential change than will be tetrodotoxin, which has a single charge. However, if inhibition is due to competitive binding, equimolar calcium ion concentrations should displace saxitoxin and tetrodotoxin to the same extent. We, therefore, compared the effects of calcium ions in displacing tetrodotoxin and saxitoxin from the solubilized garfish membrane preparation (Table 3). Both toxins were displaced from the binding site to the same extent by calcium at a concentration of 8 mm and again at a concentration of 50 mm. Clearly, the depression of toxin binding by calcium ions in the garfish membrane preparation may be explained completely by simple competitive inhibition with an average value for Kca = mm. TAiBLE 3. Toxin binding to solubilized garfish membranes in the presence of divalent cations (two experiments). The medium contained 150 mm-nacl, 10 mx-tris, ph 7-4 [Ca2+] Toxin bound Kcs [Toxin] (mm) (%) (mm) 2 nx-stx nm-stx nm-stx nm-ttx nm-ttx nm-ttx Mean±s.E However, the situation is not so simple in intact nerve. In preliminary experiments on whole nerve we have observed that the Kea calculated from inhibition of saxitoxin binding differs systematically from the Kca calculated from inhibition of tetrodotoxin binding. As the extracellular calcium concentration increases, the KCa calculated from saxitoxin binding becomes relatively smaller than the Kca calculated from tetrodotoxin binding. By attributing the difference between the two values of Kca entirely to the screening effect of calcium ions, we determined an upper limit for surface potential changes. The changes (7 mv per tenfold change of calcium ion concentration) are much lower than those calculated from measurements of the voltage shifts of sodium permeability (20 mv per tenfold change of calcium ion concentration, Frankenhaeuser & Hodgkin,, 1957; Hille, 1968b). These changes of the surface electric potential seem too small to account for the observed changes in toxin affinity caused by increasing external calcium. The major effect of calcium on toxin binding in intact nerve occurs by a binding mechanism, not by screening. Thallous ion. Experiments were done to test the effect of replacing part or all of the sodium of Locke solution by the thallous ion. Because thallous nitrate had to be used (thallous chloride being relatively insoluble) the

15 STX BINDING TO SODIUM CHANNELS 797 standard Locke solution used in these particular experiments contained 154 mm sodium nitrate instead of 154 mm sodium chloride. Changing chloride to nitrate had no effect on saxitoxin binding. At all concentrations of thallous ion tested (ionized concentration, 1I-121 mm) the uptake of saxitoxin was depressed (Table 4). With 121 mm thallous ion the total uptake of saxitoxin from an external concentration of 3-5 nm was reduced to of its control value. Much of the remaining binding must, however, have been non-specific. Non-specific TABBLE 4. Inhibition of the saturable component of saxitoxin binding by thallous ion that replaced equimolar amounts of sodium ion in the Locke solution. The ratio of the observed saxitoxin uptakes in thallous-locke and sodium-locke is the value of Stow, The ratio 688C was obtained by making the relatively small (< 10%) correction for the linear uptake to the total uptake. K for saxitoxin binding was taken as 6-7 nm. The thallous ion concentrations noted are solution concentrations calculated by accounting for incomplete dissociation of the added thallous nitrate. The inhibitory equilibrium dissociation constant for thallous ion is KT1+ = [T1+] K. 8.p&cl'j(K + [STX]) (1-6Pse)} (T1+] (STX) KTl + 1 (mm) (nm) (ma) (56.55)* Mean + s.e ± 3-20 * Value 12 s.e. from mean and therefore excluded. binding was also depressed by thallous ions. Thus, the uptake from high concentrations (560 nm) of toxin, which is mostly non-specific, was reduced to about 20% of its control value by thallous-locke (121 mm thallium). We assumed that this reduction arose from a saturable inhibition by thallous ion of the non-specific binding of the toxin. Using the calculated inhibitory constant for thallous ions for this process (which turned out to be about 30 mm) we corrected all the observed total toxin uptake in Table 2 (in varying thallium, concentrations) for the appropriately adjusted non-specific uptake. After this correction the specific saxitoxin uptake is reduced to of the control value.

16 798 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ If thallous ion is assumed to compete, like calcium, for the saxitoxin binding site, an inhibitory equilibrium dissociation constant for thallous ion can be calculated. The eleven individual values in Table 4 for which the calculation was made yielded a KT1 Of mm. The inhibition by thallium is completely reversible. Nerves soaked in thallous-locke for 3-5 hr before incubation with saxitoxin took up % of the radioactivity taken up by nerves incubated in sodium-locke during the same period. TABLE 5. Effect of various agents on uptake of saxitoxin. Column a gives the experimentally observed relative total uptake of saxitoxin. Column b shows the same data corrected (on the basis of Fig. 2) on the assumption that the test procedure only affected the specific binding Relative uptake of STX (testlcontrol) [STX] Total Specific Test compound (nm) n a b Veratrine (50 #sg/ml.) ± 0' Lidocaine (I mm) TTX (500 nm) Veratrine. The veratrum alkaloids produce a persistent component of increased sodium conductance in nodes of Ranvier of frog myelinated fibres that decays slowly on repolarization; this component is blocked by tetrodotoxin. Veratrine alters the kinetics of the sodium permeability change in nerve. It seemed worthwhile, therefore, to test the effect of veratrine on saxitoxin uptake. As Table 5 shows, are latively high concentration of mixed veratrine alkaloid (50,ug/ml.) produced only a small, and probably insignificant, reduction in saxitoxin uptake. Lidocaine. Colquhoun et al. (1972) showed that lidocaine did not affect the binding of tetrodotoxin to rabbit vagus nerves. Neither does it affect the binding of saxitoxin (Table 5). Tetrodotoxin. The uptake of saxitoxin from an external concentration of 4 nm was reduced to % of its control value by 500 mm tetrodotoxin. Much of this remaining binding is non-specific, and when the non-specific binding (obtained from Fig. 2) was subtracted from the total uptake (column b, Table 5) it was seen that the tetrodotoxin reduces the saturable saxitoxin uptake to about 5 5 % of its control value.

17 STX BINDING TO SODIUM CHANNELS 799 DISCUSSION Number of binding sites The uptake of saxitoxin by the rabbit vagus nerve, like that previously described for tetrodotoxin (Colquhoun et al. 1972) consists of a saturable component superimposed on a gradual, linear uptake that does not saturate. The dissociation constant (K) for saxitoxin is about 7 nm, similar to that found for tetrodotoxin, which is 3 nm. Since saxitoxin and tetrodotoxin, although they have some structural similarities, are entirely different molecules, there is no reason to expect the two values of K to be identical. However, if the toxins have the same site of action one would expect the total binding capacities for the two toxins to be the same: and our finding (Fig. 2) of 127 f-mole. mg dry-' for saturable saxitoxin binding is satisfactorily close to the value of 152 f-mole. mg dry-' obtained by Colquhoun et al. (1972) for tetrodotoxin. With the solubilized garfish preparation also, the agreement between the present value for saxitoxin binding (1.4 p- mole. mg protein-1) and the corresponding value obtained previously (Henderson & Wang, 1972) for tetrodotoxin binding (1.1 p-mole. mg protein-1) is satisfactory. Our preoccupation with comparing these independent measurements of the number of toxin binding sites indicates neither doubt about the accuracy of our measurements, nor doubt that both toxins act on the same sodium channels: the latter is clearly shown by the competitive binding behaviour of the two toxins (for example, Fig. 3, present experiments; Colquhoun et al. 1972; Henderson & Wang, 1972). Our main reason for making such a detailed comparison was to minimize the possibility that some very closely related radioactive impurity in the original labelled toxin had resisted our attempts at separation during the purification procedure. Should such an impurity be present, a major error could be introduced into the estimation of the number of binding sites. Colquhoun et al. (1972) had previously discussed this problem and concluded that it was an unlikely source of error because of the good agreement between their measurements on the uptake of labelled tetrodotoxin by rabbit vagus nerve and those of Keynes et al. (1971) on the uptake of unlabelled tetrodotoxin. Clearly, the present agreement (in both intact and solubilized nerves) between the amounts of labelled tetrodotoxin and labelled saxitoxin that are bound (differing by less than 30% in both cases) is further, stronger evidence of the correctness of both studies. Kinetics of binding reaction One important question is whether the binding of tetrodotoxin to the sites identified in the present experiments is indeed responsible for the

18 800 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ physiological blockage of sodium currents. It is possible, for example,that the physiological sites of action are present in even smaller concentration than the sites we observe. However, the binding parameters mimic so closely the parameters known to affect the sodium currents that this seems unlikely. Thus, as Table 1 shows, the dissociation constants for saxitoxin obtained from toxin binding data on garfish olfactory nerve (6.3 nm) agree excellently with that derived from electrophysiological measurements of the inhibition of sodium ion fluxes through the same nerve membranes (6 nm, R. Henderson & G. Strichartz, unpublished observations). Similar correlations between binding and physiological effects occur with tetrodotoxin on the garfish preparation (Table 1) and on rabbit vagus nerves (Colquhoun & Ritchie, 1972a; Colquhoun et al. 1972). The measurement (Fig. 4) of an offset rate constant of 3-2 min-' for labelled saxitoxin in its reaction with the receptor gives a value three to four times faster than that obtained in a similar manner for labelled tetrodotoxin, which was 0 95 min- (Henderson & Wang, 1972). Although equivalent rate measurements of the inhibition of sodium currents cannot be made on the same preparation (garfish olfactory nerve), Schwarz et al. (1973) and B. Hille (personal communication) have measured the rates of onset and offset of blockage of sodium currents on frog nodes of Ranvier by unlabelled toxin. The values they obtain for the offset rates at 20 C are almost identical to our measurements. In particular, B. Hille has shown that the off set rates of saxitoxin are four times faster than those for tetrodotoxin, being 1-5 and 0'35 min- respectively at 120 C. The values at 200 C would be about two times greater. This agreement in kinetic measurements, albeit on different nerves, provides strong support for the idea that the radioactive toxins are indeed binding to sodium channels, and that the structure of the binding sites in widely divergent forms of life (garfish and frogs) resemble each other closely. The onset rate constant of the saxitoxin binding reaction, 5 x 108 m-1. minor approximately 107m-l.sec-1, is not quite in the range of a diffusion limited reaction where rates are expected to be close to 109 M-1. sec-' (Gibson, 1966; Schechter, 1970). However, the exact degree of orientation of the toxin required for binding to occur is unknown, and in view of the relatively rigid structure of saxitoxin (Wong et al. 1971) it is difficult to judge whether the on reaction rate is limited by diffusion, orientation, or some more complex process.

19 STX BINDING TO SODIUM CHANNELS 801 Competition with Ca2, H+ and T1+ ions The results of Tables 2 and 3 indicate that the effect of calcium in depressing the uptake of saxitoxin can be explained in terms of a competition by calcium for the saxitoxin binding site, with an apparent Kca of mm. In their experiments with tetrodotoxin, Colquhoun et al. (1972) found a small reduction in tetrodotoxin binding to rabbit vagus nerves on addition of calcium ions or on lowering ph. However, the highest calcium ion concentration they used was 22 mm and the lowest ph 5-8, and, in fact, the binding of tetrodotoxin was reduced to about 70% under both these conditions. We have verified that these effects were indeed significant, and that at higher concentrations of calcium ions the behaviour of tetrodotoxin closely follows that reported here for saxitoxin. Protons also inhibit toxin binding. Saxitoxin binding to rabbit vagus is depressed by hydrogen ions with a calculated pka of 5-85 while tetrodotoxin binding to gar olfactory nerves is depressed by hydrogen ions with a calculated pka of In the light of these results, it is of considerable interest that Woodhull (1973) has found, in her study of frog nodes of Ranvier under voltage clamp, that both calcium ions and protons inhibit sodium currents, and do so in a manner dependent on the membrane potential. She found that calcium ions had an apparent dissociation constant of 23 mm at zero membrane potential and that this value gradually increased at larger depolarizations as if the binding site for the ion was located in the electric field of the membrane. Voltage changes at the site appeared to equal 0-26 times the total membrane potential change. She also found that the blockage of sodium currents that occurs at low ph (Hille, 1968b) had an apparent pka of 5* at zero membrane potential, which became lower at positive membrane potentials and higher at negative membrane potentials. Again, the affinity for hydrogen ion appeared to vary as though binding at the site was influenced by 0-26 times the membrane potential. If these observations are true for sodium channels in all nerves, at a resting potential of -40 mv, which is roughly that of the rabbit vagus nerves used here (Keynes & Ritchie, 1965), the pka would have been 5-9. And if the gar olfactory nerves, which we used hr after the garfish were decapitated, had become depolarized the pka would have been 5-5. The coincidence between Woodhull's values for the pka and Kca of the sodium channel determined from the effects of hydrogen and calcium ions on sodium currents, and our values for inhibition of toxin binding is remarkable. There may be other explanations for the difference between the observed values of pka for the toxin binding sites in gar olfactory and rabbit vagus

20 802 R. HENDERSON, J. M. RITCHIE AND G. R. STRICHARTZ nerves and we are presently investigating several alternatives. As we noted above, fixed charges near the binding site do appear to contribute slightly to the observed toxin binding inhibition by calcium. B. Hille (personal communication) has titrated some of the fixed charges near the voltage sensors of the sodium channel and demonstrated the presence of groups that bind hydrogen ions in the range of ph 4 to ph 6. Titration of such acid groups near the toxin binding site could affect apparent toxin affinities and we cannot exclude the possibility that the pka inferred from binding of divalent saxitoxin actually differs from that inferred from monovalent tetrodotoxin binding under identical conditions in the same tissue. The inhibition of saxitoxin binding by the thallous ion shown in Table 4 is again consistent with a competitive effect of thallium having an apparent dissociation constant of about 18 mm. In frog nodes under voltage clamp, thallous ions passing through the sodium channel appear to be one third as permeant as sodium ions if calculated by the Goldman equation at zero net current (Hille, 1972b). However, the actual sizes of the peak inward thallous currents are considerably less than one third of the peak inward sodium current. This observation may be interpreted in terms of the occupation of a site by thallous ions that causes blockage of the channels (Hille, 1972b). Hille (1972b) has also suggested that this site of blockage is actually part of the sodium channel, and that blockage may result from an ion that is delayed due to rather tight binding as it passes through the channel. An inhibitory dissociation constant for thallium can be calculated by measuring the reduction of sodium currents in a voltage clamped node of Ranvier bathed by sodium-ringer solutions containing different concentrations of thallous ion. From experiments like this, B. Hille (personal communication) has calculated a dissociation constant of about 20 mm for thallous ions in the sodium channel of frog myelinated nerves. Given the close agreement between the concentrations of calcium, thallous and hydrogen ions required to block sodium currents (in frog nodes) and to block saxitoxin binding (in rabbit vagus nerve), it is tempting to conclude that we are dealing with the same binding site in all cases, that this binding site is the principal coordination site for monovalent cations as they pass through the sodium channel, and that both tetrodotoxin and saxitoxin act by binding in the channel at this site (Kao & Nishiyama, 1965). The potential dependence of sodium current inhibition by calcium and hydrogen (Woodhull, 1973) indicates that the site is part way through the membrane. Such a hypothesis is similar to a previous speculation by Hille (1972a). Drugs that affect the gating properties of the sodium permeability change, such as batrachotoxin or veratrine, produce no change in the binding or tetrodotoxin or saxitoxin, respectively (Colquhoun et al. 1972;

21 STX BINDING TO SODIUM CHANNELS 803 this paper). This fact complements the observation that displacement currents, which have been attributed to the movement of permeability gating molecules in squid giant axon membranes, are unaffected by tetrodotoxin (Armstrong & Bezanilla, 1973; Keynes & Rojas, 1973). Furthermore, the sensors that control voltage-dependent permeability in nerve appear to be bounded by a membrane region with a rather high average density of fixed charges (Chandler et al. 1965; McLaughlin et al. 1971). But we have shown that in garfish nerves the results of calcium inhibition of toxin binding are consistent with a significantly lower density of fixed charges. Extensive studies of a wide variety of cations on whole nerve and membrane fragments, which are still being carried out, fully support these results. Thus, saxitoxin and tetrodotoxin bind to sodium channels in nerve at a site that is not identical with the region bounding the voltage sensors that regulate channel permeability. We thank Dr E. J. Schantz for supplying us with saxitoxin. This work was supported by a grant from USPHS NS One of us (R.H.) held a fellowship from the Helen Hay Whitney Foundation. REFERENCES ARMSTRONG, C. M. & BEzANILLA, F. (1973). Currents related to movement of the gating particles of the sodium channel. Nature, Lond. 242, BENZER, T. I. & RAFTERY, M. A. (1972). Partial characterization of a tetrodotoxinbinding component from nerve membrane. Proc. Natn. Acad. Sci. U.S.A. 69, BRINK, F. (1954). The role of calcium ions in neural processes. Pharmac. Rev. 6, CHANDLER, W. K., HODGKIN, A. L. & MEVES, H. (1965). The effect of changing the internal solution on sodium inactivation and related phenomena in giant axons. J. Physiol. 180, COLQUHOUN, D. & RITKI1E, J. M. (1972 a). The interaction at equilibrium between tetrodotoxin and mammalian non-myelinated nerve fibres. J. Physiol. 221, COLQUHOOUN, D. & RITCHIE, J. M. (1972b). The kinetics of the interaction between tetrodotoxin and mammalian non-myelinated fibres. Molec. Pharmac. 8, COLQUHOUN, D., HENDERSON, R. & RITCHIE, J. M. (1972). The binding of labelled tetrodotoxin to non-myelinated nerve fibres. J. Physiol. 227, COLQUIHOUN, D., RANG, H. P. & RITCHIE, J. M. (1973). The binding of labelled tetrodotoxin and cobra toxin by the rat diaphragm. Br. J. Pharmac. 47, P. DORFMAN, L. M. & WILZBACH, K. E. (1959). Tritium labelling of organic compounds by means of electric discharge. J. phys. Chem. 43, FRANKENHAEUSER, B. & HODGKIN, A. L. (1957). The action of calcium on the electrical properties of squid axons. J. Physiol. 137, GIBSON, Q. H. (1966). Biological oxidations: rapid reaction techniques. A. Rev. Biochem. 35, GILBERT, D. L. & ERRENSTEIN, G. (1970). Use of a fixed charge model to determine the pk of the negative sites on the external membrane surface. J. gen. Physiol. 55,

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