Interaction of substituted guanidines with the tetrodotoxin-binding component in Electrophorus electricus

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 195, No. 2, July, pp. 414-422, 1979

interaction

of Substituted Component

Guanidines with the Tetrodotoxin-Binding in Electrophorus electricud

JUTA K. REED AND WOUBALEM Department

of Chemistry,

University

of Toronto, Erindale

TRZOS

College, Mississauga,

Ontario,

Canada

Received September 20, 19’78;revised February 20, 1979 In efforts to understand the molecular properties of ion channels in biomembranes, we have investigated the interaction of substituted guanidines with the Na+ channel site in elect&us. This interaction was measured by membranes isolated from Electrophorus equilibrium competitive binding studies with [3H]tetrodotoxin ([3H]TTX); TTX has been shown to bind specifically to the Na+ channel in electrically excitable membranes. Although guanidine and small substituted guanidines such as methylguanidine or aminoguanidine competed with [3HJTTX for the membrane binding site, the apparent K, values for these derivatives were nearly seven orders of magnitude higher than the K, for TTX. On the other hand, the binding of the guanidines was considerably enhanced by introducing a substituent aromatic ring or aliphatic chain. Detailed analysis of the binding of aliphatic guanidines of varying chain length clearly demonstrated the contribution made by hydrophobic interactions. These results suggest that the channel site may include a hvdronhobic region in close nroximitv to the carboxylate previously postulated to be ” _ involved in TTX binding.

It is generally believed that electrical activity in biomembranes is based on transient changes in membrane permeability to specific cations (l-3). Na+ and K+ are thus postulated to permeate the membrane through specific pores or channels which display ion selectivity and voltage-sensitive gating mechanisms (see reviews, Refs. (4) and (5)). At the present time little is known about the chemical and physical properties of these channels, and clearly isolation and detailed characterization of the individual channel components is of fundamental importance in understanding the molecular events occurring during electrical activity. Although neither the Na+ nor the K+ specific channel systems have been isolated, a great deal of information about some of their chemical characteristics can still be inferred from electrophysiological studies and from other indirect methods. One of these latter approaches involves the use of specific neurotoxins as channel probes. Na+ channel-specific toxins such as tetrodotoxin * This research was supported by the National Research Council of Canada through Grant No. A0498. 0003-9861/‘79/080414-09$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

(TTX),’ saxitoxin (STX), batrachotoxin, and scorpion venom toxins have been particularly valuable in this respect (6-8). For example, by studying the interaction of radioactively labeled toxins such as TTX with electrically excitable membranes, it has been possible to gain insights into the structural features of at least part of the channel environment (9, 10). In addition, because of the remarkable specificity of these toxins, particularly TTX, they can be used to monitor the isolation of the channel components (11) either by reversible binding or as potential covalent affinity ligands. One of the difficulties in this latter approach with TTX is that even minor chemical modifications of this toxin usually produce inactive derivatives thereby limiting any effectiveness in affinity labeling studies (12). TTX also has few functional groups that can be effectively modified such that viable affinity probes can be generated. An alternative approach in synthesizing Na+ channel-specific compounds is to

* Abbreviations used: TTX, tetrodotoxin; STX, saxitoxin, ANS, 1-anilino-8-naphthalene sulfonic acid. 414

BINDING

OF GUANIDINES

design molecules with partial structural similarity to TTX. The guanidinium functional group is a logical starting point since this group is believed to be essential in the interaction of both TTX and STX with a negatively charged carboxylate at the channel opening (10, 13, 14). In this paper, we report studies on the interaction of guanidine, simple substituted guanidines, guanidine esters, and aliphatic derivatives with the Na+ channel components in plasma membranes isolated from Electrophorus electricus. This membrane preparation has been shown previously to bind [3H]TTX with affinities comparable to those observed with other nerve and muscle preparations (14). This binding represents interaction with the TTX-sensitive Na+ channels which have been characterized in not only the electroplaque but other electrically excitable tissues as well. The interaction of these guanidine derivatives has been measured directly by competition with [3H]TTX binding. Our results show that while all guanidines compete with [3H]TTX to varying degrees, those derivatives with hydrophobic substituent groups have particularly high affinities. These data may suggest the presence of a hydrophobic region in the membrane near the negatively charged carboxylate of the channel. EXPERIMENTAL

PROCEDURES

Materials. Benzoyloxypropylguanidine (I), p-nitrobenzoyloxypropylguanidine (II), and 5-hydroxy-2iminohexahydropyrimidine (III) were synthesized essentially by methods described by Schmiegel (15) with minor modifications. The esters were formed by treatment of 3-hydroxypropylguanidine with benzoyl chloride or p-nitrobenzoyl chloride. 3-Hydroxypropylguanidine was synthesized as the sulfate salt by treatment of 3-amino-1-propanol with 2-methyl-2thiopseudourea. The product was converted to the chloride salt prior to esterification. The corresponding esters were repeatedly recrystallized from 1-butanol to yield white crystals with mp 142-143°C (I) and 144- 145°C (II) consistent with literature values (15). The cyclic guanidine derivative (III) was synthesized by condensation of l,&diaminopropanol with 2-methyl2thiopseudourea. The product was recrystallized from methanol yielding white crystals with mp 243-245°C. Phenylbutylguanidine (IV) (mp 154-156”C), propylguanidine (V) (mp 242-243”C), butylguanidine (VI)

TO THE Na+ CHANNEL

415

(mp 20821o”Q hexylguanidine (VII) (mp 166-167°C) and nonylguanidine (VIII) (mp 279-281°C) were also synthesized by treatment of the corresponding amine with 2-methyl-2-thiopseudourea. The sulfate salts were recrystallized from methanol, butanol, or methanolibutanol mixtures. All compounds were characterized by ir and nmr. Guanidine hydrochloride and other simple substituted guanidines were obtained from Aldrich Chemical Co. or from Eastman Chemical Co. With the exception of guanylurea which was used as a sulfate salt, all of these guanidines were converted to the corresponding chloride salt prior to use. No differences between the chloride, sulfate, or nitrate salts were observed in the concentration range studied. Putifiation of [3H]TZ’X. [“HITTX was prepared commercially (ICN, Irvine, Calif.) by tritiation of citrate-free toxin obtained from Sankyo, Tokyo. The procedure used involved a catalytic exchange reaction using tritium gas. Because of the destructive nature of the method, radioactive decomposition products amounted to at least 95% of the total radioactivity. These were removed by repeated chromatography on BioRex 70 (NH: form) (16). Typically the crude radioactive toxin was applied to a column of resin equilibrated with 0.01 M ammonium acetate, pH 6.8. Greater than 95% of the total radioactivity eluted from the column with 0.01 M salt, representing breakdown products resulting from the tritium exchange procedure. Elution of the column with a gradient (0.01 to 0.20 M ammonium acetate, 60 ml each) removed other presumed cationic radioactive species. The radioactive material eluting between 0.05 and 0.09 M salt was pooled, lyophilized and rechromatographed on an identical column. Figure 1 shows a typical elution profile obtained for the rechromatography. Following preliminary washing of the column with 0.01 M ammonium acetate the radioactive toxin was eluted with a gradient (0.01 to 0.15 M, 60 ml each). A similar column had previously been calibrated with 1.0 ml of 1 x 1O-4M unlabeled TTX, and the elution profile for pure TTX (Fig. 1) was determined using the fluorescence assay (17). Obviously only a fraction of the apparently symetrical radioactive peak is superimposable with unlabeled TTX. The peak fractions were pooled and lyophilized, and the specific activity was determined. High-voltage electrophoresis of the final radioactive product indicated a single chemical species with a mobility comparable to that previously reported for TTX (18). Thin layer chromatography in different solvents (19) also indicated a single radioactive species. Preparation

of membranes

and binding

analysis.

Plasma membranes from the electric organ of Electrophorus electricus enriched in Na+ channels (TTX-receptor sites) were prepared as previously described (14). The binding of r3H]TTX to isolated

416

REED AND TRZOS

6

16

46

166

60

66 Fraction

126

146

160

number

FIG. 1. Elution profile for the chromatography of [3H]TTX (0) and pure unlabeled TTX (0) on BioRex ‘70. The initial radioactive peak (Fractions l-10) was eluted with 10 mM ammonium acetate; elution with the gradient (lo-150 mM ammonium acetate) is indicated by the arrow. Fractions 82-98 (bar) of the radioactive peak were pooled. These correspond to the peak of the unlabeled TTX as judged by the fluorescence assay for TTX.

membranes was determined by equilibrium dialysis at 4°C. The experimental design, treatment of data, and all other pertinent analysis were also as described (14). Typically, the equilibrium binding of [3H]TTX at five separate concentrations of toxin was determined in a medium containing 0.01 M K phosphate buffer (pH 7.0), 0.25 M sucrose, and 0.05 M NaCl. The binding of each of the model guanidine derivatives was measured by direct competition studies, varying the concentration of [3H]‘ITX at two or usually three fixed concentrations of derivative. The ionic strength was kept constant by the addition of choline chloride. Previous studies showed that choline did not bind to the channel sites in the concentration ranges studied (14). The reversible binding of [3H]TTX to the electroplaque membranes has been shown previously (14) to follow normal Langmuir hyperbolic saturation b=

PHlTTX .B Kd + [3H]TTX ’

[ll

where b is the measured binding of toxin (pmoles [3H]TTX mg protein-‘) at an equilibrium toxin concentration [3H]‘ITX, and B represents the total binding sites or binding of toxin at saturating levels. Kd is the equilibrium dissociation constant for the toxin-receptor complex. For a competitive inhibitor

of [3H]TTX binding, the following linear equation can be derived 1 - = % b

B

+ ([~YKi))~

+ f

9

VI

where Z is the concentration of inhibitor and K, is the equilibrium dissociation constant for the inhibitorreceptor complex. The K, for each compound was determined from a replot of the “apparent Kd” or Kd(l + ([II/K,)) versus [I]. The specific activity of [3H]TTX was determined in an analogous manner using unlabeled TTX as the “inhibitor” of [3H]TTX binding (14). In this case, in Eq. [2] [I] refers to the concentration of unlabeled TTX and K, = Kd where K, is in units of concentration and Kd is in units of radioactivity. From a replot of Kd(l + Z/K,) versus I, one can determine K, and Kd from the intercepts at the abscissa and ordinate, respectively. Specific activities of 410 Ci mole-’ were routinely observed. The radiopurity of [3H]TTX was determined as follows: 15 ml of membranes (3 mg protein ml-‘) in the dialysis buffer were equilibrated on ice with 3 x lo-” M [SH]lTX. Aliquots of the suspensionwere counted to accurately determine the radioactivity as well as toxin concentration. The specific activity had previously been determined. Membranes were removed by centrifugation (20 min, ZOO,OOOg), and aliquots of the supernatant

BINDING

OF GUANIDINES

TO THE Na+ CHANNEL

417

this procedure gives no indication of the radiopurity. If radioactive contaminants are still present, they either do not bind to the Na+ channel and serve only to increase the measured specific activity or if they bind to the channel they do so with affinities comparable to [3H]TTX and are reversed by unlabeled TTX. In the latter case, the radioactive contaminants may represent compounds with very close structural similarity to TTX. It is worth noting that all of the analytical methods used to assess the purity of the preparations (electrophoresis, thin-layer RESULTS chromatography) indicated a single molecular In studies reported previously (14), the species. The presence of radioactive conbinding of [3H]TTX to isolated electroplaque taminants, however, will not affect the membranes was found to be saturating with calculated value for dissociation (or inhibition) constants for the compounds to be tested, a dissociation constant of approximately 6 x 10mgM. The reversal of the binding by since these values are indirectly based on unlabeled TTX served as the basis for the binding affinities of unlabeled TTX. estimation of the specific activity of the Only the total number of binding sites will purified [3H]TTX preparation. The values be underestimated, as is the case here since obtained for the specific activity are thus radiopurities of 60-70% were routinely obtained by direct competition studies with observed. unlabeled TTX as shown in Fig. 2A. From The binding affinities of guanidine and the replot (Fig. 2B), it is possible to small substituted guanidines to the Na+ calculate the dissociation constant for the channel was also measured by competition binding of the toxin, and the apparent with [3H]TTX. Hille (20) has shown that specific activity of the radioactive toxin the channel in isolated nerves is at least preparation. It should be pointed out that somewhat permeable to guanidine and

were counted to determine radioactivity remaining. The specific activity of the [3H]TTX in this supernatant fraction was measured by standard methods, and this value used to calculate the absolute concentration of toxin in the supernatant. Knowing both the quantity of toxin as well as the radioactivity bound, one can calculate the “true” specific activity of [3H]TTX. This assumes that the only radioactivity bound is that due to TTX and not other contaminants. The radiopurity of the original [3H]TTX solution is simply the ratio of the “true” specific activity of [“H]TTX to the specific activity of the original radioactive solution. Radiopurities normally ranged between 60 an 70%.

FIG. 2. Competition between unlabeled TTX and [3H]TTX for binding to the TTX receptor site. (A) Double reciprocal plot of the binding of [3H]TTX to electroplaque membranes at varying concentrations of unlabeled TTX. (B) Replot of the calculated slopes (apparent K,) as a function of the concentration of unlabeled TTX. This analysis was used to calculate the specific activity of VH]TTX as described under Experimental Procedures.

418

REED AND TRZOS

I -

1 -

/

/

50

, -

I -

, -

Guanidine ( mM

i 0

04

02 ( 3H

TTX. nM

00

I 01

5”

mM

1 -’

100

Guanidine

FIG. 3. Competition between guanidine and [3H]TTX for the binding of the toxin to the electroplaque membranes. (A) Double reciprocal plot varying [3H]TTX at increasing concentrations of guanidine. (B) Replot of the slopes of (A) as a function of the quanidine concentration. The inhibition constant, K,, was calculated from (B) according to Eq. [3]. Experimental conditions are as described in the text.

corresponding hydroxy and amino derivatives but that the methyl derivatives were virtually impermeant. We were particularly interested in determining the equilibrium binding of these compounds to the TTXreceptor site since previous studies have suggested that a negatively charged carboxylate at the channel opening may be involved in both binding of the guanidine portion of the toxin and as a selectivity filter for ion permeation (10). It was not surprising to find that all the guanidines studied were competitive inhibitors of [3H]TTX binding. Figure 3 shows a representative plot for guanidine, showing the normal double reciprocal plot (A) and the corresponding replot (B) used to calculate K,. Similar analyses were carried out for all the guanidines; the compounds and their equilibrium inhibition constants, KI, are listed in Table I. In addition to the normal guanidines, we also examined the binding of one six-membered cyclic guanidine, 5hydroxy-2iminohexahydropyrimidine. The results show that although simple substitution of guanidine reduces the binding

affinities, the effect is not nearly as marked as expected. The differences between guanidine and methyl guanidine with respect to channel permeability is substantial (20) and is obviously not reflected in differences in equilibrium binding. It should also be pointed out that the K, values for the guanidines are higher than the Kd for TTX by nearly seven orders of magnitude. Even the cyclic guanidine, with somewhat more similarity to the ring TABLE I BINDING OF GUANIDINE AND SUBSTITUTED GUANIDINES TO THE TTX-RECEPTOR SITE

Compound Guanidine Aminoguanidine Methylguanidine 1,3-Diaminoguanidine Guanylurea l,l’-Dimethylguanidine 5-Hydroxy-Z-iminohexahydropyrimidine

K, (M) 1.0 x 2.0 x 1.6 x 2.9 x 2.0 x 7.0 x

10-z IO-2 1O-2 10-Z 10-a 10-Z

1.4 x 10-Z

BINDING

OF GUANIDINES

TO THE Na+ CHANNEL

419

TABLE III portion of TTX, had little affinity for this site. BINDING OF ALKYL GUANIDINES TO THE In efforts to obtain guanidine-containing TTX-RECEPTOR SITE compounds with enhanced affinity for the TTX-receptor, we synthesized and studied No. of carbons the binding of a series of guanidine Compounds in chain K, (M) derivatives, beginning with two guanidine 3 1.1 x 10-Z esters whose pharmacological properties had Propylguanidine Butylguanidine 4 2.5 x lo-” been described previously (21). Introducing 6 3.2 x 1O-4 an ester function was a logical approach Hexylguanidine Nonylguanidine 9 1.8 x 10-S since it was not inconceivable that the la&one form of TTX was in fact the reactive form. This aspect of the possible molecular interactions between TTX and studying the effects of various chain length the channel is outlined in more detail in alkyl guanidines on the binding of [3H]TTX. Compounds ranging in chain length from the Discussion. The two guanidine esters, benzoyloxy- three to nine carbons were synthesized as described previously. Studies with [3H]TTX propylguanidine and the p-nitro derivative were both competitive inhibitors with binding showed that all these compounds 2.2 X 10P4M were competitive inhibitors. The calculated K, values of approximately (Table II), nearly two orders of magnitude K, values are shown in Table III; enhanced less than those for simple guanidines. The binding is observed with increasing chain corresponding p-nitro derivative was investi- length with nonylguanidine binding with gated since it was felt that substitution by an inhibition constant of 1.8 x lop5 M. Figure 4 shows the variation of the an electron-withdrawing group would alter the positive charge distribution on the binding affinity (K,,,,,,) as a function of carbonyl carbon, thereby enhancing binding chain length. From the slope of this plot, it to the receptor. However, this was not the is possible to calculate the contribution of case, since both esters had nearly the same the aliphatic chain methylenes to the free inhibition constants. energy of complex formation ( AG ‘) assuming Although the enhanced binding affinities of these esters was entirely consistent with the proposed role of a lactone intermediate in TTX binding, further studies with the non-ester phenylbutylguanidine did not support this view. The phenyl derivative was found to have an inhibition constant even lower than that for the esters, i.e., 5.4 x 10-j M (Table II). The possibility that hydrophobic interactions were involved was investigated by TABLE

/jlj/

II

EFFECT OF AROMATIC RING SUBSTITUTION ON GUANIDINE BINDING TO THE TTX-RECEPTOR SITE Compound Benzoylpropylguanidine p-Nitrobenzoylpropylguanidine Phenylbutylguanidine

K, (M) 2.2 x 10-4 2.1 x 10-e 5.4 x 10-S

FIG. 4. Effect of increasing chain length of alkyl guanidines on the binding affinity (K,,,,,, ) for the TTX-receptor site. The contribution of each -CH,- to the free energy of association (AG ) was calculated from the slope assuming AG = -RT In K,,,,,,. (4°C).

420

REED

AND TRZOS

AG’ = -RT In KPSSOC,, where K,,,,,- = l/K,. The value obtained, -590 cal mole-’ for each -CH,- is well within the range normally observed for hydrophobic binding of -CH,or for transfer of -CH,- from a polar to a nonpolar environment (22, 23). Studies with longer chain alkyl guanidines (12 and 16 carbons) gave rather surprising results. Neither compound had any inhibitory effect on [3H]TTX binding at concentrations up to 3 x 1O-5M, even after incubation directly with the membranes prior to dialysis. Experiments at higher concentrations were limited by the solubility properties of the compounds. These data may suggest that although hydrophobic interactions predominate, there may be a critical size restriction on the accessibility to the binding site. It is well known that alkyl guanidines, particularly biguanidines, bind to natural and artificial membranes, affecting the membrane surface charge characteristics (24, 25). It was possible that the observed effects of some of these compounds were due not to specific interactions with the TTX-receptor site but rather to secondary effects resulting from shielding of negative charges on the membrane surface thereby reducing the concentration of TTX in the diffusion layer. To test this, we investigated the effect of the guanidines on the surface charge properties of the electroplaque membranes indirectly by measuring the effects of the compounds on the fluorescence of 1-anilino-%naphthalene sulfonic acid (ANS). Previous studies with biological membranes have shown that cations such as Na+ increase the ANS fluorescence intensity because they effectively shield negative charges on the surface of the membrane, thereby enhancing ANS binding (26). Biguanides have also been shown to increase ANS fluorescence in submitochrondrial particles (24, 25). Using the experimental approach described in (25), we found that in the absence of Na+, a small but significant increase in ANS fluorescence was observed for all the guanidines. However, the apparent dissociation constants calculated from these experiments were at least 20 to

30 times higher than those calculated from [3H]TTX binding experiments. Furthermore, in the presence of 0.05 M NaCl as routinely used in our assay, no increase in ANS fluorescence by the guanidines was observed. Although we cannot unambiguously rule out the possibility that local surface charge perturbations near the channel may still occur, it is unlikely that a general surface charge alteration is responsible for the observed competitive effects on [3H]TTX binding. DISCUSSION

In this study we have attempted to elucidate some of the key chemical features of the Na+ channel by examining the nature of the interaction of the TTX-receptor with guanidines. The obvious structural similarity (Fig. 5) between TTX and STX with respect to the guanidine moiety lends strong support to the suggestion that electrostatic interactions between the protonated guanidine and an ionized carboxylate at the channel opening contribute at least partially to the tight binding. However, other structural features of these toxins should also be noted, namely, that both have a single carbon with a high electropositive charge density. TTX has been postulated to

OH

TETRODOTOXIN

SAXITOXIN

FIG. 5. Structures of tetrodotoxin (TTX) and saxitoxin @TX). Arrows indicate the electropostive carbon in each case.

BINDING

OF GUANIDINES

exist in solution as a mixture of the hemilactal and la&one (27), while STX also exists as a mixture of the ketone and hydrated ketone (28). It is not clear which of the two species actually binds to the channel. Camougis (29) proposed that the tight binding of TTX may result from a nucleophilic attack by a group in the channel opening at the lactone to form an intermolecular hemilactal. An analogous mechanism may be proposed for STX binding. Although no experimental evidence is available to support this model, any proposed structure of the channel must obviously consider these important and unique features of the toxins. Ranney et ~2. (21) and later Spiegelstein and Kao (30) recognized the possibility that the la&one function may be important in the binding of TTX to the channel. They synthesized a number of guanidine esters and studied their effects in viva and in vitro, using either a rat diaphragm-nerve preparation or a frog sartorius fiber assay to measure effects on the action potential. Some of these compounds had measurable effects on the electrical excitability characteristics although the doses used were in general very high. These studies nevertheless prompted us to examine more closely the possible contribution of the ester moiety in binding to the TTX-receptor site. Although our approach allows us to calculate directly equilibrium dissociation constants, it does not provide any information about possible effects on spike-generating mechanisms. The two guanidine esters studied in this report, benzoyloxypropylguanidine and the p-nitro derivative, had significantly higher binding affinities to the TTX-receptor site than either free guanidine or simple substituted guanidines. The enhanced binding resulted from hydrophobic interactions with the aromatic ring and not from dipole-ion or dipole-dipole interaction between the receptor site and the ester carbonyl. This is evidenced from the fact that the non-ester, namely, phenylbutylguanidine, had an inhibition constant even lower than that for the corresponding ester. Possibly a restricted orientation around the planar ester linkage may prevent maximum

TO THE Na+ CHANNEL

421

interactions of the aromatic ring with the hydrophobic regions of the channel. The significance of hydrophobic interactions was further shown by the binding studies with alkyl guanidines of varying chain length and therefore of varying hydrophobicity. The results suggest that the main stabilization is derived from hydrophobic interactions of the alkyl chain with a nonpolar region of the channel. It is important to realize that TTX, rather than having nonpolar substituent groups, is exceeding polar containing no less than six hydroxyl groups. Although STX has only the hydrated ketone and a carbamate group (which is apparently not required for toxicity (31)) it too is a highly polar molecule. It would seem unlikely therefore that the molecular orientation of these alkyl guanidines at the channel would be identical to that of TTX or STX, and it would be difficult therefore to predict any effect on Na+ conductance in intact nerves. On the other hand, it is known that the [3H]TTX binding to nerve and electroplaque membranes is extremely sensitive to phospholipase A treatment (14, 32) or even low concentrations of detergents (11) indicating a structural role of lipids for the conformational integrity of the receptor. The interactions with the alkyl derivatives may therefore involve interactions of the chain into this nonpolar lipid region of the channel. The relationship between binding and chain length is highly reminiscent of the increased interaction of alkyl quaternary ammonium derivatives with the K+ channel (33). On the basis of these observations, Armstrong proposed the presence of a hydrophobic binding site near the axoplasm side opening of the K+ channel. Quaternary ammonium compounds have no measurable binding affinity for the TTX-receptor (14) and no effect on Na+ currents (34). Recent preliminary studies on voltageclamped squid giant axons (T. Begenisich, personal communication) indicated that nonylguanidine was an effective blocker of Na+ currents when the drug was applied internally, although little effect was seen following external application. These studies, still in progress, may raise some intriguing

422

REED

AND TRZOS

questions regarding the molecular features of the ion pore since TTX is believed to bind to the channel only from the exterior surface (35). It should be pointed out that a number of local anesthetics particularly lidocaine, affect Na+ currents also from the axoplasm side (36). However, we have been unable to detect any competition between [3H]TTX and procaine, lidocaine, butacaine, or tetracaine even at millimolar concentrations. Clearly, the mode of interaction between the membrane channel site and either the guanidines or the local anesthetics must be quite different. The membranes used in our binding experiments appear to be enclosed vesicles with some membrane fragments as indicated by electron microscopy. Nevertheless we have not been able to unambiguously determine sidedness or the degree of leakiness. It may not be unreasonable to suggest that the time involved in the dialysis (greater than 12 h) will be sufficient to allow equilibration of all intravesicle compartments, and therefore access to both faces of the channel may be possible. The mechanisms of binding of both TTX and the model guanidine compounds are still unclear and pose some interesting questions regarding the geometric and topological characteristics of the channel. These aspects of the chemical properties of the Na+ channel are currently under investigation in our laboratory. REFERENCES 1. HODGKIN, A. L., AND HUXLEY, A. F. (1952) J. Physiol. (London) 116, 449-472. 2. HODGKIN, A. L., AND HUXLEY, A. F. (1952) J. Physiol. (London) 116, 473-496. 3. COLE, K. S. (1968)Membranes IonsandImpuIses, University of California Press, Berkeley, Calif. 4. ARMSTRONG, C. M. (1975) Quart. Rev. Biophys. 7, 179-209. 5. HILLE, B. (1970) Prog. Biophys. Mol. Biol. 21, l-32. 6. CATTERALL, W. A. (1975) PTOC. Nat. Acad. Sci. USA 72, 1782-1786. 7. ALBUQUERQUE, E. X., DALY, J. W., AND WITKOP, B. (1971) Science 172, 995-1002. 8. NARAHASHI, T. (1974) Physiol. Rev. 54,813-889. 9. RITCHIE, J. M., AND ROGART, R. B. (1977) Rev. Physiol. Biochem. Pharmacol. 79, l-50. 10. HILLE, B. (1975) Biophys. J. 15, 615-619.

11. AGNEW, W. S., LEVINSON, S. R., BRABSON, J. S., AND RA~ERY, M. A. (1978) Proc. Nat. Acad. Sci. USA 75, 2606-2610. 12. DEGUCHI, T. (1967) Japan. J. Pharmacol. 17, 267-278. 13. HENDERSON, R., RITCHIE, J. M., AND STRICHARTZ, G. R. (1974) Proc. Nat. Acad. Sci. USA 71, 3936-3940. 14. REED, J. K., AND RAFTERY, M. A. (1976) Biochemistry 15, 944-953. 15. SCHMIEGEL, J. L. (1967) Ph.D. thesis, Stanford University, California. 16. BENZER, T. I., AND RAFTERY, M. A. (1972) Proc. Nat. Acad. Sci. USA 69, 3634-3637. 17. NONEZ, M. T., FISCHER, S., AND JAIMOVICH, E. (1976) Anal. Biochem. 72, x20-325. 18. COLQUHOUN, D., HENDERSON, R., AND RITCHIE, J. M. (1972) J. Physiol. (London) 227, 95-126. 19. KONOSU, S., INOUE, A., NOGUCHI, T., AND HASHIMOTO, Y. (1968) Tozicon 6, 113-117. 20. HILLE, B. (1971) J. Gen. Physiol. 58, 599-619. 21. RANNEY, B. K., FUHRMAN, F. A., SCHMIEGEL, J., AND MOSHER, H. S. (1968) Arch. Znt. Pharmacodyn. Ther. 175, 193-211. 22. TANFORD, C. (1973) in The Hydrophobic Effect. Formation of Micelles and Biological Membranes, Wiley, New York. 23. TRIGGLE, D. J. (1971) Neurotransmitter-Receptor Interactions, pp. 22-62, Academic Press, New York. 24. SCHAFER, G., AND RIEGER, E. (1974) Eur. J. Biochem. 46, 613-623. 25. SCHAFER, G., AND BOJANOWSKI, D. (1972)EtiT. J. Biochem. 27, 364-375. 26. MCLAUGHLIN, S. G. A., SZABO, G., AND EISENMAN, G. (1971) J. Gen. Physiol. 58, 667-687. 27. WOODWARD, R. B. (1964) Pure Appl. Chem. 9, 49-74. 28. SCHANTZ, E. J., GHAZOROSSIAN, V. E., SCHNOES, H. K., STRONG, F. M., SPRINGER, J. P., PEZZANITE, J. O., AND CLARDY, J. (1975) J. Amer. Chem. Sot. 97, 1238-1239. 29. CAMOUGIS, G., TAKMAN, B. H., AND TASSE, J. R. P. (1967) Science 156, 1625-1628. 30. SPIEGELSTEIN, M. Y., AND KAO, C. Y. (1971) J. Pharmacol. Exp. Ther. 177, 34-39. 31. GHAZAROSSIAN, V. E., SCHANTZ, E. J., SCHNOES, H. K., AND STRONG, F. M. (1976) Biochem. Biophys. Res. Commun. 68, 776-780. 32. VILLEGAS, R., BARNOLA, F. V., AND CAMEJO, G. (1973) Biochim. Biaphys. Acta 318, 61-68. 33. ARMSTRONG, C. M. (1971) J. Gen. Physiol. 58, 413-437. 34. HILLE, B. (1967) J. Gen. Physiot. 50, 1287-1302. 35. NARAHASHI, T., ANDERSON, N. C., AND MOORE, J. W. (1966) Science 153, 765-767. 36. STRICHARTZ, G. R. (1973) J. Gen. Physiol. 62, 37-57.