FEDERATION PROCEEDlKCS Vol. 26, No. 6, November-December, 1067

Pktea in uxz4.

Tetrodotoxin’s highly selective blockage of an ionic channel1

JOHN W. MOORE AND TOSHIO NARAHASHI

Department of Pf2ysiology and Pharmacology, Duke University,

Durflam, ATorth Carolina

A T THE CONCLUSION of Ian Fleming’s book “From Russia, With Love” (8) we see agent 007, James Bond, crumple to the floor after a minute wound from a pin- prick is delivered, of course by a beautiful woman. In “Dr. No” (g), Fleming reveals that the pinpoint was poisoned with tetrodotoxin from the fugu fish. Prompt application of artificial respiration and treatment for curare poisoning had saved Bond’s life.

Fleming spent some time in Japan and undoubtedly became acquainted with the very special place that the fugu fish, “puffer,” “balloon” or “globe” fish has in the Japanese life. The Japanese consider its meat very de- licious and have eaten it for many years in spite of their knowledge that a deadly poison is contained therein. It has often been used by individuals who wish to commit suicide. A few years ago Huntley and Brinkley showed a documentary news film (on television) depicting the collection, auctioning and preparation of this very popu- lar fish. It is now prepared for serving only at restaurants which are specially licensed after their chefs have been trained to remove the offending organs. The film clip ended with a scene showing the very characteristic Japanese insistence that others should eat first and no translation was required to reveal the thoughts expressed by gestures at the table: “after you”; “no, after you”; “no, please after you. . . .”

The Sankyo Company receives many tons of the ovaries removed from the fugu fish and extracts the poison from this tissue with a yield of about I o g/ton. The poison is called tetrodotoxin and hereafter we will sometimes refer to it using the abbreviation TTX. For many years tetrodotoxin had been studied in Japan because of its obvious importance in this nation’s economy and health. It has long been known to be a nerve blocking agent. However, the first modern electrophysiological experi- ments with TTX were not done until the late 1950’s. Furukawa et al. (IO) showed in the frog and toad that the acetylcholine response of the end-plate region was

not affected by TTX at concentrations high enough to

From the Physiology Symposium on “Ionic Permeability of Syn- I ,

aptic and Nonsynaptic Membranes” presented at the 5Ist :\nnual Meeting of the Federation of American Societies for Experimental Biology, Chicago, Ill., April 17, 1967.

1 This study was supported by National Institutes of Health Grant NBo3437 and National Science Foundation Grant GBlg67.

block nerve and muscle action potentials. Narahashi, Deguchi, and others (26) simultaneously studied the effect of TTX on the propagated action potential in skeletal muscle and found that, while the action poten- tion was blocked, the steady-state rectification persisted. They surmised that the sodium ion inflow responsible for excitation was selectively blocked. However, it was necessary to do voltage-clamp experiments to verify this suggestion. Early in 1963 just prior to Narahashi’s return to Japan, we had an opportunity to collaborate on the study of the effect of TTX on voltage clamped lobster axons. We (28) found that the previous surmise from the muscle experiments was true indeed and that the sodium current was selectively blocked by TTX.

Later in 1963 Mitsuru Takata from Osaka Univer- sity, who was visiting Duke University in the absence of Narahashi, undertook (with Moore, Kao, and Fuhrman (31)) a similar study of a toxin extracted from California salamander eggs at Stanford University. Drs. C. Y. Kao and Frederick Fuhrman (15) had shown that this ma- terial also exerted a very powerful nerve blocking action on frog sciatic nerves. This poison, called tarichatoxin, had an empirical formula which was thought to be dif- ferent from that of tetrodotoxin, tarichatoxin (2) being CnH17S308, while tetrodotoxin was C12H17N309 (34) (some workers thought that there were IO oxygens in- stead of g (35)). We undertook a more extensive series of voltage-clamp studies with tarichatoxin for compari- son with tetrodotoxin. However, before these studies were finished the group at Stanford (3, 22) showed that tarichatoxin and tetrodotoxin are identical molecules and that the formula was that originally proposed for taricha- toxin with the addition of one-half of a molecule of water of hydration.

Fortunately the results of our experiments with both toxins were also identical. Thus we can combine the discussion of all experiments done with poison materials obtained from either the salalnander or the fugu fish. Although some of our figures are labeled tarichatoxin, according to the source material, it is to be understood that the results apply fully to tetrodotoxin.

METHOD

Let us describe the method briefly. The sucrose-gap method (I 3) for obtaining membrane potentials and

1656 FEDERATION PROCEEDINGS Volume 26

current pool potential ~001

‘: --7,

_~ ~~.

05M KCI

sucrose sucrose

+ ~-- current injection L -

<

+-_- --- ~-/+v, electrodes ’ 7

area pot

clamp gain control

FIG. 1. Schematic diagram (abo~) of sucrose gap method of making an “artificial node” on a nonmyelinated fiber. Below: ap- proximate equivalent electrical circuit and the additional circuits for the voltage clamp.

inserting current at present is the only method which

can be used for voltage clamping IOO p lobster nerves.

In our laboratory, it is also the technique of choice for

voltage clamping the larger (?50400 p) squid axons.

The method is shown schematlcally in Fig. I.* Flowing

streams of sucrose insulate end regions of the axon from

the flowing seawater or test solution in the central section.

The right-hand pool is normally filled with potassium

chloride solution to reduce the membrane potential of

the axon in this pool to zero, allowing the membrane

potential across the “artificial node” in the central pool to be measured by means of gross electrodes in the right and central pool. The current density of the artificial node is measured by the operational amplifier whose input is connected to the central pool. Current may be injected between the left pool and ground for stimulation or under the control of the voltage clamp. The solution in the left pool may be seawater or potassium chloride solution. A typical family of membrane current responses to step changes in membrane potential is shown in Fig. 2. The absolute potential across the membrane during the test voltage step is shown to the right of each curve. The late steady-state current has been identified as being carried by potassium ions. This was shown by Hodgkin and Huxley (12) who did such a remarkable systelnatic

study of squid axons in the early I 950’s. They also showed that the early transient-phase current which reverses in sign when the membrane potential is about $50 mv

2 Figs. I, 4-9 reproduced by permission of the J. Gpn. Physiol. pub- lished by the Rockefeller Press (refs. ‘s,zo, 31). Figs. 1,~ reproduced by permission of Proc. Inst. Radio E++wrs. Figs. IO, 12 reproduced by permission of Science (refs. 24, 27). Copyright 1966 and 1967 by the American Association for the .\dvancernent of Science.

was carried by sodium ions. They proposed a model equivalent circuit encompassing the electrical character- istics of the squid axon membrane and this circuit is shown in a modified form in Fig. 3.

OBSERVATIONS

The effect of TTX on the membrane action potential of a lobster axon is shown in Fig. 4. From this and other experiments the concentrations required to produce a conduction block have been found to be quite low and will be expressed in nanomolar (IO-~ M). It can be seen

that the action potential is almost completely blocked within a few minutes by TTX at a concentration of go IlM. The membrane current records from voltage- clamp experiments carried out on this axon during pro- duction of this block are not shown but are similar to those given in Figs. 7 and I I. The results are summarized in Fig. 5 where the peak of the early transient current INa and the magnitude of the late steady current IK are plotted as a function of the membrane potential during the clamping pulse. The lowest curve shows the peak sodium current with the axon in seawater and the other curves show the reduction of the early transient mem- brane current phase within a few minutes after the ap- plication of go nM TTX. It is clear that it is only the transient sodium current that is affected and not the late steady potassium current. The action potential is usually blocked when the sodium current is reduced to about one-half to one-fifth that of the control. Concen-

+10

5

0

-5

7’ \

I L 0

na/cms

7 +80mv

+55mv

t20 rfi -10

/ -40

59-3

10°C

I

2 I 3

I 4 m set

FIG. 2. A family of axon membrane ionic currents as a function of time resulting from various potential steps. Value of the absolute membrane potential during the clamping pulse is given at the right of each curve.

Kozxrn ber-Decem ber 19 67 IONIC PERMEABILITY 1657

INSIDE

P

RL

cM

-I- -70 mv +55 mv -50 mv

OUTSIDE

FIG. 3. Equivalent electrical circuit representing properties of a uniform area of squid axon membrane. (Modified after Hodgkin and Huxley (I z).)

Torichotoxin

FIG. 4. Blockage of an action potential in a lobster axon over a period of a few minutes after addition of CJO no tarichatoxin.

trations of TTX as low as I O-I 5 nM are usually sufficient for blocking excitation in lobster axons.

The Hodgkin-Huxley formulation (I 2) expresses the sodium conductance as the product of an anlplitude and a transient kinetic factor. In order to see if we could distinguish whether the amplitude, the kinetic factors or both had been affected by TTX, we carried out a num- ber of studies. One of these is shown in Fig. 6, where the time for the sodium current to reach one-half of its peak value is plotted for axons in seawater under control conditions and after a few rninutcs of treatment with TTX. There is no significant difference between the experimental and control curves. The kinetics describing the turn off of the transient current was also checked and

found to be unchanged by TTX (31). Therefore we can ascribe the effect of TTX to be one of reducing the mag- nitude of the peak of the early conductance change with- out altering the kinetics involved either in the turning on or the turning off process governing this phase. This can also be rather easily seen in Fig. 7, where the effect of TTX on the membrane currents in a voltage-clamped squid axon is shown. In the upper part, at a given

I(ma/cm2)

(mv)

SW

VI

6

FIG. 5. Plot of the early transient sodium current (IN&) and late steady potassium current (1~) as a function of the voltage clamp potential. Only the transient sodium current is blocked by IJO nM tarichatoxin.

tp/2 (ms)

r.2

E(mv)

FIG. 6. Comparison of time for the sodium current to reach one- half of its peak value for the axon in seawater and in seawater plus 60 nM tarichatoxin.

I 658 FEDER,\TION PROCEEDINGS Volume 26

lllO/Cd

I- s w Recovery 7 min

0 I I I I I I 0 I 2 3 4 5 6

m set

FIG. 7. Above: records of membrane current in a squid axon at a fixed voltage by a clamp pulse taken at intervals of 15 set following application of 150 nM of tetrodotoxin. Below: similar re- cords taken at longer time intervals during wash out of the toxin.

voltage the current was sampled every 15 set following the application of the I 50 nM of TTX. The last few curves of the series were identical and two have been omitted for clarity. A corresponding family of recovery curves taken at longer time intervals between samples is shown below. Note that TTX does not affect the time course of the inward sodium current although the amplitude of this current is drastically reduced. Furthermore it may be seen that values of the late steady current can be superimposed throughout the test period. In other ex- periments we found that increased extracellular calcium gave a very limited amount of protection against the tetrodotoxin block but did improve the recovery from it.

BASIS OF TTX SELECTIVITY

Dr. M. Blaustein working at the Naval Medical Re- search Institute repeated some of our work on lobster axons. He clamped the membrane potential at a value more positive than the sodium equilibrium potential thereby producing outward transient sodium ion cur- rents. He noted that tetrodotoxin seemed less effective in blocking such outward sodium currents compared with its capacity to block the inward current obtained at lower membrane potentials. Our earlier experiments on lobster axons had not been done in this high membrane potential range, but we had conducted several experi- ments on squid axons at high positive internal potentials which indicated that TTX blocked inward and outward sodium currents equally well. Therefore, it seemed worthwhile to join forces to further investigate this prob- lem. Dr. Nels Anderson of our laboratory joined Nara- hashi, Blaustein, and myself in the following studies (20).

We reasoned that if the TTX block of the outflow of sodium was much less effective than the block of its inflow, this could mean that the blocking action of tetro- dotoxin depended on the direction of positive charge flow. On the other hand if the blockage of sodium was

direction independent then the basis of the TTX effect might be connected with some difference between sodium and potassium ions themselves. Third, the blocking action of TTX might be the result of its selective effect on the mechanism which causes the early transient con- ductance increase (through which sodium normally enters the excited axon). This phase may be contrasted with the late steady-state conductance increase (by which potassium normally leaves the excited axon). Therefore, we set out to answer the following questions. Is the selectivity of TTX due to differences between: I) Sodium and potassium ions? 2) The direction of posi- tive ion membrane current flow? 3) The early transient or the late steady ion conductance channels?

By “channels” we do not imply any particular mecha- nism by which ions traverse the membrane but use this term simply as an equivalent of “pathway” in order to differentiate between the early transient phase of con- ductance increase and the late steady conductance in- crease. Clearly axon membranes exhibit strong selectivity among the alkali cations as far as limiting their transit through these two distinct pathways or channels, hence we can devise experiments to change the amount and direction of ionic current through the two channels. The questions as posed cannot be answered by individual experiments on a one-to-one basis between an experi- ment and a question, but we do think that the cumula- tive evidence obtained from several different types of experiments can give a definitive basis for deciding among these stated possibilities or combinations thereof.

The first question can be partly answered by sub- stituting lithium for sodium in the seawater. It is well known that lithium is almost indistinguishable from sodium in terms of the generation of action potentials and of ionic currents measured in the voltage clamp. When tetrodotoxin was added to the lithium substituted seawater, the early transient current was blocked in a fashion identical with that shown for the blockage of the sodium ion influx (20).

In order to study definitively the effect of TTX on ion outflow during the early transient conductance phase, we set up experimental conditions to optimize the out- ward sodium current. This was done by substituting cesium or potassium for external sodium. Under these conditions, because cesium (19) and potassium (4, I g)

traverse the early transient pathway very sparingly, the sodium equilibrium potential is shifted from ENB = +55 mv to ENa = -20 mv (I 9). In the upper part of Fig. 8 may be seen a family of membrane currents for voltage- clamped axons bathed in cesium seawater. The corre- sponding family of curves obtained after a few minutes treatment with 150 nM TTX is shown below. It is clear that the tetrodotoxin blocks the early transient outward movement of sodium without affecting the late steady outward movement of potassium. A question remains as to the relative potency of TTX in blocking the outward movement of sodium. We estimated its effectiveness to be slightly less than its effectiveness on the inflow of sodium. An important point is that we have here a case where

.VownbPr-Dtwmber 1967 IONIC PERME.\BILITY 1%

0 I I I I

0 I 2 3

cs S.W. +I50 nM TTX +I10 mv

0 I I I I I I I 0 I 2 3

m set FIG. 8. Families of membrane currents from a voltqc-clamped

axon bathed in cesium seawater before (abooc) and (below) few exposure 150 tctrodotoxin.

both currents, the early transient and the late steady phases are outward and only one is selectively blocked by TTX.

We also produced an early transient outflow of sodium followed by a late steady inflow of potassiuili by using a “potassium seawater” and choosing a potential for the clamping pulse somewhat below zero in absolute value. Membrane currents obtained at several clamped mem- brane potentials are seen in Fig. g, in which it is also shown that the early transient outward sodium current is blocked by the application of tetrodotoxin while the late steady inward potassium current is not appreciably affected. Because of a base-line drift in this particular experiment, we have labeled the curves according to the amplitude of the potential step pulse applied to the axon since we cannot give the absolute potential at which the membrane was clamped. Nonetheless the data are perti- nent to the question of interest here. The lower part of Fig. g shows that rubidium can substitute for the potas- sium ion inflow in the late steady channel and this inflow is unaffected by tetrodotoxin.

By a proper choice of membrane potential and sodium and potassium concentrations, we also obtained an early transient inward sodium current followed by late steady inward potassium current. Again tetrodotoxin blocked only the early transient phase without appreciable effect on the late steady phase.

Additional and corroborative evidence comes from experiments by Tasaki and Singer (32) in which organic

ions were substituted for sodium. In voltage-clamp stud- ies under special conditions, these ions can generate an action potential or can provide membrane current through the early transient pathway. Inward membrane currents in axons equilibrated in solutions of hydra- zinium chloride were also blocked by tetrodotoxin. In other experiments Chandler and Meves (personal com- munication) have found that the early transient inward current in guanidinium seawater and the small potas- sium current which leaves the axon through this early transient channel were both blocked by tetrodotoxin.

From the results of all of these experiments, WC now can answer without equivocation, the questions originall) posed. The selectivity of action of tetrodotoxin cannot depend on unique properties of the sodium ion per se because lithium, hydrazinium, and guanidiniurn ions, all of which pass through the same pathway, are all blocked by TTX. Neither can the selective action of TTX be explained on the basis of the direction of the membrane current flow because we have found that sodium ion current flowing in either direction is blocked by TTX whereas the late potassium ion current is never

ma/cm2

K S.W.

7-14-65

'1, K SW.+100 nM TTX

ma/cm*

Rb S.W.+EOnM TTX

1 L -45 -0.8

FIG. 9. Membrane currents in a voltage-clamped axon bathed in the following media: aboue, potassium seawater; center, potassium seawater plus 100 no tetrodotoxin; bottom, rubidium seawater plus 150 no tetrodotoxin.

1660 FEDERATION PROCEEDINGS

I x lC?M Tetrodotoxin internally ,

(min) 0 5 IO I5 20 25 30 35 o

(ma/crf+) _2Jb---‘..‘___/+J

7-20-65 Ix 10m7M Tetrodotoxin Externally

C 2 ma/cm2

/ - 2 msec

blocked, regardless of the direction in which it is flowing. In every case the early transient current is blocked re- gardless of the direction of flow or the nature of the ion carrying it, and the steady-state current is never blocked, regardless of the direction of movement or the nature of the ion carrying it.

SITE OF TTX ACTION

We have also been able to locate the site of action of tetrodotoxin (24) by means of experiments done with internally perfused squid axons. In Fig. IO we have plotted the peak sodium current produced during a given membrane potential step applied at various times throughout the course of an experiment which lasted about 40 min. After a brief control period the axon was continuously perfused internally with a solution con- taining a very high concentration of TTX (1,000 nM). A slow decline in the sodium current was observed prob- ably all of which is attributable to a natural deteriora- tion of the axon subjected to such rigorous experimental procedures. In contrast, when only IOO nM of TTX is applied externally, the sodium current becomes com- pletely blocked within a minute or two and only a slight recovery occurs during the washout period. This ex- periment would seem to provide unequivocal evidence that tetrodotoxin acts on the exterior surface rather than on the interior surface of the axon. The fact that the mechanism responsible for excitation is located on the exterior surface of the membrane provides argument against a widely held hypothesis that an agent capable of blocking axonal conduction must also be capable of permeating lipid layers. In this hypothesis it is implied but not always explicitly stated that blocking agents, to be active, must be soluble in lipids in order that they be able to pass through lipid layers of the membrane to reach and affect sites on the inner membrane surface. The observation that procaine when applied by inter- nal perfusion blocks about as well as it does when it is

Volume 26

FIG. IO. Early transient in- ward sodium current at a con- stant potential step is plotted as a function of time in various solu- tions. A control period is followed by internal perfusion of 1,000 nM (100 M) tetrodotoxin. r\ I o-fold lower concentration of TTX applied externally for I min abruptly blocks the inward sodium current. Sample records shown below are taken at indi- cated times during the experi- ment.

applied on the exterior surface (25) allows LIS to take the above dictum and reverse it. That is to say procaine causes a block when applied intraaxonally by virtue of its ability to pass from the inside through the lipid layers of the membrane to the outer surface on which critical active sites are located.

DERIVATIVES OF TETRODOTOXIN

Samples of a number of derivatives of tetrodotoxin made by the Sankyo Co. have been graciously supplied to us by them. We have studied (27) the effect of four of these compounds. They were first tested on lobster axons simply by using changes in the amplitude of the action potential as an index of activity. The structure-activity relations we established closely parallel those recently reported by Deguchi (5) from studies with frog sciatic nerve. We also performed a nmnber of experiments on voltage-clamped squid axons to determine if the mecha- nism of blocking was in any way altered by the change in molecular structure of the toxin applied. In every case we found that, when enough of the derivative was used to block the sodium current, the mechanism of block was identical with that characteristic of TTX, the native material. An example of the action of one TTX deriva- tive is shown in Fig. I I.

The structure of the available derivatives tested by us is given in Fig. 12. There was essentially no blocking activity shown by tetrodonic acid in concentration LIP to

10-4 M (the limit of its solubility). Two other compounds which blocked conduction were

$~OO as active as TTX itself; these were tetrodamino- toxin and anhydrotetrodotoxin. A third compound, deoxytetrodotoxin, had about 4i0 the activity of TTX. We reported these findings last fall at the ?jational Acad- emy of Science meeting (27) at Duke University. How- ever, before submitting this work for publication, we had further discussions with Drs. Fuhrman and Mosher at Stanford University and they raised the possibility

IONIC PEKMEllBILITY 1661

Normal SW

~OJJM RTN 1191 4.5 min

5 ma/cm2

- I msec

FIG. I I. Falnilies of voltage-clamp currents in an axon in nor- mal seawater to which is added 20 I.LM of a tctrodotoxin derivative, anhydrotetrodotoxin.

that these derivatives, particularly deoxytetrodotoxin, might be contaminated with TTX. Sankyo Co. were aware of this possibility but apparently they had not been able to detect such contamination with TTX using the nuclear magnetic resonance equipment available to them. Drs. Durham and Mosher graciously analyzed our sample of deoxytetrodotoxin with the aid of their very sensitive nuclear magnetic spectrometer and were able to detect the contaminating presence of more than enough TTX to account for all of the biological activity of the derivative sample. Because the limit of their resolution is about 1 ‘%, it is not possible to state whether the first two derivatives mentioned, each of which had

. an activity about >{oo that of TTX, were pure or con- taminated with 1 % TTX. Thus at present, we can only speculate that these alterations of the TTX molecule reduce its toxicity by at least lOO-fold in each case.

NUMBER OF SODIUM CHANNELS

Because of the very low concentration of TTX re- quired to block electrical activity in axons and because this molecule is very selective, its action being limited to the pathway normally used by sodium ions, TTX seems to be the only presently available tool which can be used to estimate the number of sodium pathways per unit area of membrane. We have recently done some experi- ments along this line in collaboration with Dr. Trevor Shaw (21) who visited Duke University last spring. In our attempt to count the number of sodium channels, we wanted to minimize the amount of TTX used and to maximize the area of membrane exposed to the toxin. Therefore we selected the nerve trunk from the walking leg of lobsters because this nerve is made up of a large number of small fibers. External stimulating and record- ing electrodes were placed at opposite ends of the nerve which was immersed in a small pool (50 ~1) of artificial

seawater. The seawater in this pool, after addition of 300 nM tetrodotoxin, was applied successively to seven lobster nerve trunks and the uptake of the toxin during each application was detected by the reduction in the ampli- tude of the nerve action potential. On the first nerve we allowed a 6-min control period with the preparation bathed in artificial seawater to determine that an ac- ceptably stable control state had been achieved. Then TTX was added to the pool of seawater. After 10 min we siphoned off the excess solution in the pool and held it for reapplication to the next nerve (after completion of its control run of 6 min). After the seawater pool which originally contained 300 IlM TTX was applied to seven nerves in succession, considerable activity remained. We estimated the concentration of TTX remaining in solu- tion by comparing its effect with that obtained in sepa-

R= OH, Tetrodotoxin R=H. Desoxytetrodotoxin

R = OMc, Mcthoxytetrodotoxin

R=OEt, Ethoxytetrodotoxin

R=NH,. Tetrodominotoxin

R,=R,=H. Anhydrotetrodotoxin RI-H, R2=HC0, I I-Monoformylanhydrofetrodotoxin RI =R2-AC, 6,lI-Diacetylanhydrotetrodotoxin

0

CH20H

Tetrodonic acid

FIG. 12. Structures of tetrodotoxin and some of its derivatives.

1662 FEDER.\TION PROCEEDINGS Volume 26

rate experiments in which a known concentration of TTX in seawater was applied to several other nerves. From these experiments we estimated that at least IOO nM

of TTX remained

200 nM was taken up. We used light and elec- tron microscopy to estimate the total area of the axon membrane exposed to TTX and we also determined the extracellular sodium space. In order to estimate the upper limit of the number of sodium channels, we assumed that only one molecule of tetrodotoxin was required to block a single sodium channel. From the foregoing data and with this assumption, we estimated that in lobster nerve there are no more than 13 sodium channels per square micron of axon surface. All of our estimates and assumptions were on the side which would tend to in- crease the estimation of the number of sodium channels. For example if two tetrodotoxin molecules were required to block a single sodium channel, our value is too large by a factor of two. In addition, if TTX is metabolized or is adsorbed at nonspecific sites then our figure for sodium channels per unit area is correspondingly too large. The amazing thing, it seems to us, is that even on the most conservative basis the sodium channel density turns out to be so small. From this one could argue that the applied tetrodotoxin is probably not metabolized nor is it adsorbed nonspecifically. On the contrary it appears to be taken up only by a membrane mechanism concerned with excitation.

The use of tetrodotoxin on widely differing prepara- tions has been steadily increasing. A few representative (rather than exhaustive) citations have been selected to illustrate, on a variety of preparations, the unique char- acteristics of tetrodotoxin’s action. As we have already mentioned, Furukawa et al. (IO) had shown that TTX does not block the end-plate potential at a neuromuscu- lar junction. Elmqvist and Feldman (6) and Katz and Miledi (16) used TTX to block nerve impulses while studying end-plate potential phenomena initiated by local electrical depolarization of the nerve terminal. Dr. Gage (this Symposium) has reviewed his and other re- cent work with TTX in separating electrical excitation from transmitter release mechanisms. Nakamura, ?;aka- jima, and Grundfest (29) have shown that excitation in the squid axon and in the electroplax were selectively blocked by TTX in the same manner as we had observed in lobster axons. Hagiwara and Nakajima (I I) col- laborated to show that in barnacle muscle tetrodotoxin does not block the spike associated with calcium inflow. They (I I) also have shown that cardiac fibers (frog ven- tricle) are resistant to TTX except insofar as it affects the rate of rise of the action potential. Smooth muscle has also been found to be very resistant to high concen- trations of TTX by Toida and Osa (33) and rV. C. Anderson (personal communication).

Several sensory receptors appear to be resistant to tetrodotoxin. The “generator” potentials of crayfish stretch receptors have been found to be little affected by TTX in concentrations which block action potential

production (I 8, 23). The Pacinian corpuscle may be somewhat more sensitive to TTX (30) although there appears to be a difference in the experimental results reported (18). Ionophoretic application of TTX to hair cells of the guinea pig cochlea blocked nerve impulses without affecting microphonic potentials (I 7). Benolken (I) has found that an early component of the limulus photoreceptor response is blocked by TTX but a slow phase was unaffected.

Nerves from Atlantic pufferfish and California newts have been found to be highly resistant to TTX poisoning although they normally use sodium ions for excitation (Kao and Fuhrman, in press). This is interpreted to mean that there is a peculiarity in membrane structure which prevents an effective association with tetrodotoxin. The existence of such a self-protective mechanism is certainly to be expected.

Tetrodotoxin has been found to have no effect on active transport in the systems tested to date: I) Frog skin and toad bladder (Kao and Fuhrman (7); H. Frazier, personal communication; S. Solomon, personal communication). 2) Red blood cells (J. Hoffman, Ping Lee, personal communications).

T. Andreoli (personal communication) has found no effect of TTX on the electrical resistance or on the trans- ference numbers for xa and K ions in thin membranes prepared from sheep (HK and LK) red cell lipids. D. Allen (personal communication) has also found TTX to have no effect on the passive movements of Na and K in red cells. An excellent review of tetrodotoxin’s actions has been written by Kao (I 4).

SUMMARY

The more we have learned about the action of tetro- dotoxin, the more fascinated we become with its very special properties. We can epitomize them by saying that tetrodotoxin has been shown to be an exquisitely sharp electrophysiological scalpel. It can be charac- terized as follows:

I) It does not directly influence synaptic transmitter release and it does not block the action of such trans- mitters on postjunction receptor sites. Thus it is a powerful tool for the study of synaptic and neuro- muscular transmission.

2) It is relatively ineffective on cardiac muscle, smooth muscle, and on receptor transducer elements.

3) It does not block the sodium pump. 4) It is a very powerful blocking agent for axon and

skeletal muscle action potentials. 5) It acts only on the outside of axonal membranes. 6) It acts only on the voltage-sensitive early transient

conductance increase in membranes normally em- ploying sodium in the excitation process.

7) It affects the mechanism responsible for the mem- brane conductance increase and the block is inde- pendent of the direction of the ionic current or the chemical nature of the ions involved.

8) Tetrodotoxin action on nerve membrane differs

November-December 1967 IONIC PERMEABILITY 1663

from that of procaine and other local anesthetics, which have been tested in a corresponding manner. The differences involve potency, site of action, selectivity and mode of block.

g) Tetrodotoxin has assisted us in our estimation of the upper limit of the number of sodium channels per square micron of axon membrane.

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,I. 12.

‘3.

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‘7.

18.

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10) Last but not least, TTX is not only a tool of the electrophysiologist but it also is used frequently in the works of Ian Fleming (7), the most recent being a posthumous publication in Playboy, as pointed out by an industrious colleague.

The authors express their appreciation for the use of facilities

at the Marine Biological Lab., Woods Hole, Mass.