C.Y. Kao

Differentiation of the actions of tetrodotoxin and saxitoxin

Abstract While tetrodotoxin and saxitoxin are very similar in their actions, they can be differentiated biologically by the observation that newts of the genus Taricha are resistant to tetrodotoxin but not to saxitoxin. The resistance of these newts is due to a remarkable insensitivity of the somatic motor nerves to tetrodotoxin. The isolated desheathed sciatic nerve of Taricha torosa is at least 30,000 times more resistant than frog nerve. As seen in electronmicroscopic sections, there is no special enveloping structure around taricha nerves that can be considered as a particularly effective diffusion barrier. The isolated, desheathed brachial nerve of the Atlantic puffer fish, Spheroides maculatus , is about 1000 times more resistant to tetrodotoxin than frog nerve. Both taricha and tetrodon nerves are readily blocked by saxitoxin. Both nerves require external sodium ions for activity since replacement of Na + with choline, tris (hydroxymethyl) aminomethane or dimethyl-diethanol ammonium ion reduced the spike amplitudes. The resistance of these nerves to tetrodotoxin is interpreted as being a result of peculiarities in membrane structures that prevent an effective association with tetrodotoxin.

11-Oxo-tetrodotoxin and a specifically labelled 3H-Tetrodotoxin

Tetrodotoxin was oxidized to a hydrated aldehyde, 11-oxo-tetrodotoxin, which shares the specificity of tetrodotoxin for the Na+ channel of the isolated voltage-clamped frog skeletal muscle fiber, but is four to five times more potent. It binds to the solubilized Na+ channel of eel electroplax with a similarly higher potency, because of an equilibrium dissociation constant about 0.25, and a dissociation rate constant 2.4 times slower than those for tetrodotoxin. 11-Oxo-tetrodotoxin can be reduced to regenerate a tetrodotoxin, which is chemically and biologically indistinguishable from the original tetrodotoxin. By reducing with tritiated sodium borohydride, a 3H marker can be inserted regiospecifically to yield 11-[3H]-tetrodotoxin. Because it has a defined specific activity of > 2.5 Ci/mmole, and a 3H marker which does not exchange with solvent proton, 11-[3H]-tetrodotoxin is an ideal tracer for tetrodotoxin. It may enable studies of problems which require higher signals and/or better stability of the marker than those obtainable from currently available tracer Na(+)-channel ligands.

Actions of 4-epitetrodotoxin and anhydrotetrodotoxin on the squid axon

The actions of the newly discovered 4-epitetrodotoxin and of anhydrotetrodotoxin have been studied on the internally perfused squid giant axon under voltage-clamped conditions. Both compounds are selective in blocking only the sodium channel. The concentration for reducing the sodium current to one-half is 13.2 nM for 4-epitetrodotoxin and 298 nM for anhydrotetrodotoxin. Compared with tetrodotoxin, the relative potencies are 0.39 for 4-epitetrodotoxin and 0.018 for anhydrotetrodotoxin. The results suggest that hydrogen bonding at C-4 and C-9 is an important contributing force in binding to the membrane receptor site.

Actions of epimers of 12-(OH)-reduced saxitoxin and of 11-(OSO3)-saxitoxin on squid axon.

The actions of the 12 alpha-saxitoxinol, 12 beta-saxitoxinol and a C-12 ethylene thioketal derivative of saxitoxin, as well as those of 11 alpha-(OSO3)-saxitoxin, 11 beta-(OSO3)-saxitoxin and 11 alpha-(OH)-saxitoxin, have been examined on the isolated squid giant axon. Each of these analogues acted similarly to saxitoxin in blocking specifically the sodium channel. The relative potencies are: STX (1); 11 beta-(OSO3)-STX (gonyautoxin III) (0.42); 11 alpha-(OSO3)-STX (gonyautoxin II) (0.20); 11 alpha-(OH)-STX (0.10); 12 alpha-saxitoxinol (0.0021); 12 beta-saxitoxinol (0.0005). Thus, the presence of a bulky and negatively charged sulphate group on C-11 does not materially affect the biological activity of STX. Hydrogen bonding at the C-12 position is probably an important means of binding of STX to the membrane receptor site. The difference between the epimers of saxitoxinol suggests that the H in one of them may be geometrically better aligned than that in the other, with the hydrogen acceptor group in the receptor.

Actions of nortetrodotoxin on frog muscle and squid axon

Nortetrodotoxin, prepared by oxidation of the C6 end of tetrodotoxin, probably exists in solution as an equilibrium mixture of a hemilactal and a lactone form of a hydrated ketone of C6. On both frog muscle fibers under constant-current conditions and squid giant axon under voltage-clamped conditions, nortetrodotoxin selectively blocks the sodium channel. On frog muscle fibers, the solution of nortetrodotoxin used, which was several months old, was 1/13 as active as tetrodotoxin. On the squid giant axon, a freshly prepared solution of nortetrodotoxin was about 1/4 as active. These observations indicate that nortetrodotoxin possesses a substantial degree of biological activity. The suitability of nortetrodotoxin as an intermediate for synthesizing derivatives of tetrodotoxin is discussed.

Actions of decarbamoyloxysaxitoxin and decarbamoylneosaxitoxin on the frog skeletal muscle fiber

Two new analogues of the decarbamoyl series of paralytic shellfish toxins have been isolated through improved HPLC methods. In decarbamoyloxysaxitoxin (doSTX), the -OH function at C-13 of decarbamoylsaxitoxin (dcSTX) is changed to -CH3. In decarbamoylneosaxitoxin (dcneoSTX), the carbomyl side-chain of neosaxitoxin (neoSTX) has been removed. The new analogues were assayed on voltage-clamped frog skeletal muscle fiber for their potency in reducing the sodium current. Compared with neoSTX, the relative potencies of dcneoSTX are: 0.003 (at pH 6.50), 0.004 (pH 7.25), and 0.005 (pH 8.25). The influence of pH on the potency is the same in neoSTX and dcneoSTX. The fractional loss of potency caused by decarbamoylation is much greater in neoSTX than in STX, possibly because of an intramolecular interaction between the N-1 -OH in neoSTX and the -OH on C-13. Compared with STX, the ED50 for reducing INa by doSTX is 618 nM, making its relative potency 0.008 that of STX. Energetically, the decreased potency can be accounted for by the loss of two hydrogen bonds, one at the C-13 -OH of dcSTX, and the other at the amino group in the carbamoyl function of STX. These two groups resemble the C-6 and C-11 -OHs in tetrodotoxin, and probably bind to the same site-points. Thus, the near-identical actions of STX and TTX can be attributed to the common sharing of one ion-pair site and four hydrogen-bonding sites. If glutamate 387 of rat brain sodium channel II were the anionic site which ion-pairs with the 7, 8, 9 guanidinium of STX, then the carbonyl oxygen of asparagin 388 is the hydrogen-acceptor for the C-12 gem-diols.

Actions of 6-epitetrodotoxin and 11-deoxytetrodotoxin on the frog skeletal muscle fiber

6-Epitetrodotoxin (6-epiTTX) and 11-deoxytetrodotoxin (11-deoxyTTX), isolated from an Okinawan newt, Cynops ensicauda, were tested for sodium-channel blocking effects on the voltage-clamped frog skeletal muscle fiber. In 6-epiTTX, the C-6 -OH is in an epimeric position; in 11-deoxyTTX, C-11 has a methyl in place of a hydroxymethyl group. At pH 7.25, the ED50s for reducing INa are: 4.1 nM (TTX), 96 nM (6-epiTTX), and 445 nM (11-deoxyTTX). In each analogue, the lowered potency can be attributed energetically to the loss of a hydrogen bond. By complementarity, in the sodium-channel receptor for TTX, there must be a hydrogen-acceptor group for the C-6 -OH, and another for the C-11 -OH. Therefore, the TTX molecule is bound to the receptor through an ion-pair (for the guanidinium), and five hydrogen bonds, one each for the -OH on C-9, C-10, C-4, and, as now identified, for C-6 and C-11. Considering the three-dimensional structure of the toxin molecules, these binding sites must be located in a fold or a crevice of the channel protein. If glutamate 387 of rat brain sodium channel II is the ion-pairing site for the guanidinium group, then the carbonyl oxygen of asparagine 388 is the hydrogen acceptor for the C-9 and C-10 -OHs.

Diamphidia toxin, the Bushmen's arrow poison: Possible mechanism of prey-killing

The effects of a 60,000 mol. wt protein from the pupae of the beetle, Diamphidia nigro-ornata have been studied. In concentrations as high as 50 micrograms/ml, the toxin has little effect on the propagated compound action potential of isolated nerve trunks, or on the voltage-gated sodium and potassium channels of voltage-clamped single skeletal muscle fibers. In the anesthetized cat, the toxin has no specific effect on the neuro-muscular or the cardiovascular systems. It has a markedly hemolytic effect, and could reduce hemoglobin levels by as much as 75%. Plasma hemoglobin is increased, with resultant extensive hemoglobinuria and associated histopathological changes in the kidneys. Blood pressure, heart rate, PO2, PCO2, and oxygen-saturation remain essentially normal until the terminal stages of intoxication. Contrary to previous conclusions, we find no support for any particular neurotoxicity of the poison. The complex systemic effects, and possibly the prey-killing, can probably be attributed to the extensive hemolysis, reduced oxygen-carrying capacity, and generalized tissue hypoxia.

The pH dependence of the tetrodotoxin-blockade of the sodium channel and implications for toxin binding

On the internally perfused, voltage-clamped squid giant axon, the effect of pH on the potency of tetrodotoxin in blocking the sodium current has been re-examined at pH 7.8 and 8.8. Confirming previous studies, tetrodotoxin is weaker at pH 8.8, when the deprotonated C-10 -OH makes the toxin molecule a zwitterion. In contrast to previous studies, our results, based on full dose--response relations, show that the relative potency at the two pH values appreciably exceeds the ratio of the abundance of the protonated C-10 form. In a medium of lowered ionic strength, tetrodotoxin is weaker, and the relative potency at pH 7.8 and 8.8 approaches the ratio of the relative abundance of the C-10 -OH. The results are believed to support hydrogen bonding at C-10 as a contributing factor in the binding of tetrodotoxin to the membrane receptor.

Inaction of saxitoxin-oximes on the sodium channel of frog skeletal muscle fibers

Three oximes of saxitoxin, saxitoxin oxime, saxitoxin methyloxime, and saxitoxin carboxymethyloxime, were synthesized in which the oxime functions replaced the ketone function on C-12 of saxitoxin. On the voltage-clamped single frog muscle fibers these oximes were very weak or inactive in blocking the sodium channel. The results indicate that the hydrated ketone function in saxitoxin is essential for blockade of the sodium channel, probably through a hydrogen bonding mechanism with some receptor groups.