Comparison of Tetrodotoxin and Procaine in Internally Perfused Squid Giant Axons

Abstract

Squid giant axons were internally perfused with tetrodotoxin and procaine, and excitability and electrical properties were studied by means of current-clamp and sucrose-gap voltage-clamp methods. Internally perfused tetrodotoxin was virtually without effect on the resting potential, the action potential, the early transient membrane ionic current, and the late steady-state membrane ionic current even at very high concentrations (1,000-10,000 nM) for a long period of time (up to 36 min). Externally applied tetrodotoxin at a concentration of 100 r~ blocked the action potential and the early transient current in 9-3 min. Internally perfused procaine at concentrations of 1-10 mM reversibly depressed or blocked the action potential with an accompanying hyperpolarization of 2-4 mv, and inhibited both the early transient and late steady-state currents to the same extent. The time to peak early transient current was increased. The present results and the insolubility of tetrodotoxin in lipids have led to the conclusion that the gate controlling the flow of sodium ions through channels is located on the outer surface of the nerve membrane.Selective blockage of the early transient ionic conductance increase by tetrodotoxin (TTX), the active component of the toxins from the puffer fish and California newt, has been well established in giant axons of lobster (Narahashi, Moore, and Scott, 1964; Takata, Moore, Kao, and Fuhrman, 1966) and squid (Moore, Blaustein, Anderson, and Narahashi, 1967; Nakamura, Nakajima, and Grundfest, 1965 a) and in electroplaques of eel (Nakamura, Nakajima, and Grundfest, 1965 b), since this mechanism was first suggested from studies on frog muscle fibers (Narahashi, Deguchi, Urakawa, and Ohkubo, 1960; Nakajima, Iwasaki, and Obata, 1962). Because of its very strong affinity for the early transient channel, T T X has now become a powerful tool for characterizing the conductance changes responsible for excitation.In contrast, procaine blocks both the early transient and the late steadystate conductance changes (Taylor, 1959; Shanes, Freygang, Grundfest, and ~4x3 The Journal of General Physiology Amatniek , 1959; Blaustein and Goldman, 1966). Procaine differs from T T X not only in this nonselective blockage but also in its much higher threshold concentration and its effect on the kinetics of the early transient conductance increase (Takata et al., 1966). In the meantime the internal perfusion technique has been developed for squid axons (Baker, Hodgkin, and Shaw, 1961; Oikawa, Spyropoulos, Tasaki, and Teorell, 1961), giving us a straightforward approach for study of symmetry or asymmetry of the nerve membrane with respect to the action of various agents (Narahashi, 1963(Narahashi, , 1965 Baker, Hodgkin, and Shaw, 1962; Tasaki, Watanabe, and Takenaka, 1962; Tasaki and Takenaka, 1964;Rojas, 1965). Since T T X is insoluble in most organic solvents (Mosher, Fuhrman, Buchwald, and Fischer, 1964) and hence most probably so in lipids, it would be expected to block the early transient con...

Analysis of the potential‐dependent changes in optical retardation in the squid giant axon

“…On a number of occasions, the second axon from a squid whose first axon had long since gone into state 2 was still found to be in state 1 when in its turn it was taken from the refrigerator and mounted in the optical chamber. Narahashi, Anderson & Moore (1967) the sodium conductance was unimpaired. Yet within 2 min of applying 300 nM-TTX outside, the sodium conductance had fallen to zero, and soon afterwards, as may be seen in Fig.…”

Diphenylhydantoin Inhibition of Sodium Conductance in Squid Giant Axon

“…This effect (decreasing #Na, without appreciably altering m or h) is similar to the effects of saxitoxin (13) and tetrodotoxin (14,17), and is somewhat different from the effects of procaine (15). (Fig.…”

“…However, unlike diphenylhydantoin procaine requires a 100-fold greater concentration (i.e., millimolar rather than micromolar) to produce the same degree of induced inhibition of sodium current, procaine changes the potassium current, and procaine changes the time constant of the Hodgkin-Huxley parameter m (15). A closer comparison might be drawn between diphenylhydantoin and saxitoxin (13) and tetrodotoxin (14,17). The toxins and diphenylhydantoin alter #Na without modifying the relevant time constants, and it appears that both types of drug do not significantly affect the potassium currents.…”

Effects of tetrodotoxin on the slowly adapting stretch receptor neurone of lobster

“…The reversibility of the suppression of the spike activity produced by tetrodotoxin calls for some attention. This reversibility appears to be somewhat more pronounced than in excised axons (Narahashi et al 1964;Nakamura et al 1965;Takata et al 1966;Narahashi, Anderson & Moore, 1967); it became complete even after 30 min exposures to the toxin in a concentration 100 times higher than that necessary to abolish impulse initiation. From this observation it is likely that the drug produces no permanent changes in the structure of the membrane.…”

“…Narahashi, Deguchi, Urukawa & Ohkubo, 1960;Narahashi et al 1964;Nishi & Sato, 1966;Narahashi et al 1967). This may, of course, be due to a difference in reactivity of the various types of material used.…”