Effect of tetrodotoxin on the early outward currents in perfused giant axons.

Abstract

shown to vary with the log of the externial sodium concentratioin. Recently, Chandler aild Meves4 studied the effects of internal ionis On the early voltage clamp outward current with internally perfused squid axons. By varying the concentrations of monovalent cations inside and outside the axon and measuring the potentials at which the early current reversed direction, they found that internal K, Rb, and Cs ions contribute to the early outward current. The discovery that tetrodotoxin blocks the early inward current of Na+ without affecting the later outward current of K+ in the excitable 6 has naturally led to the question: Is tetrodotoxin interfering directly with the particular ion carrying this current or, more generally, with a fast mechanism by which various ions cross in the membrane? lMfoore et al.7 found that the inward currenit was blocked by tetrodotoxinl whether carried by sodium or by lithium, and that the later outward current was not affected whether carried by potassium or rubidium. The evidence, then, points to tetrodotoxin as a blocking agent specific for the early mechanism, regardless of the ions involved. The results presented here provide additional support for this hypothesis, by demonstrating that externally applied tetrodotoxin is as effective in blocking the early outward current when the current is carried by potassium ions as when it is carried by sodium or lithium ions. Methods.-The experiments were carried out on the giant axon of the Chilean squid, Dosidicus gigas. These squid were caught and killed a few miles off shore. The mantles were kept iri iced sea water during the trip to shore. Within 2 hr of capture of the animal, the manitle nier ves were removed anid the giant axonis were theni dissected from these bundles and prepared for experimenlt. AnI axonl was suspended in a temperature-cointrolled bath of K +-free artificial sea water (43() miM NaCl, 10( mM CaCl2, 50 mM MgCl2, 5 mM Tris at pH 7.5). Irnternal peifusiolis of the axonis were performed by means of a suction perfusion procedure.8 A glass outlet camiula was itmtroduced into oine enid and the axoplasm was gently removed by suction as the cannula was slowly advanced. A glass inlet cannula, connected to a perfusion solution reservoir, was introduced into the other end of the axon until the tip of the cannula was inside the outlet cannula. The axoplasm was then washed out of the outlet cannula with the perfusion solution and the outlet cannula was drawn back until about 15 mm of the axon was under perfusion. After a steady perfusion rate was obtairned, an internal electrode was introduced so that its tip was at a point midway alonig the perfused region. The membrane potential was taken between this point and an externlal reference electrode. A platinum wire was used to supply current along the part of the axon under perfusioni. The wire was insulated except for the 15-mm length in the perfused zone where it had been plated with platinium black. However, instead of introducing the current wire through thIe