Bi-ionic action potentials in squid giant axons internally perfused with sodium saltssalts.

Abstract

presence of univalent cations (such as sodium) externally is not essential for production of all-or-none action potentials under these conditions. The term action potentials has been introduced2 to designate electric responses produced under these conditions in which only two critical cations (a univalent ion internally and a divalent ion externally) are involved in the process of excitation. The present report deals mainly with the electrophysiological properties of squid giant axons internally perfused with dilute solutions of a sodium salt. With sodium as the sole intracellular cation, squid giant axons were found to maintain excitability in external media containing a calcium salt as the sole electrolyte species.2 It is to be noted that the gradient of the sodium ion concentration in these axons is opposite to that of unperfused axons immersed in sea water. These experimental findings may be explained in terms of the hypothesis postulating a conformational change (phase transition) of membrane macromolecules associated with a cooperative ion exchange process between uni- and divalent cations at negatively charged sites of the macromolecules.3, 4 Methods.-Giant axons from Loligo pealii, available at the Marine Biological Laboratory in Woods Hole, Massachusetts, were used. The major portion of small nerve fibers and other adherent tissue surrounding the giant axon were removed under a dissecting microscope during the process of dissection. The technique of internal perfusion used in the present study was substantially the same as that used in previous studies from this laboratory.1 The giant axon was mounted horizontally in a Lucite chamber (approximately 33 mm in length) containing natural sea water, and a glass cannula was inserted into each end of the preparation. Initially, the tip of the smaller, inlet cannula (150 , in diameter) was placed concentrically within the lumen of the larger, outlet cannula (250-300 A in diameter). Internal perfusion was initiated by separating the two cannulae. The inlet cannula was connected to a reservoir of internal perfusion fluid by means of a small polyethylene tubing. In most of the present experiments, the length of the internal perfusion zone was between 14 and 21 mm. Internal perfusion solutions were usually prepared by mixing a 12 (vol) % glycerol solution with a 600 mM sodium phosphate solution (pH 7.3 ? 0.1).4a In order to remove the axoplasm from the perfusion zone, perfusion was instituted with a sodium phosphate solution containing 0.05 mg/ml Pronasel, 5 (Calbiochem) for a period of 1 to 1.5 min and then switched to enzymefree solution. The flow rate of the internal perfusate, controlled by adjusting the height of the fluid reservoir, was maintained at 15-20 ,l/min. External fluid medium was, as a rule, a mixture of 400 mM CaC12 and 12 (vol) % glycerol; the pH was adjusted to 8.0 4= 0.1 with tris(hydroxymethyl)aminomethane buffer (less than 1 mM). (Note that the presence of a pH buffer in the external medium is not essential for the maintenance of bi-ionic action potentials.') Continuous flow of the external medium was maintained at 10-30 ml/min throughout the course of the experiments. A trap served to electrically isolate the suction apparatus used to drain the chamber from the external fluid medium.