Separation of sodium and calcium currents in the somatic membrane of mollusc neurones.

1. Characteristics of the transmembrane ionic currents under controlled changes in ionic composition of extra- and intracellular medium were studied in isolated neurones from the ganglia of molluscs, Helix pomatia, Limnea stagnalis and Planorbis corneus. The neurones were investigated by a new technique which allows for dialysis of their interior and for clamping of the potential at the surface membrane without using micro-electrodes.2. Replacement of K ions by Tris inside the neurones eliminated the outward K current so that the actual time course of the inward current could be measured. The latter was separated into two additive components, one of which was carried by Na ions and the other one by Ca ions.3. Both inward currents were unaltered by tetrodotoxin (TTX); however, Ca current could be separately blocked by externally applied Cd ions (K(d) = 7.2 x 10(-5)M) and by the use of fluoride as an intracellular anion.4. No reversal of Na inward current could be achieved in neurones dialysed with Na-free solution, indicating the absence of outward current carrying ions through the corresponding channels. With 5 mM-Na inside the cell, the equilibrium potential was close to the value predicted by the Nernst equilibrium.5. A non-specific outward current could be detected in K-free cells at membrane potentials exceeding 20-40 mV. Its time course was proportional to 1 - exp (-t/tau(ns)). Cd ions depressed this current. The presence of the non-specific outward current made an exact measurement of the equilibrium potential for the Ca inward current impossible.6. The kinetics of Na inward currents could be described by m(3)h and those of the Ca current by m(2)h law. The corresponding values for V(m) = 0 are: tau(m)(Na) = 1.1 +/- 0.5 msec, tau(m)(Ca) = 2.4 +/- 1.0 msec, tau(h)(Na) = 7.9 +/- 2.0 msec. The inactivation of Ca current included two first-order kinetic processes with tau(h1) = 50 +/- 10 msec and tau(h) = 320 +/- 30 msec.7. The data presented are considered to be a proof of the existence of separate systems of Na and Ca ion-conducting channels in the nerve cell membrane.