TRANSMEMBRANE ION MOVEMENTS ELICITED BY SODIUM-PUMP INHIBITION IN HELIX-ASPERSA NEURONS

1. Transmembrane ion movements upon sodium-pump inhibition were studied in identified neurons of the subesophageal ganglia of Helix aspersa. A two-microelectrode, voltage-cramp technique was used to measure transmembrane currents. Changes in intracellular Na+, K+, and Ca2+ concentrations were measured, in unclamped neurons, with Na+-sensitive microelectrodes, K+-sensitive microelectrodes, and with the fluorescent probe fura-2, respectively. 2. Inhibition of the sodium pump with ouabain (1 mM) elicited an increase in intracellular Na+ concentration, [Na+](i), at an initial rate of 0.42 +/- 0.05 mM/min (mean +/- SE; n = 27), and a membrane depolarization often followed by hyperpolarization. In cells clamped at -50 or -60 mV, ouabain produced an inward shift in membrane-hording current followed by an outward current usually having two components, transient and sustained, respectively. 3. Replacing external Na+ with either N-methyl-D-glucammonium or tetraethylammonium (TEA(+)) abolished both the ouabain-induced inward membrane current and the rise in [Na+](i), suggesting that Na+ was the charge carrier of the inward current. 4. Cd2+ (400 mu M) reduced the rate of rise of the inward current by 60% and the estimated net Na+ flux by 47%. 5. The outward current was abolished by K+-channel blockers (10 mM TEA(+) and 5 mM 4-aminopyridine or 10 nM apamin). Cd2+ (400 mu M), a Ca2+-entry blocker, also abolished the outward current. 6. Inhibition of the sodium pump elicited a fall in [K+](i) at an initial rate of 1.4 +/- 0.2 mM/min (n = 9 cells). 7. Upon inhibition of the sodium pump in neurons loaded with fura-2, [Ca2+](i) increased from an estimated resting level of 147 +/- 37 nM to a maximum of 764 +/- 248 nM (n = 12 cells). 8. The rise in [Ca2+](i) in the sustained presence of ouabain was transient, lasting 19.5 +/- 2.8 min, and could be prevented by removal of external Ca2+ before ouabain application or curtailed by removal of external Ca2+ during sustained ouabain exposure. The latter effect was not a consequence of exhaustion of caffeine-sensitive intracellular Ca2+ stores. 9. It is concluded that 1) the rise in [Ca2+](i) upon Na+-pump inhibition requires the presence of external Ca2+, 2) the outward current observed upon pump inhibition is a Ca2+-activated K+ current flowing through apamin-sensitive channels, 3)the resting Na+ permeability involves a Cd2+-sensitive component, 4) a large fraction ( similar to 30-60%) of the previously described ouabain-induced cell shrinkage may result from Ca2+-activated K+ efflux contributing to net solute and water loss.