Ionic requirements for membrane oscillations and their dependence on the calcium concentration in a molluscan...

1. Membrane currents from the bursting pace-maker neurone R-15 of Aplysia were measured under conditions designed to simulate membrane oscillations. Changes in the absorbance of the Ca(2+)-sensitive dye arsenazo III were used to monitor changes in the free intracellular Ca(2+) concentration, [Ca](i), under these conditions. In addition, changes in the extracellular K(+), concentration [K](o) were measured with K(+)-sensitive electrodes.2. In normal external ionic conditions the depolarizing phase of pace-maker activity was associated with a slow inward current and the hyperpolarizing phase with a slow outward current.3. In cells where the early inward Na(+) current was blocked by tetrodotoxin and outward K(+) currents were suppressed by intracellular EGTA and extracellular tetraethylammonium and 4-aminopyridine, the slow inward current was significantly larger in amplitude and was suppressed by removal of external Ca(2+) or the addition of external La(3+), but not by the removal of external Na(+).4. The slow inward current was increased when [Ca](o) was raised and decreased when it was reduced in the manner expected for current flow through a Ca(2+) channel. The selectivity of the slow inward current for divalent cations was [Formula: see text].5. The slow inward current was only slightly reduced by a 10 degrees C reduction in temperature.6. In normal external and internal ionic conditions changes in dye absorbance occurred when the membrane was depolarized with slow triangular voltage ramps or long depolarizing steps within the pace-maker oscillation range. The obsorbance change, and thus the increase in Ca(2+), [Ca](i), was well correlated with the appearance of the slow inward current. Moreover, the magnitude of the slow outward current was dependent upon the change in [Ca](i).7. The slow inward current and a substantial fraction of the outward current, as well as the change in [Ca](i), were reduced appreciably by the addition of La(3+) ions (3 mM) to the external medium.8. The increase in [Ca](i) during prolonged depolarization was not affected by external tetrodotoxin or by the removal of external Na(+), but was abolished by a Ca(2+)-free external medium containing EGTA. Nevertheless, significant changes occurred in [Ca](i) during depolarization in 0.1 mM-external Ca(2+).9. In normal external and internal ionic conditions extracellular K(+), [K](o), increased during the depolarizing phase of the pace-maker cycle and decayed during the hyperpolarizing phase.10. There was a measurable increase in [K](o) during small prolonged depolarizing steps which produced a net inward current, indicating that inward and outward currents overlap under normal conditions.11. In the absence of action potential discharge, [Ca](i) increased during the depolarizing phase and decreased during the hyperpolarizing phase of the membrane oscillation.12. It is proposed that pace-maker oscillations depend upon three separate but linked systems which include a voltage-dependent Ca(2+) current, the free intracellular Ca(2+) concentration and the Ca(2+)-activated K(+) current.