Calcium buffering and slow recovery kinetics of calcium-dependent outward current in molluscan neurones.

Calcium entry into molluscan neurones during depolarizing voltage-clamp steps activates an outward current which on repolarization decays over periods of more than 30 sec. This slowly decaying tail current was used to study the relation between calcium buffering in cytoplasm and the decline of a calcium-activated membrane process. Calcium-dependent outward current was also studied after injection of calcium into the cytoplasm. The time course of the fall of outward tail current was much less sensitive than tail current amplitude to the amount of calcium entry. Increasing bath temperature from 5 to 15 degrees C decreased the rate of fall of outward tail current activated by calcium entry. In contrast, outward current activated by calcium injection declined more rapidly at higher temperatures. Injection of sufficient EGTA to give maximum depression of outward current during depolarizations reduced the amplitude of outward tail current by at most 50%. After EGTA injection outward tail current declined more rapidly immediately following repolarization, but returned to base line at about the same time as the control. After injection of EGTA, outward current activated by calcium injection was reduced or completely blocked, and returned to base line more rapidly. Application of the mitochondrial uncoupler carbonyl cyanide m-chlorophenyl hydrazone (CCCP) did not alter the decay time course of outward tail current, but markedly prolonged the decline of outward current activated by calcium injection. The slow kinetics of outward tail current were compared to predictions of the concentration of calcium ions at the outermost surface of a spherical model cell following calcium influx. We conclude that after depolarization and calcium entry, the diffusion and binding of free calcium to cytoplasmic buffers plays a key role in determining the rate of fall of outward tail current. Further, different mechanisms influence the decline of calcium-dependent outward current following injection of calcium into the cytosol.