Ca2+-DEPENDENT INACTIVATION OF Ca2l CURRENT IN APLYSIA NEURONS: KINETIC STUDIES USING PHOTOLABILE

1. The kinetics and sensitivity of the Ca2"-dependent inactivation of calcium current (Ic.) were examined in intact cell bodies from the abdominal ganglion of Aplysia californica under two-electrode voltage clamp. 2. Rapid changes in the level of intracellular free calcium ([Ca2+]i) were generated at the cell surface by photolytic release of Ca2" (nitr-5 and dimethoxy nitrophen) or Ca2" buffer (diazo-4). 3. Diazo-4 increased ICa by 10-15 % and slowed the rate of Ica decay when photolysed before a test pulse or between a prepulse and a test pulse. The predominant effect of further light flashes was to increase the amount of noninactivating current (I,, ) remaining at the end of long (> 1 s) depolarizing pulses. 4. A rapid increase in [Ca2+]i buffering during Ic inactivation did not cause a rapid recovery of current but merely reduced the rate and extent of subsequent inactivation. This effect was not seen when Ba2" was the charge carrier. 5. Photolytic release of Ca2+ from nitr-5 produced estimated Ca2" jumps of 3-4 ,u1m at the front surface of the cell but failed to augment inactivation either before or during Ic. In contrast, photolysis of DM-nitrophen 10-90 ms before the test pulse decreased peak Ic by about 30 %. A flash given during Ic. rapidly blocked 41 + 3% of peak current with a time constant of 3-4 ms at 17 'C. Similar results were seen with the barium current (IBa). 6. Microinjection of the potent phosphatase inhibitor microcystin-LR (5 /IM) had variable effects on Ica inactivation and augmented the cyclic AMP-induced depression of the delayed rectifier (IKE)) by forskolin (100,UM) and 3-isobutyl-1methylxanthine (IBMX; 200 /LM). 7. Full recovery from inactivation measured in two-pulse experiments took at least 20 s. This slow recovery process was unaffected by increases in intracellular cyclic AMP elicited by direct injection or by bath application of forskolin and IBMX. It was also unaffected by decreases in cyclic AMP induced by injecting 2',5'-dideoxyadenosine (1 mM) or bath application of the Rp isomer of cyclic adenosine 3', 5'-monophosphothioate (Rp-cAMPS; 200 /uM).