Ca(2+)-dependent inactivation of Ca2+ current in Aplysia neurons: kinetic studies using photolabile Ca2+ chelators.

摘要

1. The kinetics and sensitivity of the Ca(2+)-dependent inactivation of calcium current (ICa) 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 non-inactivating current (I infinity) remaining at the end of long (> 1 s) depolarizing pulses. 4. A rapid increase in [Ca2+]i buffering during ICa 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 microM at the front surface of the cell but failed to augment inactivation either before or during ICa. In contrast, photolysis of DM-nitrophen 10-90 ms before the test pulse decreased peak ICa by about 30%. A flash given during ICa rapidly blocked 41 +/- 3% of peak current with a time constant of 3-4 ms at 17 degrees C. Similar results were seen with the barium current (IBa). 6. Microinjection of the potent phosphatase inhibitor microcystin-LR (5 microM) had variable effects on ICa inactivation and augmented the cyclic AMP-induced depression of the delayed rectifier (IK(V) by forskolin (100 microM) and 3-isobutyl-1-methylxanthine (IBMX; 200 microM). 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 microM). 8. A 'shell' model relating submembrane Ca2+ to inactivation was inconsistent with the experimental results since it greatly overestimated the effects of diazo-4 and predicted significant inactivation by nitr-5 photolysis. 9. A model linearly relating [Ca2+]i in a single Ca2+ channel 'domain' to inactivation more closely matched the experimental results with diazo-4 and DM-(ABSTRACT TRUNCATED AT 400 WORDS)