PHOTO-RELEASED INTRACELLULAR CA-2+ RAPIDLY BLOCKS BA-2+ CURRENT IN LYMNAEA NEURONS

1. The effect of intracellular Ca2+ on Ba2+ current flowing through voltage-dependent Ca2+ channels was studied using the whole-cell patch-clamp technique on isolated neurons from the snail Lymnaea stagnalis. Intracellular Ca2+ was increased by flash photolysis of the caged Ca2+ compound DM-nitrophen and measured with the optical indicator fluo-3. 2. After the highest intensity flashes, peak Ba2+ current was blocked by 42 % with a time constant of 5 ms. The onset of the block followed a similar time course whether channels were activated or closed. The Ba2+ current surviving after the flash had the same voltage dependence of activation and rate of inactivation as did the total Ba2+ current before the flash. 3. Recovery of the Ba2+ current from block was nearly complete and occurred with a time constant of 16 s. Multiple episodes of photolysis-induced block could be studied in the same cell when 7-10 min were allowed between flashes. In some cells, recovery from block was accompanied by a transient enhancement of the current above the pre-block magnitude. 4. Neurons greatly reduced the ability of photolysis to increase Ca2+ , both by unloading the DM-nitrophen before flashes were applied and by rapidly buffering the photolytically released Ca2+. Maximal flashes on extracellular droplets of the DM-Ca2+ solution created a Ca2+ jump from 110 nm to 40 muM. In contrast, the same flashes on DM-Ca2+-loaded neurons resulted in a Ca2+ transient starting from a baseline of 36 nm to a peak of 130 nm. This intracellular Ca2+ transient decayed with three time constants (120 ms, 2 s and 13 s). 5. Endogenous buffer(s) binds Ca2+ rapidly. When intracellular Ca2+ was monitored within 2 ms of the flash, no rapid Ca2+ spike due to binding of photo-released Ca2+ could be detected. Addition of dibromo-BAPTA to the intracellular solution reduced the block by one third, which is consistent with the measured reduction of intracellular Ca2+ . This indicates that the endogenous buffer can bind Ca2+ as rapidly as dibromo-BAPTA and as fast as Ca2+ is released by photolysis. 6. The Ca2+ dependence of the block, obtained by varying flash intensity, indicates some saturation by 130 nm. A simple two-state model of the block consistent with both the time course of block and recovery and the concentration dependence gave a dissociation constant of almost-equal-to 50 nm and forward rate constant of 7 x 10(8) M-1 s-1. 7. These results indicate that Lymnaea Ca2+ channels are blocked by Ca2+ in the submicromolar range at a rate near the diffusion limit. The speed of the block requires a very simple mechanism, perhaps direct binding to the channel macromolecule. These experiments also demonstrate that neuronal Ca2+ buffering is strong and rapid. Direct measurement of intracellular Ca2+ is essential if photolysis experiments are to be interpreted quantitatively.