Photo-released intracellular Ca2+ rapidly blocks Ba2+ 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 microM. In contrast, the same flashes on DM-Ca(2+)-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 approximately 50 nM and forward rate constant of 7 x 10(8) M-1 s-1.(ABSTRACT TRUNCATED AT 400 WORDS)
Johnson
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BD
Department of Biological Sciences, University of Southern California, Los Angeles 90089-2520.
Byerly
L
L
D000085:Acetates; D000818:Animals; D001464:Barium / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D005029:Ethylenediamines; D007424:Intracellular Fluid / Q000378:metabolism; D017136:Ion Transport / Q000187:drug effects*; D008195:Lymnaea / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects; D009474:Neurons / Q000378:metabolism*; D010782:Photolysis
D000085:Acetates; D005029:Ethylenediamines; C057014:DM-nitrophen; D001464:Barium; D002118:Calcium