Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina.
We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.
Akopian
A
A
Department of Ophthalmology, New York University School of Medicine, New York 10016, USA.
Gabriel
R
R
Witkovsky
P
P
D000469:Alkaline Phosphatase / Q000494:pharmacology; D000818:Animals; D001640:Bicuculline / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D019703:Calcineurin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D015220:Calcium Channels / Q000032:analysis; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D007093:Imidazoles / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D053496:Inositol 1,4,5-Trisphosphate Receptors; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011724:Pyridazines / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis; D011963:Receptors, GABA-A / Q000378:metabolism*; D012165:Retinal Ganglion Cells / Q000737:chemistry* / Q000201:enzymology*; D012433:Ryanodine / Q000494:pharmacology; D019837:Ryanodine Receptor Calcium Release Channel / Q000032:analysis; D019284:Thapsigargin / Q000494:pharmacology; D014426:Turtles; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D015220:Calcium Channels; D002614:Chelating Agents; D004791:Enzyme Inhibitors; D018756:GABA Antagonists; D007093:Imidazoles; D053496:Inositol 1,4,5-Trisphosphate Receptors; D010726:Phosphodiesterase Inhibitors; D011724:Pyridazines; D018160:Receptors, Cytoplasmic and Nuclear; D011963:Receptors, GABA-A; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D002110:Caffeine; C031938:calmidazolium; D004533:Egtazic Acid; D005680:gamma-Aminobutyric Acid; D019284:Thapsigargin; D015544:Inositol 1,4,5-Trisphosphate; C049853:gabazine; D000469:Alkaline Phosphatase; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; D001640:Bicuculline