Agonist Trapping by GABAA Receptor Channels.
GABAergic IPSCs have a relatively slow decay (deactivation) that appears to result from GABA(A) receptor channel openings that occur well beyond the predicted duration of free GABA at central synapses. Open and desensitized states have been suggested to prevent dissociation of agonist from the receptor, thus prolonging deactivation. However, simultaneous assessment of GABA binding and channel gating has not been possible. We developed a functional assay for occupancy of the GABA binding site or sites to test the GABA "trapping" hypothesis. Deactivation currents were compared in the absence and presence of bicuculline, a competitive antagonist that also allosterically inhibits GABA(A) receptors. This provided a model-independent, functional test of the hypothesis that GABA is trapped on the receptor during gating: bicuculline could only inhibit the channel if it was open but unbound by GABA. Although bicuculline inhibited spontaneous and neurosteroid-activated GABA(A) receptor currents, it failed to alter the deactivation time course of GABA-activated GABA(A) receptor currents. Protection of deactivation current from bicuculline block indicated that GABA remained bound to the receptors while the channel was open, thus suggesting that all open states, as well as all closed and desensitized states from which channel opening can occur, must be GABA liganded states. Trapping may be specific to agonists, because the positive allosteric modulator diazepam unbound from GABA(A) receptors independent of GABA binding and channel activity.
Bianchi
M T
MT
Neuroscience Graduate Program, University of Michigan, Ann Arbor, Michigan 48104-1687, USA.
Macdonald
R L
RL
D000818:Animals; D001665:Binding Sites / Q000187:drug effects / Q000502:physiology; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D018755:GABA Agonists / Q000378:metabolism* / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D058785:GABA-A Receptor Agonists; D015870:Gene Expression; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D017136:Ion Transport / Q000187:drug effects; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008024:Ligands; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D021122:Protein Subunits; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
D018755:GABA Agonists; D018756:GABA Antagonists; D058785:GABA-A Receptor Agonists; D008024:Ligands; D021122:Protein Subunits; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid