potassium-thiocyanate and 5-fluorowillardiine

AMPA receptors can be labeled using the agonist radioligands [3H](R,S)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid ([3H](R,S)-AMPA), [3H](S)-AMPA or [3H](S)-5-fluorowillardiine. In the presence of KSCN, [3H](R,S)-AMPA and [3H](S)-AMPA bind to a single population of sites in rat brain membranes, whereas [3H](S)-5-fluorowillardiine binds with two affinity components. KSCN increased the affinity of the low affinity [3H](S)-5-fluorowillardiine component > 4-fold and increased the density of both components 1.5-1.7-fold, arguing against KSCN-induced interconversion of low to high affinity states. KSCN, which promotes receptor desensitization, increased the potency of AMPA isomers, (S)-5-fluorowillardiine, quisqualate and cyclothiazide for inhibition of [3H](S)-5-fluorowillardiine binding suggesting that these ligands discriminate desensitized and nondesensitized receptors. In contrast, KSCN did not greatly affect the potency of glutamate, kainate, or competitive antagonists suggesting that these ligands do not discriminate desensitized and nondesensitized receptors. In the presence of KSCN, the rank order potency for agonists and antagonists was similar or identical in all assays indicating that the three radioligands bind identical glutamate recognition sites, a conclusion supported by their identical total receptor density. However, AMPA isomers displayed 6-10-fold higher potency for displacement of [3H](S)- or (R,S)-AMPA relative to [3H](S)-5-fluorowillardiine binding. This finding, coupled with the marked two component binding by [3H](S)-5-fluorowillardiine but not [3H](S)- or (R,S)-AMPA, suggests qualitative differences between the interaction of these ligands with the agonist recognition site.

Topics: Alanine; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Binding, Competitive; Brain; Excitatory Amino Acid Agonists; In Vitro Techniques; Ligands; Male; Pyrimidines; Rats; Receptors, AMPA; Stereoisomerism; Thiocyanates

Guanyl nucleotides inhibit the binding of the AMPA receptor agonists [3H]fluorowillardiine and [3H]AMPA and the competitive antagonist [3H]CNQX to rat brain cerebrocortical membranes. The rank order of inhibition for each of the radioligands tested was GTP > GDP > GMP. The nucleotides CTP and ATP showed no effect. GTP inhibition was unaffected by the presence or absence of NaCl and MgCl2. Pre-treatment of the membranes with GTP, and its removal before addition of radioligand, did not inhibit binding. Quantitative autoradiography demonstrated that GTP inhibition occurred throughout the brain. These results are consistent with guanyl nucleotides acting at an extracellular site present on all AMPA receptor subunits, occupation of which inhibits agonist and antagonist binding.

Topics: Alanine; Animals; Autoradiography; Brain Chemistry; Excitatory Amino Acid Agonists; Female; Guanine Nucleotides; Guanosine Triphosphate; In Vitro Techniques; Membranes; Pyrimidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, AMPA; Thiocyanates