Cyclothiazide binding to functionally active AMPA receptor reveals genuine allosteric interaction with agonist binding sites

Ilona Kovács, Ágnes Simon, Éva Szárics, Péter Barabás, László Héja, Lajos Nyikos, Julianna Kardos

Research output: Contribution to journalArticle

10 Citations (Scopus)


The agonist, [3H](-)[S]-1-(2-amino-2-carboxyethyl)-5-fluoro- pyrimidine-2,4-dione ([3H](S)F-Willardiine) binding to functional alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors of resealed plasma membrane vesicles and nerve endings freshly isolated from the rat cerebral cortex displayed two binding sites (KD1=33±7nM, BMAX1=1.6±0.3pmol/mg protein, KD2=720±250nM and BMAX2=7.8±4.0pmol/mg protein). The drug which impairs AMPA receptor desensitisation, 6-chloro-3,4-dihydro-3-(2-norbornene-5-yl)-2H-1, 2,4-benzothiadiazine-7-sulphonamide-1,1-dioxide (cyclothiazide, CTZ) fully displaced the [3H](S)F-Willardiine binding at a concentration of 500μM. In the presence of 100μM CTZ (KI(CTZ)=60±6μM), both the antagonist [3H]-1,2,3,4-tetrahydro-6-nitro-2,3-dioxo- benzo(F)quinoxaline-7-sulfonamide ([3H]NBQX: KD= 24±4nM, BMAX=12.0±0.1pmol/mg protein) and the high-affinity agonist binding showed similar affinity reduction ([ 3H](S)F-Willardiine: KD=140±19nM, B MAX=2.9±0.5pmol/mg protein; [3H]NBQX: K D=111±34nM, BMAX=12±3pmol/mg protein). To disclose structural correlates underlying genuine allosteric binding interactions, molecular mechanics calculations of CTZ-induced structural changes were performed with the use of PDB data on extracellular GluR2 binding domain dimeric crystals available by now. Hydrogen-bonding and root mean square (rms) values of amino acid residues recognising receptor agonists showed minor alterations in the agonist binding sites itself. Moreover, CTZ binding did not affect dimeric subunit structures significantly. These findings indicated that the structural changes featuring the non-desensitised state could possibly occur to a further site of the extracellular GluR2 binding domain. The increase of agonist efficacy on allosteric CTZ binding may be interpreted in terms of a mechanism involving AMPA receptor desensitisation sequential to activation.

Original languageEnglish
Pages (from-to)271-280
Number of pages10
JournalNeurochemistry international
Issue number4
Publication statusPublished - Mar 2004


  • AMPA
  • Agonist
  • Cyclothiazide

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Cell Biology

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