Solubilisation and Characterisation of a Putative Quisqualate-Type Glutamate Receptor from Chick Brain

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Abstract
The brains of 1-day-old chicks were shown to be a rich source of binding sites with the pharmacological characteristics expected of a quisqualate-type glutamate receptor. .alpha.-[3H]Amino-3-hydroxy-5-methylisoxazolepropionate ([3H]AMPA) bound with KD and Bmax values, measured at 0.degree. C in the presence of the chaotrope potassium thiocyanate, of 55 nM and 2.6 pmol/mg protein. The regional localizations of [3H]AMPA and [3H]kainate binding sites were manifestly different. The membrane-bound [3H]AMPA binding sites were efficiently solubilized by N-octyl-.beta.-D-glucopyranoside (1%) in the presence of 0.2 M thiocyanate. In the detergent extract the affinity was 69 nM and there was an apparent increase in the number of sites (Bmax, 4.6 pmol/mg protein). The rank order of potency for competitive ligands in displacing [3H]AMPA binding was quisqualate .apprxeq. AMPA > 6-cyano-7-nitroquinoxaline-2,3-dione > L-glutamate > kainate and was identical for the membrane-bound and solubilized sites. Dissociation was biphasic with rate constants of 0.117 min-1 and 0.015 min-1. The association rate constants for [3H]AMPA at the solubilized sites were 1.45 .times. 106 M-1 min-1 and 6.55 .times. 106 M-1 min-1 The kinetically derived KD values were 80.7 nM and 2.3 nM. The detection of higher affinity binding sites by kinetic analysis but not by equilibrium binding may be explained by the greater sensitivity of dissociation data to small populations of high-affinity sites. Solubilized [3H]AMPA binding sites were specifically retained on a wheat germ lectin column, but did not migrate as a single peak in sucrose density gradients or elute from a cation-exchange column at a specific NaCl concentration. The results demonstrate the solubilization of a stable [3H]AMPA binding protein and suggest that chick brain, and specifically chick telencephalon, will provide a useful system for further characterization and purification of the quisqualate-type glutamate receptor.