Solubilization of the N‐Methyl‐d‐Aspartate Receptor Channel Complex from Rat and Porcine Brain

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Abstract
The N‐methyl‐D‐aspartate (NMDA) receptor complex as defined by the binding of [3H]MK‐801 has been solubilized from membranes prepared from both rat and porcine brain using the anionic detergent deoxycholate (DOC). Of the detergents tested DOC extracted the most receptors (21% for rat, 34% for pig), and the soluble complex, stabilized by the presence of MK‐801, could be stored for up to 1 week at 4°C with (‐)‐MK‐801 = thienylcyclohexylpiperidine > dexoxadrol > SKF 10,047 > ketamine, for inhibition of [3H]MK‐801 binding, was observed in all preparations. The receptor complex in solution exhibited many of the characteristic modulations observed in membranes. After removal of small‐molecular‐weight endogenous substances by gel filtration, the following effects were observed: (1) NMDA receptor agonists such as L‐glutamate enhanced binding of [3H]MK‐801 in a concentration‐dependent manner; (2) glycine stimulated [3H]MK‐801 binding in the presence of submaximal concentrations of glutamate; (3) in the absence of glutamate and glycine, the divalent cations Mg2+, Ca2+, and Mn2+ stimulated [3H]MK‐801 binding (EC50: 10–100 μM), whereas in the presence of glutamate and glycine all divalent cations tested were inhibitory with a rank order of potency of Zn2+ > Cd2+ Mn2+ > Mg2+ > Ca2+. In summary, the soluble NMDA receptor, labelled at the cation channel site with [3H]MK‐801, maintains the ability to bind NMDA agonists, glycine, and divalent cations in a manner akin to that observed in membranes.