Probing the Mg^(2+) Blockade Site of an N-Methyl-d-aspartate (NMDA) Receptor with Unnatural Amino Acid Mutagenesis

Abstract

The N-methyl-d-aspartate (NMDA) receptor plays a central role in learning and memory in the mammalian CNS. At normal neuronal resting membrane potentials, the pore of this glutamate-gated ion channel is blocked by a Mg^(2+) ion. Previous work suggests that the Mg^(2+) binding site is quite novel, involving several asparagine residues and a cation–π interaction between Mg^(2+) and a conserved tryptophan in the pore. Using unnatural amino acid mutagenesis, we show that no such cation–π interaction exists. The implicated tryptophan instead appears to play a structural role that can only be fulfilled by a rigid, flat, hydrophobic residue. This is the first demonstration of unnatural amino acid incorporation in the NMDA receptor, and it opens the way for future investigations of this pivotal neuroreceptor.

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