An acidic amino acid in the N-methyl-D-aspartate receptor that is important for spermine stimulation.
The polyamine spermine has multiple effects on N-methyl-D-aspartate (NMDA) receptors, including "glycine-independent" stimulation, which is seen in the presence of saturating concentrations of glycine; "glycine-dependent" stimulation, which is due to an increase in the affinity of the receptor for glycine; and voltage-dependent block. These effects may involve three separate polyamine binding sites on the receptor. To identify amino acid residues that are important for spermine binding, we used site-directed mutagenesis to alter amino acids in and around a region of the NR1 subunit of the NMDA receptor that shows homology with PotD, a polyamine binding protein from Escherichia coli. Mutated subunits, expressed in heteromeric and homomeric NMDA receptors, were studied by voltage-clamp recording in Xenopus oocytes. Mutation of two acidic residues (E339-E342) to neutral amino acids reduced or abolished glycine-independent stimulation by spermine without affecting glycine-dependent stimulation or voltage-dependent block by spermine. Mutation of these residues also had modest effects on sensitivity to protons and to ifenprodil but did not alter sensitivity to glutamate and glycine or to voltage-dependent block by Mg2+. Residue E342 in NR1 appears to be critical for glycine-independent spermine stimulation. Mutations at equivalent positions in NR2A(E352Q) or NR2B(E353Q) had no effect on sensitivity to spermine, pH, or ifenprodil. Residue E342 in NR1 may form part of a discrete spermine binding site on the NMDA receptor or be involved in the mechanism of modulation by polyamines. This residue may also be involved in modulation by protons and ifenprodil.
Williams
K
K
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia 19104-6084, USA.
Kashiwagi
K
K
Fukuchi
J
J
Igarashi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018698:Glutamic Acid / Q000494:pharmacology; D005998:Glycine / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016194:Receptors, N-Methyl-D-Aspartate / Q000187:drug effects* / Q000235:genetics* / Q000378:metabolism; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D013096:Spermine / Q000378:metabolism / Q000494:pharmacology*; D013268:Stimulation, Chemical; D013329:Structure-Activity Relationship; D014981:Xenopus
D046911:Macromolecular Substances; D016194:Receptors, N-Methyl-D-Aspartate; D013096:Spermine; D018698:Glutamic Acid; D005998:Glycine