Multiple structural determinants of voltage-dependent magnesium block in recombinant NMDA receptors.
The voltage-dependent block of NMDA channels by Mg2+ is an important functional element of NMDA receptors, since relief of block by depolarization plays a key role in some forms of ischemic neurodegeneration and synaptic plasticity. To identify the relevant structural domains responsible for block by Mg2+ and TCP, we used site-directed mutagenesis to change individual amino acids of the rat NR1A subunit in a transmembrane region (599-DALTLSSAMWFSWGVLLNSGIGE-621, mutated residues underlined) previously shown to donate residues that influence ionic selectivity. Ten mutant NR1A subunits were co-expressed in Xenopus oocytes with either the epsilon 1 or NR2A subunits, and receptor properties were analyzed under two-electrode voltage clamp. The mutation N616R virtually abolished voltage-dependent Mg2+ block, reduced Zn2+ block 5-fold and greatly reduced Ba2+ permeability in confirmation of previous reports. This mutation also reduced the potency of TCP as a use-dependent blocker by 200-fold. The remaining low-affinity TCP block did not appear to be use-dependent, suggesting two blocking sites for TCP. None of the other mutations differed significantly from NR1A itself except S617N, which displayed a 6-fold reduction in Mg2+ block. A well-barrier model of permeation through the NMDA receptor channel is presented that quantitatively reproduces voltage-dependent Mg2+ block. This model demonstrates that only minimum changes energy profiles experienced by permeating ions, equivalent to the energy of a single hydrogen or ionic bond, are required to abolish Mg2+ block. These findings indicate that only small structural changes are needed to convert a Mg(2+)-insensitive ion channel to a channel with pronounced voltage-dependent Mg2+ block.
Kawajiri
S
S
Department of Pharmacology, Emory University School of Medicine, Atlanta, GA 30322.
Dingledine
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001464:Barium / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004594:Electrophysiology; D004734:Energy Metabolism; D066298:In Vitro Techniques; D008274:Magnesium / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes / Q000378:metabolism; D010622:Phencyclidine / Q000031:analogs & derivatives / Q000494:pharmacology; D016194:Receptors, N-Methyl-D-Aspartate / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology; D011995:Recombination, Genetic; D014981:Xenopus; D015032:Zinc / Q000378:metabolism
D018076:DNA, Complementary; D016194:Receptors, N-Methyl-D-Aspartate; D001464:Barium; C012058:tenocyclidine; D008274:Magnesium; D010622:Phencyclidine; D015032:Zinc