Functional effects of mutations in the putative agonist binding region of recombinant alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors.

Structural correlates of ligand binding to alpha-amino-3-hydroxy-5-methyl- 4-isoxazolepropionic acid (AMPA) receptors were examined by altering conserved lysine residues to either glutamate or glutamate at position 445 of the GluR-A subunit and position 449 of the GluR-B subunit. Receptors were expressed in human embryonic kidney 293 cells and in Xenopus oocytes as homomeric GluR-A or heteromeric combinations of GluR-A/B. The functional properties of the resulting receptors were measured with patch-clamp and voltage-clamp electrophysiology. These mutations, which reduced the total positive charge at the lysine 445 position, selectively altered the affinity of some ligands and had no obvious effect on receptor desensitization properties. The L-glutamate EC50 was 10.2 microM when wild-type GluR-A was coexpressed with GluR-B (GluR-A/B) and 38.9 microM when GluR-A445Q/B449Q receptors were tested. The AMPA EC50 was similarly reduced (wild-type, 3.71 microM; GluR-A445Q/B449Q, 21.4 microM). Receptors containing a glutamate residue at this position were even less sensitive to L-glutamate and AMPA. Whereas the apparent potency of L-glutamate varied inversely with the total positive charge regardless of the subunit, the affinity of AMPA was more sensitive to mutations in GluR-A than to those in GluR-B. Interestingly, the EC50 of kainate was unaffected by most of these mutations and actually increased slightly with GluR-A445E/B449E receptors. The affinity of 6-cyano-2,3-dihydroxy-7- nitroquinoxaline (CNQX) was slightly reduced with GluR-A445E/B449E receptors, whereas the apparent affinity of GYKI52466 was unchanged. These data confirm that structural correlates of binding to AMPA receptors vary among different ligands.