Received for publication January 30, 2006.
Revised March 3, 2006.
Accepted for publication March 14, 2006.

Determination of binding site residues responsible for the subunit selectivity of novel marine-derived compounds on kainate receptors

Abstract

Dysiherbaine (DH) and related molecules are high-affinity, subunit-selective kainate receptor (KAR) ligands originally isolated from a marine sponge. To elucidate why two compounds, DH and MSVIII-19, bind selectively to the GluR5 but not to the KA2 KAR subunit, we used molecular dynamics simulations to generate binding models that were tested experimentally in radioligand binding and electrophysiological assays. Three candidate sites, Val685, Leu735, and Ser741 in GluR5, corresponding to Ile669, Phe719, and Met725 in KA2, were predicted to underlie the distinct binding profiles of the marine toxins. Single or multiple reciprocal mutations introduced into the receptor subunits produced a variety of effects on binding affinity. Most notably, mutation of Met725 to serine in KA2 increased the affinity of DH by 350-fold; in contrast, mutation of one or more of the residues in GluR5 did not markedly alter DH binding. MSVIII-19 affinity for the KA2 subunit was significantly increased in multiple site mutants, and reciprocal mutations in the GluR5 subunit produced substantial (700-fold) reductions in MSVIII-19 affinity. Physiological characterization of the double and triple mutant subunits demonstrated altered functional behavior consistent with the changes in binding affinity. The results provide experimental support for the importance of these three LBD residues and suggest steric hindrance in the KA2 subunit LBD is largely responsible for the very low affinity for the two compounds. In this study we identified the molecular basis for subunit selectivity of these marine-derived molecules on KARs, which could facilitate the rationale design of selective ligands with distinct pharmacological profiles.

Key words: Glutamate, Molecular dynamics, Func. analysis receptor/ion channel mutants, Mutagenesis/Chimeric approaches, Receptor binding studies

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