The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 domains in the N-terminal and M3-M4 loop regions, respectively. In the present study, we sought to confirm that the conserved ligand-binding residues identified in the AMPA receptor S1S2 domains also participate in ligand binding of GluR6 kainate receptors. Amino acid substitutions were made in the GluR6 parent at R523, T690, and E738 to alter their potential interactions with ligand. Mutant receptors were expressed in human embryonic kidney 293 cells, confirmed by Western blot analysis, and tested by [

The glutamate receptor (GluR) agonist-binding site consists of amino acid residues in the extracellular S1 and S2 segments in the N-terminal and M3-M4 loop regions, respectively. Molecular and atomic level structural analyses have identified specific S1 and S2 residues that interact directly with ligands, interact with one another in a dimeric configuration, and influence channel gating and desensitization properties of AMPA receptors. Other studies suggest that KA receptor gating and desensitization may differ mechanistically. In particular, a leucine (L) to tyrosine (Y) mutation in the S1 segment of AMPA receptors is sufficient to block desensitization, whereas KA receptors naturally contain a tyrosine residue at the equivalent position (Y751 in GluR6) but retain the fast-desensitizing phenotype. We hypothesized that KA receptor desensitization is preserved by a compensatory substitution in the S2 segment. We generated a series of GluR6 mutants that converted individual S2 domain residues to their AMPA receptor equivalents. Various S2 mutations had effects on the kinetics of desensitization and recovery from desensitization, but no single amino acid substitution was found to block desensitization, as in the L/Y mutant AMPA receptors, or to prevent desensitization to KA. Other mutations designed to neutralize residues thought to interact across the dimer interface had dramatic effects on channel gating and desensitization. These results are consistent with a close but imperfect structural homology between AMPA and KA receptors and support the role of conserved S1S2 domain interactions at the dimer interface in GluR channel function.

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The adult cerebral cortex contains nicotinic acetylcholine (ACh) receptors vital to cortical function. However, little is known about the assembly of embryonic nicotinic receptor subunits into functional receptors or whether they play an active role in cortical development. We now report evidence of functional nicotinic acetylcholine receptor channels in fetal mouse cerebral cortex as early as embryonic day 10 (E10), when the cortex consists of dividing stem and progenitor cells. Patch-clamp electrophysiological measurements indicate that nicotine and ACh evoke sizable inward currents characteristic of nicotinic receptors, that are strongly rectifying with a reversal potential near 0 mV. Three different nicotinic agonists, ACh, nicotine, and dimethylphenylpiperazinium, evoked cytosolic Ca(2+) signals. Agonist-evoked Ca(2+) signals and electrophysiological responses were found in greater than 70% of all E10-E11 cells tested and were blocked by nicotinic receptor antagonists. The Ca(2+) response to nicotinic agonists was markedly prolonged in cells from early embryonic stages relative to later stages of development. alpha3, alpha4, and alpha7 receptor subunit proteins were detected immunocytochemically in cortical cells from E10 to birth. The incidence of each subunit declined with embryonic age, suggesting a role in early development. We discuss the possible function of nicotinic receptors in early cortical development and their role as a target for nicotine in the developmental pathologies associated with the fetal tobacco syndrome.

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