In the transmitter-gated ion channel class of receptors, the members of which are all believed to be heterooligomers, the number and arrangement of the subunits are only known with any certainty for the nicotinic acetylcholine receptor from Torpedo electric fish . That receptor has been shown to possess a pentameric rosette structure, with five homologous subunits (a2ßyS) arranged to enclose the central ion channel . The data were obtained by electron image analysis of two-dimensional receptor arrays, which form as a consequence of that receptor's exceptionally high abundance in the Torpedo membranes and are therefore not attainable for other receptors. We have applied another direct approach to determine the quaternary structure of native ionotropic GABA receptors. We have purified those receptors from porcine brain cortex and analysed the rotational symmetry of isolated receptors visualized by electron microscopy . The results show the receptor to have a pentameric structure with a central water-filled pore, which can now be said to be characteristic of the entire superfamily . Key Words: GABAA receptor-Electron microscopy-Image analysis-Quaternary structure. J. Neurochem . 62, 815-818 (1994) .

SummaryNeurotransmitter-gated ion channels adopt different gating modes to fine-tune signaling at central synapses. At glutamatergic synapses, high and low activity of AMPA receptors (AMPARs) is observed when pore-forming subunits coassemble with or without auxiliary subunits, respectively. Whether a common structural pathway accounts for these different gating modes is unclear. Here, we identify two structural motifs that determine the time course of AMPAR channel activation. A network of electrostatic interactions at the apex of the AMPAR ligand-binding domain (LBD) is essential for gating by pore-forming subunits, whereas a conserved motif on the lower, D2 lobe of the LBD prolongs channel activity when auxiliary subunits are present. Accordingly, channel activity is almost entirely abolished by elimination of the electrostatic network but restored via auxiliary protein interactions at the D2 lobe. In summary, we propose that activation of native AMPAR complexes is coordinated by distinct structural pathways, favored by the association/dissociation of auxiliary subunits.

AMPA-and kainate (KA)-selective ionotropic glutamate receptors (iGluRs) respond to agonist by opening (gating), then closing (desensitizing) in quick succession. Gating has been linked to agonist-induced changes within the ligand-binding domain (LBD), and desensitization to rearrangement of a dimer formed by neighboring LBDs. To explore the role of dimer conformation in both gating and desensitization, we compared the conformational effects of two kainate receptor mutants. The first, GluK2-D776K, blocks desensitization of macroscopic current responses ("macroscopic desensitization"). The second, GluK2-M770K, accelerates macroscopic desensitization and eliminates the effects of external ions on channel kinetics. Using structures determined by x-ray crystallography, we found that in both mutants the introduced lysines act as tethered cations, displacing sodium ions from their binding sites within the dimer interface. This results in new inter-and intra-protomer contacts in D776K and M770K respectively, explaining the effects of these mutations on dimer stability and desensitization kinetics. Further, chloride binding was unaffected by the M770K mutation, even though binding of sodium ions has been proposed to promote dimer stability by stabilizing anion binding. This suggests sodium binding may affect receptor function more directly than currently supposed. Notably, we also observed a ligand-specific shift in dimer conformation when comparing LBD dimers in complex with glutamate or the partial agonist KA, revealing a previously unidentified role for dimer orientation in iGluR gating.

Ionotropic glutamate receptor (iGluR) desensitization can be modulated by mutations that change the stability of a dimer formed by the agonist binding domain. Desensitization of ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors can be blocked by a single point mutation (e.g., GluR2 L483Y) that stabilizes this dimer in an active conformation. In contrast, desensitization of kainate receptors can be slowed, but not blocked, by similar dimer interface mutations. Only covalent cross-linking via introduced disulfides has been previously shown to block kainate receptor desensitization completely. We have now identified an apparently nondesensitizing GluR6 point mutant (D776K) located at the apex of the ligand binding (S1S2) domain dimer interface. Asp776 is one of a cluster of four charged residues in this region that together mediate direct dimer interactions and contribute to the binding sites for one chloride and two sodium ions. Despite the localized ϩ4 change in the net charge of the S1S2 dimer, the D776K mutation actually increased the thermodynamic stability of the dimer. Unlike GluR6 wild type, the D776K mutant is insensitive to external cations but retains sensitivity to external anions. We therefore hypothesize that the unexpected phenotype of this charge reversal mutation results from the substitution of the sodium ions bound within the dimer interface by the introduced lysine NH 3 ϩ groups. The nondesensitizing D776K mutant provides insights into kainate receptor gating and represents a potentially useful new tool for dissecting kainate receptor function.