Crystal structures of the GluR2 ligand binding core (S1S2) have been determined in the apo state and in the presence of the antagonist DNQX, the partial agonist kainate, and the full agonists AMPA and glutamate. The domains of the S1S2 ligand binding core are expanded in the apo state and contract upon ligand binding with the extent of domain separation decreasing in the order of apo > DNQX > kainate > glutamate approximately equal to AMPA. These results suggest that agonist-induced domain closure gates the transmembrane channel and the extent of receptor activation depends upon the degree of domain closure. AMPA and glutamate also promote a 180 degrees flip of a trans peptide bond in the ligand binding site. The crystal packing of the ligand binding cores suggests modes for subunit-subunit contact in the intact receptor and mechanisms by which allosteric effectors modulate receptor activity.
Ligand-gated ion channels transduce chemical signals into electrical impulses by opening a transmembrane pore in response to binding one or more neurotransmitter molecules. After activation, many ligand-gated ion channels enter a desensitized state in which the neurotransmitter remains bound but the ion channel is closed. Although receptor desensitization is crucial to the functioning of many ligand-gated ion channels in vivo, the molecular basis of this important process has until now defied analysis. Using the GluR2 AMPA-sensitive glutamate receptor, we show here that the ligand-binding cores form dimers and that stabilization of the intradimer interface by either mutations or allosteric modulators reduces desensitization. Perturbations that destabilize the interface enhance desensitization. Receptor activation involves conformational changes within each subunit that result in an increase in the separation of portions of the receptor that are linked to the ion channel. Our analysis defines the dimer interface in the resting and activated state, indicates how ligand binding is coupled to gating, and suggests modes of dimer dimer interaction in the assembled tetramer. Desensitization occurs through rearrangement of the dimer interface, which disengages the agonist-induced conformational change in the ligand-binding core from the ion channel gate.
Ionotropic glutamate receptors (iGluRs) mediate excitatory synaptic transmission in vertebrates and invertebrates through ligand-induced opening of transmembrane ion channels. iGluRs are segregated into three subtypes according to their sensitivity to the agonists AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid), kainate (a structural analogue of glutamate) or NMDA (N-methyl-D-aspartate). iGluRs are important in the development and function of the nervous system, are essential in memory and learning, and are either implicated in or have causal roles in dysfunctions ranging from Alzheimer's, Parkinson's and Huntington's diseases, schizophrenia, epilepsy and Rasmussen's encephalitis to stroke. Development of iGluR agonists and antagonists has been hampered by a lack of high-resolution structural information. Here we describe the crystal structure of an iGluR ligand-binding region in a complex with the neurotoxin (agonist) kainate. The bilobed structure shows the determinants of receptor-agonist interactions and how ligand-binding specificity and affinity are altered by remote residues and the redox state of the conserved disulphide bond. The structure indicates mechanisms for allosteric effector action and for ligand-induced channel gating. The information provided by this structure will be essential in designing new ligands.
The canonical conformational states occupied by most ligand-gated ion channels, and many cell-surface receptors, are the resting, activated, and desensitized states. While the resting and activated states of multiple receptors are well characterized, elaboration of the structural properties of the desensitized state, a state that is by definition inactive, has proven difficult. Here we use electrical, chemical, and crystallographic experiments on the AMPA-sensitive GluR2 receptor, defining the conformational rearrangements of the agonist binding cores that occur upon desensitization of this ligand-gated ion channel. These studies demonstrate that desensitization involves the rupture of an extensive interface between domain 1 of 2-fold related glutamate-binding core subunits, compensating for the ca. 21 degrees of domain closure induced by glutamate binding. The rupture of the domain 1 interface allows the ion channel to close and thereby provides a simple explanation to the long-standing question of how agonist binding is decoupled from ion channel gating upon receptor desensitization.
A vast number of proteins are involved in synaptic function. Many have been cloned and their functional role defined with varying degrees of success, but their number and complexity currently defy any molecular understanding of the physiology of synapses. A beacon of success in this medieval era of synaptic biology is an emerging understanding of the mechanisms underlying the activity of the neurotransmitter receptors for glutamate. Largely as a result of structural studies performed in the past three years we now have a mechanistic explanation for the activation of channel gating by agonists and partial agonists; the process of desensitization, and its block by allosteric modulators, is also mostly explained; and the basis of receptor subtype selectivity is emerging with clarity as more and more structures are solved. In the space of months we have gone from cartoons of postulated mechanisms to hard fact. It is anticipated that this level of understanding will emerge for other synaptic proteins in the coming decade.
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