Mei Du

Allosteric mechanism in AMPA receptors: A FRET-based investigation of conformational changes

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are the primary mediators of fast excitatory synaptic transmission in the mammalian CNS. Structures of the extracellular ligand-binding domain suggest that the extent of cleft closure in the ligand-binding domain controls the extent of activation of the receptor. Here we have developed a fluorescence resonance energy transfer-based probe that allows us to study the extent of cleft closure in the isolated ligand-binding domain in solution. These investigations show that the wild-type protein exhibits a graded cleft closure that correlates to the extent of activation, which is in qualitative agreement with the crystal structures. However, the changes in extent of cleft closure between the apo and agonist-bound states are smaller than that observed in the crystal structures. We have also used this method to study the L650T mutant and show that in solution the α-amino-5-methyl-3-hydroxy-4-isoxazole propionate-bound form of this mutant exists primarily in a conformation that is more closed than predicted based on the activity, indicating that the degree of cleft closure alone cannot be used as a measure of extent of activation of the receptor, and there are possibly other mechanisms in addition to cleft closure that mediate the subtleties in extent of activation by a given agonist.

Role of dimer interface in activation and desensitization in AMPA receptors.

The conversion of chemical to electrical signals by the AMPA receptors is the key step by which these proteins control cognitive and motor responses. Here, we have used luminescence resonance energy transfer (LRET) to gain insight into the conformational changes induced by glutamate binding in the agonist-binding domain in functional AMPA receptors expressed in oocytes and HEK-293 cells. The LRET-based distances indicate that the interface between the upper lobes of the agonist-binding domain within a dimer is in a decoupled state in the unligated Apo state of the receptor. Agonist binding results in the formation of the dimer interface in the open-channel form of the receptor. In the continued presence of glutamate when the receptor is primarily in the desensitized state, the dimer interface is decoupled, confirming that the decoupling of the dimer interface leads to channel closure. The LRET distances also indicate that the dimer interface is preformed before activation in the L484Y mutation and also is formed in the antagonist (ZK200775)-bound form of the AMPA receptor. These results suggests that, although the preformation of the interface is not sufficient to drive channel activation, it could play a role in the energetics of activation and hence modulation of the receptor by auxiliary proteins or small molecules.

LRET investigations of conformational changes in the ligand binding domain of a functional AMPA receptor.

The structural investigations using the soluble ligand binding domain of the AMPA subtype of the glutamate receptor have provided invaluable insight into the mechanistic pathway by which agonist binding to this extracellular domain mediates the formation of cation-selective channels in this protein. These structures, however, are in the absence of the transmembrane segments, the primary functional component of the protein. Here, we have used a modified luminescence resonance energy transfer based method to obtain distance changes due to agonist binding in the ligand binding domain in the presence of the transmembrane segments. These distance changes show that the cleft closure conformational change observed in the isolated ligand binding domain upon binding agonist is conserved in the receptor with the channel segments, thus establishing that the isolated ligand binding domain is a good model of the domain in the receptor containing the transmembrane segments.

Water soluble RNA based antagonist of AMPA receptors

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one of the important receptor classes involved in glutamate-mediated excitotoxicity. Although small molecule antagonists of this receptor have been shown to have neuroprotective properties, their low solubilities pose severe side effects in clinical trials. Here we have used the SELEX method to obtain water-soluble nuclease-resistant RNA ligands that bind to the agonist binding site of AMPA receptors. Using whole-cell current recordings, we have characterized the functional consequences of a representative aptamer from this class and show that it is a competitive antagonist of AMPA receptors and in the concentration range where it acts as an inhibitor of the AMPA receptor the RNA has no effect on the GluR6 homomeric kainate receptors. Additionally, using a fluorescence resonance energy transfer (FRET) probe, we show that this RNA ligand stabilizes the open cleft conformation of the ligand binding domain, consistent with the known structures of small antagonist-bound states of the soluble domain of this protein. Finally, using rat primary cortical neurons, we show that this RNA ligand significantly reduces neurotoxicity associated with oxygen glucose deprivation. The water-soluble and antagonistic properties of this aptamer coupled with its neuroprotective properties make it an excellent candidate for potential use in diseases or pathological conditions involving glutamate-mediated excitotoxicity.

Luminescence Resonance Energy Transfer Investigation of Conformational Changes in the Ligand Binding Domain of a Kainate Receptor

The apo state structure of the isolated ligand binding domain of the GluR6 subunit and the conformational changes induced by agonist binding to this protein have been investigated by luminescence resonance energy transfer (LRET) measurements. The LRET-based distances show that agonist binding induces cleft closure, and the extent of cleft closure is proportional to the extent of activation over a wide range of activations, thus establishing that the cleft closure conformational change is one of the mechanisms by which the agonist mediates receptor activation. The LRET distances also provide insight into the apo state structure, for which there is currently no crystal structure available. The distance change between the glutamate-bound state and the apo state is similar to that observed between the glutamate-bound and antagonist UBP-310-bound form of the GluR5 ligand binding domain, indicating that the cleft for the apo state of the GluR6 ligand binding domain should be similar to the UBP-310-bound form of GluR5. This observation implies that te apo state of GluR6 undergoes a cleft closure of 29–30° upon binding full agonists, one of the largest observed in the glutamate receptor family.

Vibrational spectroscopic investigation of the ligand binding domain of kainate receptors

Fourier transform infrared spectroscopy has been used to probe the agonist‐protein interactions in the ligand binding domain of the GluR6 subunit, one subunit of the kainate subtype of glutamate receptors. In order to study the changes in the interactions over a range of activations the investigations were performed using the wild type, N690S, and T661E mutations. These studies show that the strength of the interactions at the α‐amine group of the agonist, as probed by studying the environment of the nondisulphide bonded Cys 432, acts as a switch with weaker interactions at lower activations and stronger interactions at higher activations. The α‐carboxylate interactions of the agonist, however, are not significantly different over the wide range of activations, as measured by the maximum currents mediated by the receptors at saturating concentrations of agonists. Previous investigations of AMPA receptors show a similar dependence of the α‐amine interactions on activation indicating that the roles of the α‐amine interactions in mediating receptor activation are similar for both subtypes of receptors; however, in the case of the AMPA receptors a tug of war type of change was observed between the α‐amine and α‐carboxylate interactions and this is not observed in kainate receptors. This decoupling of the two interactions could arise due to the larger cleft observed in kainate receptors, which allows for a more flexible interaction for the α‐amine and α‐carboxylate groups of the agonists.