Abstract
The AMPA receptor GluA2 belongs to the family of ionotropic glutamate receptors, which are responsible for most of the fast excitatory neuronal signalling in the central nervous system. These receptors are important for memory and learning, but have also been associated with brain diseases such as Alzheimer's disease and epilepsy. Today, one drug is on the market for the treatment of epilepsy targeting AMPA receptors, i.e. a negative allosteric modulator of these receptors. Recently, crystal structures and cryo-electron microscopy (cryo-EM) structures of full-length GluA2 in the resting (apo), activated and desensitized states have been reported. Here, solution structures of full-length GluA2 are reported using small-angle neutron scattering (SANS) with a novel, fully matched-out detergent. The GluA2 solution structure was investigated in the resting state as well as in the presence of AMPA and of the negative allosteric modulator GYKI-53655. In solution and at neutral pH, the SANS data clearly indicate that GluA2 is in a compact form in the resting state. The solution structure resembles the crystal structure of GluA2 in the resting state, with an estimated maximum distance (D max) of 179 ± 11 Å and a radius of gyration (R g) of 61.9 ± 0.4 Å. An ab initio model of GluA2 in solution generated using DAMMIF clearly showed the individual domains, i.e. the extracellular N-terminal domains and ligand-binding domains as well as the transmembrane domain. Solution structures revealed that GluA2 remained in a compact form in the presence of AMPA or GYKI-53655. At acidic pH only, GluA2 in the presence of AMPA adopted a more open conformation of the extracellular part (estimated D max of 189 ± 5 Å and R g of 65.2 ± 0.5 Å), resembling the most open, desensitized class 3 cryo-EM structure of GluA2 in the presence of quisqualate. In conclusion, this methodological study may serve as an example for future SANS studies on membrane proteins.
Highlights
Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and mediates its function through interaction with metabotropic G protein-coupled receptors and ionotropic glutamate receptors
This data set was reported in a recent publication on contrast-optimized detergents (Midtgaard et al, 2018) and we showed that the solution structure of GluA2 resembles the X-ray crystal structure
In solution at 10C, we find that GluA2 primarily adopts a compact tetrameric structure both in the resting state as well as in the presence of 1 mM AMPA and 1 mM GYKI-53655, resembling the compact X-ray and cryo-electron microscopy (cryo-EM) structures determined at cryogenic temperature (Figs. 2, 3 and 5)
Summary
Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and mediates its function through interaction with metabotropic G protein-coupled receptors (mGluRs) and ionotropic glutamate receptors (iGluRs). Located in the cell membrane at the synapse, the iGluRs mediate fast synaptic transmission in the CNS and have an important role in memory and learning (Sachser et al, 2017). These receptors have been associated with brain diseases or disorders, for example epilepsy, Parkinson’s disease, Alzheimer’s disease, depression and stroke (Lee et al, 2016). IGluRs form tetrameric ion channels composed of either identical subunits (homomeric receptors) or different subunits (heteromeric receptors). The GluA2 subunit is composed of four domains (Fig. 1a): the extracellular N-terminal domain (NTD; abbreviated ATD) followed by the ligand-binding domain (LBD), the transmembrane domain (TMD) forming the ion channel and the cytosolic C-terminal domain (CTD; not included in structures)
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