Retour aux sources: defining the structural basis of glutamate receptor activation.

Ionotropic glutamate receptors (iGluRs) are the major excitatory neurotransmitter receptor in the vertebrate CNS and, as a result, their activation properties lie at the heart of much of the neuronal network activity observed in the developing and adult brain. iGluRs have also been implicated in many nervous system disorders associated with postnatal development (e.g. autism, schizophrenia), cerebral insult (e.g. stroke, epilepsy), and disorders of the ageing brain (e.g. Alzheimer's disease, Parkinsonism). In view of this, an emphasis has been placed on understanding how iGluRs activate and desensitize in functional and structural terms. Early structural models of iGluRs suggested that the strength of the agonist response was primarily governed by the degree of closure induced in the ligand-binding domain (LBD). However, recent studies have suggested a more nuanced role for the LBD with current evidence identifying the iGluR LBD interface as a "hotspot" regulating agonist behaviour. Such ideas remain to be consolidated with recently solved structures of full-length iGluRs to account for the global changes that underlie channel activation and desensitization.

Discovery of novel small-molecule antagonists for GluK2.

KA receptors have shown to be potential therapeutic targets in CNS diseases such as schizophrenia, depression, neuropathic pain and epilepsy. Through the use of our docking tool Fitted, we investigated the relationship between ligand activity towards GluK2 and the conformational state induced at the receptor level. By focusing our rational design on the interaction between the ligand and a tyrosine residue in the binding site, we synthesized a series of molecules based on a glutamate scaffold, and carried out electrophysiological recordings. The observed ability of some of these molecules to inhibit receptor activation shows the potential of our design for the development of effective antagonists with a molecular size comparable to that of the endogenous neurotransmitter L-glutamate.

Functional α6ß4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6ß4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6ß4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6ß4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6ß4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6ß4 receptor assembly. Effects on α6ß4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6ß4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6ß4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6ß4 receptor.

α6-Containing Nicotinic Acetylcholine Receptor Reconstitution Involves Mechanistically Distinct Accessory Components.

Acetylcholine gates a large family of nicotinic receptor cation channels that control neuronal excitation and neurotransmitter release. These receptors are key targets for neuropsychiatric disorders; however, difficulties in expressing nicotinic acetylcholine (nACh) receptors hamper elaboration of their pharmacology and obscure elucidation of their biological functions. Particularly intriguing are α6-containing nACh receptors, which mediate nicotine-induced dopamine release in striatum-nucleus accumbens. Using genome-wide cDNA screening, we identify three accessory proteins, ß-anchoring and -regulatory protein (BARP), lysosomal-associated membrane protein 5 (LAMP5), and SULT2B1, that complement the nACh receptor chaperone NACHO to reconstitute α6ß2ß3 channel function. Whereas NACHO mediates α6ß2ß3 assembly, BARP primarily enhances channel gating and LAMP5 and SULT2B1 promote receptor surface trafficking. BARP knockout mice show perturbations in presynaptic striatal nACh receptors that are consistent with BARP modulation of receptor desensitization. These studies unravel the molecular complexity of α6ß2ß3 biogenesis and enable physiological studies of this crucial neuropharmacological target.

α7 nicotinic acetylcholine receptor upregulation by anti-apoptotic Bcl-2 proteins.

Nicotinic acetylcholine receptors (nAChRs) mediate and modulate synaptic transmission throughout the brain, and contribute to learning, memory, and behavior. Dysregulation of α7-type nAChRs in neuropsychiatric as well as immunological and oncological diseases makes them attractive targets for pharmaceutical development. Recently, we identified NACHO as an essential chaperone for α7 nAChRs. Leveraging the robust recombinant expression of α7 nAChRs with NACHO, we utilized genome-wide cDNA library screening and discovered that several anti-apoptotic Bcl-2 family proteins further upregulate receptor assembly and cell surface expression. These effects are mediated by an intracellular motif on α7 that resembles the BH3 binding domain of pro-apoptotic Bcl-2 proteins, and can be blocked by BH3 mimetic Bcl-2 inhibitors. Overexpression of Bcl-2 member Mcl-1 in neurons enhanced surface expression of endogenous α7 nAChRs, while a combination of chemotherapeutic Bcl2-inhibitors suppressed neuronal α7 receptor assembly. These results demonstrate that Bcl-2 proteins link α7 nAChR assembly to cell survival pathways.

Nanoscale Mobility of the Apo State and TARP Stoichiometry Dictate the Gating Behavior of Alternatively Spliced AMPA Receptors.

Neurotransmitter-gated ion channels are allosteric proteins that switch on and off in response to agonist binding. Most studies have focused on the agonist-bound, activated channel while assigning a lesser role to the apo or resting state. Here, we show that nanoscale mobility of resting α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type ionotropic glutamate receptors (AMPA receptors) predetermines responsiveness to neurotransmitter, allosteric anions and TARP auxiliary subunits. Mobility at rest is regulated by alternative splicing of the flip/flop cassette of the ligand-binding domain, which controls motions in the distant AMPA receptor N-terminal domain (NTD). Flip variants promote moderate NTD movement, which establishes slower channel desensitization and robust regulation by anions and auxiliary subunits. In contrast, greater NTD mobility imparted by the flop cassette acts as a master switch to override allosteric regulation. In AMPA receptor heteromers, TARP stoichiometry further modifies these actions of the flip/flop cassette generating two functionally distinct classes of partially and fully TARPed receptors typical of cerebellar stellate and Purkinje cells.

Distinct Structural Pathways Coordinate the Activation of AMPA Receptor-Auxiliary Subunit Complexes.

Neurotransmitter-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.

Defining the structural relationship between kainate-receptor deactivation and desensitization.

Desensitization is an important mechanism curtailing the activity of ligand-gated ion channels (LGICs). Although the structural basis of desensitization is not fully resolved, it is thought to be governed by physicochemical properties of bound ligands. Here, we show the importance of an allosteric cation-binding pocket in controlling transitions between activated and desensitized states of rat kainate-type (KAR) ionotropic glutamate receptors (iGluRs). Tethering a positive charge to this pocket sustains KAR activation, preventing desensitization, whereas mutations that disrupt cation binding eliminate channel gating. These different outcomes explain the structural distinction between deactivation and desensitization. Deactivation occurs when the ligand unbinds before the cation, whereas desensitization proceeds if a ligand is bound without cation pocket occupancy. This sequence of events is absent from AMPA-type iGluRs; thus, cations are identified as gatekeepers of KAR gating, a role unique among even closely related LGICs.