Kainate receptor modulation by NETO2

Abstract

Glutamate-gated kainate receptors (KARs) are ubiquitous in the central nervous system of vertebrates, mediate synaptic transmission on post-synapse, and modulate transmitter release on pre-synapse. In the brain, the trafficking, gating kinetics, and pharmacology of KARs are tightly regulated by Neuropilin and tolloid-like proteins (Netos). Here we report cryo-EM structures of homo-tetrameric GluK2 in complex with Neto2 at inhibited and desensitized states, illustrating variable stoichiometry of GluK2-Neto2 complexes, with one or two Neto2 subunits associate with the GluK2. We find that Neto2 accesses only two broad faces of KARs, intermolecularly crosslinking the lower-lobe of ATD A/C , upper-lobe of LBD B/D , and lower-lobe of LBD A/C , illustrating how Neto2 regulates receptor-gating kinetics.The transmembrane helix of Neto2 is positioned proximal to the selectivity filter and competes with the amphiphilic H1-helix after M4 for interacting with an ICD formed by the M1-M2 linkers of the receptor, revealing how rectification is regulated by Neto2.Kainate receptors are a class of ionotropic glutamate receptors, activated by neurotransmitter glutamate 1-3 . They are not only located in the post-synapse to mediate excitatory neurotransmission in many brain regions 4,5 , but also appear in the pre-synapse to modulate transmitter release on both excitatory and inhibitory synapses 6,7 . Netos are singlepass transmembrane proteins with an extracellular domain containing two C1r/C1s-Uegf-BMP domains (CUB1 and CUB2), and a low-density lipoprotein class A domain (LDLa).These proteins have been identified as an auxiliary component of native KARs and significantly affects KARs trafficking, gating, and pharmacology 8-11 . More specifically, the Neto2 modulates KARs gating by slowing deactivation and desensitization, accelerating recovery from desensitization, and attenuating polyamine block of calcium-permeable KARs 12,13 . Despite recent progress in structural study of isolated KARs [14][15][16][17][18] , molecular basis of regulatory roles of Netos remain unclear. Here, we show the architectures of GluK2-Neto2 complex in the antagonist-bound closed-state and agonist-bound desensitized-state, illustrating interactions and stoichiometry between GluK2 and Neto2, and the modulation mechanism of Neto2 on the GluK2 receptor gating and pore properties. Moreover, a more complete pore domain, including a detailed structure of the selectivity filter (SF), is provided in our structures. Cryo-EM analysis of GluK2-Neto2 complexTo investigate the structural basis for modulation of GluK2 gating by Neto2, we coexpressed both the full length GluK2 (with Gln at the Q/R site) and Neto2 in HEK293 cells and purified the complex (Extended Data Fig. 1) 19 . Our cryo-EM studies generated three distinct assemblies of the antagonist-bound GluK2-Neto2 complexes, including GluK2 bound with one Neto2 (GluK2-1×Neto2) or two Neto2 (GluK2-1×Neto2) (Fig. 1a-1b and Extended Data Fig. 2a). The third type of complexes features a disrupted LBD dimer on one ...

Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics

“…5c ) directly contact the M2 pore loop of the selectivity filter and will thereby influence the lower half of the conduction path (Fig. 5d ; right panel), while Arg541 targets the acidic M1/2 cytoplasmic loop, implicated in AMPAR regulation 38 , 48 . In summary, contacts in the AMPAR-TARP transmembrane sector appear to be finely tuned for transmission via the M1/M4 periphery (‘transmitter’) to the ‘effector’ (the M3 gate and M2 selectivity filter) (Fig.…”

Modulatory mechanisms of TARP γ8-selective AMPA receptor therapeutics

“…5c ) directly contact the M2 pore loop of the selectivity filter and will thereby influence the lower half of the conduction path ( Fig. 5d; right panel), while Arg541 targets the acidic M1/2 cytoplasmic loop, implicated in AMPAR regulation 38,47 . In summary, contacts in the AMPAR-TARP transmembrane sector appear to be finely tuned for transmission via the M1/M4 periphery (‘transmitter’) to the ‘effector’ (the M3 gate and M2 selectivity filter) ( Fig.…”

The Antiseizure Drug Perampanel Is a Subunit-Selective Negative Allosteric Modulator of Kainate Receptors

“…We also do not understand how Neto proteins alter the binding domain to favor PMP interactions for GluK2-containing receptors, even in the absence of GluK5. The cryo-EM structures of GluK2 and Neto2 suggest that the auxiliary protein is unlikely to directly contact gating linkers (He et al, 2021); however, alteration of the M3-S2 linker changes the efficacy of Neto2 modulation (Griffith and Swanson, 2015), perhaps indirectly as a result of Neto2 interactions with adjacent membrane and ligand-binding domains. PMP inhibited central and peripheral neuronal KARs.…”