Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor.

AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop ('ß1'), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.

Mitochondrial Protein Abundance Gradients Require the Distribution of Separated Mitochondria.

Mitochondria are highly dynamic organelles that interchange their contents mediated by fission and fusion. However, it has previously been shown that the mitochondria of cultured human epithelial cells exhibit a gradient in the relative abundance of several proteins, with the perinuclear mitochondria generally exhibiting a higher protein abundance than the peripheral mitochondria. The molecular mechanisms that are required for the establishment and the maintenance of such inner-cellular mitochondrial protein abundance gradients are unknown. We verified the existence of inner-cellular gradients in the abundance of clusters of the mitochondrial outer membrane protein Tom20 in the mitochondria of kidney epithelial cells from an African green monkey (Vero cells) using STED nanoscopy and confocal microscopy. We found that the Tom20 gradients are established immediately after cell division and require the presence of microtubules. Furthermore, the gradients are abrogated in hyperfused mitochondrial networks. Our results suggest that inner-cellular protein abundance gradients from the perinuclear to the peripheral mitochondria are established by the trafficking of individual mitochondria to their respective cellular destination.

Characterizing the binding and function of TARP γ8-selective AMPA receptor modulators.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid(AMPA)-type glutamate receptors (AMPARs) are the predominant excitatory neurotransmitter receptors in the brain, where they mediate synaptic transmission and plasticity. Excessive AMPAR activation leads to diseases such as epilepsy. AMPAR properties are modulated by auxiliary proteins and foremost by the transmembrane AMPAR regulatory proteins (TARPs). These distribute in unique expression patterns across the brain, rendering AMPAR/TARP complexes promising targets for region-specific therapeutic intervention. TARP γ8 is predominantly expressed in the forebrain and is enriched in the hippocampus, a region associated with temporal lobe epilepsy. Recent high-throughput medicinal chemistry screens have identified multiple promising compounds that selectively target AMPARs associated with γ8 and hold promise for epilepsy treatment. However, how these modulators target the receptor complex is currently unknown. Here, we use a combination of ligand docking, molecular dynamics simulations, and electrophysiology to address this question. We identify a conserved oxindole isostere, shared between three structurally diverse modulators (LY-3130481, JNJ-55511118, and JNJ-61432059) as the major module engaging γ8 by an H-bond to Asn-172 (γ8). The remaining variable region of each molecule likely targets the receptor complex in ligand-selective modes. Functional data reveal parallels in the underlying modulatory action of two prominent compounds. This work will aid development of refined AMPAR epilepsy therapeutics and facilitate to uncover the mechanisms by which TARPs modulate the receptor.