Biology subjects

Plested, A.

Publications and source records attributed to Plested, A..

2 recordsLinked to original sources

bioRxiv2018

Gating modules of the AMPA receptor pore domain revealed by unnatural amino acid mutagenesis

Ionotropic glutamate receptors (iGluRs) are responsible for fast synaptic transmission throughout the nervous system. Conformational changes of the transmembrane domain (TMD) underlying ion channel activation and desensitization remain poorly understood. Here, we explored the dynamics of the TMD of AMPA-type iGluRs using genetically-encoded unnatural amino acid (UAA) photo-crosslinkers, p-benzoyl-L-phenylalanine (BzF) and p-azido-L-phenylalanine (AzF). We introduced UAAs at sites throughout the TMD of the GluA2 receptor and characterized these mutants in patch-clamp recordings, exposing them to glutamate and UV light. This approach revealed a range of optical effects on the activity of mutant receptors. We found evidence that an interaction between the Pre-M1 and the M4 TMD helix was essential for normal activation and desensitization. Photoactivation at F579AzF, a residue behind the selectivity filter, had extraordinarily broad effects on gating and desensitization. This observation suggests coupling to other parts of the receptor and like in other tetrameric channels, selectivity filter gating.

neuroscience↗

bioRxiv2017

Control Of AMPA Receptor Activity By The Extracellular Loops Of Auxiliary Proteins

At synapses throughout the mammalian brain, AMPA receptors form complexes with auxiliary proteins, including TARPs. However, how TARPs modulate AMPA receptor gating remains poorly understood. We built structural models of TARP-AMPA receptor complexes for TARPs {gamma}2 and {gamma}8, combining recent structural studies and de novo structure predictions. These models, combined with peptide binding assays, provide evidence for multiple interactions between GluA2 and variable extracellular loops of TARPs. Substitutions and deletions of these loops had surprisingly rich effects on the kinetics of glutamate-activated currents, without any effect on assembly. Critically, by altering the two interacting loops of {gamma}2 and {gamma}8, we could entirely remove all allosteric modulation of GluA2, without affecting formation of AMPA receptor-TARP complexes. Likewise, substitutions in the linker domains of GluA2 completely removed any effect of Y2 on receptor kinetics, indicating a dominant role for this previously overlooked site proximal to the AMPA receptor channel gate.

biophysics↗