Tagg, Benjamin Edward; (2023) Modulation of AMPA receptors by activity-dependent phosphorylation. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Ionotropic AMPA-type glutamate receptors underlie most fast excitatory synaptic transmission in the central nervous system. Their precise biophysical and pharmacological properties are surprisingly diverse, depending on their subunit composition and association with auxiliary proteins, including transmembrane AMPAR regulator proteins (TARPs). Previously it has been suggested that, through activity-dependent phosphorylation, AMPARs are the key effectors of learning and memory. How alteration of AMPAR function occurs mechanistically upon phosphorylation is unclear. I used outside-out patch clamp recording to examine the impact of phosphorylation on longtailed AMPAR subunits in homomeric or heteromeric receptor complexes — either in the presence or absence of the synaptic TARP γ-2. I describe how phosphorylation by protein kinase A (PKA) can enhance AMPAR conductance and, in certain cases, slow receptor deactivation. I find that phosphorylation enhances receptor function distinctly from TARP-dependent augmentation, and only when agonist efficacy is maximal. This suggests that phosphorylation enhances allosteric gating – the extent of gating between open sublevels. Since this effect only occurs when receptors are maximally activated, phosphorylation would be expected to selectively augment their response at the synapse. I report, using stochastic simulation, that transitions underlying decay of the AMPAR response can alter their characteristic current-variance relationships. The extent to which this occurs can be modulated by ‘queueing’ equilibria that communicate with open states — even indirectly — which might the underlie noncanonical current-variance relationships that are frequently observed in non-stationary fluctuation analysis of AMPAR-mediated currents. Given that augmented AMPAR function upon phosphorylation appeared not to interact with membrane potential, I sought to determine whether phosphorylation changes inter- or intra-protein interaction using tandem crosslinking mass spectrometry. I report a pipeline enabling rapid, stable, and inducible expression of multiple AMPARs, together with their auxiliary subunits.

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