A study of the molecular basis of the interaction between AMPARs and their auxiliary subunits

AMPA receptors (AMPARs) are crucial for fast excitatory synaptic transmission throughout the mammalian central nervous system (CNS). At the molecular level, these receptors are tetramers of GluA1-4 subunits, which can be homomeric or heteromeric, incorporating the Ca2+-impermeable GluA2 subunit. Although the basic tetrameric AMPAR is sufficient for expression of functional channels in a recombinant system, native AMPARs are associated with a number of auxiliary proteins, which modify their trafficking as well as their biophysical and pharmacological properties. The first AMPAR auxiliary protein discovered was stargazin (γ-2), a transmembrane protein related to the γ-1 subunit of voltage gated Ca2+- channels. The lack of γ-2 in the naturally occurring mouse mutant stargazer was found to produce a loss of AMPAR-mediated transmission at the cerebellar mossy fibre to granule cell synapse. This led to the discovery, that γ-2 is crucial for AMPAR trafficking. Subsequent studies focusing on this protein showed that it is also able to modify the functional properties of AMPARs, such as deactivation and desensitization kinetics, single-channel conductance and susceptibility of Ca2+-permeable AMPARs to block by intracellular and extracellular polyamines. In addition, a family of related proteins, known as Transmembrane AMPA Receptor Regulatory Proteins (TARPs) has been identified. As well as γ-2, this includes the subunits γ-3,-4,-5,-7 and -8. All TARPs are related to γ-2 in structure and function, although certain properties vary between the family members. Since the discovery of TARPs, more proteins modulating AMPAR function have been identified, so that a complex network of AMPAR interacting partners is apparent from the literature in the field. Surprisingly, little focus had been put on how AMPARs interact with auxiliary proteins at the molecular level. Enhancing our knowledge about these interactions could provide insights into the role of AMPARs in synaptic transmission, plasticity and neurological diseases. The aim of my thesis was thus to provide insight into the role of TARPs in modulation of AMPAR complexes and the molecular mechanisms that underlie this modulation. I used a recombinant expression system to identify the physiological relevance of different AMPAR/TARP interaction regions, as well as to investigate the role of TARP stoichiometry on AMPAR/TARP function. The initial part of my thesis focused on the molecular interactions of AMPARs with their TARP auxiliary subunits. Our experiments showed that various regions of the first extracellular loop (Ex1) of γ-2 play a role in modulation of a number of functional properties of both GluA1 and GluA2(Q) subunits. We also aimed to obtain insight into the corresponding AMPAR regions important for interaction with the TARP. We therefore focused on the possible novel role of the AMPAR N-terminal domain (NTD) in TARP-AMPAR interaction and found that, although this domain plays a role in AMPAR function, it has little effect on the modulation of AMPARs by TARPs. The first two Chapters of my thesis were aimed at answering the question - how do TARPs and AMPARs interact at the molecular level? The work presented in the final Chapter attempted to expand on this question and focused on the role of TARP stoichiometry in AMPAR function. Using tandem TARP-AMPAR constructs expressed in a recombinant system, we have studied the properties of TARP/AMPAR assemblies that contain a known number of γ-2 molecules. This allowed us to identify some of the AMPAR properties that are sensitive to TARP stoichiometry. As most neurons in the brain express more than one TARP, we also investigated the pharmacological consequences of the simultaneous presence of two TARPs (γ-2 and γ-7) within a single TARP/AMPAR assembly, a possibility that has previously received little attention