Molecular mechanisms of TARP modulation of AMPA receptor gating.

AMPA receptors are critical components of excitatory synaptic transmission and play myriad roles in brain function. AMPA receptors are nearly always accompanied by one or more types of auxiliary proteins, principal among these being the transmembrane AMPA receptor regulatory proteins (TARPs). This family of four-pass transmembrane proteins contains prominent extracellular domains that interact with AMPA receptors to modulate channel gating. However, not all TARPs modulate AMPA receptors equally. For example, the TARP γ8 alters AMPA receptor kinetics to a greater extent than γ2 (a.k.a stargazin), even inducing a striking resensitization or superactivation response. Structural and functional studies implicate the large β1-2 extracellular loop of γ8 as critical for kinetic modulation. Consistent with this, swapping the larger β1-2 loop of γ8 with the smaller loop of γ2 can broadly exchange the modulatory phenotype. Because this β1-2 loop is flexible, it remains unresolved in cryo-EM structures. Thus, the precise contacts between the loop and the AMPA receptor, and the underlying mechanism of modulation, is unclear. Interestingly, focused refinement of the β1-2 loop indicates this segment may move between the resting, active, and desensitized states, potentially producing new contacts. Here we propose to identify contact sites between the γ2 or γ8 β1-2 loop and the AMPA receptor using a combination of non-canonical amino acid crosslinking and patch clamp fluorometry. In addition, we will use patch clamp FRET to discern if the β1-2 loops of γ2 or γ8 move during activation, desensitization, and superactivation. Taken together, these studies will advance our mechanistic understanding of AMPA receptor and TARP interactions and glutamate receptor gating. Furthermore, developing these techniques will allow us and others to interrogate the relative positions and ensemble movements of the many unresolved or intrinsically disordered regions of membrane proteins. Project Number: 1R21NS146672-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: David MacLean | Institution: UNIVERSITY OF ROCHESTER, ROCHESTER, NY | Award Amount: $226,500 | Activity Code: R21 | Study Section: Biochemistry and Biophysics of Membranes Study Section[BBM] View on NIH RePORTER: https://reporter.nih.gov/project-details/11284215