Structure of transmembrane AMPA receptor regulatory protein subunit γ2

Abstract

Transmembrane AMPA receptor regulatory proteins (TARPs) are claudin-like proteins that tightly regulate AMPA receptors (AMPARs) and are fundamental for excitatory neurotransmission. With cryo-electron microscopy (cryo-EM) we reconstruct the 36 kDa TARP subunit γ2 to 2.3 Å, which points to structural diversity among TARPs. Our data reveals critical motifs that distinguish TARPs from claudins and define how sequence variations within TARPs differentiate subfamilies and their regulation of AMPARs.

Fig. 1. Structure of TARPγ2.

a Cryo-EM map of TARPγ2, colored rainbow from N-terminus, NT (blue) to C-terminus, CT (red). b Extracellular portion of the TARPγ2 model showing the β3-β4 DSB, loop anchor DSB, and TARP cleat. c Cartoon schematic of TARPγ2 structure highlighting key structural features that rigidify the entire ECD atop the tetraspanin TMD, colored as in (a).

Fig. 2. Conservation of structural features among TARP family members.

a Multiple sequence alignment demonstrating the relative conservation of the TARP Cleat Motif, β3-β4 DSB, and Loop Anchor DSB between TARP family members. Intensity of the shade of purple represents the percent identity. Type-II TARPs are labeled in green. Loop Anchor DSB is unique to type-I and excluded from type-II TARPs. b Alignment of TARPγ2 structure in cyan with other TARP family members (TARPγ3, pink, PDB: 8C2H; TARPγ5, green, PDB: 7RZ5; TARPγ8, purple, PDB: 8AYN; GSG1L, red, PDB: 7RZ9). c Zoomed in view of TARP extracellular domains illustrating differing orientations in the β1-β2 loops. d View of the TARP cleat motif illustrating conservation among all TARP family members. e Model of predicted β1-β2 loop orientations between type-I and type-II TARPs illustrating distinct potential contacts between TARP subtypes and AMPARs.

Fig. 3. The loop DSB is required for enhancement of AMPAR activation by TARPγ2.

a Schematic representation of the GluA2 (blue)-TARPγ2 (cyan) fusion construct used for electrophysiology demonstrating the location of the ΔDSB mutation (pink). b Representative traces, c current densities (two-tailed Welch’s t-test, p = 0.0026), d percentages of desensitization (two-tailed Welch’s t-test, p = 0.0014) and e time constants of desensitization kinetics of AMPARs complexed with either WT or ΔDSB TARPγ2 during a 500 ms exposure to 1 mM Glutamate (two-tailed Welch’s t-test, p = 0.33). WT, black, n = 13; ΔDSB, pink, n = 9; Bars represent mean ± SEM; **p < 0.01. Source data are provided as a Source Data File.

Fig. 4. Model of AMPAR regulation by TARPγ2.

Cartoon representation of TARPγ2 showing extracellular features that are critical for AMPAR modulation. The β1-β2 loop, the Loop Anchor DSB, and the TM3-Β5 loop (pink) are predicted to modulate AMPAR gating from TARPs in the “Y” position and, when mutated, increase the fraction of desensitized AMPARs compared to WT TARPγ2. The β4-TM2 loop (purple) is predicted to modulate AMPAR gating from the “X” position and, in addition to regulating the fraction of desensitized AMPARs, also regulates τ_des.