Cornichon homologs (CNIHs) are AMPA-type glutamate receptor (AMPAR) auxiliary subunits that modulate AMPAR ion channel function and trafficking. Mechanisms underlying this interaction and functional modulation of the receptor complex are currently unclear. Here, using proteins expressed from mouse and rat cDNA, we show that CNIH-3 forms a stable complex with tetrameric AMPARs and contributes to the transmembrane density in single-particle electron microscopy structures. Peptide array-based screening and in vitro mutagenesis identified two clusters of conserved membrane-proximal residues in CNIHs that contribute to AMPAR binding. Because CNIH-1 binds to AMPARs but modulates gating at a significantly lower magnitude compared with CNIH-3, these conserved residues mediate a direct interaction between AMPARs and CNIHs. In addition, residues in the extracellular loop of CNIH-2/3 absent in CNIH-1/4 are critical for both AMPAR interaction and gating modulation. On the AMPAR extracellular domains, the ligand-binding domain and possibly a stretch of linker, connecting the ligand-binding domain to the fourth membrane-spanning segment, is the principal contact point with the CNIH-3 extracellular loop. In contrast, the membrane-distal N-terminal domain is less involved in AMPAR gating modulation by CNIH-3 and AMPAR binding to CNIH-3. Collectively, our results identify conserved residues in the membrane-proximal region of CNIHs that contribute to AMPAR binding and an additional unique segment in the CNIH-2/3 extracellular loop required for both physical interaction and gating modulation of the AMPAR. Consistent with the dissociable properties of binding and gating modulation, we identified a mutant CNIH-3 that preserves AMPAR binding capability but has attenuated activity of gating modulation.
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