title: Molecular dissection of the interaction between the AMPA receptor and cornichon homolog-3.
abstract: 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.
authors.affiliation: Department of Molecular Physiology and Biophysics and Department of Chemistry and Biochemistry and Neurosciences Graduate Programs, University of California, San Diego, La Jolla, California 92093.
authors.forename: Natalie F
authors.initials: NF
authors.lastname: Shanks
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, CB2 0QH, Cambridge, United Kingdom.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Cais
authors.affiliation: Department of Chemistry and Biochemistry and.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Maruo
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, and.
authors.forename: Jeffrey N
authors.identifier: http://orcid.org/0000-0002-8173-5580
authors.initials: JN
authors.lastname: Savas
authors.affiliation: Department of Molecular Physiology and Biophysics and.
authors.forename: Elena I
authors.initials: EI
authors.lastname: Zaika
authors.affiliation: Department of Molecular Physiology and Biophysics and.
authors.forename: Caleigh M
authors.initials: CM
authors.lastname: Azumaya
authors.affiliation: Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California 92037, and.
authors.forename: John R
authors.initials: JR
authors.lastname: Yates
authors.affiliation: Medical Research Council Laboratory of Molecular Biology, CB2 0QH, Cambridge, United Kingdom.
authors.forename: Ingo
authors.initials: I
authors.lastname: Greger
authors.affiliation: Department of Molecular Physiology and Biophysics and Center for Structural Biology, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232, Department of Chemistry and Biochemistry and terunaga.nakagawa@vanderbilt.edu.
authors.forename: Terunaga
authors.initials: T
authors.lastname: Nakagawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D015640:Ion Channel Gating; D008297:Male; D008969:Molecular Sequence Data; D011485:Protein Binding; D051381:Rats; D018091:Receptors, AMPA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C539314:Cnih3 protein, rat; D018091:Receptors, AMPA
keywords: AMPA receptor; complex; cornichon; gating modulation; molecular mechanism; single particle EM