title: AMPA receptor/TARP stoichiometry visualized by single-molecule subunit counting.
abstract: Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R-mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R-mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Hastie
authors.forename: Maximilian H
authors.initials: MH
authors.lastname: Ulbrich
authors.forename: Hui-Li
authors.initials: HL
authors.lastname: Wang
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Arant
authors.forename: Anthony G
authors.initials: AG
authors.lastname: Lau
authors.forename: Zhenjie
authors.initials: Z
authors.lastname: Zhang
authors.forename: Ehud Y
authors.initials: EY
authors.lastname: Isacoff
authors.forename: Lu
authors.initials: L
authors.lastname: Chen
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: C491911:Cacng2 protein, rat; C522274:Cacng4 protein, rat; D015220:Calcium Channels; D046912:Multiprotein Complexes; D021122:Protein Subunits; D018091:Receptors, AMPA; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins