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-RmCherry immobilizes the stargazin-GFP spots at sites of AMPA-RmCherry, 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.single-molecule counting | TIRF | glutamate receptors | stargazin G lutamate is the main excitatory neurotransmitter in the mammalian CNS. Most fast excitatory synaptic transmission in the brain is mediated by AMPA receptors (AMPA-Rs). Four AMPA-R subunits, GluA1-4, contribute to the heterotetrameric assemblies of the AMPA-R (1-4). The localization of AMPA-Rs to the postsynaptic membrane is regulated by a large number of proteins through multiple mechanisms and plays important roles in synaptic plasticity (5-9).Transmembrane AMPA-R regulatory proteins (TARPs) represent a family of AMPA-R regulatory proteins that are tightly associated with AMPA-Rs, and can be considered auxiliary AMPA-R subunits (10-12). TARPs regulate AMPA-R function by several mechanisms. They mediate the efficient cell surface expression of AMPA-Rs, modulate gating, affect agonist efficacy, and even attenuate intracellular polyamine block of calciumpermeable AMPA-Rs (13-22). The effect of stargazin (γ-2) and other TARPs on GluA1 is not neuron specific and can be accurately mimicked in nonneuronal mammalian cells (18,(23)(24)(25), as well as Xenopus oocytes (13,14,17,26,27), suggesting that the basic mechanisms for TARP/AMPA-R interaction and TARPmediated regulation of AMPA-R trafficking are preserved in nonneuronal cells.Although much is known about the interaction domains on the classical TARPs (γ-2, γ-3, γ-4, and γ-8) and AMPA-Rs that mediate assembly and modulation (13,23,24,27,28), far less is known about the stoichiometry of the TARP/AMPA-R...

The Stoichiometry and Biophysical Properties of the Kv4 Potassium Channel Complex with K+ Channel-interacting Protein (KChIP) Subunits Are Variable, Depending on the Relative Expression Level

“…We subsequently confirmed that the stoichiometry of the Kv4.2⅐KChIP4 complex affects the properties of Kv4.2 by using tandem repeat constructs in which the stoichiometry is strictly controlled. We finally applied the subunit-counting method by single-molecule imaging (31)(32)(33)(34)(35). Although KChIP4 is a cytoplasmic protein, we could successfully apply the method to determine the number of KChIP4 subunits included in a Kv4.2⅐KChIP4 complex and confirmed that the stoichiometry changes depending on the expression level of KChIP4.…”

Protein Crowding within the Postsynaptic Density Can Impede the Escape of Membrane Proteins

“…TARP proteins have not been crystallized, but the intracellular tail (N ϭ ϳ120 amino acids) of the most studied TARP representative Stargazin is estimated to be ϳ3 nm in radius assuming an unstructured globule, as calculated from the radius of gyration of a ran- et al, 2004), where N is the number of amino acids and b is a constant that is a function of the persistent length of the polymer. Given the residue count of GluK2 cytoplasmic portion is intermediate between that of GluA1 and that of GluA2 and that full AMPARs can contain anywhere from 1 to 4 Stargazin subunits (Hastie et al, 2013), we conservatively modeled the intracellular radius of the AMPAR complex to be 8 nm. The radii of adhesion molecules were taken from a random distribution ranging from 3 to 8 nm.…”

“…Divalent PDZ-binding interactions within the synapse can stabilize the mobility of transmembrane proteins more than monovalent interactions can AMPA receptors could bear multiple TARPs capable of binding PSD scaffold molecules (Hastie et al, 2013). To test the role of divalent receptor-scaffold interactions on receptor mobility in the synapse, we developed a chemically inducible heterodimerization strategy that would permit us to acutely convert our typical monovalent binding probe to one with divalent PDZ-binding capacity.…”

Control of AMPA Receptor Activity by the Extracellular Loops of Auxiliary Proteins

“…Fourth, we show that the long extracellular loop 1 of g8 is a very strong positive modulator of AMPA receptor gating, whose influence is likely held in check by the substoichiometric combination of g8 with the AMPA receptor (Hastie et al, 2013). The subunit g8 slows receptor desensitization via L1.…”

“…On the other hand, some studies of assembly made use of functional tests to assess the strength of interaction (Shi et al, 2009). Given the variable stoichiometry of assembly between different TARP isoforms (Kim et al, 2010;Hastie et al, 2013), interpreting these data, which combine the strength of association, expression and modulation into a single metric, is difficult. Very recently, a chimeric approach confirmed impressions from structural studies that transmembrane interactions are important for proper assembly, with the TM3 and TM4 segments of g2 and the M1-M3 helices of the AMPA receptor determining complex assembly.…”