Abstract
Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This “autoinactivation” has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1–A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.
Highlights
Cellular localization and functional properties of a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are strongly influenced by transmembrane AMPA receptor regulatory proteins (TARP)
We demonstrate the presence of striking subunit- and splice variant-dependent differences in autoinactivation and present data to support the notion that autoinactivation and physical dissociation of stargazin-AMPA receptor complex are separate processes
Subunit-dependent differences in autoinactivation AMPA receptor subunits GluA1-4 were expressed together with stargazin in transiently transfected HEK293 cells, and the resulting homomeric channels were characterized by using whole cell patch clamp recordings
Summary
Cellular localization and functional properties of a-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are strongly influenced by transmembrane AMPA receptor regulatory proteins (TARP) (for reviews, see [1,2,3]). Stargazin (c2), the founding and best characterized member of TARP family, enhances AMPA receptor function by at least two distinct mechanisms. It is a key operator in AMPA receptor trafficking by promoting receptor transport to cell surface and stabilization to synaptic membrane [4,5,9]. Association with stargazin leads to profound changes in agonist and antagonist pharmacology of AMPA receptors [15,16,17,18]. Due to this profound modulation and the near-stoichiometric association of native AMPA receptors with stargazin and related TARP [19], the complex between TARP and AMPA receptor has become a critical subject for studies addressing the structure and function of AMPA receptors
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