Abstract
AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Tyr-454 and Arg-461, in its ligand binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Tyr-454/Arg-461 but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Tyr-454/Arg-461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Tyr-454 and Arg-461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway.
Highlights
␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are tetrameric ligand-gated ion channels that mediate fast excitatory neurotransmission in vertebrate brain [1, 2]
The relative strength of N-terminal domains (NTD) contacts can partly explain the strong preference of certain subunits like GluA3 for heteromeric assembly, and the ligand binding domains (LBD) harbors important structural determinants that control the biogenesis of AMPA receptors in a subunit or subunit variant-dependent manner [12,13,14]
The majority of GluA2 expressed in HEK293 cells was soluble in Triton X-100, whereas most of immunoreactive GluA3 appeared as insoluble aggregates
Summary
DNA Constructs—All cDNAs encoding full-length AMPA receptor subunits or mutants thereof represented the rat AMPA receptor subunits; the flip/flop isoform is indicated in “Results” and figure legends; GluA2 is the edited (Q607R) form. Biochemical Analyses—Transfected cells were extracted in TNE buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5 mM EDTA) containing 1% (w/v) Triton X-100 followed by ultracentrifugation at 100,000 ϫ g for 1 h. Triton X-100 extracts of transfected cells were incubated with prewashed Gamma-bind Sepharose (GE Healthcare) and appropriate antibody (at concentration detailed above) and mixed for 2 h at 4 °C. The samples used for BN-PAGE were obtained from the sucrose density gradients with the exception of the analysis of crude cell extracts. In the latter case Triton X-100 extracts prepared from transfected cells were cleared by centrifugation at 12,000 ϫ g for 20 min, mixed with native loading buffer, and used for electrophoresis. Analysis of desensitization kinetics of patch clamp recordings was done by Clampfit 10.2 software (Molecular Devices, Sunnyvale, CA) using single exponential fit paradigm
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