Agonist occupancy is essential for forward trafficking of AMPA receptors.
Regulated trafficking of AMPA receptors to cell surface and to synapses is an important determinant of neuronal excitability. In the present study, we have addressed the role of agonist binding and desensitization in the early trafficking of glutamate receptor-D (GluR-D) AMPA receptors. Analysis of point-mutated GluR-D receptors, via electrophysiology and immunofluorescence, revealed that agonist-binding activity is essential for efficient delivery to cell surface in transfected cell lines and in neurons. Cotransfection with stargazin could fully rescue the surface expression of nonbinding mutant receptors in cell lines, indicating that stargazin is able to interact with and promote exit of AMPA receptors from endoplasmic reticulum (ER) independently of agonist binding. Secretion of separately expressed ligand-binding domain constructs showed a similar dependency of agonist binding to that observed with full-length GluR-D, supporting the idea that glutamate-induced closure of the binding site cleft is registered by ER quality control as a necessary priming step for transport competence. In contrast to agonist binding, the ability of the receptor to undergo desensitization had only a minor influence on trafficking. Our results are consistent with the hypothesis that AMPA receptors are synthesized as intrinsically unstable molecules, which require glutamate binding for structural stability and for transport-competence.
Coleman
Sarah K
SK
Department of Biological and Environmental Sciences, Division of Biochemistry, Viikki Biocenter, University of Helsinki, Helsinki, Finland.
Möykkynen
Tommi
T
Jouppila
Annukka
A
Koskelainen
Susanna
S
Rivera
Claudio
C
Korpi
Esa R
ER
Keinänen
Kari
K
D000818:Animals; D001665:Binding Sites / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D019921:Biotinylation / Q000379:methods; D002461:Cell Line, Transformed; D002522:Chlorocebus aethiops; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology*; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008958:Models, Molecular; D018408:Patch-Clamp Techniques / Q000379:methods; D017354:Point Mutation / Q000235:genetics; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects / Q000235:genetics; D011810:Quinoxalines / Q000494:pharmacology; D018091:Receptors, AMPA / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods
D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D011810:Quinoxalines; D018091:Receptors, AMPA; C062865:2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline; D049452:Green Fluorescent Proteins; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase