The expression of dominant-negative subunits selectively suppresses neuronal AMPA and kainate receptors.
Glutamate-gated ion channels are widely expressed in neurons where they serve a host of cellular functions. An appealing, but yet unexplored, way to delineate the functions of particular glutamate receptor subtypes is to direct the expression of dominant-negative and gain-of-function mutant subunits. We tested the ability of two dominant-negative subunits, an alpha-amino-3-hydroxy-5-methyl-isoxazolproprionic acid receptor subunit and a kainate receptor subunit, to silence recombinant and neuronal glutamate receptors. Co-expression studies in non-neuronal cells indicated that the inclusion of a single mutant subunit was sufficient to silence the receptor. When expressed in cerebellar granule cells, the dominant-negative subunits silenced native channels in a subtype-specific fashion. Immunocytochemical staining of control and transfected neurons, as well as studies with a gain-of-function glutamate receptor-1 mutant, indicated that the mutant subunits were expressed at levels roughly equal to the total abundance of related native subunits, and both dominant-negatives suppressed native channel expression 60-65% when tested 24 h post-transfection. If co-assembly of the mutant subunits with related native subunits is combinatorial, this level of suppression gives receptor half-lives of approximately 20 h.
Robert
Antoine
A
Department of Pharmacology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520-8066, USA.
Hyde
Rhonda
R
Hughes
Thomas E
TE
Howe
James R
JR
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D002490:Central Nervous System / Q000378:metabolism*; D002525:Cerebellar Cortex / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D018690:Excitatory Amino Acid Agonists / Q000494:pharmacology; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018698:Glutamic Acid / Q000378:metabolism / Q000494:pharmacology; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins; D009154:Mutation / Q000235:genetics; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018091:Receptors, AMPA / Q000235:genetics / Q000378:metabolism*; D018092:Kainic Acid Receptors / Q000235:genetics / Q000378:metabolism*; D009435:Synaptic Transmission / Q000235:genetics*; D014162:Transfection; D000097806:GluK2 Kainate Receptor
D018076:DNA, Complementary; D018690:Excitatory Amino Acid Agonists; D018691:Excitatory Amino Acid Antagonists; D018698:Glutamic Acid; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D018091:Receptors, AMPA; D018092:Kainic Acid Receptors; D000097806:GluK2 Kainate Receptor