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Stable GABA A receptor intermediates in SF‐9 cells expressing α1, β2 and γ2 subunits
Author(s) -
Elster Lisbeth,
Schousboe Arne,
Olsen Richard W.
Publication year - 2000
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/1097-4547(20000715)61:2<193::aid-jnr10>3.0.co;2-s
Subject(s) - pentamer , muscimol , protein subunit , gabaa receptor , chemistry , receptor , spodoptera , blot , gene isoform , biochemistry , stereochemistry , microbiology and biotechnology , biology , recombinant dna , gene
Spodoptera frugiperda insect cells (Sf‐9 cells) were used to study GABA A receptor assembly. Time courses of the expression level of α1β2 and α1β2γ2 receptor protein showed [ 3 H]muscimol binding to appear 2 hr before [ 3 H]flunitrazepam and [ 35 S]TBPS binding. This indicates that muscimol may bind to pentamers with an immature conformation or to molecules smaller than the pentamer. Binding studies performed on fractions from sucrose gradients loaded with solubilized α1β2 or α1β2γ2 containing membranes revealed no binding other than to the pentameric fractions. Western blotting on fractionated sucrose gradients, however, clearly revealed the existence of GABA A receptor intermediates. The α1 subunit was seen in fractions corresponding to molecules smaller than the pentamer only when co‐expressed with γ2, indicating that the γ2 subunit is needed for the α1 to form relatively long lasting intermediates. Moreover, Western blots revealed multiple isoforms for each subunit. In general, it was primarily the lower molecular weight forms that were detected in the pentameric fractions. The exception being for the α1 and γ2 forms in subunit combinations that did not contain both of these subunits (i.e., α1, γ2, α1β2, β2γ2), where higher molecular weight forms were strongly represented. These findings show that α1 and γ2 prefer specific protein forms when expressed together. J. Neurosci. Res. 61:193–205, 2000. © 2000 Wiley‐Liss, Inc.