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Volume 17, Number 17,
Issue of September 1, 1997
pp. 6587-6596
Copyright ©1997 Society for Neuroscience
Assembly of GABAA Receptors Composed of  1 and  2
Subunits in Both Cultured Neurons and Fibroblasts
Received April 11, 1997; revised June 12, 1997; accepted June 17, 1997.
George H. Gorrie1,
Yvonne Vallis1,
Anne Stephenson3,
Jonathan Whitfield2,
Brenda Browning1,
Trevor G. Smart3, and
Stephen J. Moss1
1 Medical Research Council Laboratory of Molecular Cell
Biology and Department of Pharmacology and 2 The Eisai
Research Labs, University College, London WC1E 6BT, United Kingdom, and
3 The School of Pharmacy, London WC1N 1AX, United Kingdom
GABAA receptors are believed to be pentameric
hetero-oligomers, which can be constructed from six subunits ( ,  ,
 ,  ,  , and  ) with multiple members, generating a large
potential for receptor heterogeneity. The mechanisms used by neurons to
control the assembly of these receptors, however, remain unresolved.
Using Semliki Forest virus expression we have analyzed the assembly of
9E10 epitope-tagged receptors comprising 1 and 2 subunits in baby
hamster kidney cells and cultured superior cervical ganglia neurons.
Homomeric subunits were retained within the endoplasmic reticulum,
whereas heteromeric receptors were able to access the cell surface in
both cell types. Sucrose density gradient fractionation demonstrated
that the homomeric subunits were incapable of oligomerization, exhibiting 5 S sedimentation coefficients. Pulse-chase analysis revealed that homomers were degraded, with half-lives of ~2 hr for
both the 1(9E10) and
2(9E10) subunits. Oligomerization of the
1(9E10) and 2(9E10)
subunits was evident, as demonstrated by the formation of a stable 9 S
complex, but this process seemed inefficient. Interestingly the
appearance of cell surface receptors was slow, lagging up to 6 hr after
the formation of the 9 S receptor complex. Using metabolic labeling a
ratio of 1(9E10):2(9E10) of
1:1 was found in this 9 S fraction. Together the results suggest that
GABAA receptor assembly occurs by similar mechanisms in
both cell types, with retention in the endoplasmic reticulum featuring as a major control mechanism to prevent unassembled receptor subunits accessing the cell surface.
Key words:
Semliki Forest virus;
GABAA receptor;
density
gradients;
cultured neurons;
assembly;
endoplasmic reticulum
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