Assembly of GABAA receptors composed of alpha1 and beta2 subunits in both cultured neurons and fibroblasts.

  • Gorrie G
  • Vallis Y
  • Stephenson A
 et al. 
  • 17

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Abstract

GABAA receptors are believed to be pentameric hetero-oligomers, which can be constructed from six subunits (alpha, beta, gamma, delta, epsilon, and rho) 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 alpha1 and beta2 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 approximately 2 hr for both the alpha1((9E10)) and beta2((9E10)) subunits. Oligomerization of the alpha1((9E10)) and beta2((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 alpha1((9E10)):beta2((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.

Author-supplied keywords

  • Animals
  • Cell Membrane
  • Cell Membrane: metabolism
  • Cells
  • Chemical Fractionation
  • Cricetinae
  • Cultured
  • Fibroblasts
  • Fibroblasts: metabolism
  • GABA-A
  • GABA-A: chemistry
  • GABA-A: metabolism
  • Kidney
  • Kidney: cytology
  • Kidney: metabolism
  • Mice
  • Neurons
  • Neurons: metabolism
  • Receptors
  • Semliki forest virus
  • Semliki forest virus: metabolism
  • Superior Cervical Ganglion
  • Superior Cervical Ganglion: cytology
  • Superior Cervical Ganglion: metabolism
  • Time Factors

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Authors

  • George H Gorrie

  • Yvonne Vallis

  • Anne Stephenson

  • Jonathan Whitfield

  • Brenda Browning

  • Trevor G Smart

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