Abstract
The GABA type A receptor (GABAAR) is the major inhibitory receptor in the mammalian central nervous system and the target of numerous pharmaceuticals. The α-subunit of these pentameric Cys-loop neurotransmitter-gated ion channels contributes to the binding of both GABA and allosteric modulators such as the benzodiazepines, suggesting a role for this subunit in the conformational changes associated with activation of the receptor. Herein we use the nonsense suppression methodology to incorporate a photoactivatable unnatural amino acid and photochemically cleave the backbone of the α subunit of the α1β2 GABAAR in a linker region that is believed to span the subunit. Proteolytic cleavage impairs GABA but not pentobarbital activation, strongly suggesting that conformational changes involving this linker region are critical to the GABA activation pathway.
Footnotes
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This work was supported by the National Institutes of Health National Institute of Neurological Disorders and Stroke [Grants NS34407, NS11756].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.109.059832.
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ABBREVIATIONS:
- GABAAR
- GABA type A receptor
- BZD
- benzodiazepine
- PB
- pentobarbital
- PCR
- polymerase chain reaction
- nAChR
- nicotinic acetylcholine receptor
- Npg
- nitrophenylglycine
- tRNA
- transfer RNA
- aa
- amino acid.
- Received July 27, 2009.
- Accepted April 2, 2010.
- Copyright © 2010 The American Society for Pharmacology and Experimental Therapeutics
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