Structural Determinants of the Benzodiazepine Allosteric Regulation of GABA[sub]A[/sub] Receptor Currents
Abstract number :
1.033
Submission category :
Year :
2001
Submission ID :
632
Source :
www.aesnet.org
Presentation date :
12/1/2001 12:00:00 AM
Published date :
Dec 1, 2001, 06:00 AM
Authors :
D.M. Jones, M.S., Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI; M.T. Bianchi, Neuroscience Graduate Program, University of Michigan, Ann Arbor, MI; R.L. Macdonald, M.D., Ph.D., Neurology, Physiology, University of Michigan, Ann Arb
RATIONALE: Benzodiazepines (BZs), such as diazepam, are commonly used to treat status epilepticus. These agents act on GABA[sub]A[/sub] receptors to increase GABA[sub]A[/sub] single channel opening frequency, thereby enhancing inhibition. Several studies have implicated amino acids within the N-terminal domains of the [alpha] and [gamma] subunits of the GABA[sub]A[/sub] receptor in the binding of BZs; however, the structural basis for the coupling of BZ binding to the allosteric regulation of GABA[sub]A[/sub]R opening frequency is unclear.
METHODS: To examine the structural basis of the coupling of BZ binding to the allosteric regulation of GABA[sub]A[/sub]R opening frequency, a chimeric strategy was employed using transmembrane domain 1 (TM1) [alpha][beta][gamma]/[delta] GABA[sub]A[/sub]R chimeras. Chimeras were constructed by substitution of [delta] amino acid sequence in [alpha][beta][delta] receptors with [gamma][sub]2[/sub] amino acid sequence. Whole cell recordings were obtained from [alpha][beta][gamma]/[delta] GABA[sub]A[/sub] receptor transfected HEK293 cells in the presence of 1 [mu]M or 5 [mu]M DZP + 1 [mu]M (EC[sub]20[/sub]) GABA.
RESULTS: We confirmed our previous findings that [delta]-containing GABA[sub]A[/sub] receptors are diazepam insensitive. Substitution of the entire extracellular N-terminal domain of [delta] with [gamma][sub]2[/sub] sequence failed to confer diazepam sensitivity at the level observed in wild-type [alpha][sub]1[/sub][beta][sub]3[/sub][gamma][sub]2[/sub] receptors. Other chimeras that extended [gamma][sub]2[/sub] sequence into TM1 exhibited varying levels of diazepam sensitivity.
CONCLUSIONS: The binding site for benzodiazepines is thought to be formed by the extracellular N-terminal domains at the interface between the [alpha] and [gamma] subunits of GABA receptors. Interestingly, the substitution of the N-terminus of [gamma][sub]2[/sub] in [alpha][beta][delta] chimeras was insufficient to confer diazepam sensitivity. Our results suggest that TM1 of the GABA[sub]A[/sub] receptor [gamma][sub]2[/sub] subunit is involved in diazepam modulation of the receptor, and that it plays a role in the coupling of BZ binding to BZ potentiation of GABA[sub]A[/sub]R current at a site distinct from the BZ binding site.
Support: NIMH 5T32 MH 19547 (DMJ) and NINDS NS 33300 (RLM)