Society for Neuroscience Abstracts, 22, 509.3 (1996)
Abstract
Although GABAA receptor alpha subunits are important for binding
GABA and benzodiazepines (BZDs), the presence of a gamma subunit is required
for high affinity BZD binding (Pritchet et al., Nature 338, 1989). In
order to determine which portions of the gamma subunit confer BZD binding, we
generated chimeric protein combinations of rat gamma 2 and alpha 1 subunits
(Liu et al., Science 266, 1994) and expressed them with wild-type beta 2
and/or alpha 1 subunits. We have generated over two dozen chimeric subunits
with crossovers in the 5' extracellular region. Restriction enzyme mapping and
DNA sequencing revealed seven distinct gamma 2/alpha 1 chimeras which encode
complete ORFs. Of those expressed with wild-type alpha 1 and beta 2 subunits in
HEK 293 cells, two exhibited significant levels of [3H]flunitrazepam
binding, and when co-expressed with wild-type alpha 1 and beta 2 cRNA in Xenopus
oocytes, displayed diazepam potentiation of GABA-mediated chloride current. By
comparing where the crossover occurred in chimeras that were responsive to BZDs
with those that were not, we have identified a 45 amino acid residue region in
the 5' extracellular portion of the gamma 2 subunit that is necessary for BZD
binding and BZD potentiation of the GABA-mediated chloride current. Using the
same chimeras, we have also identified a 42 amino acid region of alpha 1 which
confers GABA responsiveness when expressed with beta 2 subunits alone.
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