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VOL. 71 (4), 821-833, 2005 CHOLINERGIC CURRENTS IN XENOPUS...

However, expression systems a7, a8 and a9 result in active homo-
oligomeric receptors (3, 5). The expression of combinations of
distinct mRNA subunits in Xenopus oocytes shows the individual
pharmacological profiles of the subunit combinations and which
makes it possible to identify the type of nicotinic receptor according
to its profile (6). The specific combination of the types of subunits
determines the pharmacology of these receptors (7).

The presence of distinct subunits in human brain has been
established by in situ hybridization techniques, but we do not know
the combination of the subunits in each native receptor. In addition
functional activity, has been tested by expressing the mRNA encoding
subunits in Xenopus oocytes. Here we assessed the physiology of the
human neuronal nicotinic acetylcholine receptor by transplanting
membranes obtained from human brain into Xenopus oocytes. This
technique alowed us to study the pharmacology of native receptors
in their original lipid and proteic environment.

MATERIALS AND METHODS

1. Subcellular fractionation protocol in human brain

Frozen brain tissue was obtained from the «Banc de Teixits
Neurològics. Serveis Científico-Tècnics. Universitat de Barcelona.
Hospital Clínic», at 7 h post-mortem from a 66-year-old male donor
with no history of neurological disorder (reference BK360). The
samples were from the frontal cortex and hippocampus.

Subcellular fractionation was performed following the method
described by Blackstone et al. (8). All procedures were performed at
4º C. About 1 g of frozen brain was homogenized 10 times at 600
r.p.m. with a glass-teflon homogenizer (Potter S, B. Braun Biotech
International) in 10 volumes with a solution 4 mM HEPES, 1 mM
EDTA (pH 7.4) with 2 µg/ml leupeptin, 1mM phenyl-methylsulfonyl
fluoride (PMSF), 20 µg/ml aprotinin, 2.5 µg/ml pepstatin. The
homogenate was agitated for 30 min and centrifuged at 1000 g for
10 min. The supernatant was then centrifuged at 25000 g for 20 min.
The resulting sediment was resuspended in the solution plus 0.25 M
sucrose and layered onto a discontinuous sucrose gradient

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