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LAURA TEIXIDÓ Y COLS. AN. R. ACAD. NAC. FARM.

containing 0.8 M/1 M/1.2 M sucrose and centrifuged for 2 h at 65000
g in a Beckman SW-28 rotor. A synaptic membrane fraction was
yielded at the 1 M/1.2 M sucrose interface, myelin in the floating
fraction and a mitochondrial fraction as the pellet. The synaptic
plasma membrane fraction was diluted 1/3 in a solution HEPES 10
mM and centrifuged for 30 min at 50000 g. The resulting sediment
was resuspended in a HEPES 10 mM, KCl 140 mM (pH 7.4) solution,
frozen with liquid nitrogen and stored at –80 ºC until use.

2. Animals and solutions

Mature females of Xenopus laevis were purchased from the «Centre
d’Elevage des Xenopes» (Montpellier, France), and were anaesthetized
by immersion in water containing 0.17 % tricaine. A few lobes were
removed from an ovary through a small incision in the abdomen.

Solutions for Xenopus oocytes: Barth’s solution contained: 88 mM
NaCl, 1 mM KCl, 0.33 mM Ca(NO3)2, 0.41 mM CaCl2, 0.82 mM MgSO4,
2.40 mM NaHCO3, and 20 mM HEPES at pH 7.4, supplemented with
100 IU/ml penicillin and 0.1 mg/ml streptomycin. Recording solution:
115 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, and 10 mM HEPES at pH
7.4. None of the Xenopus female donors used in this study exhibited
muscarinic acetylcholine receptor currents in their oocytes.

3. Oocyte preparation, membrane transplantation
and recording

Oocytes at stages V and VI (9) were removed out and kept at 15-
16º C in sterile Barth’s solution. Healthy oocytes were microinjected
with volumes within 50-100 nl of thawed suspension of human brain
membranes (3-10 mg/ml) following Marsal et al. (10), by means of an
injector (WPI, model A203XVZ). Samples were sonicated prior to
injection.

Before recording (3-4 h), oocytes were treated with collagenase
type 1A (Sigma) (0.5 mg/ml) for 45-50 min at room temperature to
remove the surrounding layers (11). They were then voltage-clamped

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