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P Charnet, C Labarca and HA Lester
CNRS-CRBM, Montpellier, France.
The nicotinic acetylcholine receptor can be expressed in Xenopus oocytes by injection of in vitro synthesized RNA for the alpha, beta, gamma, and delta mouse muscle subunits. However, detectable responses can also be obtained by injection of alpha, beta, and delta subunit RNA only. The receptors expressed in this case (gamma-less receptors) share many of the properties of the normal receptor, including relaxation time constants, Hill slope, and relative permeability for Na+, K+, Cs+, and Tris+. The major single-channel conductances of alpha beta gamma delta and alpha beta delta receptors are similar (34.2 +/- 2.9 and 38.5 +/- 0.6 pS, respectively) but clearly different from the major conductances seen after the combined injection of alpha beta delta mouse subunit RNA and Xenopus gamma subunit RNA. Mutations in the second transmembrane segment of the alpha and beta subunits, known to affect open time and blockade by QX-222, are equally effective in the gamma-less receptor. These data strongly suggest that the gamma-less receptor has the same pore diameter as the normal receptor and that alpha, beta, and delta subunits participate in its formation. Injection of alpha beta gamma delta well as alpha beta delta RNA produced additional subconductance states of around 25 pS. The low conductance state was sensitive to mutations introduced in the alpha or beta subunits with or without the gamma subunit, indicating that this channel did not need the gamma subunits but required at least the alpha and beta subunits to be produced. Injection of alpha beta delta and the adult-type epsilon subunit RNA gave rise to channels with conductances of 35 and 55 pS when the stoichiometry of the injection was 2:1:1:1, but only the 55-pS channel was recorded when the epsilon subunit RNA concentration was increased by 10-fold (stoichiometry of 2:1:1:10). The gamma-less receptor can thus be expressed even when the adult epsilon subunit is present. Whether gamma-less receptors are expressed at normal adult neuromuscular junctions remains unknown.
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