Functional and non-functional isoforms of the human muscle acetylcholine receptor.
1. The properties of a recently identified isoform of the human muscle nicotinic acetylcholine receptor (AChR) alpha subunit (alpha +), which in muscle is expressed at similar levels to the alpha subunit, were investigated by both electrophysiological and biochemical approaches following expression in Xenopus laevis oocytes. The single-channel properties of adult (alpha 2 beta delta epsilon) and fetal (alpha 2 beta delta gamma) forms of the human AChR were also investigated. 2. The mean burst duration of adult channels (4.1 +/- 0.3 ms, mean +/- S.E.M., n = 5) is half that of fetal channels (7.9 +/- 0.6 ms, n = 4), while the single-channel conductance is larger (62.2 +/- 0.8 and 37.9 +/- 1.6 pS for adult and fetal channels, respectively), comparable to the developmental changes in single-channel properties observed for other mammalian species. 3. In contrast to the alpha isoform, the alpha + subunit does not bind 125I-labelled alpha-bungarotoxin or monoclonal antibodies directed against the AChR 'main immunogenic region' (MIR), illustrating why the alpha + subunit was first detected through screening of cDNA libraries. 4. By using site-directed mutagenesis to produce subunits that conferred different single-channel conductances on the AChR, we demonstrate that the alpha + isoform is not integrated into functional AChRs. 5. The mutagenesis experiments also revealed that the two alpha subunits within an AChR pentamer are not equivalent within the pore lining region.
Newland
C F
CF
Neurosciences Group, Institute of Molecular Medicine, Oxford, UK.
Beeson
D
D
Vincent
A
A
Newsom-Davis
J
J
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002038:Bungarotoxins / Q000494:pharmacology; D018076:DNA, Complementary / Q000378:metabolism; D004594:Electrophysiology; D006801:Humans; D007457:Iodine Radioisotopes; D007536:Isomerism; D008969:Molecular Sequence Data; D009132:Muscles / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011233:Precipitin Tests; D011950:Receptors, Cholinergic / Q000235:genetics / Q000378:metabolism*; D014981:Xenopus
D000911:Antibodies, Monoclonal; D002038:Bungarotoxins; D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D011950:Receptors, Cholinergic