Role of the N-terminal alpha-helix in biogenesis of alpha7 nicotinic receptors.
We studied the role of the alpha-helix present at the N-terminus of nicotinic acetylcholine receptor (nAChR) subunits in the expression of functional channels. Deletion of this motif in alpha7 subunits abolished expression of nAChRs at the membrane of Xenopus oocytes. The same effect was observed upon substitution by homologous motifs of other ligand-gated receptors. When residues from Gln4 to Tyr15 were individually mutated to proline, receptor expression strongly decreased or was totally abolished. Equivalent substitutions to alanine were less harmful, suggesting that proline-induced break of the alpha-helix is responsible for the low expression. Steady-state levels of wild-type and mutant subunits were similar but the formation of pentameric receptors was impaired in the latter. In addition, those mutants that reached the membrane showed a slightly increased internalization rate. Expression of alpha7 nAChRs in neuroblastoma cells confirmed that mutant subunits, although stable, were unable to reach the cell membrane. Analogous mutations in heteromeric nAChRs (alpha3beta4 and alpha4beta2) and 5-HT(3A) receptors also abolished their expression at the membrane. We conclude that the N-terminal alpha-helix of nAChRs is an important requirement for receptor assembly and, therefore, for membrane expression.
Castillo
Mar
M
Instituto de Neurociencias, Universidad Miguel Hernández-CSIC, Sant Joan d'Alacant, Alicante, Spain.
Mulet
José
J
Aldea
Marcos
M
Gerber
Susana
S
Sala
Salvador
S
Sala
Francisco
F
Criado
Manuel
M
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002417:Cattle; D045744:Cell Line, Tumor; D049452:Green Fluorescent Proteins / Q000235:genetics; D007930:Leucine / Q000235:genetics; D008958:Models, Molecular; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation / Q000235:genetics; D009447:Neuroblastoma; D009865:Oocytes; D011392:Proline / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics / Q000502:physiology; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D044406:Receptors, Serotonin, 5-HT3 / Q000235:genetics; D014162:Transfection / Q000379:methods; D014981:Xenopus; D064569:alpha7 Nicotinic Acetylcholine Receptor
D002038:Bungarotoxins; D011978:Receptors, Nicotinic; D044406:Receptors, Serotonin, 5-HT3; D064569:alpha7 Nicotinic Acetylcholine Receptor; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D011392:Proline; D007930:Leucine