Charged amino acid motifs flanking each extreme of the alphaM4 transmembrane domain are involved in assembly and cell-surface targeting of the muscle nicotinic acetylcholine receptor.
The alphaM4 transmembrane domain of the nicotinic acetylcholine receptor (AChR) is flanked by two basic amino acids (His(408) and Arg(429)) located at its cytoplasmic- and extracellular-facing extremes, respectively, at the level of the phospholipid polar head regions of the postsynaptic membrane. A series of single and double alphaM4 mutants (His(408)Ala, Arg(429)Ala, Arg(429)Glu, His(408)Ala/Arg(429)Ala, and His(408)Ala/Arg(429)Glu) of the adult muscle-type AChR were produced and coexpressed with wild-type beta, delta, and epsilon subunits as stable clones in a mammalian heterologous expression system (CHO-K1 cells). The mutants were studied by alpha-bungarotoxin ([(125)I]alpha-BTX) binding, fluorescence microscopy, and equilibrium sucrose gradient centrifugation. Cell-surface [(125)I]alpha-BTX binding diminished approximately 40% in His(408)Ala and as much as 95% in the Arg(429)Ala mutant. Reversing the amino acid charge (e.g., Arg(429)Glu) abolished cell-surface expression of AChR. Fluorescence microscopy disclosed that AChR was retained at the endoplasmic reticulum, with an enhanced occurrence of unassembled AChR species in the mutant clones. Centrifugation analysis confirmed the lack of fully assembled AChR pentamers in all mutants with the exception of His(408)Ala. We conclude that His(408) and Arg(429) in alphaM4 are involved in assembly and cell-surface targeting of muscle AChR. Arg(429) plays a more decisive role in these two processes, suggesting an asymmetric weight of the charged motifs at each extreme of the alpha subunit M4 transmembrane segment. (c) 2006 Wiley-Liss, Inc.
Roccamo
A M
AM
Instituto de Investigaciones Bioquimicas and UNESCO Chair of Biophysics and Molecular Neurobiology, Bahía Blanca, Argentina.
Barrantes
F J
FJ
D020816:Amino Acid Motifs / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000502:physiology; D011485:Protein Binding; D020836:Protein Structure, Quaternary / Q000502:physiology*; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D011978:Receptors, Nicotinic