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Charged amino acid motifs flanking each extreme of the αM4 transmembrane domain are involved in assembly and cell‐surface targeting of the muscle nicotinic acetylcholine receptor
Author(s) -
Roccamo A.M.,
Barrantes F.J.
Publication year - 2006
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/jnr.21123
Subject(s) - endoplasmic reticulum , transmembrane domain , nicotinic acetylcholine receptor , acetylcholine receptor , mutant , biochemistry , transmembrane protein , biology , microbiology and biotechnology , amino acid , chemistry , receptor , gene
The αM4 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 αM4 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 β, δ, and ϵ subunits as stable clones in a mammalian heterologous expression system (CHO‐K1 cells). The mutants were studied by α‐bungarotoxin ([ 125 I]α‐BTX) binding, fluorescence microscopy, and equilibrium sucrose gradient centrifugation. Cell‐surface [ 125 I]α‐BTX binding diminished ∼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 αM4 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 α subunit M4 transmembrane segment. © 2006 Wiley‐Liss, Inc.