The αM4 transmembrane domain of the nicotinic acetylcholine receptor (AChR) is flanked by two basic amino acids (His408 and Arg429) 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 (His408Ala, Arg429Ala, Arg429Glu, His408Ala/Arg429Ala, and His408Ala/Arg429Glu) 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 ([125I]α-BTX) binding, fluorescence microscopy, and equilibrium sucrose gradient centrifugation. Cell-surface [125I]α-BTX binding diminished ˜40% in His408Ala and as much as 95% in the Arg429Ala mutant. Reversing the amino acid charge (e.g., Arg429Glu) 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 His408Ala. We conclude that His408 and Arg429 in αM4 are involved in assembly and cell-surface targeting of muscle AChR. Arg429 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.