The mechanism by which ligand binding causes channel opening in pentameric ligand-gated ion channels (pLGICs) is not yet fully understood. The outer, lipid-facing helix (M4) and the lipid bilayer in which the channel is embedded have emerged as factors that affect channel opening, though details remain to be determined. Alanine mutations in the M4 helix of anionic pLGICs M4 are neutral or detrimental to channel opening, but there has so far been no clear pattern in cationic pLGICs. Alanine mutations in the α7 nicotinic acetylcholine receptor (nAChR) M4 are generally beneficial to channel opening, but are neutral or ablate function 5-HT3A M4. This apparent discrepancy in the importance of M4 led us to assess its role in a third cationic pLGIC, the α4β2 nAChR, using fluorescence and two-electrode voltage clamp to elucidate the determinants of M4 requirement. When expressed in HEK cells, 9 alanine mutations along the length of the M4, of residues of varying charge, size, and aromaticity, ablated function. However, when expressed in Xenopus oocytes, 7 of these 9 were fully functional. Thus the cellular environment contributes to determining whether certain M4 residues are required for function, and we suggest the composition of the lipid bilayer may be of major importance here.