Endogenous charged residues in the wild-type receptor have little effect on ionic permeability. Charged amino acids at the intracellular membrane interface were neutralized to examine the role of native charges on ionic permeability. Although small elevations in relative cation permeability were evident, none of the constructs increased relative cation permeability to more than 0.15, in stark contrast to the previously described deletion of P290 and mutation of A291 to E (ΔP290/A291E), shown for comparison (Wotring et al., 2003). RRR/QQM is R286Q/R287Q/R292M, DRRR/NQQM is D285N/R286Q/R287Q/R292M, and DRR/PAD is D285P/R286A/R287D. For the latter case, the mutations were to the corresponding sequence in nAChR rather than a simple neutralization.