Variations in nAChR hα6 subunit influence the current responses of human α6β2β3V9’S-nAChRs expressed in oocytes using codon optimized nAChR hβ2 subunits. Mean (±SEM) peak inward current responses upon exposure to 100 μM nicotine (5 sec exposure; ordinate) are estimated from oocytes (n = 3-7) voltage clamped at −70 mV and heterologously expressing the indicated nAChR subunits. Current responses of hα6hβ2hβ3V9’S-nAChR are completely abolished (D57N or S156R), partially abolished (S43P, N46K, R87C, D92E or N171K), not changed (E101K, A112V, A184D, N203T or I226T) and increased (R96H, D199Y or S233C) as a result of the indicated variations in nAChR hα6 subunits. Oocytes coexpressing nAChR hα6(D199Y) subunits, codon optimized hβ2 subunits and hβ3V9’S subunits yield largest current responses to nicotine. Comparisons of peak current responses between control (hα6hβ2hβ3V9’S-nAChR) and variant nAChR groups were analyzed using one-way ANOVA with Dunnett’s multiple comparisons test (*, p < 0.05; and **, p < 0.01).