Variations in nAChR hα6 subunit influence the current responses of human α6β2β3V9’S-nAChR expressed in oocytes using WT nAChR hβ2 subunits. Attempts were made to verify the results obtained for human α6β2β3V9’S-nAChR using WT nAChR hβ2 subunits instead of codon optimized 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. (A) Oocytes coexpressing nAChR hα6(R96H, D199Y or S233C), hβ2WT and hβ3V9’S subunits yield current responses to nicotine that are higher than those expressing nAChR hα6, hβ2WT and hβ3V9’S subunits. These results are in agreement with those obtained using codon optimized hβ2 subunits. (B) Initial recordings 3 days after cRNA injection indicated that oocytes expressing WT hα6 or variant hα6(E101K, A112V, A184D, N203T or I226T) subunits along with WT hβ2 and hβ3V9’S subunits yield ~10-30 nA of current in response to 100 μM nicotine [see the hα6hβ2hβ3V9’S-nAChR response in (A)]. However recordings done after 2 additional days of waiting indicated that nicotine elicited current responses from human α6E101Kβ2β3V9’S-, α6A112Vβ2β3V9’S-, α6A184Dβ2β3V9’S-, α6N203Tβ2β3V9’S- or α6I226Tβ2β3V9’S-nAChRs are equal (p > 0.05) to those obtained from human α6β2β3V9’S-nAChRs. These results are in agreement with those obtained using codon optimized hβ2 subunits. Comparisons between groups were analyzed using one-way ANOVA with Tukey’s post hoc comparison and only those differ from the control (hα6hβ2hβ3V9’S-nAChR) are shown with asterisks. *, p < 0.05; and **, p < 0.01.