Fig. 4

Currents and number of receptors measured after the expression of concatenated human nAChRs. All concatemers have an enforced 2α:3β stoichiometry (indicated by the inset of the schematic receptor). A Wild-type receptor [F106 and S108, β2(FS), black] generates a CRC with a monophasic fit. A similar concentration–response profile was observed for concatenated α4β2 nAChRs with a S108C substitution (red, FC, substitution indicated by bold font; P3 = position 3 within the linked receptor) in a single β2 subunit. However, concatenated α4β2 nAChRs with two (green) or all three (blue) β2 subunits containing S108C in the indicated positions generate no current. CRC analysis can be found in Table 3 and Additional file 4, n = 7–14. B Raw current traces to increasing concentrations of ACh. C Concatemers where a single β2 subunit has a S108C substitution [β2(FC)] show significantly reduced currents. As stated in A, concatemers with two or three S108C-containing β2 subunits generate no current (n = 7–14). D We repeated the experiment shown in C and then measured the number of receptors in the plasma membrane in the same oocytes. Baseline recordings of maximal currents (n = 21). E Measurements of receptor number in the plasma membrane in oocytes from D (n = 3, 7 pooled oocytes in each experiment). F ACh-induced maximal currents in concatenated α4β2 nAChRs with an F106L substitution in addition to the S108C [β2(LC)] (n = 21). G Measurements of receptor number in the plasma membrane in oocytes from F (n = 3, 7 pooled oocytes in each experiment). The addition of F106L substitution to S108C [β2(LC)] did not rescue the effect of S108C. In both E and G, concatenated α4β2 nAChRs with a single S108C-containing β2 subunit have significantly reduced numbers of receptors in the plasma membrane when compared to controls. Those concatenated α4β2 nAChRs with two or three β2 subunits harboring the S108C mutation are not expressed in the plasma membrane (i.e., the values were no different from uninjected oocytes). Specific binding was measured as counts per minute (cpm). Data are shown as means ± SD and were analyzed using the Brown-Forsythe and Welch ANOVA tests, followed by Dunnett’s T3 multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001