Figure 3.
Effect of MTS reagent application on subsequent ACh-induced EC30–70 currents. A, Structure and size of the moiety that is added onto the Cys sulfur by MTSEA (left) and MTSES (right). Solvent-accessible surface shown. MTSES adds a moiety to the Cys sulfur that is slightly larger in volume than the one added by MTSEA modification. The distances between the added sulfur and the furthest atom of the added moiety are 4.8 and 5.3 Å for MTSEA and MTSES modification, respectively (Chem3D Ultra; CambridgeSoft). B–D, Sample current traces showing no effect of MTSEA application (2 mm, 2 min, at downward arrow) on ACh-induced current amplitudes for αI274Cβγδ (B), inhibition for αP272Cβγδ (C), or potentiation for αS266Cβγδ receptors (D). Current traces during reagent application not shown. E, Effects of MTSEA application (2 mm, 2 min) on wild-type and mutant receptors. Percentage effect on ACh-induced current amplitude (IACh) after MTSEA treatment is shown. Note the difference in scales for inhibitory and potentiating effect. Significance of effect was determined by one-way ANOVA with Dunnett's posttest with wild type as a control (n = 3–6, p < 0.05). F, Effect of MTSES application (5 mm, 2 min) on ACh- induced EC30–70 currents. Percentage effect on ACh-induced current amplitude (IACh) after MTSES treatment shown. The striped bar indicates the effect of MTSES application after MTSEA treatment (2 mm, 2 min) where the MTSEA treatment did not have a significant effect. Negative values represent an inhibition of IACh after MTS reaction, whereas positive values represent an increase in IACh. Data are represented by mean ± SEM. Effects that were significant different compared with wild type are indicated in dark gray or striped bars, nonsignificant effects in white bars. G, nAChR model of αE262C modified by MTSEA. αE262Cys-S-S-EA is in spacefill representation and the other 15 investigated positions are in stick representation. Hydrogens are omitted for clarity. Carbon = gray, oxygen = red, sulfur = yellow, nitrogen = blue. The addition of the aminoethanesulfide moiety increases the volume of the side chain of the engineered Cys to a size that is in between the size of a Lys or Arg side chain.