Figure 5.

MTS Probing Confirms Disulfide Bond Oxidation and Reduction. A. Sample current traces for oocytes expressing the α3L158CG181Cβ2 mutant receptor are shown for separate experiments probing oxidation (H₂O₂, left) and reduction (DTT, right) treatments by a sequential treatment with methanesulfonate dansyl (MTS). Responses evoked by application of 5 μM ACh (solid traces) or 5 μM ACh and 10 μM Mor (dotted traces) were measured before and after treatment, and then after a 2-min exposure to 50 μM MTS-dansyl; the treatments were 4.4 mM H₂O₂ + 100 μM ACh + 10 μM Mor (5 min) and 40 μM DTT (5 min). The holding potential was −60 mV. B. Collated data for a series of five experiments on the α3L158CG181Cβ2 mutant receptor similar to that described in A are shown, for responses evoked by either 5 μM ACh alone or 5 μM ACh + 10 μM Mor. The mean percentage change following MTS-dansyl treatment [(I_{post MTS}/I_{pre MTS} −1)×100] is plotted for each experiment; error bars represent the relative SEM. Control (white bars) refers to the effects (I_{post MTS}/I_{pre MTS}) of 5 min MTS-dansyl exposure. H₂O₂ w/A + M (hashed bars) is the standard treatment of 5 min 4.4 mM H₂O₂ + 100 μM ACh + 10 μM Mor, whereas H₂O₂ alone (gray bars) is simply 5 min at 4.4 mM H₂O₂. H₂O₂ w/A + M with wash (hatched bars) was an experiment in which the oxidation treatment was as described above, but oocytes were then washed for 200–300 sec with saline prior to the MTS probe (See also Figure 4). Finally, DTT (black bars) refers to treatment of a 5-min exposure at 40 μM. Replicates were n = 10 (Control); n = 20 (H₂O₂ w/A + M); n = 6 (H₂O₂ alone); n = 11 (H₂O₂ w/A + M with wash); n = 12 (DTT).