FIG. 4.

Effect of intracellular MTS reagent on the conductances of M₂ cysteine substitution mutants. (A) The conductance of only one mutant, M₂-W41C, was decreased by intracellular application of 2 mM MTSET. The M₂-H37C mutant (labeled “ia”) had activity that was too low to measure inhibition accurately in this assay. (B) M₂-W41C currents were immediately decreased upon microinjection of a concentrated solution of MTSET (40 mM) into the oocyte. The trend line shown in gray is the extrapolated current in the absence of MTSET. Note that impaling the oocyte with the injection pipette while under two-electrode voltage clamp had no significant effect on the stability of the recording. (C) Mock injection with water had no effect on the observed current. (D) M₂-G34C currents were not affected by microinjection of MTSET, in contrast to the inhibition observed when sulfhydryl-specific reagent was applied extracellularly (Fig. 1).