A disulfide bridge between cysteines engineered at positions 33 and 21′ suppresses the gain-of-function effect of the I9′A mutation on GLIC. (A–D) Macroscopic-current responses of the indicated constructs to 5-s pulses of pH 4.5 extracellular solution. The displayed traces are representative recordings obtained in the outside-out (A–C) or whole-cell (D) configuration at −80 mV. The cysteine at position 27—the only native cysteine of GLIC—was mutated to serine. The concentration of DTT was 5 mM. The traces in C and D are two examples of recordings from the same construct obtained on different cells. The recording in D was more heavily low-pass filtered than those in A–C.