Fig. 4.(A) Open channel measurements for the Leu72Cys mutant. Positive transfectants in the whole-cell clamp configuration were maintained at –40 mV with the same bath and pipette solution (140 mM NaCl, 10 mM HEPES, 1 mM CaCl₂pH 7.4). Following one rapid stimulation with 30 µM FMRF-amide, the clamped cells were perfused for 3 min with 30 µM FMRF-amide plus or minus 1 mM MTSET. Note the stability of the FMRF-amide-evoked current, and its exponential decay in the presence of MTSET. F, 30 µM FMRF; R, rinse; M, 1 mM MTSET. The same protocol has been applied for all the mutants that were shown to be accessible to MTSET in the closed configuration, and the recordings are similar, apart from mutants Ala71Cys, Leu85Cys and Ser86Cys, which inactivated upon continuous FMRF-amide stimulation. (B) Histogram summarizing the sensitivity of the mutants to 1 mM MTSET in the presence and absence of FMRF-amide. Experimental procedures were carried out as described in (A), and the results were compiled from at least six independent experiments, as described in the legend to Figure 3 and in Materials and methods. Note that the percentages of inhibition are similar in the open and closed configuration, apart from position 72, which becomes unmasked upon channel opening.