Fig. 5.Mapping of the charge selectivity filter and amiloride blockage positions. This histogram shows the accessibility of the MTSET-sensitive mutants to the anionic 10 mM MTSES in the closed configuration and the protection from 1 mM MTSET inhibition by 100 µM amiloride. For MTSES accessibility, the experimental procedure is the same as in Figure 2, with 10 mM MTSES replacing 1 mM MTSET. For amiloride protection, clamped cells were tested for FMRF-amide activation, perfused with a 100 µM amiloride solution for 3 min, rinsed, and the response to FMRF-amide was re-tested. This allowed us to verify that amiloride could be rinsed efficiently in the closed configuration. The amiloride solution was then switched after 30 s to 100 µM amiloride plus 1 mM MTSET for 3 min. The cells were then rinsed and tested for residual FMRF-amide activation. For comparison, 1 mM MTSET inhibition of the various mutants in the closed configuration is shown on the same graph.