Accessibility of MTS reagents along the lateral fenestration. (A) The mutated residues are shown in stick representation (blue, carbon atoms; red, oxygen; dark blue, nitrogen), and the remaining region is shown in cartoon representations (gray). A transmembrane pore-lining residue, T336, is shown in orange. Representative residues are labeled. (B–E) Accessibility of two different-size MTS reagents in the presence of ATP. Superimposed scaled current traces recorded in response to ATP without (black) and with (blue) MTSET or MTS-TPAE applications (blue bars) show no modification for Q52C (B) and S326C (C), and modification for I328C (D) and T336C (E). (F) TR-MTSEA is accessible to I328C in the open state but not in the closed state. Exposing the channels to TR-MTSEA for 10 s in the closed state did not modify the ATP-induced current, whereas a 6-s exposure in the presence of ATP modified the current.