Fig. 5.

Reactivity of strategically introduced cysteines to MTSET⁺. Summary data for the block of MTSET⁺ in the absence (A) and presence (B) of cytoplasmic Na⁺ with Ca²⁺ present in the pipette (extracellular). In the absence of Na⁺ (replaced with Cs⁺), the ion binding sites are exposed to the cytoplasm (inward state configuration). MTSET⁺ (5 mM) was applied from the intracellular side of the membrane, and the percent of block was measured after reagent washout following up to 1 min continuous exposure, such as in the case of unresponsive mutants. A large number of residues lining TMS2 and -7 were inactivated by MTSET⁺, indicating that these two TMSs are part of the cytoplasmic ionic pathway. The speed of the current block while NCX1.1 was held in the inward-facing state is summarized in C, whereas mean rate values during transport are shown in D. ^#Values represent an upper limit; they cannot be accurately measured because of our limited temporal resolution, and faster kinetics may occur. Mutant exchangers that were not modified by MTSET⁺ are indicated as no block (N.B.).