Fig. 2.

Rate constants for reactivity of cytoplasmic pathway mutants with MTS reagents. (Upper) Calculated rate constants for inactivation of β-CIT binding to the indicated mutants by MTSEA (and MTSES for TMs 6 and 8). Bottom axes are SERT positions, and corresponding residues in LeuT are shown on the top axes (15). C and E indicate the cytoplasmic and extracellular ends, respectively, of each region. The green line indicates the accessibility in the cytoplasm-facing state model. The solvent-accessible surface area (SASA) for each residue was calculated as a percentage of the SASA of the same amino acid type (X) in a reference gly-X-gly tripeptide. From this, the equivalent percentage in the crystal structure was subtracted, so that the accessibility is positive only for residues that are exposed in the cytoplasm-facing state model; for all other residues, the accessibility is zero. Mutants with the highest rates of inactivation were further tested by measuring inactivation rates in the presence of 10 μM cocaine or ibogaine. (Lower) Rates of inactivation, relative to rates with the MTS reagent alone, for these selected positions. Error bars indicate the standard error of the means from three independent experiments. Asterisks indicate a significant difference from the control rate with MTS reagent alone (P < 0.05, paired Student's t test). TM5 data are from (20, 21).