Functional assessment of PCFT aspartate mutants. [³H]-MTX influx was assessed at pH 5.5 and 37°C over 1 minute at a concentration of 0.5μM in Hela-R1-11 transfectants. (A) Initial screening of Asp mutants. (B) Studies focused on D109, D156, and D331 residues replaced with Glu, Asp, or Ala. Data are the mean ± SEM from 3 independent experiments.