Figure 5: Leu99 in hSERT is crucial for the optimal transport rate.
From: A conserved leucine occupies the empty substrate site of LeuT in the Na+-free return state
Leu99hSERT is equivalent to Leu25LeuT (see Fig. 1). (a,b), [3H]-5-HT uptake experiments in HEK-293-MSR cells expressing hSERT with Leu99 mutations show that the apparent substrate affinity is largely unaffected by mutations of Leu99 (a), whereas the maximum uptake rates are dramatically reduced for even the most conservative mutations (b,c), Transport rates show no systematic correlation with side chain volumes but rather a highly specific requirement for Leu, further supporting a specific two-site function of Leu99. For a–c, bars or points represent the mean from at least three independent experiments and the error bars the resulting s.e.m. A one-way ANOVA was used to compare KM (a) or Vmax (b) of each mutant to wt hSERT without any significant changes found for KM. All mutant Vmax values were found to be significantly decreased relative to wt hSERT. *P<0.05, **P<0.01.
