Effect of pH on transport activity of Nramp2 mutants at conserved histidine residues in TM6.

(A) Fe²⁺ transport in the various negative (pCB6) and positive controls (N2.3, N2GG) as well as in CHO clones expressingNramp2 mutants at the plasma membrane (His267Ala, His272Ala, His267/272Ala, His267Cys, His272Cys, His267Arg, His272Arg, His267/272Arg) was studied by the calcein-quenching assay as described in the legend to Figure 5A, with the following modifications. After loading with calcein-am at pH 7.4, cells were washed and resuspended in transport buffer at different pH (indicated). Fluorescence was allowed to stabilize for 1 minute, followed by addition of 20 μM Fe²⁺. (B) Summary of transport results obtained for the histidine mutants at pH 5.0.