Relative Fe²⁺ and Co²⁺ transport activity of Nramp2 mutations affecting conserved charged residues in TM domains.

(A) Control CHO cells (pCB6) as well as CHO transfectants expressing wild-type Nramp2 (N2.3) were loaded with the metal-sensitive fluorescent dye calcein (introduced as 0.250 μM calcein-am) during 10 minutes at 37°C. Cells were washed, resuspended in 500 μL transport buffer (pH 6.0), and fluorescence recorded with an LS-50B fluorescence spectrometer. When fluorescence stabilized, divalent metals (20 μM final concentration of Fe²⁺ or Co²⁺) were added to the cell suspension (arrow 1), and fluorescence was continuously monitored for an additional 3 minutes. For iron transport studies (right panel), the membrane-impermeable iron chelator HES-DFO (200 μM) was added at 4 minutes (arrow 2) to reveal cell-associated extracellular Fe-calcein complexes. At 5 minutes, the membrane-permeant iron chelator SIH (250 μM) was added (arrow 3) and revealed intracellular Fe-calcein complexes (see “Materials and methods”). The transport activity of Nramp2 was measured as an initial rate (slope) during the early portion of the calcein-quenching curve and is shown as a shaded area (slope interval). (B) Relative transport activities of wild-type (N2.3, N2GG) and mutant Nramp2 variants (identified) were calculated from the initial slope of calcein-quenching curves as shown in panel A and are expressed as a relative transport activity with transport in the positive control N2.3 set at 100%. Error bars represent standard errors on the means of 3 or more independent experiments. *Transport activities significantly different (ie, more than 2 standard deviations) from those detected in the negative control, vector-transfected (pCB6) cells.