Comparative assessment of the transport properties of WT and Nramp2^G185R. (A) Measurements of calcein fluorescence in cells expressing WT Nramp2 or Nramp2^G185R. Cells were initially bathed at pH 7.3 and pH was lowered to 5.5 where indicated. Finally, 100 μMCo²⁺ or Fe²⁺ was added as noted. Representative raw data are illustrated. (B) Co²⁺ uptake determinations by cells transfected with WT or Nramp2^G185R, calculated from experiments like that in panel A. Data are presented as the ratio of the initial fluorescence (F₀) divided by the fluorescence (F) recorded at the indicated times. (C) The initial rates of Co²⁺ transport, measured as in panel B, were normalized by the number of surface-exposed Nramp2 molecules, determined in parallel with anti-HA antibodies, as described in “Materials and methods.” To facilitate comparison, the specific transport activity of the mutants is expressed as a fraction of the WT. Data are means plus or minus SE of 6 individual determinations. ^*P < .05, determined by Student t test. (D) Eadie-Hofstee plot of the transport activity of WT (▪) and Nramp2^G185R (○) as a function of the concentration of Co²⁺. Data are means plus or minus SE of 6 determinations. (E) pH dependence of Co²⁺ transport in cells transfected with WT or Nramp2G185R. Measurements of the specific transport activity are presented as a fraction of the activity of WT Nramp2 at pH 5.5. Data are means plus or minus SE of 4 determinations.