pH-dependence of metal transport by Nramp1HA and Nramp2HA expressed in CHO transfectants. CHO control cells along with Nramp1HA (N1-94) and Nramp2HA (N2-310a) expressing CHO transfectants were loaded with Fura2-AM, and the quenching of intracellular Fura2 fluorescence by extracellular Mn²⁺ ions was determined as described in “Materials and methods” and in the legend to Figure 4. Transport was conducted in buffer systems adjusted to pH 5, 6, or 7. Data were collected (0.5-second intervals) for 200 seconds. Typical fluorescence quenching traces for each cell line are shown for pH 5.0 (A), 6.0 (B), and 7.0 (C). The average rate (with standard errors) of Nramp1/2-dependent Fura2 quenching by Mn²⁺ was calculated at each pH from the initial slope of 3-5 independent quenching curves (D). These rate data are also expressed relative to background metal uptake in CHO-negative controls (E). The means of normalized quenching rates (E) of Nramp1/2HA transfectants were statistically different than those of CHO controls at pH 5, pH 6 (P < .0005), and pH 7.0 (P = .0015 and P = .015, respectively) as determined using the Student t test. The normalized mean of Nramp1HA transport (E) at pH 5 was not statistically different than that of its transport at pH 6 (P = .1). However, the normalized means of Nramp1HA transport (E) at both pH 5 (P = .0006) and pH 6 (P = .015) were significantly different than that of its transport at pH 7.0. The mean of normalized Nramp2HA transport (E) at pH 5 was statistically different than those of its transport at pH 6.0 and 7.0 (P < .0001). Likewise, the mean of Nramp2HA transport (E) at pH 6.0 was statistically different than that of its transport at pH 7.0 (P = .012).