Fig. 9. Kinetic analysis of I^- uptake in transfected COS cells. (A) Initial rates (4-minute time points) of I^- uptake were determined at the indicated concentrations of I^-, as described in Materials and Methods. Calculated curves (smooth lines) were generated using the equation: v([I^-])=(V_maxx[I^-])/(K_m+[I^-])+0.03xI^-]+0.37. The terms 0.03x[I^-]+0.37 correspond to background adjusted by least squares to the data obtained with non-transfected cells. V_max-I^- and K_m-I^- values are indicated in Table 1. Symbols: non-transfected cells (black circles), WT NIS (red squares), rQ267E (green triangles), rQ267A (blue diamonds) and rQ267N (gray circles). (B) Na⁺-dependent kinetic analysis. To assess Na⁺ dependence of I^- uptake, cells were incubated for 4 minutes with the indicated concentrations of Na⁺; isotonicity was maintained constant with choline chloride. Na⁺ dependence data were analyzed using the equation v=(V_maxx[Na⁺]²)/(K_m+[Na⁺]²)-0.001x[Na⁺]+0.87. The term 0.001x[Na⁺]+0.87 corresponds to the background adjusted by lineal regression analysis obtained with non-transfected cells. Data were fitted by non-linear least squares using the Marquard-Levenberg algorithm (Press et al., 1986). V_max-Na⁺ and K_m-Na⁺ values are indicated in Table 1.