On the Ca2+ dependence of non-transferrin-bound iron uptake in PC12 cells

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pa thways, Ca2+-dependent and Ca2+-independent (Wright, T. L., Brissot, P,, Ma , W, L,, and Weisiger, R. A. (1986) J. Biol. Chern, 261, 10909-10914; Sturr ock, k, Alexander, J,, Lamb, J,, Craven, C, M., and Kaplan, J, (1990) J, Bi ol. Chem. 265, 3139-3145). Studies reporting the two pathways have ignored the weak interactions of Ca2+ with the chelator nitrilotriacetate (NTA) and the reducing agent ascorbate. These studies used a constant ratio of total Fe2+ to NTA with and without Ca2+, We observed Ca2+ activation of NTBI upt ake in PC12 cells with the characteristics reported for other cells upon us ing 1 mM ascorbate and a constant ratio of total Fe2+ to NTA with or withou t Ca2+. However, Ca2+ did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca2and Fe2+ by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe2+, and total Ca2+ concentr ations). In solutions based on these constants and taking Ca2+ chelation in to account, Ca2+ did not affect NTBI uptake over a range of free Fe2+ conce ntrations. Thus, the Ca2+ activation of NTBI uptake observed using the cons tant total Fe2+ to NTA ratio was because of Ca2+-NTA chelation rather than an activation of the NTBI transporter itself, It is suggested that the prev iously reported Ca2+ dependence of NTBI uptake be re-evaluated.