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

Abstract

Non-transferrin-bound iron (NTBI) uptake has been reported to follow two pathways, Ca2+-dependent and Ca2+-independent (Wright, T. L., Brissot, P., Ma, W. L., and Weisiger, R. A. (1986) J. Biol. Chem. 261, 10909-10914; Sturrock, A., Alexander, J., Lamb, J., Craven, C. M., and Kaplan, J. (1990) J. Biol. 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 uptake in PC12 cells with the characteristics reported for other cells upon using 1 mM ascorbate and a constant ratio of total Fe2+ to NTA with or without Ca2+. However, Ca2+ did not affect NTBI uptake in solutions without NTA. We then determined conditional stability constants for NTA binding to Ca2+ and Fe2+ by potentiometry under conditions of NTBI uptake experiments (pH, ionic strength, temperature, ascorbate, total Fe2+, and total Ca2+ concentrations). In solutions based on these constants and taking Ca2+ chelation into account, Ca2+ did not affect NTBI uptake over a range of free Fe2+ concentrations. Thus, the Ca2+ activation of NTBI uptake observed using the constant 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 previously reported Ca2+ dependence of NTBI uptake be re-evaluated.