Evidence for pH dependent Zn2+ influx in K562 erythroleukemia cells:: Studies using ZnAF-2F fluorescence and 65Zn2+ uptake

Using both ZnAF-2F (a Zn2+ specific fluorophore) and Zn-65(2+), we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 mu m), the initial rate of Zn-65(2+) accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 mu m for the K-m and 0.203 nmol/mg/s for the V-max were obtained for Zn2+ influx. Zn-65(2+) uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pK(a) for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect. (c) 2005 Elsevier Inc. All rights reserved.