title: Evidence for pH dependent Zn2+influx in K562 erythroleukemia cells: studies using ZnAF-2F fluorescence and 65Zn2+ uptake.
abstract: Using both ZnAF-2F (a Zn2+ specific fluorophore) and 65Zn2+, 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 microM), the initial rate of 65Zn2+ 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 microM for the Km and 0.203 nmol/mg/s for the Vmax were obtained for Zn2+ influx. 65Zn2+ 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 pKa for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect.
authors.affiliation: Department of Biological Sciences, OHIO University, Athens, OH 45701, USA. colvin@ohio.edu
authors.forename: Robert A
authors.initials: RA
authors.lastname: Colvin
authors.forename: Charles P
authors.initials: CP
authors.lastname: Fontaine
authors.forename: Dustin
authors.initials: D
authors.lastname: Thomas
authors.forename: Tomoya
authors.initials: T
authors.lastname: Hirano
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Nagano
authors.forename: Kazuya
authors.initials: K
authors.lastname: Kikuchi
mesh_terms: D027682:Cation Transport Proteins / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000502:physiology; D020014:K562 Cells; D007700:Kinetics; D015032:Zinc / Q000032:analysis / Q000378:metabolism*
chemical_list: D027682:Cation Transport Proteins; D005456:Fluorescent Dyes; C403517:SLC39A1 protein, human; D006639:Histidine; D015032:Zinc