Functional expression of a low-affinity zinc uptake transporter (FrZIP2) from pufferfish (Takifugu rubripes) in MDCK cells

Abstract

Zinc is a vital micronutrient to all organisms and it is therefore very important to determine the mechanisms that regulate cellular zinc uptake. Previously, we reported on zinc uptake transporters from zebrafish (Danio rerio; DrZIP1) and Fugu pufferfish (Takifugu rubripes; FrZIP1) that facilitated cellular zinc uptake of high affinity (Km<0.5 μM) in both CHSE214 [chinook salmon (Oncorhynchus tshawytscha) embryonic 214] cells and Xenopus laevis oocytes. To investigate additional biochemical pathways of zinc uptake in fish, we molecularly cloned the second fish member (FrZIP2) of the SLC39 subfamily II from Fugu pufferfish gill. Functional characterization suggests that FrZIP2 stimulated zinc uptake in a temperature-, time-, concentration- and pH-dependent manner when overexpressed in MDCK cells (Madin–Darby canine kidney cells). In comparison with FrZIP1 and DrZIP1 (<0.5 μM), FrZIP2 appears to represent a low-affinity zinc uptake transporter (Km=13.6 μM) in pufferfish. FrZIP2 protein was selective for zinc, but it might also transport Cu2+, since 20 times excess of Cu2+ completely abolished its zinc uptake activity. The zinc uptake by FrZIP2 was stimulated in a slightly acidic medium (pH 5.5–6.5) and was completely blocked at pH 7.5 and above, suggesting that an inward H+ gradient might provide a driving force for zinc transport by FrZIP2. Furthermore, FrZIP2-mediated zinc uptake activity was slightly inhibited by 0.5 mM HCO3−, indicating that FrZIP2 may employ a different mechanism of zinc translocation from the assumed HCO3−-coupled zinc transport used by human SLC39A2. The FrZIP2 gene was expressed in all the tissues studied herein, with especially high levels in the ovary and intestines. Thus FrZIP2 may be a prominent zinc uptake transporter of low affinity in many cell types of Fugu pufferfish.