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Biology

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Published: June 2nd, 2023

DOI:

10.3791/65217

1Department of Life Sciences and National Institute for Biotechnology in the Negev, Ben-Gurion University, 2Department of Physiology and Cell Biology, Faculty of Health Sciences, Ben-Gurion University

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Zinc is an essential trace element in the cellular milieu. It incorporates one-third of all proteins and is involved in various cellular processes, such as catalysis1, transcription2, and structural motifs3. However, despite being redox-inert, high zinc concentrations are toxic to the cell, which is why no mammalian organism has survived without the presence of mechanisms regulating zinc homeostasis. In mammals, three mechanisms are responsible for this process: (1) metallothioneins, which are cytosolic cysteine-rich proteins that bind zinc at a high affinity, thus preventing excess free cytosolic....

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1. Cell transfection

  1. Culture HEK293T cells in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, and 1x penicillin/streptomycin (see Table of Materials) in a humidified incubator at 37 °C/5% CO2 until confluence on a 10 cm plate (8.8 x 106 total cells).
  2. Place one 13 mm coverslip in each of the wells of a 12-well plate. Dilute 0.44 x 106 trypsinized cells from step 1.1 in.......

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ZnT1 is a mammalian zinc transporter located on the cell plasma membrane13. It is a member of the cation diffusion facilitator (CDF) protein family that extrudes zinc from the cytosol to the extracellular millieu14. ZnT1 has a two-domain architecture: the transmembrane domain, which transports the ions across the membrane, and a C-terminal domain14. Unlike other known CDF proteins, ZnT1 has an extended unstructured C-terminal domain (USCTD). The role.......

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The above-described method allows for the direct measurement of the intracellular zinc concentration with high temporal resolution. Compared to other methods, this method involving monitoring changes in intracellular Zn2+ can substantially decrease background noise. In addition, the dye's selectivity for zinc eliminates potential cross-interactions with other metal cations18,19. Finally, its lack of immediate cytotoxicity enables the testing of liv.......

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Raz Zarivach is supported by the Israel Science Foundation (grant no. 163/22). Tomer Eli Ben Yosef and Arie Moran are supported by the Israel Science Foundation (grant no. 2047/20). We would like to thank Daniel Gitler and his group at Ben-Gurion University for their cooperation, support, and expertise.

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Name Company Catalog Number Comments
10 cm plate greiner bio-one 664160
12-well cell culture plate greiner bio-one 665180
13 mm coverslips Superior Marienfeld 111530
22 mm cover slides Superior Marienfeld 101050
6-well culture plate greiner bio-one 657160
Bovine serum albumin bioWorld 22070008
Calcium chloride anhydrous, granular Sigma Aldrich C1016 Concentration in Ringer solution: 1 mM
D-(+)-Glucose Glentham Life Science GC6947 Concentration in Ringer solution: 10 mM
Dubelco’s Modified Eagle Media (DMEM)  Sartorius 01-055-1A
Eclipse Ti inverted microscope Nikon TI-DH Discontinued. Replaced by Eclipse Ti2
Fetal Bovine Serum (FBS) Cytiva SH30088.03
Fine tweezers Dumont 0203-55-PS
Fluozin-3AM Invitrogen F24195
HyClone Penicillin-Streptomycin 100x solution Cytiva SV30010 
LED illumination system CoolLED pE-4000
L-glutamine Biological Industries 03-020-1B
Magnesium chloride hexahydrate Merck 1.05833 Concentration in Ringer solution: 0.8 mM
N[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES) Formedium HEPES10 Concentration in Ringer solution: 10 mM
Neo 5.5 sCMOS camera ANDOR DC-152Q-FI
NIS-Elements imaging software Nikon AR
Pluronic acid F-127 Millipore 540025
Pottasium chloride Bio-Lab 163823 Concentration in Ringer solution: 5.4 mM
Pyrithione Sigma Aldrich H3261 Concentration in Ringer zinc solution: 7 μM
Silicone Grease Kit Warner Instruments W4 64-0378
Sodium chloride Bio-Lab 190305 Concentration in Ringer solution: 120 mM
Zinc sulfate Concentration in Ringer zinc solution: 7 μM
Sigma Aldrich 31665

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