Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Summary

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.

Abstract

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.

Introduction

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....

Protocol

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.......

Discussion

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 cations^18,19. Finally, its lack of immediate cytotoxicity enables the testing of liv.......

Materials

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