An Iodide-Yellow Fluorescent Protein-Gap Junction-Intercellular Communication Assay

Summary

Here, we present a protocol for a novel gap junction intercellular communication assay designed for the high-throughput screening of gap junction-modulating chemicals for drug discovery and toxicological assessment.

Abstract

Gap junctions (GJs) are cell membrane channels that allow diffusion of molecules smaller than 1 kDa between adjacent cells. As they have physiological and pathological roles, there is need of high-throughput screening (HTS) assays to identify GJ modulators in drug discovery and toxicology assays. A novel iodide-yellow fluorescent protein-gap junction-intercellular communication (I-YFP-GJIC) assay fulfills this need. It is a cell-based assay including acceptor and donor cells that are engineered to stably express a yellow fluorescent protein (YFP) variant, whose fluorescence is sensitively quenched by iodide, or SLC26A4, an iodide transporter, respectively. When iodide is added to a mixed culture of the two cell types, they enter the donor cells via the SLC26A4 transporter and diffuse to the adjacent acceptor cells via GJs where they quench the YFP fluorescence. YFP fluorescence is measured well by well in a kinetic mode. The YFP quenching rate reflects GJ activity. The assay is reliable and rapid enough to be used for HTS. The protocol for the I-YFP-GJIC assay using the LN215 cells, human glioma cells, is described.

Introduction

Gap junctions (GJs) act as intercellular channels to allow the diffusion of small molecules of <1 kDa such as nutrients, metabolites, and signaling molecules between adjacent cells. The junctional elements include a hemichannel or connexon in each cell, and each connexon constitutes six connexins (Cxs)¹. GJs and Cxs have been used in toxicology assays of carcinogens such as polycyclic aromatic hydrocarbons (PAH), which are GJ inhibitors^2,3,4. Disrupted GJIC has been associated with nongenotoxic carcinogenesis^5,

Protocol

1. Generation of lentiviruses expressing YFP^QL and SLC26A4

1. Grow HEK293T human embryonic kidney cells to 80% confluency on 100 mm culture plates. Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin is the culture medium used throughout the protocol to maintain HEK293T and other cells mentioned below.
2. Coat 6-well culture plates by adding 2 mL of 0.005% of sterile poly-L-lysine (PLL) solution to each well for 10 min. Aspirate the PLL solution and rinse the surface twice with 2 mL of sterile water.
3. Wash the HEK293T cells with 10 mL of phosphate....

Results

Twenty-nine 96-well culture plates were used to screen 2,320 chemicals to identify novel GJIC modulators by I-YFP GJIC assay using the LN215-YFP^QL and LN215-I⁻ cells. The results obtained with a representative plate are shown in Figure 2. The percentage of YFP fluorescence in each well is shown as a line graph in Figure 2A and the percentage of GJIC activity in each well is shown in th.......

Discussion

The I-YFP-GJIC assay can be used for HTS because it is robust, rapid, and inexpensive. An HTS assay is considered robust if the Z'-factor is above 0.5²⁵. See Zhang et al. for a description of the statistical analysis used to assessing the suitability of HTS assays²⁵. When LN215 cells were used, the Z'-factor was >0.5 without any assay optimization. If other cell types are used in the assay and its Z'-factor is <0.5, the robustness can be improved by exte.......

Materials

Name	Company	Catalog Number	Comments
96-well plate	SPL	30096
Calcium chloride (CaCl2)	Sigma	C5670	I-solution
D-(+)-Glucose	Sigma	G7021	C-solution, I-solution
Dimethyl sulfoxide (DMSO)	sigma	276855
HEPES	Sigma	RES6003H-B7	C-solution, I-solution
Lipofectamine 2000	Invitrogen	11668-027	transfection reagent
Magnesium chloride hexahydrate (MgCl2 6H2O)	Sigma	M2393	C-solution
Microplate reader	BMG LabTech	POLARstar Omega 415-1618
pMD2.G	Addgene	#12259
Polybrene	sigma	H9268
Poly-L-lysine solution	sigma	P4707
Potassium chloride (KCl)	Sigma	P5405	C-solution, I-solution
psPAX2	Addgene	#12260
Puromycin Dihydrochloride	sigma	P8833
Sodium chloride (NaCl)	Sigma	S5886	C-solution, I-solution
Sodium hyroxide (NaOH)	Sigma	S2770
Sodium Iodide (NaI)	Sigma	383112	I-solution