Identification of Cx45 as a Major Component of GJs in HeLa Cells.

Nicolás Palacios-Prado,Juan C Sáez,Jinu Lee,Eun Ju Choi

doi:10.3390/biom10101389

Abstract

Gap junctions (GJs) are intercellular channels that connect adjacent cells electrically and metabolically. The iodide-yellow fluorescent protein (I-YFP) gap junctional intercellular communication (GJIC) assay is a recently developed method with high sensitivity. HeLa cells have been widely used as GJ-deficient cells for GJ-related research. Herein, we present evidence showing that HeLa cells have functional GJs comprising connexin (Cx) 45 using the I-YFP GJ assay and CRISPR/Cas9 system. We conducted the I-YFP GJIC assay in HeLa cells, which revealed a weak level of GJIC that could not be detected by the Lucifer yellow scrape-loading assay. The mRNA expression of GJB5 (Cx31.1), GJA1 (Cx43), and GJC1 (Cx45) was detected in HeLa cells by RT-PCR analysis. Knocking out GJC1 (Cx45) abolished GJIC, as analyzed by the I-YFP assay and dual whole-cell patch-clamp assay. These results suggest that HeLa cells express Cx45-based GJs and that the I-YFP GJIC assay can be used for cells with weak GJIC, such as Cx45-expressing HeLa cells. Further, GJC1 (Cx45)-knockout HeLa cells are more suitable as a GJ-null cell model for transfection experiments than wild-type HeLa cells. This experimental design was successfully applied to knock out Cx43 expression and GJIC in A549 lung cancer cells and can thus be used to identify major Cxs in other cell types and to establish GJ assay systems for different Cxs.

Highlights

Gap junctions (GJs) are intercellular channels located on cell-to-cell interfaces through which ions and metabolites less than about 1 kDa can diffuse between cells [1]
We used the iodide-yellow fluorescent protein (I-YFP) Gap junctional intercellular communication (GJIC) assay to examine whether HeLa cells have GJ activity [18,27,28,29]
If HeLa cells express functional GJs, the iodides enter donor cells via SLC26A4 when iodides are added to the co-culture of the donor and acceptor HeLa cells, diffuse to the acceptor cells through the GJs, and quench YFPQL (Figure 1A, right box)

Summary

Introduction

Gap junctions (GJs) are intercellular channels located on cell-to-cell interfaces through which ions and metabolites less than about 1 kDa can diffuse between cells [1]. The Cx gene family consists of 21 members in the human genome, and it is divided into five subfamilies (GJA, GJB, GJC, GJD, and GJE) according to their sequence homology [3]. Their proteins are named based on the approximate molecular weight in kDa, i.e., Cx43 for GJA1. GJs play an important role in pathological conditions, including ischemia/reperfusion injury [6,7,8], epilepsy [9,10], inflammation [11,12], and neurodegenerations [13]

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Results

Conclusion