Confirmation of Connexin45 Underlying Weak Gap Junctional Intercellular Coupling in HeLa Cells

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.

Stretch-Induced Activation of Pannexin 1 Channels Can Be Prevented by PKA-Dependent Phosphorylation

“…Upon mechanical stretch, HeLa-P cells did not show significant changes in DAPI uptake rate, but HeLa Panx1 cells showed a significant increase in the DAPI uptake rate compared with basal conditions ( Figure 1B and Supplementary Figure S1B,C). Although the dye uptake of HeLa-P cells was much less sensitive to mechanical stretch, a small and variable baseline uptake of DAPI was detected, which could be explained by very low expression of endogenous connexin45 hemichannels [37]. After performing Figure 1.…”

“…Parental HeLa cells (HeLa-P) were obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured as described in [37]. HeLa cells transfected with the Panx1 cDNA fused to EGFP (HeLa Panx1) were donated by Dr. Felixas Bukaukas (Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY, USA).…”

Functional Evaluation of a Rare Variant c.516G>C (p.Trp172Cys) in the GJB2 (Connexin 26) Gene Associated with Nonsyndromic Hearing Loss

“…HeLa cell line was chosen as an experimental model. Since the current data on the level of endogenous GJB2 (Cx26) expression in HeLa cell lines are ambiguous [ 40 , 58 , 59 , 60 , 61 , 62 ], we primarily established the GJB2 knockout HeLa cells (HeLa Cx26-KO) with deletion of whole GJB2 coding region for subsequent generation of transgenic cell lines to avoid any GJB2 expression in the used HeLa cell line.…”

The Roles of Calmodulin and CaMKII in Cx36 Plasticity

“…In general, Hela cells and Neuro2a cells are not thought to express significant amounts of endogenous connexins or form gap junctions. However, coupling in HeLa cells is documented [68,69], which could confound the interpretation of the results. It stands to be demonstrated whether the different intracellular environments and degrees of endogenous gap junction coupling of human cervical carcinoma (Hela) and rodent neuroblastoma (Neuro2a) cell lines account for the reported opposite outcomes.…”