College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon 21983, Korea.
Departamento de Fisiología, Pontificia Universidad Católica de Chile, Santiago 6513677, Chile.
Biomolecules. 2020 Sep 29;10(10):1389. doi: 10.3390/biom10101389.
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 (Cx31.1), (Cx43), and (Cx45) was detected in HeLa cells by RT-PCR analysis. Knocking out (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, (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.
间隙连接（GJ）是连接相邻细胞的电和代谢的细胞间通道。碘黄色荧光蛋白（I-YFP）间隙连接细胞间通讯（GJIC）测定法是一种最近开发的高灵敏度方法。HeLa 细胞已被广泛用作 GJ 相关研究的 GJ 缺陷细胞。在此，我们提供证据表明，HeLa 细胞具有功能性 GJ，包括使用 I-YFP GJ 测定法和 CRISPR/Cas9 系统的连接蛋白（Cx）45。我们在 HeLa 细胞中进行了 I-YFP GJIC 测定，结果显示 GJIC 水平较弱，无法通过 Lucifer yellow 刮除加载测定法检测到。通过 RT-PCR 分析检测到 HeLa 细胞中 （Cx31.1）、 （Cx43）和 （Cx45）的 mRNA 表达。通过 I-YFP 测定法和双全细胞膜片钳测定法分析，敲除 （Cx45）可消除 GJIC。这些结果表明，HeLa 细胞表达基于 Cx45 的 GJ，并且 I-YFP GJIC 测定法可用于具有较弱 GJIC 的细胞，例如表达 Cx45 的 HeLa 细胞。此外，与野生型 HeLa 细胞相比，Cx45 敲除 HeLa 细胞更适合作为转染实验的 GJ 缺失细胞模型。该实验设计成功地应用于敲除 A549 肺癌细胞中的 Cx43 表达和 GJIC，因此可用于鉴定其他细胞类型中的主要 Cx，并为不同的 Cx 建立 GJ 测定系统。
