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Mutations in more than half of human connexin genes encoding gap junction subunits have been linked to inherited human diseases. Functional studies of human gap junction (GJ) channels are essential for revealing mechanistic insights on the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels.
Figure 1. Coupling status of genetically engineered HEK293 cells.(A) Dual whole cell patch clamp technique was used to measure transjunctional current (Ij) in response to indicated Vj in HEK293 cell pairs. Representative Ij recordings obtained from wildtype (WT), Cx45 knockout (Cx45KO), Cx43 knockout (Cx43KO), and double knockout with both Cx45 and Cx43 ablated (DKO) HEK293 cell pairs. (B) Bar graphs summarize the median percentage (the error bars indicate IQR) of coupled cell pairs (coupling%) in different HEK293 cells. Data points represent the number of transfections. (C) Bar graph illustrates the median coupling conductance (Gj) of HEK293 cell pairs. Data points represent the number of cell pairs. Kruskal–Wallis test followed by Dunn's post hoc test was used to compare different groups for all bar graphs. The statistical significance was indicated (**P < 0.01, and ***P < 0.001).
Figure 2. Vj-gating properties of genetic engineered HEK293 cells.Superimposed transjunctional currents (Ijs) in response to the indicated Vj pulses (±20 to ±100 mV, in the case of Cx43KO ±5 mV Vj pulses were also added and in gray color) for cell pairs of WT (open circles), Cx45KO (open triangles), and Cx43KO (open inverse triangles). Depending on the cells recorded, the absolute Ij amplitude values were either remained steady during the Vj pulse (at Vjs of ±20 to ±40 mV for WT and Cx45KO cells and at Vj of ±5 mV for Cx43KO cells) or decreased mirror symmetrically during the Vj pulses (at the rest of the tested Vjs). Normalized steady-state conductance (Gj,ss) was obtained and plotted against tested Vjs (right panel). The Gj,ss – Vj relationship could be fitted well by Boltzmann equations and the smooth lines in each plot represent Boltzmann fittings for both Vj polarities. The error bars represent the standard error of mean (±SEM). The number of cells pairs is indicated.
Figure 3. Single channel analysis of genetically engineered HEK293 cells.(A) Single channel currents (ijs) recordings were shown in response to the indicated Vjs (−Vj in black and +Vj in gray) from Cx45KO (top) and Cx43KO (bottom) cell pairs. The Cx45KO ijs showed subconductance state (or residue conductance state, open arrows) and fully closed state (filled arrows). All point histograms (gray) were constructed in the boxed regions on the original ijs shown on the left panels and Gaussian fitting curves (black smooth curves) were used to measure the amplitude of the ijs at the main open state. (B) The ijs from Cx45KO (open triangles) and Cx43KO (inverse open triangles) were plotted against Vjs. Linear regressions were used to determine the slope single channel conductance (γj) for Cx45KO and Cx43KO cell pairs. The γjs and the number of cell pairs are indicated.
Figure 4. Expression of human connexins formed functional GJs in DKO HEK293 cells with signature Vj-gating properties.Superimposed transjunctional currents (Ijs) in response to the indicated Vj pulses (±20 to ±100 mV, in some cases also with ±5 mV) for DKO cell pairs expressing GFP, Cx46, Cx50, Cx45, Cx37, or Cx36 as indicated. Expression of GFP alone failed to form any functional GJ channels, while expression of Cx46, Cx50, Cx45, Cx37, or Cx36 formed functional GJs with their signature Vj-gating properties.
Figure 5. Normalized steady-state junctional conductance (Gj,ss) was plotted against Vj in cell pairs expressing Cx45, Cx37, and Cx36.The Gj,ss – Vj plots of both Cx45 and Cx37 could be well fitted by Boltzmann equations for each of their Vjs, while in the case of Cx36 we were unable to fit the data with Boltzmann equations (the line was simply connecting the averaged data points). The number of cell pairs are indicated for each plot.
Figure 6. Single Cx46 and Cx26 GJ currents could be studied in DKO HEK293 cells.Top panel shows the unitary transjunctional currents (ijs) recorded in response to the indicated Vj pulses in a DKO HEK293 cell pair expressing Cx46-IRES-GFP and Cx26-IRES-GFP. The amplitude of ijs increased with the increase of Vj in each case and the linear regression of the ij – Vj plot (bottom panel) for each of these cell pairs was used to obtain slope single channel conductance of Cx46 GJ (γj = 213 pS) and Cx26 GJ (γj = 156 pS). Open arrows on the ijs indicate residual conductance state, and the black filled arrow indicates fully closed state.
Figure 7. Cx45 R184G failed to form functional GJs in DKO HEK293 cells likely due to failed clustering at the cell-cell junctions.(A) Dual whole cell patch clamp technique was used to measure transjunctional current (Ij) in response to indicated Vj in DKO HEK293 cell pairs. Representative Ij recordings obtained from cell pairs expressing untagged Cx45 or Cx45 R184G. (B) Bar graph on the left panel summarizes the median percentage (the error bars indicate IQR) of coupled cell pairs (coupling%) in cell pairs expressing Cx45 (filled circles) or Cx45 R184G (open circles). Data points represent the number of transfections. Bar graph on the right panel illustrates the median coupling conductance (Gj) of cell pairs expressing Cx45 and Cx45 R184G. Data points represent the number of cell pairs. Mann–Whitney test was used to compare different groups for these bar graphs. The statistical significance was indicated (**P < 0.01, and ***P < 0.001). (C) Confocal images of DKO HEK293 cells expressing GFP tagged Cx45 (Cx45-GFP) or Cx45-R184G (R184G-GFP). GJ plaque like clusters of Cx45-GFP could be observed at the cell-cell junctions, whereas R184G-GFP failed to form any clusters at the cell-cell junctions. Fluorescence, differential interference contrast (DIC), and superimposed images are shown as indicated. These are representative images from three independent experiments. Scale bar = 10 μm.
Figure 8. Design of sgRNA to knockout GJA1 (encoding Cx43) and GJC1 (encoding Cx45) genes in HEK293 cells.(A) and (B) panels illustrate the gene structure of GJA1 and GJC1 and the designed single guide RNA (sgRNA) targeting at the beginning of the coding regions (filled green and blue bars) of these genes. (C) Western blot shows that Cx43 is expressed and showed typical multiple bands in wildtype (WT) HEK293 cells. CRISPR-Cas9 gene ablation targeting the GJA1 gene (encoding Cx43) was successful in deleting Cx43 (Cx43KO) in this clone of HEK293 cells. GAPDH was used as a loading control.
