Human KCNK2 Stable Cell Line - CHO-K1

Studies have indicated that the two-pore-domain potassium channel TREK-1 is involved in the proliferation processes of neural stem cells, astrocytes, and human osteoblasts. Here, researchers established a CHO cell line stably expressing hTREK-1 (designated as CHO/hTREK-1 cells). Compared to control cells, CHO/hTREK-1 cells exhibited high levels of TREK-1 expression, reaching up to 320% ± 16%. Upon treatment with arachidonic acid (10 μmol/L), chloroform (1 mmol/L), or etomidate (10 μmol/L), the TREK-1 channel currents within CHO/hTREK-1 cells were significantly enhanced. Overexpression of TREK-1 resulted in the arrest of CHO cells in the G1 phase and significantly downregulated the expression levels of Cyclin D1. The TREK-1 inhibitor l-butylphthalide (1-100 μmol/L) was able to alleviate the TREK-1-induced G1-phase cell arrest in a dose-dependent manner. Furthermore, TREK-1 overexpression significantly reduced the phosphorylation levels of Akt (at S473), glycogen synthase kinase-3β (at S9), and cAMP-response element-binding protein (CREB, at S133); concurrently, it enhanced the phosphorylation levels of p38 (at T180/Y182); however, it had no effect on the phosphorylation or expression levels of signal transducer and activator of transcription 3 (STAT3). These results suggest that TREK-1 overexpression inhibits the proliferation of CHO cells by inducing G1-phase cell arrest, a process mediated through the suppression of PKA and p38/MAPK signaling pathway activities.

To evaluate the biological function of TREK-1 in cell proliferation, researchers utilized flow cytometry to analyze the cell cycle distribution of CHO/hTREK-1 and CHO/EGFP cells. The results indicated that the overexpression of human TREK-1 inhibited the proliferation of CHO cells. The proportion of CHO/hTREK-1 cells entering the G1 phase was 59.3% ± 2.6% (Figure 1B), which was significantly higher than that of the CHO/EGFP cell group (46.2% ± 0.3%, Figure 1A), and resulted in a corresponding reduction in the number of cells in the G2/M phase. Consequently, these results suggest that TREK-1 may inhibit the growth of CHO cells by suppressing the transition from the G1 phase to the S phase within the cell cycle. Subsequently, the researchers employed l-NBP as a TREK-1 inhibitor for further investigation. As shown in Figure 1C, following a 72-hour treatment with l-NBP, the number of cells in the G1 phase was significantly reduced compared to the CHO/hTREK-1 cell control group. These findings demonstrate that TREK-1 indeed inhibits CHO cell proliferation, but the specific TREK-1 blocker l-NBP is capable of reversing this inhibitory effect of TREK-1 on cell proliferation. Consistent with the flow cytometry results, MTT assays revealed that cell viability was significantly enhanced following l-NBP treatment compared to the control group (Figure 1D).

Figure 1. TREK-1 increased the percentage of cells in the G1 phase, and l-NBP reversed the effect of TREK-1 on the cell cycle distribution. (Zhang M, et al., 2016)