Human PIEZO1 Stable Cell Line - HEK293

The human mechanosensitive ion channel PIEZO1 is regulated by membrane tension and participates in important biological processes such as angiogenesis and erythrocyte volume homeostasis. Currently, little is known about the localization and structure of PIEZO1 on the plasma membrane. Here, researchers used the PIEZO1-GFP fusion protein to investigate whether the enrichment or depletion of cholesterol by methyl-β-cyclodextrin (MBCD) and the disruption of membrane cholesterol structure by dynasore affect the response of PIEZO1-GFP to mechanical forces. Cell-attachment electrophysiological recordings showed that MBCD caused a rightward shift in the pressure-response curve of PIEZO1-GFP, increased the latency of the channel's response to mechanical stimulation, and significantly slowed channel inactivation. The same effect was observed in native PIEZO1 in N2A cells. STORM super-resolution imaging revealed that at the nanoscale, PIEZO1-GFP channels on the membrane aggregate in clusters and are sensitive to membrane manipulation. MBCD (5 mM) treatment affected the distribution and diffusion rate of these clusters. Supplementation with polyunsaturated fatty acids appears to enhance the stress response of PIEZO1-GFP. Therefore, these results suggest that the function of PIEZO1 is directly dependent on membrane composition and the lateral alignment of membrane cholesterol domains, which coordinate the activity of aggregated PIEZO1 channels.

Here, researchers measured the effects of cholesterol depletion, supplementation, and dynasore (a compound known to disrupt cholesterol-rich domains) on PIEZO1-GFP channels in PIEZO1-GFP stable HEK293T cells. These cells expressed very low levels of STOML3. Compared to control cells, the sensitivity and kinetics of the channels were significantly altered when cholesterol was removed using MBCD or dynasore was added (Figure 1A-D). High doses of the water-soluble MBCD/cholesterol complex did not affect the gating of PIEZO1-GFP (Figure 1B). However, treatment with 5 mM MBCD significantly reduced the stress sensitivity of PIEZO1-GFP (Figure 1E and F), but this was not observed at lower doses and had no significant effect on cell viability. Desensitization to applied stress was accompanied by a significant increase in response latency, both at the initial opening and at the occurrence of maximum current (Figure 1G). None of the compounds altered the mean maximum current of PIEZO1-GFP (Figure 1H). Both MBCD and dynasore cause delayed activation of PIEZO1-GFP, but only MBCD shifts the midpoint of channel activation pressure to the right by about 40%. Interestingly, both MBCD and dynasore treatments significantly reduce channel inactivation, which can be quantified by normalizing the steady-state current (Figure 1I).

Figure 1. Modulation of PIEZO1-GFP activity in cell-attached patches of HEK293T cells in response to manipulation of cholesterol domains in the plasma membrane. (Ridone P, et al., 2020)