Disruption of membrane cholesterol organization impairs the concerted activity of PIEZO1 channel clusters

Abstract

The essential mammalian mechanosensitive channel PIEZO1 organizes in the plasma membrane into nanometric clusters which depend on the integrity of cholesterol domains to rapidly detect applied force and especially inactivate syncronously, the most commonly altered feature of PIEZO1 in pathology. ABSTRACTThe human mechanosensitive ion channel PIEZO1 is gated by membrane tension and regulates essential biological processes such as vascular development and erythrocyte volume homeostasis. Currently, little is known about PIEZO1 plasma membrane localization and organization. Using a PIEZO1-GFP fusion protein, we investigated whether cholesterol enrichment or depletion by methyl-β-Cyclodextrin (MBCD) and disruption of membrane cholesterol organization by dynasore affects PIEZO1-GFP's response to mechanical force. Electrophysiological recordings in the cell-attached configuration revealed that MBCD caused a rightward shift in the PIEZO1-GFP pressure-response curve, increased channel latency in response to mechanical stimuli and markedly slowed channel inactivation. The same effects were seen in native PIEZO1 in N2A cells. STORM super-resolution imaging revealed that, at the nano-scale, PIEZO1-GFP channels in the membrane associate as clusters sensitive to membrane manipulation. Both cluster distribution and diffusion rates were affected by treatment with MBCD (5 mM). Supplementation of polyunsaturated fatty acids appeared to sensitize the PIEZO1-GFP response to applied pressure. Together, our results indicate that PIEZO1 function is directly dependent on the membrane mechanical properties and lateral organization of membrane cholesterol domains which coordinates the concerted activity of PIEZO1 channels.

Force Sensing by Piezo Channels in Cardiovascular Health and Disease

“…Consistent with these observations, studies of overexpressed channels have also shown no inactivation or slow inactivation. 100 It would seem, therefore, that inactivation is a variable property; its extent depending on context and probably also the type of stimulus. It is also unclear whether the rapid pressure pulses and indentation pulses commonly used in experimental studies have physiological correlates—in physiology, it is more likely that the stimuli are relatively slow changes in membrane tension or fluid flow.…”

“…It is also unclear whether the rapid pressure pulses and indentation pulses commonly used in experimental studies have physiological correlates—in physiology, it is more likely that the stimuli are relatively slow changes in membrane tension or fluid flow. How inactivation is controlled or avoided (inactivation of inactivation) in native systems is not yet clear, but one possibility is regulation by local lipid composition 29,100 (Text Box 5). Lipids could conceivably interact with and disable an inactivation gate, creating a pool of available channels that is variable in number and contribution.…”

Computational reconstruction of the complete Piezo1 structure reveals a unique footprint and specific lipid interactions

“…In other studies, cholesterol depletion 54 reduces the activity of ENaC (Balut et al, 2005) and TRPC (Bergdahl et al, 2003) channels. 55 In a recent preprint study, lateral organization of cholesterol in the membrane is reported to 56 regulate Piezo1 activity (Ridone et al, 2019). Further indirect links between cholesterol and 57…”

Piezo1 Forms Specific, Functionally Important Interactions with Phosphoinositides and Cholesterol

“…Functional data point to a role for various lipids in mechanical gating of Piezo1. For example, methyl-b-cyclodextrin, a drug commonly used to deplete cholesterol, had a minor influence on Piezo1 clustering (41), reduced mechanically induced Piezo1 currents in response to cell indentation (42), and increased the pressure required to open the channel in cell-attached patch clamping (41). Depletion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) via activation of TRPV1 inhibits Piezo1 activity (43).…”