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

Abstract

The 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 superresolution imaging revealed that, at the nanoscale, 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 composition and lateral organization of membrane cholesterol domains, which coordinate the activity of clustered PIEZO1 channels.

Modified N-linked glycosylation status predicts trafficking defective human Piezo1 channel mutations

“…In integrins, N-glycans are important for clustering. Hence future studies could explore whether higher order Piezo1 glycosylation is involved in the previously identified clustering of Piezo1 in the plasma membrane 9 . If N-glycans are a means of interactions with other membrane proteins, or the ECM , this gives cells an extra dynamic mechanism to regulate mechanosensitivity.…”

“…These are large membrane proteins with more than 30 transmembrane helices that decode mechanical cues. Piezo1 in particular appears to be a central mechanotransducer in the cardiovascular system 3,4 and is sensitive to membrane forces 2,[5][6][7] and thus lipid composition [8][9][10][11][12] . Like all other integral membrane proteins, Piezo1 channels undergo biosynthetic quality control in the endoplasmic reticulum (ER) and Golgi 13 .…”

Enhanced Ca2+ Influx in Mechanically Distorted Erythrocytes: Measurements with 19F Nuclear Magnetic Resonance Spectroscopy

“…Given that Piezo1 is directly influenced by the surrounding membrane, it is much more likely that the change in leaflet composition can influence Piezo1 activity. For example, this channel can gate in lipids alone [47] and polyunsaturated lipids can modify the channel kinetics [48], as can cholesterol [49]. And more importantly there is even precedence for a flippase being directly involved in Piezo1 regulation in myoblasts [50].…”

Membrane cholesterol regulates TRPV4 function, cytoskeletal expression, and the cellular response to tension

“…It is worth noting that cholesterol enrichment does not affect lateral diffusion of membrane lipids or proteins(59. 60) and that, in contrast to the effects of MβCD, MβCD:cholesterol micelles do not affect the gating of mechanosensitive Piezo1 channels(61).If cholesterol regulated ion channels principally by altering the bulk mechanical properties of the membrane, its effects should be translatable across experimental systems yet in contrast to our observations in TM cells, MβCD suppresses TRPV4 activation in retinal glia(62), inhibits TRPV4-mediated Ca 2+ influx in mesenteric endothelial cells(29), reduces TRPA1, TRPC1, TRPC6 activation in mammalian cells(63 -65), and blocks TRPL currents in fly photoreceptors(66). Visoelastic "force-from-lipid" models(54,67,68) cannot explain why cholesterol enantiomers with similar effects on membrane properties have dramatically different effects on channel function(69).…”