SUMMARY Piezo proteins form mechanically activated ion channels that are responsible for our sense of light touch, proprioception, and vascular blood flow. Upon activation by mechanical stimuli, Piezo channels rapidly inactivate in a voltage-dependent manner through an unknown mechanism. Inactivation of Piezo channels is physiologically important, as it modulates overall mechanical sensitivity, gives rise to frequency filtering of repetitive mechanical stimuli, and is itself the target of numerous human disease-related channelopathies that are not well understood mechanistically. Here, we identify the globular C-terminal extracellular domain as a structure that is sufficient to confer the time course of inactivation and a single positively charged lysine residue at the adjacent inner pore helix as being required for its voltage dependence. Our results are consistent with a mechanism for inactivation that is mediated through voltage-dependent conformations of the inner pore helix and allosteric coupling with the C-terminal extracellular domain.

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An essential component of mammalian cells, cholesterol exerts significant influence on the physical properties of the cell membrane and in turn its constituents, including membrane proteins. Although sparse, polar amino acid residues are highly conserved in membrane proteins and play pivotal roles in determining specific structural and functional properties. To improve our understanding of particular polar residues in the membrane environment, we have examined two specific “guest” Arg residues within a well-defined and deuterium-labeled “host” framework provided by the transmembrane helical peptide GWALP23 (acetyl-GGALWLALALALALALALWLAGA-amide). Solid-state 2H nuclear magnetic resonance (NMR) spectra from aligned bilayer membrane samples effectively report changes in the host helix properties because of the incorporation of the guest residues. The focus of this work is two-pronged. First, GWALP23-R14 was examined over a pH range of 2–13 in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) ester- or ether-linked bilayer membranes. Our findings indicate that the Arg guanidinium side chain remains charged over this entire range, in agreement with numerous molecular dynamics simulations. Second, GWALP23-R12 and GWALP23-R14 peptides were characterized in DOPC bilayers with varying cholesterol content. Our findings suggest that 10 or 20% cholesterol content has minimal impact on the orientation of the R14 peptide. Although the NMR signals are broader and weaker in the presence of 20% cholesterol, the deuterium quadrupolar splittings for [2H]Ala residues in GWALP23-R14 change very little. Conversely, cholesterol appears to modulate the multistate behavior of GWALP23-R12 and to favor a major interfacial state for the helix, bound at the bilayer surface. These results indicate a conditional sensitivity of a complex multistate transmembrane Arg-containing peptide helix to the presence of cholesterol.