Piezo1 ion channels are capable of conformational signaling.

Piezo1 is a mechanically activated ion channel that senses forces with short latency and high sensitivity. Piezos undergo large conformational changes, induce far-reaching deformation onto the membrane, and modulate the function of two-pore potassium (K2P) channels. Taken together, this led us to hypothesize that Piezos may be able to signal their conformational state to other nearby proteins. Here, we use chemical control to acutely restrict Piezo1 conformational flexibility and show that Piezo1 conformational changes, but not ion permeation through it, are required for modulating the K2P channel TREK1. Super-resolution imaging and stochastic simulations further reveal that both channels do not co-localize, which implies that modulation is not mediated through direct binding interactions; however, at high Piezo1 densities, most TREK1 channels are within the predicted Piezo1 membrane footprint, suggesting the footprint may underlie conformational signaling. We speculate that physiological roles originally attributed to Piezo1 ionotropic function could, alternatively, involve conformational signaling.

Labeling PIEZO2 activity in the peripheral nervous system.

Sensory neurons detect mechanical forces from both the environment and internal organs to regulate physiology. PIEZO2 is a mechanosensory ion channel critical for touch, proprioception, and bladder stretch sensation, yet its broad expression in sensory neurons suggests it has undiscovered physiological roles. To fully understand mechanosensory physiology, we must know where and when PIEZO2-expressing neurons detect force. The fluorescent styryl dye FM 1-43 was previously shown to label sensory neurons. Surprisingly, we find that the vast majority of FM 1-43 somatosensory neuron labeling in mice in vivo is dependent on PIEZO2 activity within the peripheral nerve endings. We illustrate the potential of FM 1-43 by using it to identify novel PIEZO2-expressing urethral neurons that are engaged by urination. These data reveal that FM 1-43 is a functional probe for mechanosensitivity via PIEZO2 activation in vivo and will facilitate the characterization of known and novel mechanosensory processes in multiple organ systems.

The energetics of rapid cellular mechanotransduction.

Cells throughout the human body detect mechanical forces. While it is known that the rapid (millisecond) detection of mechanical forces is mediated by force-gated ion channels, a detailed quantitative understanding of cells as sensors of mechanical energy is still lacking. Here, we combine atomic force microscopy with patch-clamp electrophysiology to determine the physical limits of cells expressing the force-gated ion channels (FGICs) Piezo1, Piezo2, TREK1, and TRAAK. We find that, depending on the ion channel expressed, cells can function either as proportional or nonlinear transducers of mechanical energy and detect mechanical energies as little as ~100 fJ, with a resolution of up to ~1 fJ. These specific energetic values depend on cell size, channel density, and cytoskeletal architecture. We also make the surprising discovery that cells can transduce forces either nearly instantaneously (<1 ms) or with a substantial time delay (~10 ms). Using a chimeric experimental approach and simulations, we show how such delays can emerge from channel-intrinsic properties and the slow diffusion of tension in the membrane. Overall, our experiments reveal the capabilities and limits of cellular mechanosensing and provide insights into molecular mechanisms that different cell types may employ to specialize for their distinct physiological roles.

Physics of mechanotransduction by Piezo ion channels.

Piezo ion channels are sensors of mechanical forces and mediate a wide range of physiological mechanotransduction processes. More than a decade of intense research has elucidated much of the structural and mechanistic principles underlying Piezo gating and its roles in physiology, although wide gaps of knowledge continue to exist. Here, we review the forces and energies involved in mechanical activation of Piezo ion channels and their functional modulation by other chemical and physical stimuli including lipids, voltage, and temperature. We compare the three predominant mechanisms likely to explain Piezo activation-the force-from-lipids mechanism, the tether model, and the membrane footprint theory. Additional sections shine light on how Piezo ion channels may affect each other through spatial clustering and functional cooperativity, and how substantial functional heterogeneity of Piezo ion channels arises as a byproduct of the precise physical environment each channel experiences. Finally, our review concludes by pointing out major research questions and technological limitations that future research can address.

Piezo1 ion channels inherently function as independent mechanotransducers.

Piezo1 is a mechanically activated ion channel involved in sensing forces in various cell types and tissues. Cryo-electron microscopy has revealed that the Piezo1 structure is bowl-shaped and capable of inducing membrane curvature via its extended footprint, which indirectly suggests that Piezo1 ion channels may bias each other's spatial distribution and interact functionally. Here, we use cell-attached patch-clamp electrophysiology and pressure-clamp stimulation to functionally examine large numbers of membrane patches from cells expressing Piezo1 endogenously at low levels and cells overexpressing Piezo1 at high levels. Our data, together with stochastic simulations of Piezo1 spatial distributions, show that both at endogenous densities (1-2 channels/µm2), and at non-physiological densities (10-100 channels/µm2) predicted to cause substantial footprint overlap, Piezo1 density has no effect on its pressure sensitivity or open probability in the nominal absence of membrane tension. The results suggest that Piezo channels, at densities likely to be physiologically relevant, inherently behave as independent mechanotransducers. We propose that this property is essential for cells to transduce forces homogeneously across the entire cell membrane.

Cells can sense a range of mechanical forces both inside and outside the body, such as the stroke of a fingertip or the filling of a lung. Pores on the surface of the cell called Piezo channels open up in response to this pressure. This allows ions to flood in to the cell and trigger a series of biochemical reactions that alter the cell's behavior. Piezo channels have a unique bowl-like structure that transforms the shape of the cell surface around them, potentially affecting how nearby proteins behave. Previous research had suggested that these channels might be unevenly distributed across the cell surface, and were predicted to modify each other's behaviors when tightly packed together. This cooperative response would have a significant impact on how cells sense mechanical force. To investigate if this was the case, Lewis and Grandl studied a mouse cell called Neuro2A which naturally produces Piezo ion channels. In the experiment, pressure was applied to different parts of the cell and the electric current generated by ions moving across the surface was recorded: the higher the electrical activity, the more ion channels present. This showed that Piezo channels are randomly distributed across the cell surface and do not tend to cluster together. The same Neuro2A cells were then engineered to produce up to one hundred times more Piezo proteins. Despite the channels being more densely packed together, how they responded to mechanical force remained the same. These results suggest that Piezo channels act independently and are not influenced by close proximity to one another. Lewis and Grandl propose that this property may ensure that all parts of the cell surface react to mechanical force in the same way. Further work is needed to see if this finding applies to other cell types that produce Piezo proteins.

Stretch and poke stimulation for characterizing mechanically activated ion channels.

Quantitative functional characterization of mechanically activated ion channels is most commonly achieved by a combination of patch-clamp electrophysiology and stimulation by stretch (or pressure-clamp) and poke (or cell-indentation). A variety of stretch and poke protocols can be used to make measurements of many ion channel properties, including channel number, unitary conductance, ion selectivity, stimulus threshold and sensitivity, stimulus adaptation, and gating kinetics (activation, deactivation, inactivation, recovery from inactivation). Here, we review the general methods of stretch and poke stimulation and discuss the advantages and disadvantages of each. We use the vertebrate mechanically activated ion channel Piezo1 to explain equipment components and calibration, demonstrate experimental protocols and data analyses, and discuss underlying concepts and mechanistic interpretations.

An Internal Dial for Sensitivity and Gain of Rapid Mechanotransduction.

Piezos are ion channels that are activated by mechanical force. Now, 10 years after their initial discovery, Geng et al. (2020) reports, in this issue of Neuron, a novel Piezo1 variant with distinct functional properties, providing key insights into Piezo's structure and function that were fully unexpected.

Inactivation Kinetics and Mechanical Gating of Piezo1 Ion Channels Depend on Subdomains within the Cap.

Piezo1 ion channels are activated by mechanical stimuli and mediate the sensing of blood flow. Although cryo-electron microscopy (cryo-EM) structures have revealed the overall architecture of Piezo1, the precise domains involved in activation and subsequent inactivation have remained elusive. Here, we perform a targeted chimeric screen between Piezo1 and the closely related isoform Piezo2 and use electrophysiology to characterize their inactivation kinetics during mechanical stimulation. We identify three small subdomains within the extracellular cap that individually can confer the distinct kinetics of inactivation of Piezo2 onto Piezo1. We further show by cysteine crosslinking that conformational flexibility of these subdomains is required for mechanical activation to occur and that electrostatic interactions functionally couple the cap to the extensive blades, which have been proposed to function as sensors of membrane curvature and tension. This study provides a demonstration of internal gating motions involved in mechanotransduction by Piezo1.

Inactivation of Mechanically Activated Piezo1 Ion Channels Is Determined by the C-Terminal Extracellular Domain and the Inner Pore Helix.

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.

Transduction of Repetitive Mechanical Stimuli by Piezo1 and Piezo2 Ion Channels.

Several cell types experience repetitive mechanical stimuli, including vein endothelial cells during pulsating blood flow, inner ear hair cells upon sound exposure, and skin cells and their innervating dorsal root ganglion (DRG) neurons when sweeping across a textured surface or touching a vibrating object. While mechanosensitive Piezo ion channels have been clearly implicated in sensing static touch, their roles in transducing repetitive stimulations are less clear. Here, we perform electrophysiological recordings of heterologously expressed mouse Piezo1 and Piezo2 responding to repetitive mechanical stimulations. We find that both channels function as pronounced frequency filters whose transduction efficiencies vary with stimulus frequency, waveform, and duration. We then use numerical simulations and human disease-related point mutations to demonstrate that channel inactivation is the molecular mechanism underlying frequency filtering and further show that frequency filtering is conserved in rapidly adapting mouse DRG neurons. Our results give insight into the potential contributions of Piezos in transducing repetitive mechanical stimuli.

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization.

A gold standard for characterizing mechanically activated (MA) currents is via heterologous expression of candidate channels in naive cells. Two recent studies described MA channels using this paradigm. TMEM150c was proposed to be a component of an MA channel partly based on a heterologous expression approach (Hong et al., 2016). In another study, Piezo1's N-terminal "propeller" domain was proposed to constitute an intrinsic mechanosensitive module based on expression of a chimera between a pore-forming domain of the mechanically insensitive ASIC1 channel and Piezo1 (Zhao et al., 2016). When we attempted to replicate these results, we found each construct conferred modest MA currents in a small fraction of naive HEK cells similar to the published work. Strikingly, these MA currents were not detected in cells in which endogenous Piezo1 was CRISPR/Cas9 inactivated. These results highlight the importance of choosing cells lacking endogenous MA channels to assay the mechanotransduction properties of various proteins. This Matters Arising paper is in response to Hong et al. (2016) and Zhao et al. (2016) in Neuron. See also the response papers by Hong et al. (2017) and Zhao et al. (2017) published concurrently with this Matters Arising.

Touch, Tension, and Transduction - The Function and Regulation of Piezo Ion Channels.

In 2010, two proteins, Piezo1 and Piezo2, were identified as the long-sought molecular carriers of an excitatory mechanically activated current found in many cells. This discovery has opened the floodgates for studying a vast number of mechanotransduction processes. Over the past 6 years, groundbreaking research has identified Piezos as ion channels that sense light touch, proprioception, and vascular blood flow, ruled out roles for Piezos in several other mechanotransduction processes, and revealed the basic structural and functional properties of the channel. Here, we review these findings and discuss the many aspects of Piezo function that remain mysterious, including how Piezos convert a variety of mechanical stimuli into channel activation and subsequent inactivation, and what molecules and mechanisms modulate Piezo function.

Mechanical sensitivity of Piezo1 ion channels can be tuned by cellular membrane tension.

Piezo1 ion channels mediate the conversion of mechanical forces into electrical signals and are critical for responsiveness to touch in metazoans. The apparent mechanical sensitivity of Piezo1 varies substantially across cellular environments, stimulating methods and protocols, raising the fundamental questions of what precise physical stimulus activates the channel and how its stimulus sensitivity is regulated. Here, we measured Piezo1 currents evoked by membrane stretch in three patch configurations, while simultaneously visualizing and measuring membrane geometry. Building on this approach, we developed protocols to minimize resting membrane curvature and tension prior to probing Piezo1 activity. We find that Piezo1 responds to lateral membrane tension with exquisite sensitivity as compared to other mechanically activated channels and that resting tension can drive channel inactivation, thereby tuning overall mechanical sensitivity of Piezo1. Our results explain how Piezo1 can function efficiently and with adaptable sensitivity as a sensor of mechanical stimulation in diverse cellular contexts.

Balboa binds to pickpocket in vivo and is required for mechanical nociception in Drosophila larvae.

The Drosophila gene pickpocket (ppk) encodes an ion channel subunit of the degenerin/epithelial sodium channel (DEG/ENaC) family. PPK is specifically expressed in nociceptive, class IV multidendritic (md) neurons and is functionally required for mechanical nociception responses. In this study, in a genome-wide genetic screen for other ion channel subunits required for mechanical nociception, we identify a gene that we name balboa (also known as CG8546, ppk26). Interestingly, the balboa locus encodes a DEG/ENaC ion channel subunit highly similar in amino acid sequence to PPK. Moreover, laser-capture isolation of RNA from larval neurons and microarray analyses reveal that balboa is also highly enriched in nociceptive neurons. The requirement for Balboa and PPK in mechanical nociception behaviors and their specific expression in larval nociceptors led us to hypothesize that these DEG/ENaC subunits form an ion channel complex in vivo. In nociceptive neurons, Balboa::GFP proteins distribute uniformly throughout dendrites but remarkably localize to discrete foci when ectopically expressed in other neuron subtypes (where PPK is not expressed). Indeed, ectopically coexpressing ppk transforms this punctate Balboa::GFP expression pattern to the uniform distribution observed in its native cell type. Furthermore, ppk-RNAi in class IV neurons alters the broad Balboa::GFP pattern to a punctate distribution. Interestingly, this interaction is mutually codependent as balboa-RNAi eliminates Venus::PPK from the sensory dendrites of nociceptors. Finally, using a GFP-reconstitution approach in transgenic larvae, we directly detect in vivo physical interactions among PPK and Balboa subunits. Combined, our results indicate a critical mechanical nociception function for heteromeric PPK and Balboa channels in vivo.

Resurgent current of voltage-gated Na(+) channels.

Resurgent Na(+) current results from a distinctive form of Na(+) channel gating, originally identified in cerebellar Purkinje neurons. In these neurons, the tetrodotoxin-sensitive voltage-gated Na(+) channels responsible for action potential firing have specialized mechanisms that reduce the likelihood that they accumulate in fast inactivated states, thereby shortening refractory periods and permitting rapid, repetitive, and/or burst firing. Under voltage clamp, step depolarizations evoke transient Na(+) currents that rapidly activate and quickly decay, and step repolarizations elicit slower channel reopening, or a 'resurgent' current. The generation of resurgent current depends on a factor in the Na(+) channel complex, probably a subunit such as NaVß4 (Scn4b), which blocks open Na(+) channels at positive voltages, competing with the fast inactivation gate, and unblocks at negative voltages, permitting recovery from an open channel block along with a flow of current. Following its initial discovery, resurgent Na(+) current has been found in nearly 20 types of neurons. Emerging research suggests that resurgent current is preferentially increased in a variety of clinical conditions associated with altered cellular excitability. Here we review the biophysical, molecular and structural mechanisms of resurgent current and their relation to the normal functions of excitable cells as well as pathophysiology.

Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVß4 open-channel blocking peptide.

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVß4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVß4 (the "ß4 peptide"). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; "CW" channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the ß4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

Iberiotoxin-sensitive and -insensitive BK currents in Purkinje neuron somata.

Purkinje cells have specialized intrinsic ionic conductances that generate high-frequency action potentials. Disruptions of their Ca or Ca-activated K (KCa) currents correlate with altered firing patterns in vitro and impaired motor behavior in vivo. To examine the properties of somatic KCa currents, we recorded voltage-clamped KCa currents in Purkinje cell bodies isolated from postnatal day 17-21 mouse cerebellum. Currents were evoked by endogenous Ca influx with approximately physiological Ca buffering. Purkinje somata expressed voltage-activated, Cd-sensitive KCa currents with iberiotoxin (IBTX)-sensitive (>100 nS) and IBTX-insensitive (>75 nS) components. IBTX-sensitive currents activated and partially inactivated within milliseconds. Rapid, incomplete macroscopic inactivation was also evident during 50- or 100-Hz trains of 1-ms depolarizations. In contrast, IBTX-insensitive currents activated more slowly and did not inactivate. These currents were insensitive to the small- and intermediate-conductance KCa channel blockers apamin, scyllatoxin, UCL1684, bicuculline methiodide, and TRAM-34, but were largely blocked by 1 mM tetraethylammonium. The underlying channels had single-channel conductances of ∼150 pS, suggesting that the currents are carried by IBTX-resistant (ß4-containing) large-conductance KCa (BK) channels. IBTX-insensitive currents were nevertheless increased by small-conductance KCa channel agonists EBIO, chlorzoxazone, and CyPPA. During trains of brief depolarizations, IBTX-insensitive currents flowed during interstep intervals, and the accumulation of interstep outward current was enhanced by EBIO. In current clamp, EBIO slowed spiking, especially during depolarizing current injections. The two components of BK current in Purkinje somata likely contribute differently to spike repolarization and firing rate. Moreover, augmentation of BK current may partially underlie the action of EBIO and chlorzoxazone to alleviate disrupted Purkinje cell firing associated with genetic ataxias.

Cross-species conservation of open-channel block by Na channel ß4 peptides reveals structural features required for resurgent Na current.

Voltage-gated Na channels in many neurons, including several in the cerebellum and brainstem, are specialized to allow rapid firing of action potentials. Repetitive firing is facilitated by resurgent Na current, which flows upon repolarization as Na channels recover through open states from block by an endogenous protein. The best candidate blocking protein to date is Na(V)ß4. The sequence of this protein diverges among species, however, while high-frequency firing is maintained, raising the question of whether the proposed blocking action of the Na(V)ß4 cytoplasmic tail has been conserved. Here, we find that, despite differences in the Na(V)ß4 sequence, Purkinje cells isolated from embryonic chick have resurgent currents with kinetics and amplitudes indistinguishable from those in mouse Purkinje cells. Furthermore, synthetic peptides derived from the divergent Na(V)ß4 cytoplasmic tails from five species have the capacity to induce resurgent current in mouse hippocampal neurons, which lack a functional endogenous blocking protein. These data further support a blocking role for Na(V)ß4 and also indicate the relative importance of different residues in inducing open-channel block. To investigate the contribution of the few highly conserved residues to open-channel block, we synthesized several mutant peptides in which the identities and relative orientations of a phenylalanine and two lysines were disrupted. These mutant peptides produced currents with vastly different kinetics than did the species-derived peptides, suggesting that these residues are required for an open-channel block that approximates physiological resurgent Na current. Thus, if other blocking proteins exist, they may share these structural elements with the Na(V)ß4 cytoplasmic tail.