Search

Opioids are widely used for pain relief; however, chronic opioid use causes a paradoxical state of enhanced pain sensitivity, termed “Opioid-induced hyperalgesia (OIH).” Despite the clinical importance of OIH, the detailed mechanism by which it enhances pain sensitivity remains unclear. In this study, we tested whether repeated morphine induces a neuronal circuit polarization in the mouse spinal dorsal horn (SDH). Transgenic mice expressing GFP to neurokinin 1 receptor-expressing neurons (sNK1Rn) and GABAergic interneurons (sGABAn) that received morphine [20 mg/kg, once daily for four consecutive days (i.p.)] developed mechanical hypersensitivity. Repeated morphine altered synaptic strengths in the SDH as a specific cell-type but not in a gender-dependent manner. In sNK1Rn and non-tonic firing neurons, repeated morphine treatment significantly increased frequency of spontaneous excitatory postsynaptic current (sEPSC) and evoked EPSC (eEPSC). In addition, repeated morphine treatment significantly decreased evoked inhibitory postsynaptic current (eIPSC) in sNK1Rn. Conversely, in sGABAn and tonic firing neurons, repeated morphine treatment significantly decreased sEPSC frequency and eEPSC, but had no change of eIPSC in sGABAn. Interestingly, repeated morphine treatment significantly decreased neuronal rheobase of sNK1Rn but had no effect on sGABAn. These findings suggest that spinal neuronal circuit polarization maybe the mechanism of OIH and identify a potential therapeutic mechanism to prevent or treat opioid-induced pain.

Chronic pain is the most common neurological disorder of HIV patients. Multiple neuropathologies were identified in the pain pathway. Among them is the prominent astrocytic reaction (also know an astrogliosis). However, the pathogenic role and mechanism of the astrogliosis are unclear. Here, we show that the astrogliosis is crucial for the pain development induced by a key neurotoxic HIV protein gp120 and that a neuron-to-astrocyte Wnt5a signal controls the astrogliosis. Ablation of astrogliosis blocked the development of gp120-induced mechanical hyperalgesia, and concomitantly the expression of neural circuit polarization in the spinal dorsal horn. We demonstrated that conditional knockout of either Wnt5a in neurons or its receptor ROR2 in astrocytes abolished not only gp120-induced astrogliosis but also hyperalgesia and neural circuit polarization. Furthermore, we found that the astrogliosis promoted expression of hyperalgesia and NCP via IL-1β regulated by a Wnt5a-ROR2-MMP2 axis. Our results shed light on the role and mechanism of astrogliosis in the pathogenesis of HIV-associated pain.

Opioid analgesics are the frontline pain medicine for managing various types of pain. Paradoxically, repeated use of opioid analgesics may cause an exacerbated pain state known as opioid-induced hyperalgesia (OIH). OIH significantly contributes to dose escalation and consequently opioid overdose. In addition to neuronal malplasticity, emerging evidence suggests a critical role of reactive glia in OIH development. A potential astrocytic underpinning of OIH pathogenesis is indicated by their prominently activation in OIH animal models. However, this hypothesis has not been conclusively tested and the mechanism underlying the astrocyte activation remains unclear. Here, we show that reactive astrocytes (a.k.a. astrogliosis) are critical for OIH development in mice. Genetic ablation of astrogliosis inhibited the expression of OIH and morphine-induced neural circuit polarization (NCP) in the spinal dorsal horn (SDH). We also found that Wnt5a is a neuron-to-astrocyte signal that is required for morphine-induced astrogliosis. Conditional knock-out of Wnt5a in neurons or its co-receptor ROR2 in astrocytes blocked not only morphine-induced astrogliosis but also OIH and NCP. Furthermore, we showed that the Wnt5a-ROR2 signaling-dependent astrogliosis contributes to OIH via inflammasome-regulated IL-1β. Our results reveal an important role of morphine-induced astrogliosis in OIH pathogenesis and elucidate a neuron-to-astrocyte intercellular Wnt signaling pathway that controls the astrogliosis. One sentence summary Neuron-to-astrocyte Wnt5a signaling controls the pathogenesis of opioid-induced hyperalgesia via astrogliosis

HIV-1 infection of the nervous system causes various neurological diseases, and synaptic degeneration is likely a critical step in the neuropathogenesis. Our prior studies revealed a significant decrease of synaptic protein, specifically in the spinal dorsal horn of patients with HIV-1 in whom pain developed, suggesting a potential contribution of synaptic degeneration to the pathogenesis of HIV-associated pain. However, the mechanism by which HIV-1 causes the spinal synaptic degeneration is unclear. Here, we identified a critical role of microglia in the synaptic degeneration. In primary cortical cultures (dayin vitro14) and spinal cords of 3- to 5-month-old mice (both sexes), microglial ablation inhibited gp120-induced synapse decrease. Fractalkine (FKN), a microglia activation chemokine specifically expressed in neurons, was upregulated by gp120, and knockout of the FKN receptor CX3CR1, which is predominantly expressed in microglia, protected synapses from gp120-induced toxicity. These results indicate that the neuron-to-microglia intercellular FKN/CX3CR1 signaling plays a role in gp120-induced synaptic degeneration. To elucidate the mechanism controlling this intercellular signaling, we tested the role of the Wnt/β–catenin pathway in regulating FKN expression. Inhibition of Wnt/β-catenin signaling blocked both gp120-induced FKN upregulation and synaptic degeneration, and gp120 stimulated Wnt/β-catenin-regulated FKN expression via NMDA receptors (NMDARs). Furthermore, NMDAR antagonist APV, Wnt/β-catenin signaling suppressor DKK1, or knockout of CX3CR1 alleviated gp120-induced mechanical allodynia in mice, suggesting a critical contribution of the Wnt/β–catenin/FKN/CX3R1 pathway to gp120-induced pain. These findings collectively suggest that HIV-1 gp120 induces synaptic degeneration in the spinal pain neural circuit by activating microglia via Wnt3a/β-catenin-regulated FKN expression in neurons.SIGNIFICANCE STATEMENTSynaptic degeneration develops in the spinal cord dorsal horn of HIV patients with chronic pain, but the patients without the pain disorder do not show this neuropathology, indicating a pathogenic contribution of the synaptic degeneration to the development of HIV-associated pain. However, the mechanism underlying the synaptic degeneration is unclear. We report here that HIV-1 gp120, a neurotoxic protein that is specifically associated with the manifestation of pain in HIV patients, induces synapse loss via microglia. Further studies elucidate that gp120 activates microglia by stimulating Wnt/β-catenin-regulated fractalkine in neuron. The results demonstrate a critical role of microglia in the pathogenesis of HIV-associated synaptic degeneration in the spinal pain neural circuit.

Since 1967, spinal cord stimulation (SCS) has been used to manage chronic intractable pain of the trunk and limbs. Compared to traditional high-intensity, low-frequency (100 Hz) SCS that is thought to produce paresthesia and pain relief by stimulating large myelinated fibers in the dorsal column (DC), low-intensity, high-frequency (10 kHz) SCS has demonstrated long-term pain relief without generation of paresthesia. To understand this paresthesia-free analgesic mechanism of 10 kHz SCS, we examined whether 10 kHz SCS at intensities below sensory thresholds would modulate spinal dorsal horn (DH) neuronal function in a neuron type-dependent manner. By using in vivo and ex vivo electrophysiological approaches, we found that low-intensity (sub-sensory threshold) 10 kHz SCS, but not 1 kHz or 5 kHz SCS, selectively activates inhibitory interneurons in the spinal DH. This study suggests that low-intensity 10 kHz SCS may inhibit pain sensory processing in the spinal DH by activating inhibitory interneurons without activating DC fibers, resulting in paresthesia-free pain relief.

Reactive oxygen species has been suggested as a key player in neuropathic pain, causing central sensitization by changing synaptic strengths in spinal dorsal horn neurons. However, it remains unclear as to what type of reactive oxygen species changes what aspect of synaptic strengths for central sensitization in neuropathic pain conditions. In this study, we investigated whether mitochondrial superoxide affects both excitatory and inhibitory synaptic strengths in spinal dorsal horn neurons after peripheral nerve injury. Upregulation of mitochondrial superoxide level by knockout of superoxide dismutase-2 exacerbated neuropathic mechanical hypersensitivity caused by L5 spinal nerve ligation, whereas downregulation of mitochondrial superoxide level by overexpression of superoxide dismutase-2 alleviated the hypersensitivity. In spinal nerve ligation condition, the frequency of miniature excitatory postsynaptic currents increased, while that of miniature inhibitory postsynaptic currents decreased in spinal dorsal horn neurons. Superoxide dismutase-2-knockout augmented, whereas superoxide dismutase-2-overexpression prevented, the spinal nerve ligation-increased miniature excitatory postsynaptic currents frequency. However, superoxide dismutase-2-knockout had no effect on the spinal nerve ligation-decreased miniature inhibitory postsynaptic current frequency, and superoxide dismutase-2-overexpression unexpectedly decreased miniature inhibitory postsynaptic current frequency in the normal condition. When applied to the spinal cord slice during in vitro recordings, mitoTEMPO, a specific scavenger of mitochondrial superoxide, reduced the spinal nerve ligation-increased miniature excitatory postsynaptic currents frequency but failed to normalize the spinal nerve ligation-decreased miniature inhibitory postsynaptic current frequency. These results suggest that in spinal dorsal horn neurons, high levels of mitochondrial superoxide increase excitatory synaptic strength after peripheral nerve injury and contribute to neuropathic mechanical hypersensitivity. However, mitochondrial superoxide does not seem to be involved in the decreased inhibitory synaptic strength in this neuropathic pain condition.

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.

Mechanosensitive ion channels are force-transducing enzymes that couple mechanical stimuli to ion flux. Understanding the gating mechanism of mechanosensitive channels is challenging because the stimulus seen by the channel reflects forces shared between the membrane, cytoskeleton and extracellular matrix. Here we examine whether the mechanosensitive channel PIEZO1 is activated by force-transmission through the bilayer. To achieve this, we generate HEK293 cell membrane blebs largely free of cytoskeleton. Using the bacterial channel MscL, we calibrate the bilayer tension demonstrating that activation of MscL in blebs is identical to that in reconstituted bilayers. Utilizing a novel PIEZO1–GFP fusion, we then show PIEZO1 is activated by bilayer tension in bleb membranes, gating at lower pressures indicative of removal of the cortical cytoskeleton and the mechanoprotection it provides. Thus, PIEZO1 channels must sense force directly transmitted through the bilayer., PIEZO1 is a mechanosensitive ion channel, but the mechanism of force transduction is unknown. Here Cox and Bae et al. disrupt the cortical cytoskeleton in HEK293 cells to show that PIEZO1 is gated directly by membrane tension.

Piezo1 is a eukaryotic cation-selective mechanosensitive ion channel. To understand channel function in vivo, we first need to analyze and compare the response in the whole cell and the patch. In patches, Piezo1 inactivates and the current is fit well by a 3-state model with a single pressure-dependent rate. However, repeated stimulation led to an irreversible loss of inactivation. Remarkably, the loss of inactivation did not occur on a channel-by-channel basis but on all channels at the same time. Thus, the channels are in common mechanical domain. Divalent ions decreased the unitary conductance from ~68 pS to ~37 pS, irrespective of the cation species. Mg and Ca did not affect inactivation rates, but Zn caused a 3-fold slowing. CytochalasinD (cytoD) does not alter inactivation rates or the transition to the non-inactivating mode but does reduce the steady-state response. Whole-cell currents were similar to patch currents but also had significant differences. In contrast to the patch, cytoD inhibited the current suggesting that the activating forces were transmitted through the actin cytoskeleton. Hypotonic swelling that prestressed the cytoskeleton and the bilayer greatly increased the sensitivity of both control and cytoD cells so there are two pathways to transmit force to the channels. In contrast to patch, removing divalent ions decreased the whole-cell current. The difference between whole cell and patch properties provide new insights into our understanding of the Piezo1 gating mechanisms and cautions against generalization to in situ behavior.

It is important to know the mechanism of cell membrane fluctuation because it can be readout for the nanomechanical interaction between cytoskeleton and plasma membrane. Traditional techniques, however, have drawbacks such as probe contact with the cell surface, complicate analysis, and limit spatial and temporal resolution. In this study, we developed a new system for non-contact measurement of nano-scale localized-cell surface dynamics using modified-scanning ion-conductance microscopy. With 2 nm resolution, we determined that endothelial cells have local membrane fluctuations of ~20 nm, actin depolymerization causes increase in fluctuation amplitude, and ATP depletion abolishes all membrane fluctuations.

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the "split protein" is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone.

The ability to form gigaseals is essential for patch-clamp electrophysiology; however, ion channels located in the seal can produce measureable currents. To explore the expected properties of channels in the seal (i.e., rim channels), we created a mathematical model. The seal was a two-dimensional cable filled with saline and bounded on one side by membrane (with resistance and capacitance) and on the other side by glass (nonconductive and noncapacitive). We included ion depletion/accumulation around the channels. The channels were ohmic with a conductance that increased with the concentration of permeant ions. The aqueous seal thickness was set nominally to 1 nm. Imaging with fluorescent dyes in the pipette showed that the hydrophilic dye Alexa 488 is impermeant, but lipophilic FM1-43 labels the seal. The model showed that to obtain high-resistance seals, the conductivity of the seal media has to be

Cells dynamically interact with their physical micro-environment through the assembly of nascent focal contacts and focal adhesions. The dynamics and mechanics of these contact points are controlled by transmembrane integrins and an array of intracellular adaptor proteins. In order to study the mechanics and dynamics of focal adhesion assembly, we have developed a technique for the timed induction of a nascent focal adhesion. Bovine aortic endothelial cells were approached at the apical surface by a nanoelectrode whose position was controlled with a resolution of 10s of nanometers using changes in electrode current to monitor distance from the cell surface. Since this probe was functionalized with fibronectin, a focal contact formed at the contact location. Nascent focal adhesion assembly was confirmed using time-lapse confocal fluorescent images of red fluorescent protein (RFP) – tagged talin, an adapter protein that binds to activated integrins. Binding to the cell was verified by noting a lack of change of electrode current upon retraction of the electrode. This study demonstrates that functionalized nanoelectrodes can enable precisely-timed induction and 3-D mechanical manipulation of focal adhesions and the assay of the detailed molecular kinetics of their assembly.

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ∼80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ∼155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ∼30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.

PIEZO1 and PIEZO2 are mechanosensitive channels (MSCs) important for cellular function and mutations in them lead to human disorders. We examined how functional heteromers form between subunits of PIEZO1 using the mutants E2117K, E2117D, and E2117A. Homomers of E2117K do not conduct. E2117A homomers have low conductance with rapid inactivation, and those of E2117D have high conductance with slow inactivation. Pairing E2117K with E2117D or E2117A with E2117D gave rise to new channel species representing heteromers with distinct conductances. Whole-cell currents from co-expression of E2117A and E2117D fit well with a linear-combination model of homomeric channel currents suggesting that functional channels do not form from freely-diffusing, randomly-mixed monomers in-vitro. Whole-cell current from coexpressed PIEZO1/PIEZO2 also fit as a linear combination of homomer currents. High-resolution optical images of fluorescently-tagged channels support this interpretation because coexpressed subunits segregate into discrete domains.

Electric fields can be focused by micropipette-based electrodes to induce stresses on cell membranes leading to tension and poration. To date, however, these membrane stress distributions have not been quantified. In this study, we determine membrane tension, stress, and strain distributions in the vicinity of a microelectrode using finite element analysis of a multiscale electro-mechanical model of pipette, media, membrane, actin cortex, and cytoplasm. Electric field forces are coupled to membranes using the Maxwell stress tensor and membrane electrocompression theory. Results suggest that micropipette electrodes provide a new non-contact method to deliver physiological stresses directly to membranes in a focused and controlled manner, thus providing the quantitative foundation for micreoelectrotension, a new technique for membrane mechanobiology.

PIEZO1 is a recently cloned eukaryotic cation-selective channel that opens with mechanical force. We found that extracellular protonation inhibits channel activation by ≈90% by increased occupancy in the closed or the inactivated state. Titration between pH 6.3 and 8.3 exhibited a pK of ≈6.9. The steepness of the titration data suggests positive cooperativity, implying the involvement of at least two protonation sites. Whole-cell recordings yielded results similar to patches, and pH 6.5 reduced whole-cell currents by >80%. The effects were reversible. To assess whether pH acts on the open or the inactivated state, we tested a double-mutant PIEZO1 that does not inactivate. Cell-attached patches and whole-cell currents from this mutant channel were pH-insensitive. Thus, protonation appears to be associated with domain(s) of the channel involved with inactivation. pH also did not affect mutant channels with point mutations at position 2456 that are known to exhibit slow inactivation. To determine whether the physical properties of the membrane are altered by pH and thereby affect channel gating, we measured patch capacitance during mechanical stimuli at pH 6.5 and 7.3. The rate constants for changes in patch capacitance were independent of pH, suggesting that bilayer mechanics are not involved. In summary, low pH stabilizes the inactivated state. This effect may be important when channels are activated under pathological conditions in which the pH is reduced, such as during ischemia.

Members of the eukaryotic PIEZO family (the human orthologs are noted hPIEZO1 and hPIEZO2) form cation-selective mechanically-gated channels. We characterized the selectivity of human PIEZO1 (hPIEZO1) for alkali ions: K+, Na+, Cs+ and Li+; organic cations: TMA and TEA, and divalents: Ba2+, Ca2+, Mg2+ and Mn2+. All monovalent ions permeated the channel. At a membrane potential of -100 mV, Cs+, Na+ and K+ had chord conductances in the range of 35–55 pS with the exception of Li+, which had a significantly lower conductance of ~ 23 pS. The divalents decreased the single-channel permeability of K+, presumably because the divalents permeated slowly and occupied the open channel for a significant fraction of the time. In cell-attached mode, 90 mM extracellular divalents had a conductance for inward currents carried by the divalents of: 25 pS for Ba2+ and 15 pS for Ca2+ at -80 mV and 10 pS for Mg2+ at -50 mV. The organic cations, TMA and TEA, permeated slowly and attenuated K+ currents much like the divalents. As expected, the channel K+ conductance increased with K+ concentration saturating at ~ 45 pS and the KD of K+ for the channel was 32 mM. Pure divalent ion currents were of lower amplitude than those with alkali ions and the channel opening rate was lower in the presence of divalents than in the presence of monovalents. Exposing cells to the actin disrupting reagent cytochalasin D increased the frequency of openings in cell-attached patches probably by reducing mechanoprotection.

Eukaryotic PIEZO1 ion channels are cation selective and open with membrane tension. The channel is further characterized by voltage dependent inactivation and a conductance of ∼45 pS. We mutated the glutamic acid at position 2117 to an aspartic acid and this increased channel conductance to 72 pS. The mutations E2117A or E2117K lowered channel conductance. These mutations did not alter the current-voltage relationship or the ion selectivity, suggesting that the site is not in the selectivity filter but possibly near the pore. This is consistent with the cryo EM structure of mPiezo1 where the homologous residue is near, but not in the pore located toward the C-terminus. These mutations also affected the rates of activation and inactivation implying that the C-terminus is involved in channel gating. The mutation E2117D slowed the inactivation rate 8-fold. We made heteromeric channels in HEK cells by co-transfecting with high and low conductance mutants. This created active channels with two new unitary conductances indicating that pore formation occurs at the subunits’ interface. The number of new conducting states supports the trimer structure as revealed by Cryo EM. This is the first demonstration of functional heteromers of Piezo channels. Heteromeric formation is likely to be important for understanding the physiological activity of these channels.

Intradermally injected capsaicin induces secondary mechanical hyperalgesia and allodynia outside the primary (i.e., capsaicin-injected) site. This secondary mechanical hypersensitivity is attributed to central sensitization in which reactive oxygen species (ROS) play a key role. We examined whether ROS would be differentially involved in secondary mechanical hyperalgesia and allodynia using a mouse intraplantar capsaicin injection model. In mice, capsaicin-induced secondary mechanical hyperalgesia outlasted its allodynia counterpart. Unlike the hyperalgesia, the allodynia was temporarily abolished by an anesthetic given at the capsaicin-injected site. The ROS scavenger phenyl-N-tert-butylnitrone slowed the development of both secondary mechanical hyperalgesia and allodynia when administered before intraplantar capsaicin injection, whereas it inhibited only the allodynia when administered after capsaicin had already induced secondary mechanical hyperalgesia and allodynia. Intrathecal injection of the ROS donor KO2 induced both mechanical hyperalgesia and allodynia with the former outlasting the latter. Metformin, an activator of redox-sensitive adenosine monophosphate-activated protein kinase, selectively inhibited capsaicin-induced secondary mechanical allodynia and intrathecal KO2-induced mechanical allodynia. These results suggest that ROS is required for rapid activation of central sensitization mechanisms for both secondary mechanical hyperalgesia and allodynia after intraplantar capsaicin injection. Once activated, the mechanism for the hyperalgesia is long-lasting without being critically dependent on ongoing afferent activities arising from the capsaicin-injected site and the continuous presence of ROS. On the contrary, the ongoing afferent activities, ROS presence and adenosine monophosphate-activated protein kinase inhibition are indispensable for the maintenance mechanism for capsaicin-induced secondary mechanical allodynia.

We created clones that covalently linked a fluorescent protein to PIEZO1 in order to image PIEZO1 on the cell surface. Inserting mCherry1 in internal positions of the PIEZO1 protein, we found a construct at position 1591 that yielded a protein whose channel kinetics were nearly identical to wild type. Through cotransfection, we imaged PIEZO1 (mCherry) and TREK-1 (GFP) on HEK cells. These two mechanosensitive channels were at the cell surface in completely different physical domains. PIEZO1 appeared as random clusters but TREK tended to follow cytoskeleton tracks.Given the stability of the PEIZO1 protein with a fluorescent group at 1591, we asked if the PIEZO1 protein could be split at position 1591 and still function. Fragment 1 (1-1591) was fused to mCherry, and fragment 2 (1592-2520) to GFP. Co-expression of the two segments showed currents in whole cell recording, cell-attached patches, and outside-out patches. The biophysical characteristics were nearly identical to the wild type channel, having a reversal potential around 0 mV and voltage dependent inactivation in whole cell and cell attached mode.We next asked if either fragment alone was functional. The C-terminal fragment produced mechanosensitive currents in outside-out patches but the N terminal segment (1-1591) did not in any recording mode. We then shortened the C terminal fragment from its C- and N- termini using the outside-out patches to screen for current. We obtained mechanosensitive currents from a fragment of ∼ 450 amino acids, so the fundamental properties of the giant PIEZO1 channel are contained in this small fragment. This protein segment appears to contain the mechanosensor and the gate, and establishes that eight putative transmembrane domains are required for pore formation. None of the shortened fragments displayed whole cell currents.

Familial xerocytosis (HX) in humans is an autosomal disease that causes dehydration of red blood cells resulting in hemolytic anemia which has been traced to two individual mutations in the mechanosensitive ion channel, PIEZO1. Each mutation alters channel kinetics in ways that can explain the clinical presentation. Both mutations slowed inactivation and introduced a pronounced latency for activation. A conservative substitution of lysine for arginine (R2456K) eliminated inactivation and also slowed deactivation, indicating that this mutant's loss of charge is not responsible for HX. Fitting the current vs. pressure data to Boltzmann distributions showed that the half-activation pressure, P1/2, for M2225R was similar to that of WT, whereas mutations at position 2456 were left shifted. The absolute stress sensitivity was calibrated by cotransfection and comparison with MscL, a well-characterized mechanosensitive channel from bacteria that is driven by bilayer tension. The slope sensitivity of WT and mutant human PIEZO1 (hPIEZO1) was similar to that of MscL implying that the in-plane area increased markedly, by ∼6-20 nm(2) during opening. In addition to the behavior of individual channels, groups of hPIEZO1 channels could undergo simultaneous changes in kinetics including a loss of inactivation and a long (∼200 ms), silent latency for activation. These observations suggest that hPIEZO1 exists in spatial domains whose global properties can modify channel gating. The mutations that create HX affect cation fluxes in two ways: slow inactivation increases the cation flux, and the latency decreases it. These data provide a direct link between pathology and mechanosensitive channel dysfunction in nonsensory cells.

The ability to form gigaseals is essential for patch clamp, but the physics of how membranes form gigaseals cannot analyzed spectroscopically because the seal region is too thin. However, ion channels located in the seal can produce measureable currents that contribute to patch recordings and potentially their kinetics can elucidate properties of the seal. We modeled the seal using a finite element simulation of the 2D cable equation with the membrane (resistance and capacitance) as one side and nonconductive/noncapacitative glass as the other, and a resistive and diffusive aqueous solution in between. The channel current was calculated as I=g0∗C (x,y)/C0∗(V(x,y)-Vr) where g0 is the free channel conductance, C0 is the solution concentration of the source permeant ions that yields g0, C(x,y), and V(x,y) are respectively the concentration of source permeant ions and the potential at position (x,y),and Vr is the reversal potential calculated from the Nernst potential. Simulations showed that to obtain 10 GΩ seals, conductivity of the seal space had to be

Piezo1 is a cation selective channel in eukaryotes, and Xerocytosis is a disease of red cells that has been mapped to mutations in the gene encoding Piezo1. We introduced these mutations into a cloned human Pieoz1 and measured their effects on the channel kinetics. Substituting arginine for methionine at position 2225 (M2225R), or a hisitidine for arginine at position 2455 (R2455H) slowed inactivation. This behavior is true in whole cell recordings and cell-attached and outside-out current patches. At position 2455, introduction of lysine, a conservative amino acid change for arginine, we were unable to restore inactivation, indicating that a charge movement at that location in the membrane field is not involved in voltage-dependent inactivation. Cotransfection of cells with Piezo1 and MscL allowed us to estimate the relative stress sensitivity, equivalent to the mean area change, of both channels in a single patch. Surprisingly, the slope sensitivities of both channels were similar, indicating that the dimensional changes for opening Piezo1 is close to that of MscL at 6.5 nm2. We also demonstrate that the inhibitor of mechanical channels, GsMTx4, also blocks currents from mutant channels. The biophysics of Piezo dysfunction shows that the channel stays open longer than in the wild type and likely affects red cell volume regulation by allowing an excess influx of calcium.Supported by the National Institutes of Health and the Department of Defense

Thermally and actively driven cell membrane fluctuations are known to be readouts for the nanomechanical interaction between the cortical cytoskeleton and the plasma membrane and the membrane. In this study, we developed a non-contact method to measure cell surface fluctuations through measurements of resistance between a microelectrode tip and the cell membrane. The system resolution was < 2 nm tested by using 2-10 Hz sinusoidal piezo stage motion with amplitudes ranging from 2 nm to 100 nm. We found that endothelial cells exhibited local membrane fluctuations of ∼20 nm at a number of characteristic frequencies. To determine the role of actin in membrane fluctuation, we treated cells with 2 μM of actin depolymerizing drug, cytochalasin D, and we found that actin depolymerization increased in fluctuation amplitude up to 2 times at all frequencies. Finally, to determine role of ATP in membrane fluctuations, we treated cells with ATP depletion drug cocktail which consisted of 25nM Antimycin A + 2mg/ml 2-Deoxy-D-Glucose, and we found that ATP depletion abolished all membrane fluctuations. Therefore, actin cytoskeleton and dynamic processes facilitated by ATP may modulate membrane functions through mechanical effects on membrane fluctuations

Piezo1 channels gate with mechanical stress in the membrane and gating involves both activation and inactivation. In HEK293 cells transfected with Piezo1 and subjected to pressure stimuli, cell-attached patch recordings showed that the inactivation rate slowed as extracellular divalent ions were reduced. With >1mM Mg+2, activation had no measurable latency and the inactivation rate was rapid but stress dependent, suggesting that Mg+2 may act as an open channel blocker (the effects of Ca+2 are in progress). Without divalents there was no inactivation, but surprisingly, activation now had a pronounced latency (∼500 ms). Inactivation may actually represent adaptation of the local stimulus by the cytoskeleton and not overt channel closure. To disrupt the cytoskeleton we treated cells with cytochalasin D before patching and found inactivation was unaffected suggesting cytoskeletal adaption was not the cause. Attempting the inverse experiment, we increased cytoskeletal stress by swelling the cells osmotically, but that too didn't affect the inactivation rate.For analogy close to the in situ situation, we evoked whole cell Piezo1 currents by indenting cells with a glass probe. Like the patch, removing extracellular divalent ions reversibly reduced the inactivation rate. However, in contrast to patch recordings, Cytochalasin D caused a loss of whole cell current and cell swelling increased the evoked currents. These results suggest that the forces that gate Piezo1 in whole cell mode propagate through the cytoskeleton, and that divalent ion block may be responsible for inactivation.Supported by the NIH and CDMRP

Piezo1 is a eukaryotic mechanosensitive cation channel. We characterized its permeability to monovalent and divalent cations. The i-V relationships for 150 mM Na+, K+ and Cs+ ions were similar and they showed non-linearity (for 150 mM K+, the conductance was 36 pS at −40 mV and 47 pS at −100 mV). This similarity implies that the ions permeate without requiring much dehydration (hydrated radius of ions: Na+: 276 pm; K+: 232 pm; Cs+: 226 pm). However, conductance of Li+ was lower (27 pS at −100 mV) implying that some of the hydrated shell had to be shed in order for the ion to permeate the channel. At 150 mM, the impermeant occluding ions TMA, TEA and Tris with hydrated diameters of 550, 660, and 580 pm, respectively, severely attenuated K+ currents through the channel. Extracellular Tris attenuated the K+ current in a dose-dependent manner with an equilibrium constant of 30 mM. Permeation of monovalent ions and occlusion by Tris suggests that the radius of the pore is between 340 pm and 580 pm. Recording with pure divalent pipette solutions established the conductance of Mg2+, Ca2+ and Ba2+ ions. The conductances of inward currents at a membrane potential of 50 mV were 9 pS, 14 pS and 24 pS for Mg2+, Ca2+ and Ba2+ repectively. In general, divalent currents were rarely elicited compared to monovalent currents. Pre-exposing cells to cytochalasinD, a known actin disruptor, enhanced the probability of divalent-based currents. By maintaining K+ as the main cation and titrating it with divalents of increasing concentration we determined that divalents decreased the single-channel conductance of K+ based currents as though the ions blocked the open channel for a fraction of the time.

Piezo1 is a mechanically activated nonselective cation channel isolated from NEURO2A cell line (1). We measured the kinetic response to mechanical stimuli using a high speed pressure clamp. Expressing a clone of Piezo1 (courtesy Armand Patapoutian) in BAEC cells we compared them the responses to endogenous channels in the NEURO2A cells. The transfected BAEC cells responded to mechanical stress in cell-attached mode consistent with expression of Piezo1, having similar pressure sensitivity and voltage dependent inactivation. We modeled channel activity using a linear 3 state model of closed, open and inactivated with the closed to open state transition as pressure sensitive. Outside-out patches from transfected cells behaved similarly suggesting that the details of cytoskeletal structure are not critical to channel function. With outside-outs the pressure activation curve right shifted to a increased pressure as expected since the radius of curvature of outside-outs is smaller than inside-outs. We also showed that GsMTx4, an inhibitor of mechanical channels, inhibited Piezo1 currents in Neuro2A transfected cells. At a 2.5 µM we observed 75% inhibition of peak channel activity, and the effect was reversible with wash out rates on the order of seconds.1. Coste et al. Piezo1 and Piezo2 Are Essential Components of Distinct Mechanically Activated Cation Channels (2010) Sci 330 55-60.

Piezo1 is a cation selective channel isolated from eukaryotes which shows voltage dependent inactivation. Xerocytosis is a condition that causes hemolytic anemia of red blood cells associated with either of two individual mutations in the gene encoding Piezo1. In this work we have incorporated both mutations together into the Piezo1 gene and characterized the properties of the new channel. The protein has a substitution of arginine for methionine at position 2225 (M2225R) and a lysine for arginine at position 2455 (R2455K). The double mutant showed no voltage dependent inactivation, a property also seen for the single mutant R2455K. We measured the pressure dependence of the channel in cell attached mode and found it to be shifted significantly leftward (Boltzmann). The ability to activate Piezo1 at low pressure has allowed us to determine more accurately the kinetics of Piezo1 channel gating. The conductance of the double mutant is ∼ 30 pS. Whole cell currents were significantly larger, indicating that the double-mutant channel is more sensitive to cell mechanical stimulation. We have co-transfected Piezo1 and MscL and sequentially activated both channels with increasing pressure, allowing us to compare area changes within a single patch. The double mutated channel appears to have changes similar to those found for MscL. We also show that the channel is blocked by the addition of GsMTx4, a peptide inhibitor for mechanical channels.