Jörg Grandl

Piezo proteins are pore-forming subunits of mechanically activated channels

Mechanotransduction has an important role in physiology. Biological processes including sensing touch and sound waves require as-yet-unidentified cation channels that detect pressure. Mouse Piezo1 (MmPiezo1) and MmPiezo2 (also called Fam38a and Fam38b, respectively) induce mechanically activated cationic currents in cells; however, it is unknown whether Piezo proteins are pore-forming ion channels or modulate ion channels. Here we show that Drosophila melanogaster Piezo (DmPiezo, also called CG8486) also induces mechanically activated currents in cells, but through channels with remarkably distinct pore properties including sensitivity to the pore blocker ruthenium red and single channel conductances. MmPiezo1 assembles as a ∼1.2-million-dalton homo-oligomer, with no evidence of other proteins in this complex. Purified MmPiezo1 reconstituted into asymmetric lipid bilayers and liposomes forms ruthenium-red-sensitive ion channels. These data demonstrate that Piezo proteins are an evolutionarily conserved ion channel family involved in mechanotransduction.

Temperature-induced opening of TRPV1 ion channel is stabilized by the pore domain

TRPV1 is the founding and best-studied member of the family of temperature-activated transient receptor potential ion channels (thermoTRPs). Voltage, chemicals and heat allosterically gate TRPV1. Molecular determinants of TRPV1 activation by capsaicin, allicin, acid, ammonia and voltage have been identified. However, the structures and mechanisms mediating TRPV1s pronounced temperature sensitivity remain unclear. Recent studies of the related channel TRPV3 identified residues in the pore region that are required for heat activation. We used both random and targeted mutagenesis screens of rat TRPV1 and identified point mutations in the outer pore region that specifically impair temperature activation. Single-channel analysis indicated that TRPV1 mutations disrupted heat sensitivity by ablating long channel openings, which are part of the temperature-gating pathway. We propose that sequential occupancy of short and long open states on activation provides a mechanism for enhancing temperature sensitivity. Our results suggest that the outer pore is important for the heat sensitivity of thermoTRPs.

Pore region of TRPV3 ion channel is specifically required for heat-activation

Ion channels can be activated (gated) by a variety of stimuli, including chemicals, voltage, mechanical force or temperature. Although molecular mechanisms of ion channel gating by chemical and voltage stimuli are understood in principal, the mechanisms of temperature activation remain unknown. The transient receptor potential channel TRPV3 is a nonselective cation channel that is activated by warm temperatures and sensory chemicals such as camphor. Here we screened ∼14,000 random mutant clones of mouse TRPV3 and identified five single point mutations that specifically abolish heat activation but do not perturb chemical activation or voltage modulation. Notably, all five mutations are located in the putative sixth transmembrane helix and the adjacent extracellular loop in the pore region of mouse TRPV3. Although distinct in sequence, we found that the corresponding loop of frog TRPV3 is also specifically required for heat activation. These findings demonstrate that the temperature sensitivity of TRPV3 is separable from all other known activation mechanisms and implicate a specific region in temperature sensing.

Synergy between Piezo1 and Piezo2 channels confers high-strain mechanosensitivity to articular cartilage.

Significance Cartilage, a mechanically sensitive tissue that covers joints, is essential for vertebrate locomotion by sustaining skeletal mobility. Transduction of mechanical stimuli by cartilage cells, chondrocytes, leads to biochemical–metabolic responses. Such mechanotransduction can be beneficial for tissue maintenance when evoked by low-level mechanical stimuli, or can have health-adverse effects via cartilage-damaging high-strain mechanical stress. Thus, high-strain mechanotransduction by cartilage mechanotrauma is relevant for the pathogenesis of osteoarthritis. Molecular mechanisms of high-strain mechanotransduction of chondrocytes have been elusive. Here we identify Piezo1 and Piezo2 mechanosensitive ion channels in chondrocytes as transduction channels for high-strain mechanical stress. We verify their functional link to the cytoskeleton as important for their concerted function and offer a remedial strategy by application of a Piezo1/2 blocking peptide, GsMTx4, from tarantula venom. Diarthrodial joints are essential for load bearing and locomotion. Physiologically, articular cartilage sustains millions of cycles of mechanical loading. Chondrocytes, the cells in cartilage, regulate their metabolic activities in response to mechanical loading. Pathological mechanical stress can lead to maladaptive cellular responses and subsequent cartilage degeneration. We sought to deconstruct chondrocyte mechanotransduction by identifying mechanosensitive ion channels functioning at injurious levels of strain. We detected robust expression of the recently identified mechanosensitive channels, PIEZO1 and PIEZO2. Combined directed expression of Piezo1 and -2 sustained potentiated mechanically induced Ca2+ signals and electrical currents compared with single-Piezo expression. In primary articular chondrocytes, mechanically evoked Ca2+ transients produced by atomic force microscopy were inhibited by GsMTx4, a PIEZO-blocking peptide, and by Piezo1- or Piezo2-specific siRNA. We complemented the cellular approach with an explant-cartilage injury model. GsMTx4 reduced chondrocyte death after mechanical injury, suggesting a possible therapy for reducing cartilage injury and posttraumatic osteoarthritis by attenuating Piezo-mediated cartilage mechanotransduction of injurious strains.

Two amino acid residues determine 2-APB sensitivity of the ion channels TRPV3 and TRPV4.

Temperature-activated transient receptor potential ion channels (thermoTRPs) are polymodal detectors of various stimuli including temperature, voltage, and chemicals. To date, it is not known how TRP channels integrate the action of such disparate stimuli. Identifying specific residues required for channel-activation by distinct stimuli is necessary for understanding overall TRP channel function. TRPV3 is activated by warm temperatures and various chemicals, and is modulated by voltage. One potent activator of TRPV3 is 2-aminoethyl diphenylborinate (2-APB), a synthetic chemical that modulates many TRP channels. In a high-throughput mutagenesis screen of ≈14,000 mutated mouse TRPV3 clones, we found 2 residues (H426 and R696) specifically required for sensitivity of TRPV3 to 2-APB, but not to camphor or voltage. The cytoplasmic N-terminal mutation H426N in human, dog, and frog TRPV3 also effectively abolished 2-APB activation without affecting camphor responses. Interestingly, chicken TRPV3 is weakly sensitive to 2-APB, and the equivalent residue at 426 is an asparagine (N). Mutating this residue to histidine induced 2-APB sensitivity of chicken TRPV3 to levels comparable for other TRPV3 orthologs. The cytoplasmic C-terminal mutation R696K in the TRP box displayed 2-APB specific deficits only in the presence of extracellular calcium, suggesting involvement in gating. TRPV4, a related thermoTRP, is 2-APB insensitive and has variant sequences at both residues identified here. Remarkably, mutating these 2 residues in TRPV4 to TRPV3 sequences (N426H and W737R) was sufficient to induce TRPV3-like 2-APB sensitivity. Therefore, 2-APB activation of TRPV3 is separable from other activation mechanisms, and depends on 2 cytoplasmic residues.

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. DOI: http://dx.doi.org/10.7554/eLife.12088.001

Directionality of Temperature Activation in Mouse TRPA1 Ion Channel Can Be Inverted by Single-Point Mutations in Ankyrin Repeat Six

Several transient receptor potential (TRP) ion channels are activated with high sensitivity by either cold or hot temperatures. However, structures and mechanism that determine temperature directionality (cold versus heat) are not established. Here we screened 12,000 random mutant clones of the cold-activated mouse TRPA1 ion channel with a heat stimulus. We identified three single-point mutations that are individually sufficient to make mouse TRPA1 warm activated, while leaving sensitivity to chemicals unaffected. Mutant channels have high temperature sensitivity of voltage activation, specifically of channel opening, but not channel closing, which is reminiscent of other heat-activated TRP channels. All mutations are located in ankyrin repeat six, which identifies this domain as a sensitive modulator of thermal activation. We propose that a change in the coupling of temperature sensing to channel gating generates this sensitivity to warm temperatures. Our results demonstrate that minimal changes in protein sequence are sufficient to generate a wide diversity of thermal sensitivities in TRPA1.

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.

Single residues in the outer pore of TRPV1 and TRPV3 have temperature-dependent conformations.

Thermosensation is mediated by ion channels that are highly temperature-sensitive. Several members of the family of transient receptor potential (TRP) ion channels are activated by cold or hot temperatures and have been shown to function as temperature sensors in vivo. The molecular mechanism of temperature-sensitivity of these ion channels is not understood. A number of domains or even single amino acids that regulate temperature-sensitivity have been identified in several TRP channels. However, it is unclear what precise conformational changes occur upon temperature activation. Here, we used the cysteine accessibility method to probe temperature-dependent conformations of single amino acids in TRP channels. We screened over 50 amino acids in the predicted outer pore domains of the heat-activated ion channels TRPV1 and TRPV3. In both ion channels we found residues that have temperature-dependent accessibilities to the extracellular solvent. The identified residues are located within the second predicted extracellular pore loop. These residues are identical or proximal to residues that were shown to be specifically required for temperature-activation, but not chemical activation. Our data precisely locate conformational changes upon temperature-activation within the outer pore domain. Collectively, this suggests that these specific residues and the second predicted pore loop in general are crucial for the temperature-activation mechanism of these heat-activated thermoTRPs.

Localized force application reveals mechanically sensitive domains of Piezo1

Piezos are mechanically activated ion channels that function as sensors of touch and pressure in various cell types. However, the precise mechanism and structures mediating mechanical activation and subsequent inactivation have not yet been identified. Here we use magnetic nanoparticles as localized transducers of mechanical force in combination with pressure-clamp electrophysiology to identify mechanically sensitive domains important for activation and inactivation.