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Mechanotransduction plays a crucial role in vascular biology. One example of this is local regulation of vascular resistance via flow-mediated vasodilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction, and a precursor to a wide array of vascular diseases such as hypertension and atherosclerosis. And yet, the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium, and is a critical signaling component in cardiovascular pathophysiology.

International audience; Mechanotransduction plays a crucial role in vascular biology. One example of this is the local regulation ofvascular resistance via flow-mediated dilation (FMD). Impairment of this process is a hallmark of endothelial dysfunction and a precursor to a wide array of vascular diseases, such as hypertension and atherosclerosis. Yet the molecules responsible for sensing flow (shear stress) within endothelial cells remain largely unknown. We designed a 384-well screening system that applies shear stress on cultured cells. We identified a mechanosensitive cell line that exhibits shear stress-activated calcium transients, screened a focused RNAi library, and identified GPR68 as necessary and sufficient for shear stress responses. GPR68 is expressed in endothelial cells of small-diameter (resistance) arteries. Importantly, Gpr68-deficient mice display markedly impaired acute FMD and chronic flow-mediated outward remodeling in mesenteric arterioles. Therefore, GPR68 is an essential flow sensor in arteriolar endothelium and is a critical signaling component in cardiovascular pathophysiology.

Summary Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro ; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro . The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development.

SummarySeveral 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.

Mechanotransduction, the pathway by which mechanical forces are translated to biological signals, plays important but poorly characterized roles in physiology. PIEZOs are recently identified, widely expressed, mechanically activated ion channels that are hypothesized to play a role in mechanotransduction in mammals. Here, we describe two distinct PIEZO2 mutations in patients with a subtype of Distal Arthrogryposis Type 5 characterized by generalized autosomal dominant contractures with limited eye movements, restrictive lung disease, and variable absence of cruciate knee ligaments. Electrophysiological studies reveal that the two PIEZO2 mutations affect biophysical properties related to channel inactivation: both E2727del and I802F mutations cause the PIEZO2-dependent, mechanically activated currents to recover faster from inactivation, while E2727del also causes a slowing of inactivation. Both types of changes in kinetics result in increased channel activity in response to a given mechanical stimulus, suggesting that Distal Arthrogryposis Type 5 can be caused by gain-of-function mutations in PIEZO2 . We further show that overexpression of mutated PIEZO2 cDNAs does not cause constitutive activity or toxicity to cells, indicating that the observed phenotype is likely due to a mechanotransduction defect. Our studies identify a type of channelopathy and link the dysfunction of mechanically activated ion channels to developmental malformations and joint contractures.

Although certain ion channels can be gated by temperature, it is currently unknown how this occurs. Here, the authors identify regions of TRPV3 that are critical for heat sensation, yet independent of other channel gating mechanisms. 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.

Six highly temperature-sensitive ion channels of the transient receptor potential (TRP) family have been implicated to mediate temperature sensation. These channels, expressed in sensory neurons innervating the skin or the skin itself, are active at specific temperatures ranging from noxious cold to burning heat. In addition to temperature sensation thermoTRPs are the receptors of a growing number of environmental chemicals (chemesthesis). Recent studies have provided some striking new insights into the molecular mechanism of thermal and chemical activation of these biological thermometers.

Piezo1 ion channels are mediators of mechanotransduction in several cell types including the vascular endothelium, renal tubular cells and erythrocytes. Gain-of-function mutations in PIEZO1 cause an autosomal dominant haemolytic anaemia in humans called dehydrated hereditary stomatocytosis. However, the phenotypic consequence of PIEZO1 loss of function in humans has not previously been documented. Here we discover a novel role of this channel in the lymphatic system. Through whole-exome sequencing, we identify biallelic mutations in PIEZO1 (a splicing variant leading to early truncation and a non-synonymous missense variant) in a pair of siblings affected with persistent lymphoedema caused by congenital lymphatic dysplasia. Analysis of patients' erythrocytes as well as studies in a heterologous system reveal greatly attenuated PIEZO1 function in affected alleles. Our results delineate a novel clinical category of PIEZO1-associated hereditary lymphoedema., Lukacs et al. identify mutations in the PIEZO1 gene in patients with congenital lymphatic dysplasia. The study also characterizes the functional consequence of the disease-associated Piezo1 mutant proteins and show attenuated ion channel function in cellular context.

Red blood cells (RBCs) experience significant mechanical forces while recirculating, but the consequences of these forces are not fully understood. Recent work has shown that gain-of-function mutations in mechanically activated Piezo1 cation channels are associated with the dehydrating RBC disease xerocytosis, implicating a role of mechanotransduction in RBC volume regulation. However, the mechanisms by which these mutations result in RBC dehydration are unknown. In this study, we show that RBCs exhibit robust calcium entry in response to mechanical stretch and that this entry is dependent on Piezo1 expression. Furthermore, RBCs from blood-cell-specific Piezo1 conditional knockout mice are overhydrated and exhibit increased fragility both in vitro and in vivo. Finally, we show that Yoda1, a chemical activator of Piezo1, causes calcium influx and subsequent dehydration of RBCs via downstream activation of the KCa3.1 Gardos channel, directly implicating Piezo1 signaling in RBC volume control. Therefore, mechanically activated Piezo1 plays an essential role in RBC volume homeostasis. DOI: http://dx.doi.org/10.7554/eLife.07370.001, eLife digest Within our bodies, cells and tissues are constantly being pushed and pulled by their surrounding environment. These mechanical forces are then transformed into electrical or chemical signals by cells. This process is crucial for many biological structures, such as blood vessels, to develop correctly, and is also a key part of our senses of touch and hearing. In 2010, researchers discovered a group of ion channels—proteins embedded in the membrane that surrounds a cell—that open up when a force is applied and allow calcium and other ions to enter the cell. This movement of ions generates the electrical response of the cell to the applied force. However, not much is known about the roles of these ‘Piezo’ ion channels. Red blood cells experience significant forces when they pass through narrow blood vessels. In a disease called xerocytosis, the red blood cells become severely dehydrated and shrink. In 2013, researchers discovered that patients with this disease have mutations in the gene that codes for the Piezo1 protein: a Piezo protein that has also been linked to a role in blood vessel development in embryos. This suggested that Piezo1 may regulate the volume of red blood cells. Cahalan, Lukacs et al.—including some of the researchers who worked on the 2010 and 2013 studies—have now investigated the role of Piezo1 in red blood cells in more detail. Applying strong forces to red blood cells from mice caused calcium to rapidly enter cells through Piezo1 channels. Cahalan, Lukacs et al. then deleted the Piezo1 gene from red blood cells. This made the cells larger and more fragile than normal cells because they contained too much water. To investigate how Piezo1 regulates water content, the cells were treated with a chemical compound called Yoda1. This compound was shown in a separate study by Syeda et al. to activate Piezo1 channels. Activating Piezo1 caused a second type of ion channel to open up as well, which allowed potassium ions and water molecules to leave the cell. This resulted in the cell becoming dehydrated. This work raises the possibility that Piezo proteins are involved in other diseases where red blood cell volume is altered. In particular, many believe that Piezo1 may be involved in sickle cell disease, a possibility that can now be tested using the tools described in this study. DOI: http://dx.doi.org/10.7554/eLife.07370.002

Within our bodies, cells and tissues are constantly being pushed and pulled by their surrounding environment. These mechanical forces are then transformed into electrical or chemical signals by cells. This process is crucial for many biological structures, such as blood vessels, to develop correctly, and is also a key part of our senses of touch and hearing. In 2010, researchers discovered a group of ion channels—proteins embedded in the membrane that surrounds a cell—that open up when a force is applied and allow ions such as calcium, potassium, and sodium to flow. This movement of ions generates the electrical response of the cell to the applied force. However, not much is known about how these ‘Piezo’ ion channels work. To investigate this, it is important to be able to precisely control how and when the Piezo channels open. Many other ion channels are studied by using small chemical compounds to activate them, but there were none that were known to act on Piezo proteins. Syeda et al.—including some of the researchers involved in the 2010 work—screened over three million compounds for their ability to cause calcium ions to flow into human cells to try to identify chemicals that activate the Piezo channels. This revealed one promising candidate named Yoda1, which specifically activated Piezo1: a Piezo protein that had previously been linked to a role in blood vessel development in embryos. To investigate how Yoda1 activates Piezo1, Syeda et al. placed Piezo1 in an artificial cell membrane that did not contain any other cellular components. When Yoda1 was added to this set up, the Piezo1 channels opened up. This suggests that Piezo1 and Yoda1 interact in a manner that does not require additional cellular components other than a cell membrane. Separate work by Cahalan, Lukacs et al. uses Yoda1 to reveal that Piezo1 helps to control the volume of red blood cells, showing that in the future, Yoda1 could be valuable in research that investigates the roles of Piezo1.

Fatty acid amide hydrolase (FAAH) is an integral membrane enzyme that catabolizes several bioactive lipids in vivo. Most of the physiological substrates of FAAH characterized to date belong to the N-acyl ethanolamine (NAE) class of fatty acid amides, including the endocannabinoid anandamide, the anti-inflammatory lipid N-palmitoyl ethanolamine, and the satiating factor N-oleoyl ethanolamine. We recently identified a second structural class of fatty acid amides regulated by FAAH in vivo: the N-acyl taurines (NATs). Global metabolite profiling revealed high concentrations of long chain (or = C20) saturated NATs in the central nervous system (CNS) of FAAH(-/-) mice. Here, we use metabolite profiling to characterize the FAAH-NAT system in peripheral mouse tissues. Livers and kidneys of FAAH(-/-) mice possessed dramatic elevations in NATs, which, in contrast to those detected in the CNS, were enriched in polyunsaturated acyl chains (e.g., C20:4, C22:6). Peripheral NATs rose more than 10-fold within 1 h following pharmacological inactivation of FAAH and reached levels up to approximately 5000 pmol/g tissue (C22:6 in kidney), implicating a constitutive and highly active pathway for NAT metabolism in which FAAH plays an integral part. Interestingly, NATs were found to activate multiple members of the transient receptor potential (TRP) family of calcium channels, including TRPV1 and TRPV4, which are both expressed in kidney. The dramatic elevation in endogenous levels of NATs following acute or chronic inactivation of FAAH, in conjunction with the pharmacological effects of these lipids on TRP channels, suggests the existence of a second major lipid signaling system regulated by FAAH in vivo.

Menthol is a cooling compound derived from mint leaves and is extensively used as a flavoring chemical. Menthol activates transient receptor potential melastatin 8 (TRPM8), an ion channel also activated by cold, voltage and phosphatidylinositol-4,5-bisphosphate (PIP2). Here we investigated the mechanism by which menthol activates mouse TRPM8. Using a new high-throughput approach, we screened a random mutant library consisting of approximately 14,000 individual TRPM8 mutants for clones that are affected in their response to menthol while retaining channel function. We identified determinants of menthol sensitivity in two regions: putative transmembrane segment 2 (S2) and the C-terminal TRP domain. Analysis of these mutants indicated that activation by menthol involves a gating mechanism distinct and separable from gating by cold, voltage or PIP2. Notably, TRP domain mutations mainly attenuated menthol efficacy, suggesting that this domain influences events downstream of initial binding. In contrast, S2 mutations strongly shifted the concentration dependence of menthol activation, raising the possibility that S2 influences menthol binding.

SummaryGarlic’s pungent flavor has made it a popular ingredient in cuisines around the world and throughout history. Garlic’s health benefits have been elevated from folklore to clinical study [1–5]. Although there is some controversy as to the efficacy of garlic, garlic products are one of the most popular herbal supplements in the U.S. [6]. Chemically complex, garlic contains different assortments of sulfur compounds depending on whether the cloves are intact, crushed, cooked, or raw [7]. Raw garlic, when cut and placed on the tongue or lips, elicits painful burning and prickling sensations through unknown mechanisms. Here, we show that raw but not baked garlic activates TRPA1 and TRPV1, two temperature-activated ion channels that belong to the transient receptor potential (TRP) family [8–12]. These thermoTRPs are present in the pain-sensing neurons that innervate the mouth. We further show that allicin, an unstable component of fresh garlic, is the chemical responsible for TRPA1 and TRPV1 activation and is therefore likely to cause garlic’s pungency.

How do we feel temperature? A study now finds that a recently discovered anion channel is a temperature sensor that, like previously identified cation channels, mediates the perception of noxious hot temperatures.

A subset of Transient Receptor Potential (TRP) ion channels is expressed in sensory neurons and enables thermosensation in mammals. These TRP channels are activated with high sensitivity either by hot or cold temperatures. The molecular mechanism of this temperature-directionality (heat vs. cold) is not understood. TRPA1 is special in this respect, because several TRPA1 orthologues are activated by either heat or cold or are temperature-insensitive. We hypothesized that specific amino-acid residues in TRP channels determine temperature-directionality and that mutation of these residues might shift the thermal-activation profile. To test this hypothesis, we generated a library of cold-activated mouse TRPA1 containing ∼12,000 random mutant clones and stimulated it with hot temperatures, while measuring channel activity in a FLIPR calcium assay.We discovered three single-point mutations in mouse TRPA1 that each cause strong heat-sensitivity in TRPA1. All three mutations are located in ankyrin repeat six. The sensitivity to chemical agonists remained unaffected in all three mutants. Electrophysiological characterization of one point-mutant revealed a strong temperature-dependence of voltage-sensitivity, the rate of channel activation but not deactivation, and the open-probability but not unitary conductance. Taken together, our data demonstrate that single-point mutations are sufficient to make mouse TRPA1 strongly heat-sensitive and that chemical activation can be uncoupled from temperature activation. We propose that a change in the coupling between temperature-sensing and channel-gating causes this change in temperature-directionality.

The itch sensation results from the excitation of primary sensory nerve endings in the skin, but the underlying molecular mechanisms are not completely understood. Liu et al. (2009) now report that some members of the Mrgpr class of G protein-coupled receptors mediate the itch caused by the antimalarial drug chloroquine.

The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR2-COO- [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-18146]. In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues. Modification of the OH or the COO- groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers. The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group. However, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups. These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP. The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.

The plasma membrane H+-ATPase of yeast assumes distinct conformational states during its catalytic cycle. To better understand structural changes in the LOOP1 domain, a catalytically important cytoplasmic loop segment linking transmembrane segments 2 and 3, surface epitopes were examined at different stages of catalysis. A polyclonal rabbit antibody was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein. This antibody was affinity-purified to produce a LOOP1-specific fraction that could be used in competition enzyme-linked immunosorbent assays to assess surface exposure of the LOOP1 epitopes. It was found that in an E1 conformation stabilized with either adenosine 5'-(beta,gamma -imino)triphosphate (AMP-PNP) or ADP, less than 10% of the LOOP1 epitopes were accessible on native enzyme. However, when the enzyme was stabilized in an E2-state with ATP plus vanadate, approximately 40% of the surface epitopes on LOOP1 became accessible to antibody. The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure. Enzyme-linked immunosorbent assays utilizing fusion proteins consisting of LOOP1 subdomains demonstrated that all of the available epitopes were contained in the beta-strand region (Glu-195-- Val-267) of LOOP1. The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TGES sequence. Our results suggest that during catalysis either the beta-strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.

Citrate metabolism in the lactic acid bacterium Leuconostoc mesenteroides generates an electrochemical proton gradient across the membrane by a secondary mechanism (C. Marty-Teysset, C. Posthuma, J. S. Lolkema, P. Schmitt, C. Divies, and W. N. Konings, J. Bacteriol. 178:2178–2185, 1996). Reports on the energetics of citrate metabolism in the related organism Lactococcus lactis are contradictory, and this study was performed to clarify this issue. Cloning of the membrane potential-generating citrate transporter (CitP) of Leuconostoc mesenteroides revealed an amino acid sequence that is almost identical to the known sequence of the CitP of Lactococcus lactis . The cloned gene was expressed in a Lactococcus lactis Cit − strain, and the gene product was functionally characterized in membrane vesicles. Uptake of citrate was counteracted by the membrane potential, and the transporter efficiently catalyzed heterologous citrate-lactate exchange. These properties are essential for generation of a membrane potential under physiological conditions and show that the Leuconostoc CitP retains its properties when it is embedded in the cytoplasmic membrane of Lactococcus lactis . Furthermore, using the same criteria and experimental approach, we demonstrated that the endogenous CitP of Lactococcus lactis has the same properties, showing that the few differences in the amino acid sequences of the CitPs of members of the two genera do not result in different catalytic mechanisms. The results strongly suggest that the energetics of citrate degradation in Lactococcus lactis and Leuconostoc mesenteroides are the same; i.e., citrate metabolism in Lactococcus lactis is a proton motive force-generating process.

Membrane potential generation via malate/lactate exchange catalyzed by the malate carrier (MleP) of Lactococcus lactis, together with the generation of a pH gradient via decarboxylation of malate to lactate in the cytoplasm, is a typical example of a secondary proton motive force-generating system. The mleP gene was cloned, sequenced, and expressed in a malolactic fermentation-deficient L. lactis strain. Functional analysis revealed the same properties as observed in membrane vesicles of a malolactic fermentation-positive strain. MleP belongs to a family of secondary transporters in which the citrate carriers from Leuconostoc mesenteroides (CitP) and Klebsiella pneumoniae (CitS) are found also. CitP, but not CitS, is also involved in membrane potential generation via electrogenic citrate/lactate exchange. MleP, CitP, and CitS were analyzed for their substrate specificity. The 2-hydroxycarboxylate motif R1R2COHCOOH, common to the physiological substrates, was found to be essential for transport although some 2-oxocarboxylates could be transported to a lesser extent. Clear differences in substrate specificity among the transporters were observed because of different tolerances toward the R substituents at the C2 atom. Both MleP and CitP transport a broad range of 2-hydroxycarboxylates with R substituents ranging in size from two hydrogen atoms (glycolate) to acetyl and methyl groups (citromalate) for MleP and two acetyl groups (citrate) for CitP. CitS was much less tolerant and transported only citrate and at a low rate citromalate. The substrate specificities are discussed in the context of the physiological function of the transporters.

Nature Communications 4: Article number: 1884 (2013); Published: 21 May 2013; Updated: 23 September 2013. In the first paragraph of the Methods section in this Article, case 8 (K8 in Table 1) was incorrectly referred to as case 4. The following is the correct sentence: ‘Case 8 (ref. 3) and family 3 (refs 32, 33) were case reports, these patients expressed a moderate form of anaemia and haemolysis without initial evidence of perinatal oedema or pseudohyperkalaemia.

International audience; Dehydrated hereditary stomatocytosis is a genetic condition with defective red blood cell membrane properties that causes an imbalance in intracellular cation concentrations. Recently, two missense mutations in the mechanically activated PIEZO1 (FAM38A) ion channel were associated with dehydrated hereditary stomatocytosis. However, it is not known how these mutations affect PIEZO1 function. Here, by combining linkage analysis and whole-exome sequencing in a large pedigree and Sanger sequencing in two additional kindreds and 11 unrelated dehydrated hereditary stomatocytosis cases, we identify three novel missense mutations and one recurrent duplication in PIEZO1, demonstrating that it is the major gene for dehydrated hereditary stomatocytosis. All the dehydrated hereditary stomatocytosis-associated mutations locate at C-terminal half of PIEZO1. Remarkably, we find that all PIEZO1 mutations give rise to mechanically activated currents that inactivate more slowly than wild-type currents. This gain-of-function PIEZO1 phenotype provides insight that helps to explain the increased permeability of cations in red blood cells of dehydrated hereditary stomatocytosis patients. Our findings also suggest a new role for mechanotransduction in red blood cell biology and pathophysiology.

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 for TRPV1 activation by capsaicin, allicin, acid, ammonia, and voltage have been identified. However, many years after the discovery of TRPV1, the structures and mechanisms mediating temperature-sensitivity remain unclear. Recent studies of the related channel TRPV3 identified residues within the pore region required for heat activation. Here we describe both random and targeted mutagenesis screens of TRPV1 to identify single point-mutations that specifically affect temperature-activation. The mutations found are all located in the outer pore region, in close proximity to but distinct from residues previously implicated in acid-activation. Electrophysiological analysis shows that mutations affect a temperature-independent step that is part of the temperature-gating pathway. These results suggest that the outer pore plays a general role in heat-sensitivity of thermoTRPs.

Zinc is an essential biological trace element. It is required for the structure or function of over 300 proteins, and it is increasingly recognized for its role in cell signaling. However, high concentrations of zinc have cytotoxic effects, and overexposure to zinc can cause pain and inflammation through unknown mechanisms. Here we show that zinc excites nociceptive somatosensory neurons and causes nociception in mice through TRPA1, a cation channel previously shown to mediate the pungency of wasabi and cinnamon through cysteine modification. Zinc activates TRPA1 through a unique mechanism that requires zinc influx through TRPA1 channels and subsequent activation via specific intracellular cysteine and histidine residues. TRPA1 is highly sensitive to intracellular zinc, as low nanomolar concentrations activate TRPA1 and modulate its sensitivity. These findings identify TRPA1 as an important target for the sensory effects of zinc and support an emerging role for zinc as a signaling molecule that can modulate sensory transmission.

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.

Maintaining physiological pH is required for survival, and exposure to alkaline chemicals such as ammonia (smelling salts) elicits severe pain and inflammation through unknown mechanisms. TRPV1, the capsaicin receptor, is an integrator of noxious stimuli including heat and extracellular acidic pH. Here, we report that ammonia activates TRPV1, TRPA1 (another polymodal nocisensor), and other unknown receptor(s) expressed in sensory neurons. Ammonia and intracellular alkalization activate TRPV1 through a mechanism that involves a cytoplasmic histidine residue, not used by other TRPV1 agonists such as heat, capsaicin or low pH. Our studies show that TRPV1 detects both acidic and basic deviations from homeostatic pH.

The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism by which CitP transports both a divalent and a monovalent substrate was the subject of this investigation. Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR(2)-COO(-). CitP has a high affinity for substrates that have a "second" carboxylate at one of the R groups, such as divalent citrate and (S)-malate (Bandell, M., and Lolkema, J. S. (1999) Biochemistry 38, 10352-10360). Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity. In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue. By using two substrates, i.e. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not. The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold. Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively. It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates, and thus plays an important role in the generation of the membrane potential.

The membrane potential-generating transporters CitP of Leuconostoc mesenteroides and MleP of Lactococcus lactis are homologous proteins with 48% identical residues that catalyze citrate-lactate and malate-lactate exchange, respectively. The two transporters are highly specific for substrates containing a 2-hydroxycarboxylate motif (HO-CR(2)-COO(-)) in which substitutions of the R groups are tolerated well. Differences in substrate specificity between MleP and CitP are based on subtle changes in the interaction of the protein with the R groups affecting both binding and translocation properties. The conserved, 46-residue long C-terminal region of the transporters containing the C-terminal putative transmembrane segment XI was investigated for its role in substrate recognition by constructing chimeric transporters. Replacement of the C-terminal region of MleP with that of CitP and vice versa did not alter the exchange kinetics with the substrates malate and citrate, indicating that the main interactions between the proteins and di- and tricarboxylate substrates were not altered. In contrast, the interaction of the proteins with the monocarboxylate substrates mandelate and 2-hydroxyisovalerate changed in a complementary manner. The affinity of CitP for the S-enantiomers of these substrates was at least 1 order of magnitude lower than observed for MleP. Introduction of the C-terminal residues of MleP in CitP resulted in a higher affinity and vice versa. Interchanging the C-termini had a more complicated effect on the R-enantiomers, affecting different kinetic parameters with different substrates, indicating multiple interactions of the R groups at this side of the binding pocket. It is suggested that the binding pocket is located between transmembrane segment XI and the other transmembrane segments of the transporters.

The cytoplasmic domain linking transmembrane segments 2 and 3 (LOOP1) of the yeast H + -ATPase was probed by the introduction of unique factor Xa recognition sites. Three sites, I 170 EGR, I 254 EGR and I 275 EGR, representing different structural regions of the LOOP1 domain, were engineered by site-specific mutagenesis of the PMA1 gene. In each case, multiple amino acid substitutions were required to form the factor Xa sites, which enabled an analysis of clustered mutations. Both I 170 EGR and I 275 EGR-containing mutants grew at normal rates, but showed prominent growth resistance to hygromycin B and sensitivity to low external pH. The engineered I 254 EGR site within the predicted β-strand region produced a recessive lethal phenotype, indicating that mutations G 254 I and F 257 R were not tolerated. Mutant I 170 EGR- and I 275 EGR-containing enzymes showed relatively normal K m and V max values, but they displayed a strong insensitivity to inhibition by vanadate. An I 170 EGR/I 275 EGR double mutant was more significantly perturbed showing a reduced V max and pronounced vanadate insensitivity. The I 170 EGR site within the putative α-helical stalk region was cleaved to a maximum of 10% by factor Xa under non-denaturing conditions resulting in a characteristic 81 kDa fragment, whereas the I 275 EGR site, near the end of the β-strand region, showed about 30–35% cleavage with the appearance of a 70 kDa fragment. A I 170 EGR/I 275 EGR double mutant enzyme showed about 55–60% cleavage. The cleavage profile for the mutant enzymes was enhanced under denaturing conditions, but was unaffected by MgATP or MgATP plus vanadate. Cleavage at the I 275 EGR position had no adverse effects on ATP hydrolysis or proton transport by the H + -ATPase making it unlikely that this localized region of LOOP1 influences coupling. Overall, these results suggest that the local region encompassing I 275 EGR is accessible to factor Xa, while the region around I 170 EGR appears buried. Although there is no evidence for gross molecular motion at either site, the effects of multiple amino acid substitutions in these regions suggest that the LOOP1 domain is conformationally active, and that perturbations in this domain affect the distribution of conformational intermediates during steady-state catalysis.

Six members of the mammalian transient receptor potential (TRP) ion channels respond to varied temperature thresholds. The natural compounds capsaicin and menthol activate noxious heat-sensitive TRPV1 and cold-sensitive TRPM8, respectively. The burning and cooling perception of capsaicin and menthol demonstrate that these ion channels mediate thermosensation. We show that, in addition to noxious cold, pungent natural compounds present in cinnamon oil, wintergreen oil, clove oil, mustard oil, and ginger all activate TRPA1 (ANKTM1). Bradykinin, an inflammatory peptide acting through its G protein-coupled receptor, also activates TRPA1. We further show that phospholipase C is an important signaling component for TRPA1 activation. Cinnamaldehyde, the most specific TRPA1 activator, excites a subset of sensory neurons highly enriched in cold-sensitive neurons and elicits nociceptive behavior in mice. Collectively, these data demonstrate that TRPA1 activation elicits a painful sensation and provide a potential molecular model for why noxious cold can paradoxically be perceived as burning pain.