C-C motif chemokine receptor-like 2 (CCRL2) is a non-signaling 7 transmembrane receptor that binds chemotactic ligands to shape leukocyte recruitment to sites of inflammation. However, there is a lack of consensus on the ligands that directly bind CCRL2 or their functional impact. Studies with CCRL2 knockout mice have demonstrated that neutrophils have impaired degranulation and migration in response to CXCL8, where the underlying molecular mechanism is proposed to be due to the formation of CCRL2 heterodimers with the chemokine receptor CXCR2. Herein, we characterized the ligands that bind directly to CCRL2 and interrogated the impact of CCRL2 neutralization on CXCL8 signaling in neutrophils using pharmacological antibody tools. Using flow cytometry and Surface Plasmon Resonance microscopy (SPRm) cell binding experiments, we confirmed that chemerin, but not previously reported C-C chemokines, binds CCRL2. Furthermore, we identified human and mouse CCRL2 antibodies that neutralized chemerin binding to CCRL2. Unexpectedly, we found that neutralization of CCRL2 with these antibodies did not attenuate CXCL8-induced human neutrophil degranulation nor CXCL8-induced murine neutrophil recruitment to the peritoneum. Based on the observed differences in modulating CCRL2 function with neutralizing antibodies compared to the reported CCRL2 deficient murine models, we hypothesize that the ligand binding function of CCRL2 is dispensable for CXCL8 signaling in neutrophils. Finally, extensive profiling of CCRL2 expression on peripheral blood leukocytes revealed monocytes, dendritic cells (DC), and subpopulations of natural killer T (NKT) cells as additional targets, highlighting potential roles for CCRL2 in human cell types beyond neutrophils that warrants future investigation.

Hunger and pain are two competing signals that individuals must resolve to ensure survival. However, the neural processes that prioritize conflicting survival needs are poorly understood. We discovered that hunger attenuates behavioral responses and affective properties of inflammatory pain without altering acute nociceptive responses. This effect is centrally controlled, as activity in hunger-sensitive agouti-related protein (AgRP)-expressing neurons abrogates inflammatory pain. Systematic analysis of AgRP projection subpopulations revealed that the neural processing of hunger and inflammatory pain converge in the hindbrain parabrachial nucleus (PBN). Strikingly, activity in AgRP → PBN neurons blocked the behavioral response to inflammatory pain as effectively as hunger or analgesics. The anti-nociceptive effect of hunger is mediated by neuropeptide Y (NPY) signaling in the PBN. By investigating the intersection between hunger and pain, we have identified a neural circuit that mediates competing survival needs and uncovered NPY Y1 receptor signaling in the PBN as a target for pain suppression.

The brain regulates food intake by processing sensory cues and peripheral physiological signals, but the neural basis of this integration remains unclear. Hypothalamic, agouti-related protein (AgRP)-expressing neurons are critical regulators of food intake. AgRP neuron activity is high during hunger and is rapidly reduced by the sight and smell of food. Here, we reveal two distinct components of AgRP neuron activity regulation: a rapid but transient sensory-driven signal and a slower, sustained calorie-dependent signal. We discovered that nutrients are necessary and sufficient for sustained reductions in AgRP neuron activity and that activity reductions are proportional to the calories obtained. This change in activity is recapitulated by exogenous administration of gut-derived satiation signals. Furthermore, we showed that the nutritive value of food trains sensory systems-in a single trial-to drive rapid, anticipatory AgRP neuron activity inhibition. Together, these data demonstrate that nutrients are the primary regulators of AgRP neuron activity.

Significance Humans express nearly 80 K + channels. This large diversity of K + channels (compare 10 Na + channels and 10 Ca 2+ channels) reflects their importance in tying many different biochemical processes to electrical signaling and to shaping and fine-tuning the electrical waveforms produced by excitable cells. We understand the biological roles of only a tiny fraction of the K + channels because, for many of them, good pharmacological tools do not exist. To remedy this situation and thus open up a new avenue to study K + channels’ biological roles, we developed a new cell-free assay that enables the discovery of inhibitors and activators for almost any K + channel. It also provides a new high-throughput human ether-à-go-go-related gene safety assay.

Mechanosensitive ion channels underlie neuronal responses to physical forces in the sensation of touch, hearing, and other mechanical stimuli. The fundamental basis of force transduction in eukaryotic mechanosensitive ion channels is unknown. Are mechanical forces transmitted directly from membrane to channel as in prokaryotic mechanosensors or are they mediated through macromolecular tethers attached to the channel? Here we show in cells that the K+ channel TRAAK (K2P4.1) is responsive to mechanical forces similar to the ion channel Piezo1 and that mechanical activation of TRAAK can electrically counter Piezo1 activation. We then show that the biophysical origins of force transduction in TRAAK and TREK1 (K2P2.1) two-pore domain K+ (K2P) channels come from the lipid membrane, not from attached tethers. These findings extend the "force-from-lipid" principle established for prokaryotic mechanosensitive channels MscL and MscS to these eukaryotic mechanosensitive K+ channels.

TRPV4 is a mechanically activated Ca(2+)-passing channel implicated in the sensing of forces, including those acting on bones. To date, 33 mutations are known to affect human bone development to different extents. The spectrum of these skeletal dysplasias (SD) ranges from dominantly inherited mild brachylomia (BO) to neonatal lethal forms of metatropic dysplasia (MD). Complexities of the results from fluorescence and electrophysiological studies have led to questions on whether channel activity is a good predictor of disease severity. Here we report on a systematic examination of 14 TRPV4 mutant alleles covering the entire SD spectrum. Expressed in Xenopus oocyte and without any stimulation, the wild-type channel had a ~1% open probability (Po) while those of most of the lethal MD channels approached 100%. All mutant channels had higher basal open probabilities, which limited their further increase by agonist or hypotonicity. The magnitude of this limitation revealed a clear correlation between the degree of over-activity (the molecular phenotype) and the severity of the disease over the entire spectrum (the biological phenotype). Thus, while other factors are at play, our results are consistent with the increased TRPV4 basal activity being a critical determinant of the severity of skeletal dysplasia. We discuss how the channel over-activity may lead to the "gain-of-function" phenotype and speculate that the function of wild-type TRPV4 may be secondary in normal bone development but crucial in an acute process such as fracture repair in the adult.

Stretch-activated conductances are commonly encountered in careful electric recordings. Those of known proteins (TRP, MscL, MscS, K2p, Kv, etc.) all share a core, which houses the ion pathway and the gate, but no recognizable force-sensing domain. Like animal TRPs, the yeast TRPY1 is polymodal, activated by stretch force, Ca2+, etc. To test whether its S5–S6 core senses the stretch force, we tried to uncouple it from the peripheral domains by strategic peptide insertions to block the covalent core–periphery interactions. Insertion of long unstructured peptides should distort, if not disrupt, protein structures that transmit force. Such insertions between S6 and the C-terminal tail largely removed Ca2+ activation, showing their effectiveness. However, such insertions as well as those between S5 and the N-terminal region, which includes S1–S4, did not significantly alter mechanosensitivity. Even insertions at both locations flanking the S5–S6 core did not much alter mechanosensitivity. Tryptophan scanning mutations in S5 were also constructed to perturb possible noncovalent core–periphery contacts. The testable tryptophan mutations also have little or no effects on mechanosensitivity. Boltzmann fits of the wild-type force–response curves agree with a structural homology model for a stretch-induced core expansion of ∼2 nm2 upon opening. We hypothesize that membrane tension pulls on S5–S6, expanding the core and opening the TRPY1 gate. The core being the major force sensor offers the simplest, though not the only, explanation of why so many channels of disparate designs are mechanically sensitive. Compared with the bacterial MscL, TRPY1 is much less sensitive to force, befitting a polymodal channel that relies on multiple stimuli.

TRPV4 is a polymodal cation channel gain-of-function (GOF) allele which causes skeletal dysplasia in humans. To better understand its gating, we screened for additional GOF alleles based on their ability to block yeast proliferation. Repeatedly, only a limited number of such growth-blocking mutations were isolated. Expressed in oocytes, wild-type channels can be strongly activated by either hypotonicity or exposure to the potent agonist 4alphaPDD, although the GOF channels behaved as if they were fully prestimulated as well as lacking a previously uncharacterized voltage-dependent inactivation. Five of six mutations occurred at or near the inner ends of the predicted core helices, giving further direct evidence that this region indeed forms the main intracellular gate in TRP channels. Surprisingly, both wild-type channels as well as these GOF channels maintain strong steady-state outward rectification that is not due to a Ca(2+) block, as has been proposed elsewhere. We conclude that TRPV4 contains an additional voltage-dependent gating mechanism in series with the main intracellular gate.

Whether animal ion channels functioning as mechanosensors are directly activated by stretch force or indirectly by ligands produced by the stretch is a crucial question. TRPV4, a key molecular model, can be activated by hypotonicity, but the mechanism of activation is unclear. One model has this channel being activated by a downstream product of phospholipase A2, relegating mechanosensitivity to the enzymes or their regulators. We expressed rat TRPV4 in Xenopus oocytes and repeatedly examined >200 excised patches bathed in a simple buffer. We found that TRPV4 can be activated by tens of mm Hg pipette suctions with open probability rising with suction even in the presence of relevant enzyme inhibitors. Mechanosensitivity of TRPV4 provides the simplest explanation of its various force-related physiological roles, one of which is in the sensing of weight load during bone development. Gain-of-function mutants cause heritable skeletal dysplasias in human. We therefore examined the brachyolmia-causing R616Q gain-of-function channel and found increased whole-cell current densities compared with wild-type channels. Single-channel analysis revealed that R616Q channels maintain mechanosensitivity but have greater constitutive activity and no change in unitary conductance or rectification.

Though clear for the prokaryotic mechanosensitive (MS) MscL and MscS, how mechanical force activates eukaryotic MS channels remains poorly understood. Several members of transient receptor potential (TRP) channel family are apparently involved in animal mechanosensations and are therefore promising MS-channel candidates. The yeast homolog, TRPY1, expressed in the yeast vacuolar membrane, can clearly be gated by hyper-osmotic shock in vivo and by directly stretching excised vacuolar membrane patches under patch clamp. Here, we investigated the structure-function relationship of TRPY1 by mutagenesis, aiming at dissecting how force is sensed by TRPY1 and how force opens the channel gate. TRP channels share general organization with well-studied voltage-gated potassium channels, being tetramers with each subunit consisting of six transmembrane helices (S1-S6) and N- and C-terminal cytoplamic domains. We found that the C-terminal cytoplasmic domains of TRPY1 harbor Ca2+ binding motifs, which confer TRPY1 Ca2+ activation. We demonstrated that the Ca2+ activation and the force activation are synergistic and the two gating mechanisms act in parallel. We proposed that force is perceived by the transmembrane domains. Our further in-depth analyses showed that strategic insertions of long peptide linkers before S4-S5 linker and after S6 can surprisingly yield functional channels with largely intact mechanosensitivity, highlighting the crucial roles of the pore module in TRPY1 mechanosensitivity. Together with our detailed scanning mutagenesis, we will discuss possible molecular mechanisms on how force activates TRPY1.

Transient‐receptor‐potential channels (TRPs) underlie the sensing of chemicals, heat, and mechanical force. We expressed the rat TRPV1 and TRPV4 subtypes in yeast and monitored their activities in vivo as Ca2+ rise using transgenic aequorin. Heat and capsaicin activate TRPV1 but not TRPV4 in yeast. Hypotonic shocks activate TRPV4 but not TRPV1. Osmotic swelling is modeled to activate enzyme(s), producing polyunsaturated fatty acids (PUFAs) to open TRPV4 in mammalian cells. This model relegates mechanosensitivity to the enzyme and not the channel. Yeast has only a single Δ9 fatty‐acid monodesaturase and cannot make PUFAs suggesting an alternative mechanism for TRPV4 activation. We discuss possible explanations of this difference.

Mechanosensitive (MS) ion channels likely underlie myriad force-sensing processes, from basic osmotic regulation to specified sensations of animal hearing and touch. Albeit important, the molecular identities of many eukaryotic MS channels remain elusive, let alone their working mechanisms. This is in stark contrast to our advanced knowledge on voltage- or ligand-sensitive channels. Several members of transient receptor potential (TRP) ion channel family have been implicated to function in mechanosensation and are recognized as promising candidate MS channels. The yeast TRP homolog, TRPY1, is clearly a first-line force transducer. It can be activated by hypertonic shock in vivo and by membrane stretch force in excised patches under patch clamp, making it a useful model for understanding TRP channel mechanosensitivity in general. TRPY1 offers two additional research advantages: (1) It has a large (∼300 pS) unitary conductance and therefore a favorable S/N ratio. (2) Budding yeast allows convenient and efficient genetic and molecular manipulations. In this review, we focus on the current research of TRPY1 and discuss its prospect. We also describe the use of yeast as a system to express and characterize animal TRP channels.

The ability to sense mechanical and osmotic stimuli is vital to all organisms from mammals to bacteria. Members of the transient receptor potential (TRP) ion-channel family have attracted intense attention for their involvement in mechanosensation. The yeast homologue TRPY1 can clearly be activated by hypertonic shock in vivo and by stretch force under patch clamp. Like its animal counterparts, TRPY1 is polymodal, being gated by membrane stretch force and by cytoplasmic Ca2+. Here, we investigated how these two gating principles interact. We found that stretch force can induce some channel activation without cytoplasmic Ca2+. Tens of micromolar Ca2+ greatly enhance the observed force-induced activities, with open probabilities following well the Boltzmann distribution, in which the two gating energies are summed as exponents. To map this formalism to structures, we found Ca2+-binding proteins such as calmodulin or calcineurin to be unnecessary. However, removing a dense cluster of negative charges in the C-terminal cytoplasmic domain of TRPY1 greatly diminishes the Ca2+ activation as well as its influence on force activation. We also found a strategic point upstream of this charge cluster, at which insertion of amino acids weakens Ca2+ activation considerably but leaves the mechanosensitivity nearly intact. These results led to a structure–function model in which Ca2+ binding to the cytoplasmic domain and stretching of the membrane-embedded domain both generate gating force, reaching the gate in parallel.

The complexity of animals and plants is due largely to cellular arrangement. The structures and activities of macromolecules had, however, evolved in early microbes long before the appearance of this complexity. Among such molecules are those that sense light, heat, force, water, and ligands. Though historically and didactically associated with the nervous system, ion channels also have deep evolutionary roots. For example, force sensing with channels, which likely began as water sensing through membrane stretch generated by osmotic pressure, must be ancient and is universal in extant species. Extant microbial species, such as the model bacterium Escherichia coli and yeast Saccharomyces cerevisiae, are equipped with stretch-activated channels. The ion channel proteins MscL and MscS show clearly that these bacterial channels receive stretch forces from the lipid bilayer. TRPY1, the mechanosensitive channel in yeast, is being developed towards a similar basic understanding of channels of the TRP (transientreceptor- potential) superfamily. TRPY1 resides in the vacuolar membrane and releases Ca2+ from the vacuole to the cytoplasm upon hyperosmotic shock. Unlike in most TRP preparations from animals, the mechanosensitivity of TRPY1 can be examined directly under patch clamp in either whole-vacuole mode or excised patch mode. The combination of direct biophysical examination in vitro with powerful microbial genetics in vivo should complement the study of mechanosensations of complex animals and plants.

The yeast TRPY1 (Yvc1p) channel is activated by membrane stretch to release vacuolar Ca2+ into the cytoplasm upon osmotic upshock. Exogenously added indole greatly enhances the upshock‐induced Ca2+ release in vivo. Indole also reversibly activates the channels under patch clamp. A minimum of 10−6 M Ca2+ is needed for membrane stretch force to open TPRY1, but indole activation appears to be Ca2+ independent. A deletion of 30 residues at the predicted cytoplasmic domain, 570–600Δ, renders TRPY1 insensitive to stretch force upto 10−3 M Ca2+. Nonetheless, indole readily activates this mutant channel. Several other aromatic compounds, e.g. the antimicrobial pbens, also activate TRPY1. These compounds likely alter the innate forces in the lipid bilayer received by the channel.

Transient receptor potential (TRP) channels are first elements in sensing chemicals, heat, and force and are widespread among protists and fungi as well as animals. Despite their importance, the arrangement and roles of the amino acids that constitute the TRP channel gate are unknown. The yeast TRPY1 is activated in vivo by osmotically induced vacuolar membrane deformation and by cytoplasmic Ca 2+ . After a random mutagenesis, we isolated TRPY1 mutants that responded more strongly to mild osmotic upshocks. One such gain-of-function mutant has a Y458H substitution at the C terminus of the predicted sixth transmembrane helix. Direct patch-clamp examination of vacuolar membranes showed that Y458H channels were already active with little stimulus and showed marked flickers between the open and intraburst closed states. They remained responsive to membrane stretch force and to Ca 2+ , indicating primary defects in the gate region but not in the sensing of gating principles. None of the other 18 amino acid replacements engineered here showed normal channel kinetics except the two aromatic substitutions, Y458F and Y458W. The Y458 of TRPY1 has its aromatic counterpart in mammalian TRPM. Furthermore, conserved aromatics one α-helical turn downstream from this point are also found in animal TRPC, TRPN, TRPP, and TRPML, suggesting that gate anchoring with aromatics may be common among many TRP channels. The possible roles of aromatics at the end of the sixth transmembrane helix are discussed.

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across subfamilies. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550I) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of phototaxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure–function principles that can be generalized across different TRP subfamilies. It appears that the conserved aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed.

Automated visual inspection (AVI) systems have been extended to many fields, such as agriculture and the food, plastic and textile industries. Generally, most visual systems only inspect product defects, and then analyze and grade them due to the lack of any sorting function. This main reason rests with the difficulty of using the image data in real time. However, it is increasingly important to either sort good products from bad or grade products into separate groups using AVI systems. This article describes the development of a mechatronic sorting system and its integration with a vision system for automatically removing contaminants from wool in real time. The integration is implemented by a personal computer, which continuously processes live images under the Windows 2000 operating system. The developed real-time sorting approach is also applicable to many other AVI systems.

In the textile industry, scoured wool contains different types of foreign materials (contaminants) that need to be separated out before it goes into further processing, so that the textile machines are protected from damage and the quality of the final woollen products is ensured. This paper presents an automated visual inspection (AVI) system for detecting and sorting contaminants from wool in real time. The techniques were first developed in the lab and subsequently applied to a large-scale factory system. The combinative use of image processing algorithms in RGB and HSV colour spaces can segment 96% of contaminant types (minimum size around 4 cm long and 5 mm in diameter) in real-time on the lab test rig. One of the most important aspects of the system is to use the non-linear colour space transformation and merge the threshold algorithm in HSV colour space into the image processing algorithms in RGB colour space to enhance the contaminant identification in real time. The real-time capability of the system is also analysed in detail. The experimental results demonstrate that the factory AVI system could identify and remove the contaminants at a camera speed of around 800 lines/s and the conveyor speed of 20 m/min in real time.

This paper describes the research approach to identify the contaminants which have similar color to the background wool and their removal from wool in real time. First, different light source is sought for getting the high contrast image between wool and contaminants. Second, different CCD detector including infrared camera, monochrome area scan camera was tried for identification of white contaminants. Relative infrared theory and spectral theory are also presented. Third, different image processing algorithms including threshold in HSV color space, local adaptive threshold, region-growing algorithm and their comparisons are presented. The combination of local adaptive threshold and global threshold algorithms can well identify most of white contaminants. At last, a research approach on contaminant removal from wool by the image processing algorithm in real time is presented. Both software and hardware approach are reported.