GPR15L is an epithelial inflammation-derived pruritogen.

Itch is an unpleasant sensation that often accompanies chronic dermatological conditions. Although many of the itch receptors and the neural pathways underlying this sensation are known, the identity of endogenous ligands is still not fully appreciated. Using an unbiased bioinformatic approach, we identified GPR15L as a candidate pruritogen whose expression is robustly up-regulated in psoriasis and atopic dermatitis. Although GPR15L was previously shown to be a cognate ligand of the receptor GPR15, expressed in dermal T cells, here we show that it also contributes to pruritogenesis by activating Mas-related G protein-coupled receptors (MRGPRs). GPR15L can selectively stimulate mouse dorsal root ganglion neurons that express Mrgpra3 and evokes intense itch responses. GPR15L causes mast cell degranulation through stimulation of MRGPRX2 and Mrgprb2. Genetic disruption of GPR15L expression attenuates scratch responses in a mouse model of psoriasis. Our study reveals unrecognized features of GRP15L, showing that it is a potent itch-inducing agent.

Molecular Genetics of Kappa Opioids in Pain and Itch Sensations.

The opioid peptides and their receptors have been linked to multiple key biological processes in the nervous system. Here we review the functions of the kappa opioid receptor (KOR) and its endogenous agonists dynorphins (Goldstein A, Tachibana S, Lowney LI, Hunkapiller M, Hood L, Proc Natl Acad Sci U S A 76:6666-6670, 1979) in modulating itch and pain (nociception). Specifically, we discuss their roles relative to recent findings that tell us more about the cells and circuits which are impacted by this opioid and its receptor and present reanalysis of single-cell sequencing data showing the expression profiles of these molecules. Since the KOR is relatively specifically activated by peptides derived from the prodynorphin gene and other opioid peptides that show lower affinities, this will be the only interactions we consider (Chavkin C, Goldstein A, Nature 291:591-593, 1981; Chavkin C, James IF, Goldstein A, Science 215:413-415, 1982), although it was noted that at higher doses peptides other than dynorphins might stimulate KOR (Lai J, Luo MC, Chen Q, Ma S, Gardell LR, Ossipov MH, Porreca F, Nat Neurosci 9:1534-1540, 2006). This review has been organized based on anatomy with each section describing the effect of the kappa opioid system in a specific location but let us not forget that most of these circuits are interconnected and are therefore interdependent.

Interactions of the Neuro‒Immune‒Stromal Triad in Itch.

This review focuses on recent advances in understanding the mechanisms involved in itch signaling in the skin and how these new findings fit into the wider picture of the expression of itch mediators and their receptors in the dermal layer. Because at present studies mostly concentrate on single cellular compartments (e.g., neural alone), we suggest that they may miss important interactions with other compartments. Therefore, to fully appreciate pruritus, we propose that studies should consider (e.g., using transcriptomic information) signal transmission within the entire neuro‒immune‒stromal triad.

Specific ß-Defensins Stimulate Pruritus through Activation of Sensory Neurons.

Pruritus is a common symptom of dermatological disorders and has a major negative impact on QOL. Previously, it was suggested that human ß-defensin peptides elicit itch through the activation of mast cells. In this study, we investigated in more detail the mechanisms by which ß-defensins induce itch by defining the receptors activated by these peptides in humans and mice, by establishing their action in vivo, and by examining their expression in inflammatory dermal diseases. We found that elevated expression of DEFB103 is highly correlated with skin lesions in psoriasis and atopic dermatitis. We showed that the peptide encoded by this gene and related genes activate Mas-related G protein-coupled receptors with different potencies that are related to their charge density. Furthermore, we establish that although these peptides can activate mast cells, they also activate sensory neurons, with the former cells being dispensable for itch reactions in mice. Together, our studies highlight that specific ß-defensins are likely endogenous pruritogens that can directly stimulate sensory neurons.

Oncostatin M can sensitize sensory neurons in inflammatory pruritus.

Chronic itch is a major symptom of many inflammatory skin diseases. This type of pruritus is thought to be facilitated by cytokines that activate cutaneous nerve fibers; however, the molecular components and mechanisms involved are poorly understood. We found that the cytokine oncostatin M (OSM) is highly up-regulated in psoriasis, atopic dermatitis, and cutaneous T cell lymphoma, diseases associated with chronic itch. OSM receptor (OSMR) is expressed by itch-selective natriuretic polypeptide B (Nppb) neurons, and single-cell sequencing showed that OSM is mainly produced by dermal T cells and monocytes. Unlike canonical pruritogens, OSM does not activate sensory neurons. Instead, it sensitizes neurons by potentiating neural responses to pruritogens and by enhancing neural excitability. Knockout of OSMR in sensory neurons attenuated OSM-sensitized itch and inflammatory itch in mice, and pharmacological antagonism of the OSMR complex effectively alleviated pruritus in experimental inflammatory dermatitis in a rodent model. Together, our results uncover OSM as an itch neuromodulator and reveal OSM signal transduction as a potential target for antipruritic therapy.

The Cytokine TGF-ß Induces Interleukin-31 Expression from Dermal Dendritic Cells to Activate Sensory Neurons and Stimulate Wound Itching.

Cutaneous wound healing is associated with the unpleasant sensation of itching. Here we investigated the mechanisms underlying this type of itch, focusing on the contribution of soluble factors released during healing. We found high amounts of interleukin 31 (IL-31) in skin wound tissue during the peak of itch responses. Il31-/- mice lacked wound-induced itch responses. IL-31 was released by dermal conventional type 2 dendritic cells (cDC2s) recruited to wounds and increased itch sensory neuron sensitivity. Transfer of cDC2s isolated from late-stage wounds into healthy skin was sufficient to induce itching in a manner dependent on IL-31 expression. Addition of the cytokine TGF-ß1, which promotes wound healing, to dermal DCs in vitro was sufficient to induce Il31 expression, and Tgfbr1f/f CD11c-Cre mice exhibited reduced scratching and decreased Il31 expression in wounds in vivo. Thus, cDC2s promote itching during skin would healing via a TGF-ß-IL-31 axis with implications for treatment of wound itching.

α-Actinin-1 promotes activity of the L-type Ca2+ channel Cav 1.2.

The L-type Ca2+ channel CaV 1.2 governs gene expression, cardiac contraction, and neuronal activity. Binding of α-actinin to the IQ motif of CaV 1.2 supports its surface localization and postsynaptic targeting in neurons. We report a bi-functional mechanism that restricts CaV 1.2 activity to its target sites. We solved separate NMR structures of the IQ motif (residues 1,646-1,664) bound to α-actinin-1 and to apo-calmodulin (apoCaM). The CaV 1.2 K1647A and Y1649A mutations, which impair α-actinin-1 but not apoCaM binding, but not the F1658A and K1662E mutations, which impair apoCaM but not α-actinin-1 binding, decreased single-channel open probability, gating charge movement, and its coupling to channel opening. Thus, α-actinin recruits CaV 1.2 to defined surface regions and simultaneously boosts its open probability so that CaV 1.2 is mostly active when appropriately localized.

Nppb Neurons Are Sensors of Mast Cell-Induced Itch.

Itch is an unpleasant skin sensation that can be triggered by exposure to many chemicals, including those released by mast cells. The natriuretic polypeptide b (Nppb)-expressing class of sensory neurons, when activated, elicits scratching responses in mice, but it is unclear which itch-inducing agents stimulate these cells and the receptors involved. Here, we identify receptors expressed by Nppb neurons and demonstrate the functional importance of these receptors as sensors of endogenous pruritogens released by mast cells. Our search for receptors in Nppb neurons reveals that they express leukotriene, serotonin, and sphingosine-1-phosphate receptors. Targeted cell ablation, calcium imaging of primary sensory neurons, and conditional receptor knockout studies demonstrate that these receptors induce itch by the direct stimulation of Nppb neurons and neurotransmission through the canonical gastrin-releasing peptide (GRP)-dependent spinal cord itch pathway. Together, our results define a molecular and cellular pathway for mast cell-induced itch.

MrgprX1 Mediates Neuronal Excitability and Itch Through Tetrodotoxin-Resistant Sodium Channels.

In this study, we sought to elucidate the molecular mechanism underlying human Mas-related G protein-coupled receptor X1 (MrgprX1) mediated itch sensation. We found that activation of MrgprX1 by BAM8-22 triggered robust action potential discharges in dorsal root ganglion (DRG) neurons. This neuronal excitability is not mediated by Transient receptor potential (TRP) cation channels, M-type potassium channels, or chloride channels. Instead, activation of MrgprX1 lowers the activation threshold of TTX-resistant sodium channels and induces inward sodium currents. These MrgprX1-elicited action potential discharges can be blocked by Pertussis toxin (PTX) and a Gßγ inhibitor - Gallein. Behavioral results showed that Nav1.9 knockout but not Trpa1 knockout significantly reduced BAM8-22 evoked scratching behavior. Collectively, these data suggest that activation of MrgprX1 triggers itch sensation by increasing the activity of TTX-resistant voltage-gated sodium channels.

Author Correction: Circuit dissection of the role of somatostatin in itch and pain.

In the version of this article initially published online, the labels were switched for the right-hand pair of bars in Fig. 4e. The left one of the two should be Chloroquine + veh, the right one Chloroquine + CNO. The error has been corrected in the print, HTML and PDF versions of the article.

Circuit dissection of the role of somatostatin in itch and pain.

Stimuli that elicit itch are detected by sensory neurons that innervate the skin. This information is processed by the spinal cord; however, the way in which this occurs is still poorly understood. Here we investigated the neuronal pathways for itch neurotransmission, particularly the contribution of the neuropeptide somatostatin. We find that in the periphery, somatostatin is exclusively expressed in Nppb+ neurons, and we demonstrate that Nppb+somatostatin+ cells function as pruriceptors. Employing chemogenetics, pharmacology and cell-specific ablation methods, we demonstrate that somatostatin potentiates itch by inhibiting inhibitory dynorphin neurons, which results in disinhibition of GRPR+ neurons. Furthermore, elimination of somatostatin from primary afferents and/or from spinal interneurons demonstrates differential involvement of the peptide released from these sources in itch and pain. Our results define the neural circuit underlying somatostatin-induced itch and characterize a contrasting antinociceptive role for the peptide.

α-Actinin Promotes Surface Localization and Current Density of the Ca2+ Channel CaV1.2 by Binding to the IQ Region of the α1 Subunit.

The voltage-gated L-type Ca2+ channel CaV1.2 is crucial for initiating heartbeat and control of a number of neuronal functions such as neuronal excitability and long-term potentiation. Mutations of CaV1.2 subunits result in serious health problems, including arrhythmia, autism spectrum disorders, immunodeficiency, and hypoglycemia. Thus, precise control of CaV1.2 surface expression and localization is essential. We previously reported that α-actinin associates and colocalizes with neuronal CaV1.2 channels and that shRNA-mediated depletion of α-actinin significantly reduces localization of endogenous CaV1.2 in dendritic spines in hippocampal neurons. Here we investigated the hypothesis that direct binding of α-actinin to CaV1.2 supports its surface expression. Using two-hybrid screens and pull-down assays, we identified three point mutations (K1647A, Y1649A, and I1654A) in the central, pore-forming α11.2 subunit of CaV1.2 that individually impaired α-actinin binding. Surface biotinylation and flow cytometry assays revealed that CaV1.2 channels composed of the corresponding α-actinin-binding-deficient mutants result in a 35-40% reduction in surface expression compared to that of wild-type channels. Moreover, the mutant CaV1.2 channels expressed in HEK293 cells exhibit a 60-75% decrease in current density. The larger decrease in current density as compared to surface expression imparted by these α11.2 subunit mutations hints at the possibility that α-actinin not only stabilizes surface localization of CaV1.2 but also augments its ion conducting activity.

Targeting human Mas-related G protein-coupled receptor X1 to inhibit persistent pain.

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Discovery and characterization of 2-(cyclopropanesulfonamido)-N-(2-ethoxyphenyl)benzamide, ML382: a potent and selective positive allosteric modulator of MrgX1.

Previous studies have shown that the activation of mouse MrgC11, a G-protein-coupled receptor, by its peptide ligand BAM8-22 can inhibit chronic pain. A large-scale screen has been carried out to isolate small-molecule allosteric agonists of MrgX1, the human homologue of MrgC11. The goal of this study is to improve the efficacy and potency of positive allosteric modulators (PAMs) with therapeutic implications in combating chronic pain. Herein we report an iterative parallel synthesis effort and a structure-activity relationship study of a series of arylsulfonamides which led to the discovery of the first PAM of MrgX1, ML382.

Competition between α-actinin and Ca²âº-calmodulin controls surface retention of the L-type Ca²âº channel Ca(V)1.2.

Regulation of neuronal excitability and cardiac excitation-contraction coupling requires the proper localization of L-type Ca²âº channels. We show that the actin-binding protein α-actinin binds to the C-terminal surface targeting motif of α11.2, the central pore-forming Ca(V)1.2 subunit, in order to foster its surface expression. Disruption of α-actinin function by dominant-negative or small hairpin RNA constructs reduces Ca(V)1.2 surface localization in human embryonic kidney 293 and neuronal cultures and dendritic spine localization in neurons. We demonstrate that calmodulin displaces α-actinin from their shared binding site on α11.2 upon Ca²âº influx through L-type channels, but not through NMDAR, thereby triggering loss of Ca(V)1.2 from spines. Coexpression of a Ca²âº-binding-deficient calmodulin mutant does not affect basal Ca(V)1.2 surface expression but inhibits its internalization upon Ca²âº influx. We conclude that α-actinin stabilizes Ca(V)1.2 at the plasma membrane and that its displacement by Ca²âº-calmodulin triggers Ca²âº-induced endocytosis of Ca(V)1.2, thus providing an important negative feedback mechanism for Ca²âº influx.

Dominantly inherited myotonia congenita resulting from a mutation that increases open probability of the muscle chloride channel CLC-1.

Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (P (o)) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common-gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased P (o) of the channel's fast-gate, from ~0.4 in the wild type to >0.9 in the mutant at -90 mV. In contrast, the mutant exhibits a minimal effect on the P (o) of the common-gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast-gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a fivefold increase in chloride conductance in the wild type to a twofold increase in the mutant-this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.

Binding of ATP to the CBS domains in the C-terminal region of CLC-1.

The common gating of CLC-1 has been shown to be inhibited by intracellular adenosine triphosphate (ATP) in acidic pH conditions. Such modulation is thought to be mediated by direct binding of ATP to the cystathionine ß-synthase (CBS) domains at the C-terminal cytoplasmic region of CLC-1. Guided by the crystal structure of the C-terminal domain of CLC-5, we constructed a homology model of CLC-1's C terminus and mutated critical amino acid residues lining the potential ATP-binding site. The CLC-1 mutations V634A and E865A completely abolished the ATP inhibition of CLC-1, consistent with the loss of ATP binding seen with the corresponding mutations in CLC-5. Mutating two other residues, V613 and V860, also disrupted the ATP modulation of CLC-1. However, placing aromatic amino acids at position 634 increases the apparent ATP affinity. Mutant cycle analyses showed that the modulation effects of ATP and cytidine triphosphate on wild-type CLC-1 and the V634F mutant were nonadditive, suggesting that the side chain of amino acid at position 634 interacts with the base moiety of the nucleotide. The mutation effects of V634F and V613A on the ATP modulation were also nonadditive, which is consistent with the assertion suggested from the homology model that these two residues may both interact with the bound nucleotide. These results provide evidence for a direct ATP binding for modulating the function of CLC-1 and suggest an overall conserved architecture of the ATP-binding sites in CLC-1 and CLC-5. This study also demonstrates that CLC-1 is a convenient experimental model for studying the interaction of nucleotides/nucleosides with the CBS domain.

Blocking pore-open mutants of CLC-0 by amphiphilic blockers.

The blockade of CLC-0 chloride channels by p-chlorophenoxy acetate (CPA) has been thought to be state dependent; the conformational change of the channel pore during the "fast gating" alters the CPA binding affinity. Here, we examine the mechanism of CPA blocking in pore-open mutants of CLC-0 in which the residue E166 was replaced by various amino acids. We find that the CPA-blocking affinities depend upon the volume and the hydrophobicity of the side chain of the introduced residue; CPA affinity can vary by three orders of magnitude in these mutants. On the other hand, mutations at the intracellular pore entrance, although affecting the association and dissociation rates of the CPA block, generate only a modest effect on the steady-state blocking affinity. In addition, various amphiphilic compounds, including fatty acids and alkyl sulfonates, can also block the pore-open mutants of CLC-0 through a similar mechanism. The blocking affinity of fatty acids and alkyl sulfonates increases with the length of these amphiphilic blockers, a phenomenon similar to the block of the Shaker K(+) channel by long-chain quaternary ammonium (QA) ions. These observations lead us to propose that the CPA block of the open pore of CLC-0 is similar to the blockade of voltage-gated K(+) channels by long-chain QAs or by the inactivation ball peptide: the blocker first uses the hydrophilic end to "dock" at the pore entrance, and the hydrophobic part of the blocker then enters the pore to interact with a more hydrophobic region of the pore. This blocking mechanism appears to be very general because the block does not require a precise structural fit between the blocker and the pore, and the blocking mechanism applies to the cation and anion channels with unrelated pore architectures.

ATP inhibition of CLC-1 is controlled by oxidation and reduction.

The effect of intracellular adenosine triphosphate (ATP) on the "common gating" of the CLC-1 chloride channel has been studied by several laboratories with controversial results. Our previous study on the channel expressed in Xenopus oocytes using excised inside-out patch-clamp methods showed a robust effect of ATP in shifting the open probability curve of the common gate toward more depolarizing voltages (Tseng, P.Y., B. Bennetts, and T.Y. Chen. 2007. J. Gen. Physiol. 130:217-221). The results were consistent with those from studying the channel expressed in mammalian cells using whole cell recording methods (Bennetts, B., M.W. Parker, and B.A. Cromer. 2007. J. Biol. Chem. 282:32780-32791). However, a recent study using excised-patch recording methods for channels expressed in Xenopus oocytes reported that ATP had no direct effect on CLC-1 (Zifarelli, G., and M. Pusch. 2008. J. Gen. Physiol. 131:109-116). Here, we report that oxidation of CLC-1 may be the culprit underlying the controversy. When patches were excised from mammalian cells, the sensitivity to ATP was lost quickly--within 2-3 min. This loss of ATP sensitivity could be prevented or reversed by reducing agents. On the other hand, CLC-1 expressed in Xenopus oocytes lost the ATP sensitivity when patches were treated with oxidizing reagents. These results suggest a novel view in muscle physiology that the mechanisms controlling muscle fatigability may include the oxidation of CLC-1.