Rosa Planells-Cases

Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration

ClC‐7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H+‐ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC‐7 or the H+‐ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC‐7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC‐7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron‐dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC‐3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H+‐ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H+‐ATPase and ClC‐7 can underlie human osteopetrosis.

Attenuation of thermal nociception and hyperalgesia by VR1 blockers

Vanilloid receptor subunit 1 (VR1) appears to play a critical role in the transduction of noxious chemical and thermal stimuli by sensory nerve endings in peripheral tissues. Thus, VR1 antagonists are useful compounds to unravel the contribution of this receptor to pain perception, as well as to induce analgesia. We have used a combinatorial approach to identify new, nonpeptidic channel blockers of VR1. Screening of a library of trimers of N-alkylglycines resulted in the identification of two molecules referred to as DD161515 {N-[2-(2-(N-methylpyrrolidinyl)ethyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide} and DD191515 {[N-[3-(N,N-diethylamino)propyl]glycyl]-[N-[2,4-dichlorophenethyl]glycyl]-N-(2,4-dichlorophenethyl)glycinamide} that selectively block VR1 channel activity with micromolar efficacy, rivaling that characteristic of vanilloid-related inhibitors. These compounds appear to be noncompetitive VR1 antagonists that recognize a receptor site distinct from that of capsaicin. Intraperitoneal administration of both trialkylglycines into mice significantly attenuated thermal nociception as measured in the hot plate test. It is noteworthy that these compounds eliminated pain and neurogenic inflammation evoked by intradermal injection of capsaicin into the animal hindpaw, as well as the thermal hyperalgesia induced by tissue irritation with nitrogen mustard. In contrast, responses to mechanical stimuli were not modified by either compound. Modulation of sensory nerve fibers excitability appears to underlie the peptoid analgesic activity. Collectively, these results indicate that blockade of VR1 activity attenuates chemical and thermal nociception and hyperalgesia, supporting the tenet that this ionotropic receptor contributes to chemical and thermal sensitivity and pain perception in vivo. These trialkylglycine-based, noncompetitive VR1 antagonists may likely be developed into analgesics to treat inflammatory pain.

Identification of a Tetramerization Domain in the C Terminus of the Vanilloid Receptor

TRPV1 (transient receptor potential vanilloid receptor subtype 1) is a member of the TRP channel family gated by vanilloids, protons, and heat. Structurally, TRPV1 appears to be a tetramer formed by the assembly of four identical subunits around a central aqueous pore. The molecular determinants that govern its subunit oligomerization remain elusive. Here, we report the identification of a segment comprising 684Glu-721Arg (referred to as the TRP-like domain) in the C terminus of TRPV1 as an association domain (AD) of the protein. Purified recombinant C terminus of TRPV1 (TRPV1-C) formed discrete and stable multimers in vitro. Yeast two-hybrid and pull-down assays showed that self-association of the TRPV1-C is blocked when segment 684Glu-721Arg is deleted. Biochemical and immunological analysis indicate that removal of the AD from full-length TRPV1 monomers blocks the formation of stable heteromeric assemblies with wild-type TRPV1 subunits. Deletion of the AD in a poreless TRPV1 subunit suppressed its robust dominant-negative phenotype. Together, these findings are consistent with the tenet that the TRP-like domain in TRPV1 is a molecular determinant of the tetramerization of receptor subunits into functional channels. Our observations suggest that the homologous TRP domain in the TRP protein family may function as a general, evolutionary conserved AD involved in subunit multimerization.

Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6

Mammalian CLC proteins function as Cl− channels or as electrogenic Cl−/H+ exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6−/− neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia

Neurogenic inflammation is produced by overstimulation of peripheral nociceptor terminals by injury or inflammation of tissues. Excessive activity of sensory neurons produces vasodilation, plasma extravasation and hypersensitivity. Mechanistically, neurogenic inflammation is due to the release of substances from primary sensory nerve terminals that act directly or indirectly at the peripheral terminals, either activating or sensitizing nociceptors, endothelial cells and immunocytes. Notably, small-diameter sensory neurons that are sensitive to capsaicin play a key role in the generation of neurogenic inflammation. The cloning of the vanilloid receptor 1 (TRPV1) has been a breakthrough that has propelled our understanding of the molecular mechanisms involved in neurogenic inflammation. TRPV1 pivotally contributes to the integration of various stimuli and modulates nociceptor excitability, thus making it a true gateway for pain transduction. In addition, TRPV1 is the endpoint target of intracellular signalling pathways triggered by inflammatory mediators. Phosphorylation-induced potentiation of TRPV1 channel activity, along with an incremented TRPV1 surface expression are major events underlying the nociceptor activation and sensitization that leads to thermal hyperalgesia. The important contribution of TRPV1 receptor to the onset and maintenance of neurogenic inflammation has validated it as a therapeutic target for inflammatory pain management. As a result, the development of specific TRPV1 antagonists is a central focus of current drug discovery programs.

Physiology and pharmacology of the vanilloid receptor.

The identification and cloning of the vanilloid receptor 1 (TRPV1) represented a significant step for the understanding of the molecular mechanisms underlying the transduction of noxious chemical and thermal stimuli by peripheral nociceptors. TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 channel activity is remarkably potentiated by pro-inflammatory agents, a phenomenon that is thought to underlie the peripheral sensitisation of nociceptors that leads to thermal hyperalgesia. Cumulative evidence is building a strong case for the involvement of this receptor in the etiology of both peripheral and visceral inflammatory pain, such as inflammatory bowel disease, bladder inflammation and cancer pain. The validation of TRPV1 receptor as a key therapeutic target for pain management has thrust intensive drug discovery programs aimed at developing orally active antagonists of the receptor protein. Nonetheless, the real challenge of these drug discovery platforms is to develop antagonists that preserve the physiological activity of TRPV1 receptors while correcting over-active channels. This is a condition to ensure normal pro-prioceptive and nociceptive responses that represent a safety mechanism to prevent tissue injury. Recent and exciting advances in the function, dysfunction and modulation of this receptor will be the focus of this review.

Subunit composition of VRAC channels determines substrate specificity and cellular resistance to Pt‐based anti‐cancer drugs

Although platinum‐based drugs are widely used chemotherapeutics for cancer treatment, the determinants of tumor cell responsiveness remain poorly understood. We show that the loss of subunits LRRC8A and LRRC8D of the heteromeric LRRC8 volume‐regulated anion channels (VRACs) increased resistance to clinically relevant cisplatin/carboplatin concentrations. Under isotonic conditions, about 50% of cisplatin uptake depended on LRRC8A and LRRC8D, but neither on LRRC8C nor on LRRC8E. Cell swelling strongly enhanced LRRC8‐dependent cisplatin uptake, bolstering the notion that cisplatin enters cells through VRAC. LRRC8A disruption also suppressed drug‐induced apoptosis independently from drug uptake, possibly by impairing VRAC‐dependent apoptotic cell volume decrease. Hence, by mediating cisplatin uptake and facilitating apoptosis, VRAC plays a dual role in the cellular drug response. Incorporation of the LRRC8D subunit into VRAC substantially increased its permeability for cisplatin and the cellular osmolyte taurine, indicating that LRRC8 proteins form the channel pore. Our work suggests that LRRC8D‐containing VRACs are crucial for cell volume regulation by an important organic osmolyte and may influence cisplatin/carboplatin responsiveness of tumors.

Differential contribution of SNARE-dependent exocytosis to inflammatory potentiation of TRPV1 in nociceptors

Potentiation of the pain‐integrator ion channel transient receptor potential vanilloid type 1 (TRPV1) underlies thermal hyperalgesia mediated by a variety of proinflammatory factors. Two complemen‐ tary mechanisms of TRPV1 inflammatory sensitization have been proposed, namely a decrease of its activation threshold and an increment of its surface expression in nociceptors. Here we investigated the involvement of regulated exocytosis to the inflammatory sensitization of TRPV1 in rat neonatal dorsal root ganglion neurons by proalgesic agents. The contribution of soluble N‐ ethylmaleimide‐sensitive factor attachment protein re‐ ceptor (SNARE)‐dependent exocytosis was evaluated using a small peptide patterned after the synaptosomal‐ associated protein of 25 kDa (SNAP‐25) protein that acts as a specific and potent inhibitor of neuronal exocytosis. We found that TRPV1 sensitization medi‐ ated by nerve growth factor, ATP, and IGF‐I was accompanied by a higher channel expression in the neuronal plasma membrane, which was prevented by blockade of regulated exocytosis. In contrast, TRPV1 sensitization caused by bradykinin, IL‐1β, and artemin was insensitive to inhibition of SNARE‐dependent ve‐ sicular fusion and was not due to an increase in TRPV1 surface expression. Therefore, it appears that some, but not all, proinflammatory agents sensitize rat noci‐ ceptors by promoting the recruitment of TRPV1 chan‐ nels to the neuronal surface. These findings support the tenet that SNARE complex‐mediated exocytosis of TRPV1 may be a valid therapeutic target to treat inflammatory pain.—Camprubi‐Robles, M., Planells‐ Cases, R., Ferrer‐Montiel, A. Differential contribution of SNARE‐dependent exocytosis to inflammatory po‐ tentiation of TRPV1 in nociceptors. FASEB J. 23, 3722‐3733 (2009). www.fasebj.org

Advances in modulating thermosensory TRP channels

Introduction: Thermosensory channels are a subfamily of the transient receptor potential (TRP) channel family that are activated by changes in the environmental temperature. These channels, known as thermoTRPs, cover the entire spectrum of temperatures, from noxious cold (< 15°C) to injurious heat (> 42°C). In addition, dysfunction of these channels contributes to the thermal hypersensitivity that accompanies painful conditions. Moreover, because of their wide tissue and cellular distribution, thermoTRPs are also involved in the pathophysiology of several diseases, from inflammation to cancer. Areas covered: Although the number of thermoTRPs is increasing with the identification of novel members such as TRPM3, we will cover the recent advances in the pharmacology of the classical thermosensory channels, namely TRPV1, TRPV2, TRPV3, TRPV4, TRPM8 and TRPA1. This review will focus on the therapeutic progress carried out for all these channels and will highlight the tenet that TRPV1, TRPM8 and TRPA1 are the most exploited channels, and that the interest on TRPV3 and TRPV4 is growing with the first TRPV3 antagonist that moves into Phase-II clinical trials. In contrast, the pharmacology of TRPV2 is yet in its infancy. Expert opinion: Despite the tremendous academic and industrial investment to develop therapeutic modulators of thermoTRPs, it apparently seems that we are still far from the first successful product, although hope is maintained high for all compounds currently in clinical trials. A major concern has been the appearance of side effects. A better knowledge of the thermosensory protein networks (signal-plexes), along with the application of system biology approaches may provide novel strategies to modulate thermoTRPs activity with improved therapeutic index. A case in point is TRPV1, where acting on interacting proteins is providing new therapeutic opportunities.

Arginine-rich peptides are blockers of VR-1 channels with analgesic activity

Vanilloid receptors (VRs) play a fundamental role in the transduction of peripheral tissue injury and/or inflammation responses. Molecules that antagonize VR channel activity may act as selective and potent analgesics. We report that synthetic arginine‐rich hexapeptides block heterologously expressed VR‐1 channels with submicromolar efficacy in a weak voltage‐dependent manner, consistent with a binding site located near/at the entryway of the aqueous pore. Dynorphins, natural arginine‐rich peptides, also blocked VR‐1 activity with micromolar affinity. Notably, synthetic and natural arginine‐rich peptides attenuated the ocular irritation produced by topical capsaicin application onto the eyes of experimental animals. Taken together, our results imply that arginine‐rich peptides are VR‐1 channel blockers with analgesic activity. These findings may expand the development of novel analgesics by targeting receptor sites distinct from the capsaicin binding site.