Properties and Therapeutic Potential of Transient Receptor Potential Channels with Putative Roles in Adversity: Focus on TRPC5, TRPM2 and TRPA1

Mammals contain 28 genes encoding Transient Receptor Potential (TRP) proteins. The proteins assemble into cationic channels, often with calcium permeability. Important roles in physiology and disease have emerged and so there is interest in whether the channels might be suitable therapeutic drug targets. Here we review selected members of three subfamilies of mammalian TRP channel (TRPC5, TRPM2 and TRPA1) that show relevance to sensing of adversity by cells and biological systems. Summarized are the cellular and tissue distributions, general properties, endogenous modulators, protein partners, cellular and tissue functions, therapeutic potential, and pharmacology. TRPC5 is stimulated by receptor agonists and other factors that include lipids and metal ions; it heteromultimerises with other TRPC proteins and is involved in cell movement and anxiety control. TRPM2 is activated by hydrogen peroxide; it is implicated in stress-related inflammatory, vascular and neurodegenerative conditions. TRPA1 is stimulated by a wide range of irritants including mustard oil and nicotine but also, controversially, noxious cold and mechanical pressure; it is implicated in pain and inflammatory responses, including in the airways. The channels have in common that they show polymodal stimulation, have activities that are enhanced by redox factors, are permeable to calcium, and are facilitated by elevations of intracellular calcium. Developing inhibitors of the channels could lead to new agents for a variety of conditions: for example, suppressing unwanted tissue remodeling, inflammation, pain and anxiety, and addressing problems relating to asthma and stroke

Itchy channels and where to find them

Itch (pruritus) is an unpleasant sensory experience that triggers a desire to scratch. It is induced by activation of a subset of cutaneous C fibers in response to environmental irritants and some endogenous mediators (such as histamine

Probing the Light Pseudoscalar Window

Very light pseudoscalars can arise from the symmetry-breaking sector in many extensions of the Standard Model. If their mass is below 200 MeV, they can be long-lived and have interesting phenomenology. We discuss the experimental constraints on several models with light pseudoscalars, including one in which the pseudoscalar is naturally fermiophobic. Taking into account the stringent bounds from rare K and B decays, we find allowed parameter space in each model that may be accessible in direct production experiments. In particular, we study the photoproduction of light pseudoscalars at Jefferson Lab and conclude that a beam dump experiment could explore some of the allowed parameter space of these models.Comment: 22 pages, 4 figure

Drug Delivery Micropump with Built-In Monitoring

AbstractWe report on the fabrication of the first MEMS micropump including an integrated pressure sensor. The signal of this sensor allows direct insight into the pump's operating dynamics and enables real-time self-monitoring. We demonstrate the changes in the sensor signal under different pumping conditions, including the presence of air in the pumping chamber and a downstream occlusions. Deviations from normal operating conditions create clear and characteristic deviations from the normal signal. These deviations will be exploited to detect extraordinary or faulty pumping conditions during use

Sequential therapy of anti-Nogo-A antibody treatment and treadmill training leads to cumulative improvements after spinal cord injury in rats

Intense training is the most clinically successful treatment modality following incomplete spinal cord injuries (SCIs). With the advent of plasticity enhancing treatments, understanding how treatments might interact when delivered in combination becomes critical. Here, we investigated a rational approach to sequentially combine treadmill locomotor training with antibody mediated suppression of the fiber growth inhibitory protein Nogo-A. Following a large but incomplete thoracic lesion, rats were immediately treated with either anti-Nogo-A or control antibody (2 weeks) and then either left untrained or step-trained starting 3 weeks after injury for 8 weeks. It was found that sequentially combined therapy improved step consistency and reduced toe dragging and climbing errors, as seen with training and anti-Nogo-A individually. Animals with sequential therapy also adopted a more parallel paw position during bipedal walking and showed greater overall quadrupedal locomotor recovery than individual treatments. Histologically, sequential therapy induced the greatest corticospinal tract sprouting caudally into the lumbar region and increased the number of serotonergic synapses onto lumbar motoneurons. Increased primary afferent sprouting and synapse formation onto lumbar motoneurons observed with anti-Nogo-A antibody were reduced by training. Animals with sequential therapy also showed the highest reduction of lumbar interneuronal activity associated with walking (c-fos expression). No treatment effects for thermal nociception, mechanical allodynia, or lesion volume were observed. The results demonstrate that sequential administration of anti-Nogo-A antibody followed in time with intensive locomotor training leads to superior recovery of lost locomotor functions, which is probably mediated by changes in the interaction between descending sprouting and local segmental networks after SCI

Delineating an extracellular redox-sensitive module in T-type Ca2+ channels

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch-clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing i) a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I, and ii) a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide (NEM), as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His-191 in Cav3.2 reduced the channel’s sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to both MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2, such that it become sensitive to ambient zinc

Auxiliary subunits control biophysical properties and response to compound NS5806 of the Kv4 potassium channel complex

Kv4 pore‐forming subunits co‐assemble with β‐subunits including KChIP2 and DPP6 and the resultant complexes conduct cardiac transient outward K+ current (Ito). Compound NS5806 has been shown to potentate Ito in canine cardiomyocytes; however, its effects on Ito in other species yet to be determined. We found that NS5806 inhibited native Ito in a concentration‐dependent manner (0.1~30 μM) in both mouse ventricular cardiomyocytes and human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs), but potentiated Ito in the canine cardiomyocytes. In HEK293 cells co‐transfected with cloned Kv4.3 (or Kv4.2) and β‐subunit KChIP2, NS5806 significantly increased the peak current amplitude and slowed the inactivation. In contrast, NS5806 suppressed the current and accelerated inactivation of the channels when cells were co‐transfected with Kv4.3 (or Kv4.2), KChIP2 and another β‐subunit, DPP6‐L (long isoform). Western blot analysis showed that DPP6‐L was dominantly expressed in both mouse ventricular myocardium and hiPSC‐CMs, while it was almost undetectable in canine ventricular myocardium. In addition, low level of DPP6‐S expression was found in canine heart, whereas levels of KChIP2 expression were comparable among all three species. siRNA knockdown of DPP6 antagonized the Ito inhibition by NS5806 in hiPSC‐CMs. Molecular docking simulation suggested that DPP6‐L may associate with KChIP2 subunits. Mutations of putative KChIP2‐interacting residues of DPP6‐L reversed the inhibitory effect of NS5806 into potentiation of the current. We conclude that a pharmacological modulator can elicit opposite regulatory effects on Kv4 channel complex among different species, depending on the presence of distinct β‐subunits. These findings provide novel insight into the molecular design and regulation of cardiac Ito. Since Ito is a potential therapeutic target for treatment of multiple cardiovascular diseases, our data will facilitate the development of new therapeutic Ito modulators

Characterization of the arcD Arginine:Ornithine Exchanger of Pseudomonas aeruginosa. Localization in the Cytoplasmic Membrane and a Topological Model

The arcDABC operon of Pseudomonas aeruginosa encodes the enzymes of the arginine deiminase pathway and is induced by oxygen limitation. The arcD gene specifies a 53-kDa protein with arginine: ornithine exchange activity. The ArcD protein of P. aeruginosa, like the LysI lysine transporter of Corynebacterium glutamicum, has 13 hydrophobic regions which could span the cytoplasmic membrane. Fusion of a Caa (colicin A) epitope to the N-terminal part of ArcD permitted the localization, by immunoblotting, of the hybrid protein in the inner membrane of P. aeruginosa. Fusion of PhoA (alkaline phosphatase) to the very C terminus of ArcD produced another hybrid protein, which exhibited PhoA activity. Both ArcD hybrid proteins retained arginine transport activity and served to support a topological model which proposes that the N terminus is oriented toward the cytoplasm and the C terminus faces the periplasm. Further ArcD-PhoA fusions were consistent with this model. When the Caa epitope was fused to a C-terminal ArcD fragment consisting of only 5 hydrophobic domains, the resulting hybrid protein could be recovered intact from the inner membrane, suggesting that the C-terminal part of ArcD contains sufficient information for insertion into the membrane. This study illustrates the utility of the Caa epitope to tag membrane proteins