Curcumin inhibits activation of TRPM2 channels in rat hepatocytes

Oxidative stress is a hallmark of many liver diseases including viral and drug-induced hepatitis, ischemia-reperfusion injury, and non-alcoholic steatohepatitis. One of the consequences of oxidative stress in the liver is deregulation of Ca2+ homeostasis, resulting in a sustained elevation of the free cytosolic Ca2+ concentration ([Ca2+]c) in hepatocytes, which leads to irreversible cellular damage. Recently it has been shown that liver damage induced by paracetamol and subsequent oxidative stress is, in large part, mediated by Ca2+ entry through Transient Receptor Potential Melastatin 2 (TRPM2) channels. Involvement of TRPM2 channels in hepatocellular damage induced by oxidative stress makes TRPM2 a potential therapeutic target for treatment of a range of oxidative stress-related liver diseases. We report here the identification of curcumin ((1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione), a natural plant-derived polyphenol in turmeric spice, as a novel inhibitor of TRPM2 channel. Presence of 5 µM curcumin in the incubation medium prevented the H2O2- and paracetamol-induced [Ca2+]c rise in rat hepatocytes. Furthermore, in patch clamping experiments incubation of hepatocytes with curcumin inhibited activation of TRPM2 current by intracellular ADPR with IC50 of approximately 50 nM. These findings enhance understanding of the actions of curcumin and suggest that the known hepatoprotective properties of curcumin are, at least in part, mediated through inhibition of TRPM2 channels

Bile acids modulate store-operated CA2+ channels and stromal interaction molecule1 (STIM1) distribution in liver cells

E.C. Aromataris, J. Castro, G.Y. Rychkov, G.J. Barrit

Permeation and Block of the Skeletal Muscle Chloride Channel, ClC-1, by Foreign Anions

A distinctive feature of the voltage-dependent chloride channels ClC-0 (the Torpedo electroplaque chloride channel) and ClC-1 (the major skeletal muscle chloride channel) is that chloride acts as a ligand to its own channel, regulating channel opening and so controlling the permeation of its own species. We have now studied the permeation of a number of foreign anions through ClC-1 using voltage-clamp techniques on Xenopus oocytes and Sf9 cells expressing human (hClC-1) or rat (rClC-1) isoforms, respectively. From their effect on channel gating, the anions presented in this paper can be divided into three groups: impermeant or poorly permeant anions that can not replace Cl− as a channel opener and do not block the channel appreciably (glutamate, gluconate, HCO3−, BrO3−); impermeant anions that can open the channel and show significant block (methanesulfonate, cyclamate); and permeant anions that replace Cl− at the regulatory binding site but impair Cl− passage through the channel pore (Br−, NO3−, ClO3−, I−, ClO4−, SCN−). The permeability sequence for rClC-1, SCN− ∼ ClO4− > Cl− > Br− > NO3− ∼ ClO3− > I− >> BrO3− > HCO3− >> methanesulfonate ∼ cyclamate ∼ glutamate, was different from the sequence determined for blocking potency and ability to shift the Popen curve, SCN− ∼ ClO4− > I− > NO3− ∼ ClO3− ∼ methanesulfonate > Br− > cyclamate > BrO3− > HCO3− > glutamate, implying that the regulatory binding site that opens the channel is different from the selectivity center and situated closer to the external side. Channel block by foreign anions is voltage dependent and can be entirely accounted for by reduction in single channel conductance. Minimum pore diameter was estimated to be ∼4.5 Å. Anomalous mole-fraction effects found for permeability ratios and conductance in mixtures of Cl− and SCN− or ClO4− suggest a multi-ion pore. Hydrophobic interactions with the wall of the channel pore may explain discrepancies between the measured permeabilities of some anions and their size

Orai1- and Orai2-, but not Orai3-mediated ⁄(CRAC) is regulated by intracellular pH

Three Orai (Orai1, Orai2, and Orai3) and two stromal interaction molecule (STIM1 and STIM2) mammalian protein homologues constitute major components of the store-operated Ca2+ entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca2+ channels with properties of Ca2+ release-activated Ca2+ (CRAC) channels. Despite the high level of homology between Orai proteins, CRAC channels formed by different Orai isoforms have distinctive properties, particularly with regards to Ca2+-dependent inactivation, inhibition/potentiation by 2-aminoethyl diphenylborinate and sensitivity to reactive oxygen species. This study characterises and compares the regulation of Orai1, Orai2- and Orai3-mediated CRAC current (ICRAC) by intracellular pH (pHi). Using whole-cell patch clamping of HEK293T cells heterologously expressing Orai and STIM1, we show that ICRAC formed by each Orai homologue has a unique sensitivity to changes in pHi. Orai1-mediated ICRAC exhibits a strong dependence on pHi of both current amplitude and the kinetics of Ca2+-dependent inactivation. In contrast, Orai2 amplitude, but not kinetics, depends on pHi, whereas Orai3 shows no dependence on pHi at all. Investigation of different Orai1–Orai3 chimeras suggests that pHi dependence of Orai1 resides in both the N-terminus and intracellular loop 2, and may also involve pH-dependent interactions with STIM1.Grigori Y. Rychkov, Fiona H. Zhou, MelissaK.Adams, StuartM.Brierley, Linlin Ma, and Greg J. Barrit

Extracellular cGMP, the downstream effector released in response to linaclotide-induced activation of guanylate cyclase-C, reduces excitability of murine and human dorsal root ganglion neurons

Poster presentation PP096J. Castro, G. Y. Rychkov, A. Ghetti, A. M. Harrington, C. Kurtz, A. Silos-Santiago and S. M. Brierle

Drosophila expressing mutant human KCNT1 transgenes make an effective tool for targeted drug screening in a whole animal model of KCNT1-epilepsy

Mutations in the KCNT1 potassium channel cause severe forms of epilepsy which are poorly controlled with current treatments. In vitro studies have shown that KCNT1-epilepsy mutations are gain of function, significantly increasing K+ current amplitudes. To investigate if Drosophila can be used to model human KCNT1 epilepsy, we generated Drosophila melanogaster lines carrying human KCNT1 with the patient mutation G288S, R398Q or R928C. Expression of each mutant channel in GABAergic neurons gave a seizure phenotype which responded either positively or negatively to 5 frontline epilepsy drugs most commonly administered to patients with KCNT1-epilepsy, often with little or no improvement of seizures. Cannabidiol showed the greatest reduction of the seizure phenotype while some drugs increased the seizure phenotype. Our study shows that Drosophila has the potential to model human KCNT1- epilepsy and can be used as a tool to assess new treatments for KCNT1- epilepsy

NaV1.1 inhibition can reduce visceral hypersensitivity

Published: June 7, 2018Functional bowel disorder patients can suffer from chronic abdominal pain, likely due to visceral hypersensitivity to mechanical stimuli. As there is only a limited understanding of the basis of chronic visceral hypersensitivity (CVH), drug-based management strategies are ill defined, vary considerably, and include NSAIDs, opioids, and even anticonvulsants. We previously reported that the 1.1 subtype of the voltage-gated sodium (NaV; NaV1.1) channel family regulates the excitability of sensory nerve fibers that transmit a mechanical pain message to the spinal cord. Herein, we investigated whether this channel subtype also underlies the abdominal pain that occurs with CVH. We demonstrate that NaV1.1 is functionally upregulated under CVH conditions and that inhibiting channel function reduces mechanical pain in 3 mechanistically distinct mouse models of chronic pain. In particular, we use a small molecule to show that selective NaV1.1 inhibition (a) decreases sodium currents in colon-innervating dorsal root ganglion neurons, (b) reduces colonic nociceptor mechanical responses, and (c) normalizes the enhanced visceromotor response to distension observed in 2 mouse models of irritable bowel syndrome. These results provide support for a relationship between NaV1.1 and chronic abdominal pain associated with functional bowel disorders.Juan Salvatierra, Joel Castro, Andelain Erickson, Qian Li, Joao Braz, John Gilchrist, Luke Grundy, Grigori Y. Rychkov, Annemie Deiteren, Rana Rais, Glenn F. King, Barbara S. Slusher, Allan Basbaum, Pankaj J. Pasricha, Stuart M. Brierley, and Frank Bosman

Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction

Irritable bowel syndrome (IBS) patients suffer from chronic abdominal pain and extraintestinal comorbidities, including overactive bladder (OAB) and interstitial cystitis/painful bladder syndrome (IC-PBS). Mechanistic understanding of the cause and time course of these comorbid symptoms is lacking, as are clinical treatments. Here, we report that colitis triggers hypersensitivity of colonic afferents, neuroplasticity of spinal cord circuits, and chronic abdominal pain, which persists after inflammation. Subsequently, and in the absence of bladder pathology, colonic hypersensitivity induces persistent hypersensitivity of bladder afferent pathways, resulting in bladder-voiding dysfunction, indicative of OAB/IC-PBS. Daily administration of linaclotide, a guanylate cyclase-C (GC-C) agonist that is restricted to and acts within the gastrointestinal tract, reverses colonic afferent hypersensitivity, reverses neuroplasticity-induced alterations in spinal circuitry, and alleviates chronic abdominal pain in mice. Intriguingly, daily linaclotide administration also reverses persistent bladder afferent hypersensitivity to mechanical and chemical stimuli and restores normal bladder voiding. Linaclotide itself does not inhibit bladder afferents, rather normalization of bladder function by daily linaclotide treatment occurs via indirect inhibition of bladder afferents via reduced nociceptive signaling from the colon. These data support the concepts that cross-organ sensitization underlies the development and maintenance of visceral comorbidities, while pharmaceutical treatments that inhibit colonic afferents may also improve urological symptoms through common sensory pathways.Luke Grundy, Andrea M. Harrington, Joel Castro, Sonia Garcia-Caraballo, Annemie Deiteren ... Grigori Y. Rychkov ... at al