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.

Functional Effects of Epilepsy Associated Mutations Suggest Pathogenesis via Aberrant Inhibitory Neuronal Activity.

KCNT1 (K channel subfamily T member 1) is a sodium-activated potassium channel highly expressed in the nervous system which regulates neuronal excitability by contributing to the resting membrane potential and hyperpolarisation following a train of action potentials. Gain of function mutations in the gene are the cause of neurological disorders associated with different forms of epilepsy. To gain insights into the underlying pathobiology we investigated the functional effects of 9 recently published mutations, 4 previously studied mutations, and one previously unpublished variant of unknown significance.

Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca-induced proteostasis disease stress.

The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement.

Orai1- and Orai2-, but not Orai3-mediated I 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 Ca entry mechanism. When co-expressed with STIM1, Orai1, Orai2 and Orai3 form highly selective Ca channels with properties of Ca release-activated Ca (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 Ca -dependent inactivation, inhibition/potentiation by 2-aminoethyl diphenylborinate and sensitivity to reactive oxygen species.

TRPM2 Non-Selective Cation Channels in Liver Injury Mediated by Reactive Oxygen Species.

TRPM2 channels admit Ca and Na across the plasma membrane and release Ca and Zn from lysosomes. Channel activation is initiated by reactive oxygen species (ROS), leading to a subsequent increase in ADP-ribose and the binding of ADP-ribose to an allosteric site in the cytosolic NUDT9 homology domain. In many animal cell types, Ca entry via TRPM2 channels mediates ROS-initiated cell injury and death.

Targeting Ca Signaling in the Initiation, Promotion and Progression of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a considerable health burden worldwide and a major contributor to cancer-related deaths. HCC is often not noticed until at an advanced stage where treatment options are limited and current systemic drugs can usually only prolong survival for a short time. Understanding the biology and pathology of HCC is a challenge, due to the cellular and anatomic complexities of the liver.

Deranged hepatocyte intracellular Ca homeostasis and the progression of non-alcoholic fatty liver disease to hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths in men, and the sixth in women. Non-alcoholic fatty liver disease (NAFLD) is now one of the major risk factors for HCC. NAFLD, which involves the accumulation of excess lipid in cytoplasmic lipid droplets in hepatocytes, can progress to non-alcoholic steatosis, fibrosis, and HCC.

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.

Measurement of the CRAC Channel Fast Ca-Dependent Inactivation (FCDI).

Fast Ca-dependent inactivation (FCDI) is a safety mechanism limiting Ca entry through some types of Ca channels, including Ca release-activated Ca (CRAC) channels. This type of inactivation is caused by Ca, which passes through Ca channel and binds to a specific site within a short distance from the inner mouth of the pore, causing channel to shut.The main technique that is used to investigate FCDI is whole-cell patch clamping.

Oxidative stress promotes redistribution of TRPM2 channels to the plasma membrane in hepatocytes.

Transient Receptor Potential Melastatin (TRPM) 2 is a non-selective Ca permeable cation channel and a member of the Transient Receptor Potential (TRP) channel family. TRPM2 has unique gating properties; it is activated by intracellular ADP-ribose (ADPR), whereas Ca plays a role of an important co-factor in channel activation, increasing TRPM2 sensitivity to ADPR. TRPM2 is highly expressed in rat and mouse hepatocytes, where it has been shown to contribute to oxidative stress-induced cell death and liver damage due to paracetamol-overdose.

NaV1.1 inhibition can reduce visceral hypersensitivity.

Functional 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.

Cyclic analogues of α-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain.

Background And Purpose: Patients with irritable bowel syndrome suffer from chronic visceral pain (CVP) and limited analgesic therapeutic options are currently available. We have shown that α-conotoxin Vc1.1 induced activation of GABA receptors on the peripheral endings of colonic afferents and reduced nociceptive signalling from the viscera.

Metabolic Disorders and Cancer: Hepatocyte Store-Operated Ca Channels in Nonalcoholic Fatty Liver Disease.

In steatotic hepatocytes, intracellular Ca homeostasis is substantially altered compared to normal. Decreased Ca in the endoplasmic reticulum (ER) can lead to ER stress, an important mediator of the progression of liver steatosis to nonalcoholic steatohepatitis, type 2 diabetes, and hepatocellular carcinoma. Store-operated Ca channels (SOCs) in hepatocytes are composed principally of Orai1 and STIM1 proteins.

Modulation of TRP Channel Activity by Hydroxylation and Its Therapeutic Potential.

Two transient receptor potential (TRP) channels-TRPA1 and TRPV3-are post-translationally hydroxylated, resulting in oxygen-dependent regulation of channel activity. The enzymes responsible are the HIF prolyl hydroxylases (PHDs) and the asparaginyl hydroxylase factor inhibiting HIF (FIH). The PHDs and FIH are well characterized for their hydroxylation of the hypoxic inducible transcription factors (HIFs), mediating their hypoxic regulation.

Multiple sodium channel isoforms mediate the pathological effects of Pacific ciguatoxin-1.

Human intoxication with the seafood poison ciguatoxin, a dinoflagellate polyether that activates voltage-gated sodium channels (Na), causes ciguatera, a disease characterised by gastrointestinal and neurological disturbances. We assessed the activity of the most potent congener, Pacific ciguatoxin-1 (P-CTX-1), on Na1.1-1.

Selective spider toxins reveal a role for the Nav1.1 channel in mechanical pain.

Voltage-gated sodium (Nav) channels initiate action potentials in most neurons, including primary afferent nerve fibres of the pain pathway. Local anaesthetics block pain through non-specific actions at all Nav channels, but the discovery of selective modulators would facilitate the analysis of individual subtypes of these channels and their contributions to chemical, mechanical, or thermal pain. Here we identify and characterize spider (Heteroscodra maculata) toxins that selectively activate the Nav1.

The glucagon-like peptide-1 analogue exendin-4 reverses impaired intracellular Ca(2+) signalling in steatotic hepatocytes.

The release of Ca(2+) from the endoplasmic reticulum (ER) and subsequent replenishment of ER Ca(2+) by Ca(2+) entry through store-operated Ca(2+) channels (SOCE) play critical roles in the regulation of liver metabolism by adrenaline, glucagon and other hormones. Both ER Ca(2+) release and Ca(2+) entry are severely inhibited in steatotic hepatocytes. Exendin-4, a slowly-metabolised glucagon-like peptide-1 (GLP-1) analogue, is known to reduce liver glucose output and liver lipid, but the mechanisms involved are not well understood.

Steatosis inhibits liver cell store-operated Ca²⁺ entry and reduces ER Ca²⁺ through a protein kinase C-dependent mechanism.

Lipid accumulation in hepatocytes can lead to non-alcoholic fatty liver disease (NAFLD), which can progress to non-alcoholic steatohepatitis (NASH) and Type 2 diabetes (T2D). Hormone-initiated release of Ca²⁺ from the endoplasmic reticulum (ER) stores and subsequent replenishment of these stores by Ca²⁺ entry through SOCs (store-operated Ca²⁺ channels; SOCE) plays a critical role in the regulation of liver metabolism. ER Ca²⁺ homoeostasis is known to be altered in steatotic hepatocytes.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD.

Neurogenic potential of dental pulp stem cells isolated from murine incisors.

Introduction: Interest in the use of dental pulp stem cells (DPSC) to enhance neurological recovery following stroke and traumatic injury is increasing following successful pre-clinical studies. A murine model of autologous neural stem cell transplantation would be useful for further pre-clinical investigation of the underlying mechanisms. However, while human-derived DPSC have been well characterised, the neurogenic potential of murine DPSC (mDPSC) has been largely neglected.

TRPM2 channels mediate acetaminophen-induced liver damage.

Acetaminophen (paracetamol) is the most frequently used analgesic and antipyretic drug available over the counter. At the same time, acetaminophen overdose is the most common cause of acute liver failure and the leading cause of chronic liver damage requiring liver transplantation in developed countries. Acetaminophen overdose causes a multitude of interrelated biochemical reactions in hepatocytes including the formation of reactive oxygen species, deregulation of Ca(2+) homeostasis, covalent modification and oxidation of proteins, lipid peroxidation, and DNA fragmentation.

Structural and stoichiometric determinants of Ca2+ release-activated Ca2+ (CRAC) channel Ca2+-dependent inactivation.

Depletion of intracellular Ca(2+) stores in mammalian cells results in Ca(2+) entry across the plasma membrane mediated primarily by Ca(2+) release-activated Ca(2+) (CRAC) channels. Ca(2+) influx through these channels is required for the maintenance of homeostasis and Ca(2+) signaling in most cell types. One of the main features of native CRAC channels is fast Ca(2+)-dependent inactivation (FCDI), where Ca(2+) entering through the channel binds to a site near its intracellular mouth and causes a conformational change, closing the channel and limiting further Ca(2+) entry.

Glu¹⁰⁶ in the Orai1 pore contributes to fast Ca²⁺-dependent inactivation and pH dependence of Ca²⁺ release-activated Ca²⁺ (CRAC) current.

FCDI (fast Ca²⁺-dependent inactivation) is a mechanism that limits Ca²⁺ entry through Ca²⁺ channels, including CRAC (Ca²⁺ release-activated Ca²⁺) channels. This phenomenon occurs when the Ca²⁺ concentration rises beyond a certain level in the vicinity of the intracellular mouth of the channel pore. In CRAC channels, several regions of the pore-forming protein Orai1, and STIM1 (stromal interaction molecule 1), the sarcoplasmic/endoplasmic reticulum Ca²⁺ sensor that communicates the Ca²⁺ load of the intracellular stores to Orai1, have been shown to regulate fast Ca²⁺-dependent inactivation.

TRPA1 contributes to specific mechanically activated currents and sensory neuron mechanical hypersensitivity.

The mechanosensory role of TRPA1 and its contribution to mechanical hypersensitivity in sensory neurons remains enigmatic. We elucidated this role by recording mechanically activated currents in conjunction with TRPA1 over- and under-expression and selective pharmacology. First, we established that TRPA1 transcript, protein and functional expression are more abundant in smaller-diameter neurons than larger-diameter neurons, allowing comparison of two different neuronal populations.

Respiratory distress and perinatal lethality in Nedd4-2-deficient mice.

The epithelial sodium channel (ENaC) is essential for sodium homoeostasis in many epithelia. ENaC activity is required for lung fluid clearance in newborn animals and for maintenance of blood volume and blood pressure in adults. In vitro studies show that the ubiquitin ligase Nedd4-2 ubiquitinates ENaC to regulate its cell surface expression.