Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca2+-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. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquitinated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorders.

Orai1- and Orai2-, but not Orai3-mediated ICRAC 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. KEY POINTS: It has been shown previously that Orai1/stromal interaction molecule 1 (STIM1)-mediated Ca2+ release-activated Ca2+ current (ICRAC ) is inhibited by intracellular acidification and potentiated by intracellular alkalinisation. The present study reveals that CRAC channels formed by each of the Orai homologues Orai1, Orai2 and Orai3 has a unique sensitivity to changes in intracellular pH (pHi ). The amplitude of Orai2 current is affected by the changes in pHi  similarly to the amplitude of Orai1. However, unlike Orai1, fast Ca2+ -dependent inactivation of Orai2 is unaffected by acidic pHi . In contrast to both Orai1 and Orai2, Orai3 is not sensitive to pHi  changes. Domain swapping between Orai1 and Orai3 identified the N-terminus and intracellular loop 2 as the molecular structures responsible for Orai1 regulation by pHi . Reduction of ICRAC dependence on pHi seen in a STIM1-independent Orai1 mutant suggested that some parts of STIM1 are also involved in ICRAC modulation by pHi .

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 Nav1.1 subtype, the role of which in nociception and pain has not been elucidated. We use these probes to show that Nav1.1-expressing fibres are modality-specific nociceptors: their activation elicits robust pain behaviours without neurogenic inflammation and produces profound hypersensitivity to mechanical, but not thermal, stimuli. In the gut, high-threshold mechanosensitive fibres also express Nav1.1 and show enhanced toxin sensitivity in a mouse model of irritable bowel syndrome. Together, these findings establish an unexpected role for Nav1.1 channels in regulating the excitability of sensory nerve fibres that mediate mechanical pain.

Conversion of Human Fibroblasts into Induced Neural Stem Cells by Small Molecules.

Induced neural stem cells (iNSCs) reprogrammed from somatic cells hold great potentials for drug discovery, disease modelling and the treatment of neurological diseases. Although studies have shown that human somatic cells can be converted into iNSCs by introducing transcription factors, these iNSCs are unlikely to be used for clinical application due to the safety concern of using exogenous genes and viral transduction vectors. Here, we report the successful conversion of human fibroblasts into iNSCs using a cocktail of small molecules. Furthermore, our results demonstrate that these human iNSCs (hiNSCs) have similar gene expression profiles to bona fide NSCs, can proliferate, and are capable of differentiating into glial cells and functional neurons. This study collectively describes a novel approach based on small molecules to produce hiNSCs from human fibroblasts, which may be useful for both research and therapeutic purposes.

Functional Effects of Epilepsy Associated KCNT1 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 KCNT1 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 KCNT1 mutations, 4 previously studied KCNT1 mutations, and one previously unpublished KCNT1 variant of unknown significance. We analysed the properties of KCNT1 potassium currents and attempted to find a correlation between the changes in KCNT1 characteristics due to the mutations and severity of the neurological disorder they cause. KCNT1 mutations identified in patients with epilepsy were introduced into the full length human KCNT1 cDNA using quick-change site-directed mutagenesis protocol. Electrophysiological properties of different KCNT1 constructs were investigated using a heterologous expression system (HEK293T cells) and patch clamping. All mutations studied, except T314A, increased the amplitude of KCNT1 currents, and some mutations shifted the voltage dependence of KCNT1 open probability, increasing the proportion of channels open at the resting membrane potential. The T314A mutation did not affect KCNT1 current amplitude but abolished its voltage dependence. We observed a positive correlation between the severity of the neurological disorder and the KCNT1 channel open probability at resting membrane potential. This suggests that gain of function KCNT1 mutations cause epilepsy by increasing resting potassium conductance and suppressing the activity of inhibitory neurons. A reduction in action potential firing in inhibitory neurons due to excessively high resting potassium conductance leads to disinhibition of neural circuits, hyperexcitability and seizures.

Conversion of human urine-derived cells into neuron-like cells by small molecules.

Neural cell transplantation is an effective way for treatment of neurological diseases. However, the absence of transplantable human neurons remains a barrier for clinical therapies. Human urine-derived cells, namely renal cells and urine stem cells, have become a good source of cells for reprogramming or trans-differentiation research. Here, we show that human urine-derived cells can be partially converted into neuron-like cells by applying a cocktail of small molecules. Gene expression analysis has shown that these induced cells expressed some neuron-specific genes, and a proportion of the cells are GABAergic neurons. Moreover, whole-cell patch clamping recording has shown that some induced cells have neuron-specific voltage gated Na+ and K+ currents but have failed to generate Ca2+ currents and action potentials. Taken together, these results suggest that induced neuronal cells from human urine-derived cells may be useful for neurological disease modelling, drug screening and cell therapies.

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

Transient Receptor Potential Melastatin (TRPM) 2 is a non-selective Ca2+ 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 Ca2+ 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. The mechanisms regulating the activity of TRPM2 channels in hepatocytes, however, are not well understood. In this paper, we investigate the localisation of TRPM2 protein in hepatocytes. The presented results demonstrate that in rat hepatocytes under normal conditions, most of the TRPM2 protein is localised intracellularly. This was determined by confocal microscopy using TRPM2-and plasma membrane (PM)-specific antibodies and immunofluorescence, and biotinylation studies followed by western blotting. Interestingly, in hepatocytes treated with either H2O2 or paracetamol, the amount of TRPM2 co-localised with PM is significantly increased, compared to the untreated cells. It is concluded that trafficking of TRPM2 to the PM could potentially contribute to a positive feedback mechanism mediating Ca2+ overload in hepatocytes under conditions of oxidative stress.

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. The aim of this study was to determine whether exendin-4 alters intracellular Ca(2+) homeostasis in steatotic hepatocytes, and to evaluate the mechanisms involved. Exendin-4 completely reversed lipid-induced inhibition of SOCE in steatotic liver cells, but did not reverse lipid-induced inhibition of ER Ca(2+) release. The action of exendin-4 on Ca(2+) entry was rapid in onset and was mimicked by GLP-1 or dibutyryl cyclic AMP. In steatotic liver cells, exendin-4 caused a rapid decrease in lipid (half time 6.5min), inhibited the accumulation of lipid in liver cells incubated in the presence of palmitate plus the SOCE inhibitor BTP-2, and enhanced the formation of cyclic AMP. Hormone-stimulated accumulation of extracellular glucose in glycogen replete steatotic liver cells was inhibited compared to that in non-steatotic cells, and this effect of lipid was reversed by exendin-4. It is concluded that, in steatotic hepatocytes, exendin-4 reverses the lipid-induced inhibition of SOCE leading to restoration of hormone-regulated cytoplasmic Ca(2+) signalling. The mechanism may involve GLP-1 receptors, cyclic AMP, lipolysis, decreased diacylglycerol and decreased activity of protein kinase C.

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. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Metabolic Disorders and Cancer: Hepatocyte Store-Operated Ca2+ Channels in Nonalcoholic Fatty Liver Disease.

In steatotic hepatocytes, intracellular Ca2+ homeostasis is substantially altered compared to normal. Decreased Ca2+ 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 Ca2+ channels (SOCs) in hepatocytes are composed principally of Orai1 and STIM1 proteins. Their main role is the maintenance of adequate Ca2+ in the lumen of the ER. In steatotic hepatocytes, store-operated Ca2+ entry (SOCE) is substantially inhibited. This inhibition is associated with a decrease in Ca2+ in the ER. Lipid-induced inhibition of SOCE is mediated by protein kinase C (PKC) and may involve the phosphorylation and subsequent inhibition of Orai1. Experimental inhibition of SOCE enhances lipid accumulation in normal hepatocytes incubated in the presence of exogenous fatty acids. The antidiabetic drug exendin-4 reverses the lipid-induced inhibition of SOCE and decreases liver lipid with rapid onset. It is proposed that lipid-induced inhibition of SOCE in the plasma membrane and of SERCA2b in the ER membrane leads to a persistent decrease in ER Ca2+, ER stress, and the ER stress response, which in turn enhances (amplifies) lipid accumulation. A low level of persistent SOCE due to chronic ER Ca2+ depletion in steatotic hepatocytes may contribute to an elevated cytoplasmic-free Ca2+ concentration leading to the activation of calcium-calmodulin kinase II (CaMKII), decreased lipid removal by autophagy, and insulin resistance. It is concluded that lipid-induced inhibition of SOCE plays an important role in the progression of liver steatosis to insulin insensitivity and hepatocellular carcinoma.

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. Although an increase in intracellular Ca(2+) concentration in hepatocytes is a known consequence of acetaminophen overdose, its importance in acetaminophen-induced liver toxicity is not well understood, primarily due to lack of knowledge about the source of the Ca(2+) rise. Here we report that the channel responsible for Ca(2+) entry in hepatocytes in acetaminophen overdose is the Transient Receptor Potential Melanostatine 2 (TRPM2) cation channel. We show by whole-cell patch clamping that treatment of hepatocytes with acetaminophen results in activation of a cation current similar to that activated by H2O2 or the intracellular application of ADP ribose. siRNA-mediated knockdown of TRPM2 in hepatocytes inhibits activation of the current by either acetaminophen or H2O2. In TRPM2 knockout mice, acetaminophen-induced liver damage, assessed by the blood concentration of liver enzymes and liver histology, is significantly diminished compared with wild-type mice. The presented data strongly suggest that TRPM2 channels are essential in the mechanism of acetaminophen-induced hepatocellular death.

Steatosis inhibits liver cell store-operated Ca²âº entry and reduces ER Ca²âº 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 Ca²âº from the endoplasmic reticulum (ER) stores and subsequent replenishment of these stores by Ca²âº entry through SOCs (store-operated Ca²âº channels; SOCE) plays a critical role in the regulation of liver metabolism. ER Ca²âº homoeostasis is known to be altered in steatotic hepatocytes. Whether store-operated Ca²âº entry is altered in steatotic hepatocytes and the mechanisms involved were investigated. Lipid accumulation in vitro was induced in cultured liver cells by amiodarone or palmitate and in vivo in hepatocytes isolated from obese Zucker rats. Rates of Ca²âº entry and release were substantially reduced in lipid-loaded cells. Inhibition of Ca²âº entry was associated with reduced hormone-initiated intracellular Ca²âº signalling and enhanced lipid accumulation. Impaired Ca²âº entry was not associated with altered expression of stromal interaction molecule 1 (STIM1) or Orai1. Inhibition of protein kinase C (PKC) reversed the impairment of Ca²âº entry in lipid-loaded cells. It is concluded that steatosis leads to a substantial inhibition of SOCE through a PKC-dependent mechanism. This enhances lipid accumulation by positive feedback and may contribute to the development of NASH and insulin resistance.

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. Early studies suggested that FCDI of CRAC channels was mediated by calmodulin. However, since the discovery of STIM1 and Orai1 proteins as the basic molecular components of the CRAC channel, it has become apparent that FCDI is a more complex phenomenon. Data obtained using heterologous overexpression of STIM1 and Orai1 suggest that, in addition to calmodulin, several cytoplasmic domains of STIM1 and Orai1 and the selectivity filter within the channel pore are required for FCDI. The stoichiometry of STIM1 binding to Orai1 also has emerged as an important determinant of FCDI. Consequently, STIM1 protein expression levels have the potential to be an endogenous regulator of CRAC channel Ca(2+) influx. This review discusses the current understanding of the molecular mechanisms governing the FCDI of CRAC channels, including an evaluation of further experiments that may delineate whether STIM1 and/or Orai1 protein expression is endogenously regulated to modulate CRAC channel function, or may be dysregulated in some pathophysiological states.

Nedd4-2 (NEDD4L) controls intracellular Na(+)-mediated activity of voltage-gated sodium channels in primary cortical neurons.

Nedd4-2, a HECT (homologous with E6-associated protein C-terminus)-type ubiquitin protein ligase, has been implicated in regulating several ion channels, including Navs (voltage-gated sodium channels). In Xenopus oocytes Nedd4-2 strongly inhibits the activity of multiple Navs. However, the conditions under which Nedd4-2 mediates native Nav regulation remain uncharacterized. Using Nedd4-2-deficient mice, we demonstrate in the present study that in foetal cortical neurons Nedd4-2 regulates Navs specifically in response to elevated intracellular Na(+), but does not affect steady-state Nav activity. In dorsal root ganglia neurons from the same mice, however, Nedd4-2 does not control Nav activities. The results of the present study provide the first physiological evidence for an essential function of Nedd4-2 in regulating Navs in the central nervous system.

Mutations of the Ca2+-sensing stromal interaction molecule STIM1 regulate Ca2+ influx by altered oligomerization of STIM1 and by destabilization of the Ca2+ channel Orai1.

A drop of endoplasmic reticulum Ca(2+) concentration triggers its Ca(2+) ssensor protein stromal interaction molecule 1 (STIM1) to oligomerize and accumulate within endoplasmic reticulum-plasma membrane junctions where it activates Orai1 channels, providing store-operated Ca(2+) entry. To elucidate the functional significance of N-glycosylation sites of STIM1, we created different mutations of asparagine-131 and asparagine-171. STIM1 NN/DQ resulted in a strong gain of function. Patch clamp, Total Internal Reflection Fluorescent (TIRF) microscopy, and fluorescence recovery after photobleaching (FRAP) analyses revealed that expression of STIM1 DQ mutants increases the number of active Orai1 channels and the rate of STIM1 translocation to endoplasmic reticulum-plasma membrane junctions with a decrease in current latency. Surprisingly, co-expression of STIM1 DQ decreased Orai1 protein, altering the STIM1:Orai1 stoichiometry. We describe a novel mathematical tool to delineate the effects of altered STIM1 or Orai1 diffusion parameters from stoichiometrical changes. The mutant uncovers a novel mechanism whereby "superactive" STIM1 DQ leads to altered oligomerization rate constants and to degradation of Orai1 with a change in stoichiometry of activator (STIM1) to effector (Orai1) ratio leading to altered Ca(2+) homeostasis.

Comparative localization of colorectal sensory afferent central projections in the mouse spinal cord dorsal horn and caudal medulla dorsal vagal complex.

The distal colon and rectum (colorectum) are innervated by spinal and vagal afferent pathways. The central circuits into which vagal and spinal afferents relay colorectal nociceptive information remain to be comparatively assessed. To address this, regional colorectal retrograde tracing and colorectal distension (CRD)-evoked neuronal activation were used to compare the circuits within the dorsal vagal complex (DVC) and dorsal horn (thoracolumbar [TL] and lumbosacral [LS] spinal levels) into which vagal and spinal colorectal afferents project. Vagal afferent projections were observed in the nucleus tractus solitarius (NTS), area postrema (AP), and dorsal motor nucleus of the vagus (DMV), labeled from the rostral colorectum. In the NTS, projections were opposed to catecholamine and pontine parabrachial nuclei (PbN)-projecting neurons. Spinal afferent projections were labeled from rostral through to caudal aspects of the colorectum. In the dorsal horn, the number of neurons activated by CRD was linked to pressure intensity, unlike in the DVC. In the NTS, 13% ± 0.6% of CRD-activated neurons projected to the PbN. In the dorsal horn, at the TL spinal level, afferent input was associated with PbN-projecting neurons in lamina I (LI), with 63% ± 3.15% of CRD-activated neurons in LI projecting to the PbN. On the other hand, at the LS spinal level, only 18% ± 0.6% of CRD-activated neurons in LI projected to the PbN. The collective data identify differences in the central neuroanatomy that support the disparate roles of vagal and spinal afferent signaling in the facilitation and modulation of colorectal nociceptive responses.

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.

Glu¹°6 in the Orai1 pore contributes to fast Ca²âº-dependent inactivation and pH dependence of Ca²âº release-activated Ca²âº (CRAC) current.

FCDI (fast Ca²âº-dependent inactivation) is a mechanism that limits Ca²âº entry through Ca²âº channels, including CRAC (Ca²âº release-activated Ca²âº) channels. This phenomenon occurs when the Ca²âº 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 Ca²âº sensor that communicates the Ca²âº load of the intracellular stores to Orai1, have been shown to regulate fast Ca²âº-dependent inactivation. Although significant advances in unravelling the mechanisms of CRAC channel gating have occurred, the mechanisms regulating fast Ca²âº-dependent inactivation in this channel are not well understood. We have identified that a pore mutation, E106D Orai1, changes the kinetics and voltage dependence of the ICRAC (CRAC current), and the selectivity of the Ca²âº-binding site that regulates fast Ca²âº-dependent inactivation, whereas the V102I and E190Q mutants when expressed at appropriate ratios with STIM1 have fast Ca²âº-dependent inactivation similar to that of WT (wild-type) Orai1. Unexpectedly, the E106D mutation also changes the pH dependence of ICRAC. Unlike WT ICRAC, E106D-mediated current is not inhibited at low pH, but instead the block of Na⁺ permeation through the E106D Orai1 pore by Ca²âº is diminished. These results suggest that Glu¹°6 inside the CRAC channel pore is involved in co-ordinating the Ca²âº-binding site that mediates fast Ca²âº-dependent inactivation.

Movement of hClC-1 C-termini during common gating and limits on their cytoplasmic location.

Functionally, the dimeric human skeletal muscle chloride channel hClC-1 is characterized by two distinctive gating processes, fast (protopore) gating and slow (common) gating. Of these, common gating is poorly understood, but extensive conformational rearrangement is suspected. To examine this possibility, we used FRET (fluorescence resonance energy transfer) and assessed the effects of manipulating the common-gating process. Closure of the common gate was accompanied by a separation of the C-termini, whereas, with opening, the C-termini approached each other more closely. These movements were considerably smaller than those seen in ClC-0. To estimate the C-terminus depth within the cytoplasm we constructed a pair of split hClC-1 fragments tagged extracellularly and intracellularly respectively. These not only combined appropriately to rescue channel function, but we detected positive FRET between them. This restricts the C-termini of hClC-1 to a position close to its membrane-resident domain. From mutants in which fast or common gating were affected, FRET revealed a close linkage between the two gating processes with the carboxyl group of Glu²³² apparently acting as the final effector for both.

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. Utilising whole cell patch clamping, we applied calibrated displacements to neurites of dorsal root ganglion (DRG) neurons in short-term culture and recorded mechanically activated currents termed intermediately (IAMCs), rapidly (RAMCs) or slowly adapting (SAMCs). Trpa1 deletion (­/­) significantly reduced maximum IAMC amplitude by 43% in small-diameter neurons compared with wild-type (+/+) neurons. All other mechanically activated currents in small- and large-diameter Trpa1−/− neurons were unaltered. Seventy-three per cent of Trpa1+/+ small-diameter neurons responding to the TRPA1 agonist allyl-isothiocyanate (AITC) displayed IAMCs to neurite displacement, which were significantly enhanced after AITC addition. The TRPA1 antagonist HC-030031 significantly decreased Trpa1+/+ IAMC amplitudes, but only in AITC responsive neurons. Using a transfection system we also showed TRPA1 over-expression in Trpa1+/+ small-diameter neurons increases IAMC amplitude, an effect reversed by HC-030031. Furthermore, TRPA1 introduction into Trpa1−/− small-diameter neurons restored IAMC amplitudes to Trpa1+/+ levels, which was subsequently reversed by HC-030031. In summary our data demonstrate TRPA1 makes a contribution to normal mechanosensation in a specific subset of DRG neurons. Furthermore, they also provide new evidence illustrating mechanisms by which sensitisation or over-expression of TRPA1 enhances nociceptor mechanosensitivity. Overall, these findings suggest TRPA1 has the capacity to tune neuronal mechanosensitivity depending on its degree of activation or expression.