Дисертації з теми "Chloride channels"

Onchocerca volvulus, a filarial nematode, is the causative agent of onchocerciasis.<br>O. volvulus is a human parasite with no animal model host and is endemic in the tropics. O. volvulus material is scarce and must be conserved as part of the Onchocerciasis Control Program. A genomic library was constructed to provide a substantial source of renewable genetic material, in place of original parasite DNA.<br>Currently there is only one glutamate-gated chloride channel that has been sequenced from O. volvulus, but this has not yet been characterized. This GluClx partial cDNA sequence isolated by Cully et al., 1997, may be found in GenBank, accession number U59745. Specific primers were designed to amplify this gene from the genomic library. A fragment of this gene was isolated but the primers were non-specific, amplifying genes in addition to GluClx.<br>A motif is a short recognition sequence within a protein that may allow the modification of the protein. The cysteine loop in the N-terminal of all the ligand-gated ion channels is interesting because it contains the neurotransmitter-gated ion channel signature sequence. (Abstract shortened by UMI.)

Glutamate-gated chloride channels (GluCl) belong to then icotinic ligand-gated ion channel family and are thus assumed to be heteropentamers. Each subunit contains a large extracellular N-terminal domain, four transmembrane domains (TM1--TM4), and an extracellular C terminal. Caenorhabditis elegans expresses various GluCl channels formed by alpha1, alpha2, alpha3, alpha4 and beta subunits. The best understood GluCl channel is expressed in pharyngeal muscle cells where it mediates response to the M3 motor neuron. alpha2 forms this channel, probably in association with beta. The alpha2 mutant lacks M3 neurotransmission which can be rescued by pharynx-specific alpha2 expression. My results show that alpha1 and alpha3 subunits cannot substitute for alpha2. Formation of chimeric constructs of alpha1, alpha2 and alpha3 pinpoints the M1--M3 transmembrane region of alpha2 as the minimal rescuing domain. This region may therefore be important for localization or, in association with another subunit, in the formation of the active channel.

In vomeronasal sensory neurons, signal transduction occurs in microvilli that are present at the neuron’s apical surface. The binding of pheromone to vomeronasal receptors causes an increase of the intracellular calcium concentration by calcium entry through TRPC2. An important issue is the impact of Ca2+ entry in pheromonal transduction. In the first part of this thesis we have investigated the functional role played by the increase in intracellular Ca2+ concentration in the apical region of vomeronasal sensory neurons. By taking advantage of flash photolysis of caged calcium restricted to the apical region of neurons, we have measured a calcium-activated current with the whole-cell voltage-clamp technique. Our results demonstrated that a large current is indeed activated by calcium in the apical region of mouse vomeronasal sensory neurons and our immunohistochemistry data has revealed the presence of the proteins TMEM16A and TMEM16B, responsible for calcium-activated chloride channels, in the microvilli of vomeronasal sensory neurons. Therefore we have concluded that calcium-activated chloride channels are present at high density in the region where signal transduction occurs and therefore may play an important role in vomeronasal transduction. In the second part of this thesis we have characterized in more detail the calcium activated currents in mouse vomeronasal sensory neurons using the whole-cell voltage-clamp technique in the presence of various intracellular Ca2+ concentrations. From the dose-response relation we determined that the Ca2+ concentration necessary to activate 50% of the maximal current was 1.4 µM at -100 mV and 0.6 µM at +100 mV. From ion selectivity experiments, we found that the current is carried by anions. Moreover, we demonstrated that some of the commonly used Cl- channel blockers, NFA and CaCCinh-A01, do inhibit the Ca2+-activated current in vomeronasal sensory neurons. Further studies with knockout mice for TMEM16A or TMEM16B will be necessary to establish the physiological role of these channels in vomeronasal transduction.

In embryonic neurons, the intracellular chloride concentration is elevated, making GABA and glycine depolarizing. Later in development, coincident with neuronal maturation, the extruding potassium-chloride co-transporter KCC2 is expressed. It reverses the chloride gradient, rendering it hyperpolarizing. Early depolarization is assumed to play trophic roles during nervous system development. I thus decided to investigate the effects of the depolarizing chloride gradient on development in vivo in the zebrafish embryo. I first determined the temporal pattern of KCC2 expression in zebrafish and found it was absent in the embryo. I then over-expressed wild-type, gain-of-function and loss-of-function variants of human KCC2, using GFP-tagged constructs for detection purposes. Over-expression of functional hKCC2 perturbed the morphology and motor behaviours of the embryos. At the cellular level, KCC2 impaired axonal growth and affected the neuronal populations in the brain, hindbrain and spinal cord. This suggests the depolarizing effects of glycine are critical for neurogenesis.

The objective of this thesis was to understand the regulation of islet Cl⁻ current by cAMP. This current, known as Icl,islet flew is the first Cl⁻ channel current characterized in pancreatic 𝛃 cells. Icl,islet has been hypothesized to modulate insulin secretion through changes in islet electrical activity. Both 5 𝛍M forskolin and 100 𝛍M IBMX (3-isobutyl-1-methylxanthine), agents that increase intracellular cAMP, were shown to activate an outwardly-rectifying ionic current in HIT cells that closely resembled Icl,islet. The current was blocked when iodide was substituted for external Cl⁻ or when the Cl⁻ channel blocker niflumic acid was applied to cells. In contrast, removal of [Na⁺]O did not inhibit the current. In many cells, Cl⁻ current activated and then spontaneously deactivated following cAMP stimulation, suggesting the possibility that the channel desensitizes to [cAMP]i. Exposing cells to multiple cAMP activators revealed that Cl⁻ current declined because it became refractory to increased [cAMP]i. The implication of these results to islet physiology is discussed.

Calcium-activated chloride channels (CaCCs) are ubiquitously expressed in a plethora of cell types and, consequently, are involved in numerous cellular processes as diverse as epithelial secretion, regulation of cardiac excitability and smooth muscle contraction. Current pharmacology of CaCCs is limited to compounds with low potency and poor selectivity. The lack of knowledge surrounding the molecular identity of the CaCC has greatly hindered the development of more specific drugs and has impaired our understanding of the channel physiology and biophysics. The recent discovery that the TMEM16A gene codes for CaCCs has offered hope for new developments in these areas. CaCCs have been suggested as possible targets to treat a variety of conditions including asthma as well as pulmonary and systemic hypertension. Due to the ubiquitous expression of CaCCs and the ability of the channel to interact with a number of pharmacological compounds with diverse chemical structures however, it was hypothesised that TMEM16A could be a possible source for off-target drug effects and may represent a concern for safety pharmacology. The principal aim of this thesis was to assess the functional significance of TMEM16A in the cardiovascular system, as this is one of the major systems of concern for safety pharmacology and accounts for the largest number of post-market drug withdrawals. The main findings of this study can be summarised as follows: 1) RT-PCR analysis revealed a ubiquitous expression of TMEM16A in tissues of the rat and human cardiovascular systems, including systemic and pulmonary arteries as well as cardiac tissue. Analysis also revealed the presence of multiple TMEM16A splice variants in all rat tissues examined, in addition to a number of other TMEM16x family members. 2) Myography experiments using the “classical” CaCC blocker niflumic acid and newly identified TMEM16A blockers confirmed a functional role for TMEM16A in phenylephrine-induced vascular smooth muscle contraction. 3) The suitability of currently available Cl- channel blockers for use as pharmacological tools for TMEM16A research was assessed using conventional whole-cell patch clamp and high-throughput electrophysiology techniques to respectively compare their potencies and selectivity over other cardiovascular ion channels. Of the compounds tested, DIDS and T16Ainh-A01 appeared the most suitable blockers; however all compounds had a degree of non-selectivity, raising concerns for their use in functional studies. In conclusion, these findings provide evidence for the ubiquitous expression and functional significance of TMEM16A within the cardiovascular system and support the hypothesis that TMEM16A is a concern for safety pharmacology and should be included into future pre-clinical safety assays. The inadequacy of current inhibitors however highlights the urgency for the development of novel potent and selective channel modulators for future TMEM16A research.

The Best PhD Thesis in the Faculties of Dentistry, Engineering, Medicine and Science (University of Hong Kong), Li Ka Shing Prize,2007-2008<br>published_or_final_version<br>Chemistry<br>Doctoral<br>Doctor of Philosophy

Glutamate-gated chloride channels (GluCls) are the proposed site of action for macrocyclic lactone anthelminthics, such as IVM, and the milbemycins, such as MOX. The objective of this thesis was to determine whether Haemonchus contortus GluCls are important targets for these anthelminthics. To begin to address this we cloned a full length GluCl alpha-subunit cDNA from H. contortus (HcGluCla). This subunit shares a high homology with GluCl subunits from Caenhorhabditis elegans that have been shown to be important targets for IVM, suggesting that HcGluCla is also an IVM target. However, if HcGluCla is an IVM receptor then it should contain an IVM binding site. To investigate this, the HcGluCla gene was expressed in COS-7 cells. The resulting subunit bound [3H]IVM and [ 3H]MOX with affinities sufficiently high enough to explain their high in vivo potency. Interestingly, glutamate was an allosteric modulator of [ 3H]MOX and [3H]IVM binding where it increased the affinity of these drugs to HcGluCla. To gain further insight into the potentiation of IVM, various glutamatergic and non-glutamatergic ligands were screened for their ability to enhance [3H]IVM binding to HcGluCla. Of the ligands tested, only the GluCl agonists glutamate and ibotenate potentiated [3H]IVM binding. It is possible therefore that if IVM interacts with GluCls in vivo then IVM efficacy may be enhanced by GluCl agonists. To examine this, we tested whether ibotenate could enhance IVM efficacy in gerbils infected with H. contortus. In in vivo efficacy studies, ibotenate (at 1 mg/kg) increased IVM efficacy by 15% (p = 0.048). The enhancement of IVM efficacy in vivo by a GluCl agonist suggests that one of the IVM targets in H. contortus is the GluCl. Finally, to determine the potential physiological response from an interaction between IVM and H. contortus GluCls, we expressed HcGluCla in Xenopus oocytes. HcGluCla expressed in oocytes formed a homomeric channel that responded to

Thesis (Ph. D.)--University of Missouri-Columbia, 2006.<br>The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on August 10, 2007) Vita. Includes bibliographical references.

Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive type of glial brain tumour found in the pons region of the brainstem. DIPG accounts for about 10% of childhood central nervous system tumours and the prognosis for these children is poor. Resistance to radiation, the only current available therapy for DIPG, is one of the biggest challenges. This resistance could be due to the plasticity of DIPG cells, allowing them to rapidly adapt in response to different conditions. Preliminary RNA sequencing analyses of patient tumours identified the expression of ion channel genes including the GABA family. It is known that ion channels regulate tumour plasticity in other cancers and as such we aimed to investigate and characterise the role of ion channels in DIPG. This study aimed to validate the mRNA and protein expression of the GABA-A receptors associated with ligand-gated chloride channels, in DIPG patient-derived cell lines. The results of the study validated mRNA expression of the GABA-A receptor subunits using semi-quantitative and quantitative RT-PCR. Protein expression of three of the most highly expressed subunits (GABRA2, GABRA4, and GABRA5) was also demonstrated using western blotting and immunocytochemistry. Furthermore, a drug screen and titration showed that some GABA-A receptor modulators significantly inhibited proliferation of DIPG cells. This work confirmed that GABA-A subunits are expressed in DIPG cells and that blocking these ion channels inhibits DIPG cell proliferation. These findings form the foundation for future studies that will investigate GABA-A receptor drugs as potential treatments for DIPG using preclinical models.

Les canaux chlore volume-sensibles (VRACs) régulent les activités différenciatives, migratoires et prolifératives des fibroblastes et pourraient donc être impliqués dans la pathobiologie de l’hypertension artérielle pulmonaire (HTAP) et la fibrose pulmonaire interstitielle (FPI). Diverses études antérieures ont montré que l’endothéline-1 (ET1) a des propriétés pro-fibrosantes, en plus cette molécule participe au remodelage des artérioles pulmonaires dans l’HTAP. D’autre part, ces pathologies d’HTAP et de fibrose pulmonaire peuvent être antagonisées par la mélatonine et par l’interaction entre protéines de morphogenèse (BMPs: bone morphogenetic proteins) et leur récepteur 2 (BMPR2). <p>La première partie de mon travail s’est intéressée aux effets de la BMP2 et de l’endothéline1 sur les canaux chlore volume-sensibles de fibroblastes pulmonaires. <p>La stimulation hypotonique du courant a été inhibée par la BMP2 en dépendance de la dose appliquée et de la durée d’exposition à la molécule. Un maximum d’effet de la BMP2 a été observé avec une concentration de 10ng/ml pendant 45min de prétraitement. En plus, les courants chlore volume-sensibles, inhibés par la BMP2, se sont restaurés en présence de l’inhibiteur spécifique de la voie de la protéine kinase C (PKC), le GFX. D’autre part, le prétraitement des fibroblastes avec l’ET1 à 100μM pendant 2heures a induit l’apparition d’un courant activable par l’acide lysophosphatidique (ICl-LPA) (marqueur de la différenciation des fibroblastes) et l’expression de l’α-sma (alpha smooth muscle actin, marqueur classique des myofibroblastes). La migration des fibroblastes a été aussi induite en présence de l’ET1, alors que l’inhibition des canaux chlore par le DIDS (Diisothiocyanatostilbene-disulfonic acid) a bloqué cet effet. La BMP2 s’est opposée à l'effet de l’ET1 sur la différenciation des fibroblastes par l’inhibition de l’induction du courant ICl-LPA et de l’expression génique de l’α-sma. En plus, la migration des fibroblastes, induite par l’ET1, a été inhibée par la BMP2. Nous avons aussi montré que l’expression de gène du canal anoctamine6 a été stimulée par l’ET1, alors la BMP2 s’est opposée à cet effet, ce qui suggère que l’anoctamine6 est le canal responsable de la différenciation des fibroblastes marquée par l’apparition du courant ICl-LPA. Il apparaît donc que l’ET1 et la BMP2 ont des effets opposés sur la différenciation et la migration des fibroblastes pulmonaires via leurs effets sur l’activité et l’expression des canaux chlore volume-sensibles. <p>La deuxième partie du travail s’est intéressée à l’effet de la mélatonine sur les canaux chlore volume-sensibles de fibroblastes L929 et aux conséquences de cet effet sur la migration et la prolifération de ces cellules. Le prétraitement des fibroblastes avec 100μM de mélatonine pendant 30min a inhibé significativement l’activation des canaux chlore volume-sensibles. En plus, une concentration de 100 nM pendant une nuit a donné le même effet observé avec la mélatonine à 100μM pendant 30 min. Nous avons aussi constaté que l’inhibition des VRACs par la mélatonine a été dose-dépendante. L’effet de la mélatonine sur les VRACs a été inhibé en présence de l’antagoniste non sélectif des récepteurs de la mélatonine (Luzindole) et l’antagoniste sélectif pour le récepteur 2 (MT2) de la mélatonine (K185). En plus, l’inhibiteur de la voie de la PKC (GFX) a empêché la mélatonine d’agir sur les canaux chlore volume-sensibles. Ces résultats suggèrent que la mélatonine agit sur les VRACs en se fixant sur MT2 et en activant la voie de la PKC. L’inhibition des VRACs par la mélatonine a eu pour conséquence l’inhibition du phénomène de RVD (regulatory volume decrease), qui suit le gonflement hypotonique. Nous avons aussi montré que la migration des fibroblastes L929 a été inhibée par la mélatonine à 100μM et cela via l’inhibition des VRACs, puisque la mélatonine s’est montrée incapable d’induire une inhibition supplémentaire de la migration en présence de l’inhibiteur des canaux chlore volume-sensibles (DIDS). En plus, l’antagoniste non sélectif des récepteurs de la mélatonine (luzindole), l’antagoniste sélectif pour MT2 (K185) et l’inhibiteur de la voie de PKC (GFX) ont provoqué la disparition de l’effet de la mélatonine sur la migration. Cela suggère que la mélatonine agit sur la migration via les voies empruntées pour l’inhibition des VRACs. L’inhibition des VRACs, par la mélatonine et le DIDS, n'a pas induit d'inhibition significative sur la prolifération des fibroblastes L929, ce qui veut dire que l’inhibition des VRACs est insuffisante pour induire une inhibition significative de la prolifération. Donc, la mélatonine inhibe les canaux chlore volume-sensibles via sa fixation sur MT2 et l’activation de la voie de la PKC. Cela a pour conséquence l’inhibition du RVD et de la migration des fibroblastes L929, mais cette inhibition des VRACs est insuffisante pour inhiber la prolifération de ces cellules. <p>En conclusion, j’ai pu montrer l’importance des canaux chlore volume-sensibles dans la régulation de la physiologie des fibroblastes et leurs interactions avec des médiateurs d’affections pulmonaires à composante fibrosante, telles que l’HTAP et la FPI./<p>Volume-regulated anion channels (VRACs) regulate fibroblast differentiation, migration and proliferation. Fibroblasts have been shown to be involved in several pathologic states including pulmonary arterial hypertension (PAH) and interstitial pulmonary fibrosis (IPF). A number of previous studies have shown that endothelin-1 (ET1) has pro-fibrotic properties and participates in the remodeling of pulmonary arterioles in PAH. On the other hand, PAH and IPF may be controlled by melatonin and bone morphogenetic protein receptor 2 (BMPR2) signaling. <p>The first part of my work described the effects of BMP2 and ET1 on the VRAC in the pulmonary fibroblasts and the consequences of these effects on differentiation and migration of these cells. Pretreatment of fibroblasts with BMP2 inhibited hypotonic current stimulation and this effect was dependent on the BMP2 concentration and on the time of exposition to the molecule. The maximum effect of BMP2 was observed at a concentration of 10ng/ml for 45 min of pretreatment. In addition, volume-sensitive chloride current, inhibited by BMP2, was restored in presence of PKC (protein kinase C) pathway inhibitor (GFX). On the other hand, the pretreatment of fibroblasts with100μM of ET1 for 2 hours, induced the appearance of a lysophosphatidic acid-activable chloride current (ICl-LPA) (a marker of fibroblast differentiation) and stimulated the expression of the smooth muscle actin alpha (α-sma) (the classical marker of myofibroblasts). ET1 also stimulated fibroblast migration, while the inhibition of chloride channels by (DIDS) (Diisothiocyanatostilbene disulfonic acid) bloked this effect. The BMP2 opposed the effect of ET1 on fibroblast differentiation by preventing the induction of ICl-LPA current and α-sma gene expression. In addition, BMP2 inhibited the fibroblast migration induced by ET1. We have also shown that ET1 stimulated anoctamin6 channel gene expression and that BMP2 opposed this effect, which suggests the implication of anoctamin6 on fibroblast differentiation marked by the appearance of ICl-LPA current. Thus, ET1 and BMP2 have opposite effects on pulmonary fibroblast differentiation and migration via their effects on the activity and expression of volume-regulated anion channels. <p>The second part of the work focused on the effect of melatonin, which is a vasorelaxant and antifibrotic agent, on the volume-sensitive chloride channels in L929 fibroblasts and primary rat fibroblasts and on the consequences of this effect on migration and proliferation of these cells. Fibroblast pretreatment with 100μM of melatonin for 30 min significantly inhibited the activation of volume-sensitive chloride channels. In addition, a concentration of 100 nM of melatonin overnight produced the same effect observed with melatonin at 100μM for 30 min. The effect of melatonin on VRAC current was dose-dependent. Inhibition of VRACs by melatonin resulted the inhibition of the RVD phenomenon (Regulatory Volume Decrease) following the hypotonic swelling. The effect of melatonin on VRACs was inhibited in the presence of the non-selective antagonist of melatonin receptors (Luzindole) and the selective antagonist of the melatonin receptor 2 (MT2), the K185. In addition, the PKC pathway inhibitor (GFX) inhibited the effect of melatonin on the volume-sensitive chloride channels. These results suggest that, melatonin acts on the VRACs by binding to MT2 and by activating the PKC pathway. We have also shown that the L929 fibroblast migration was inhibited by melatonin (100μM) via inhibition of VRAC channels, since melatonin was unable to induce further inhibition of migration in the presence of the volume-sensitive chloride channels inhibitor (DIDS). In addition, the non-selective melatonin receptors antagonist (luzindole), the selective antagonist for MT2 (K185) and the PKC pathway inhibitor (GFX), blocked the effect of melatonin on migration, which suggests that melatonin acts on migration via the same pathways that inhibit VRAC channels. Inhibition of VRACs by melatonin and DIDS have not shown any significant inhibition of L929 fibroblast proliferation, which means that the VRAC inhibition is not sufficient to induce a significant inhibition of proliferation. Thus, melatonin inhibits volume-sensitive chloride channels via its binding to MT2 and activation of the PKC pathway. This has as consequences the inhibition of RVD and migration of L929 fibroblasts but insufficient to inhibit the proliferation of these cells. <p>In conclusion, I have shown the importance of volume-sensitive chloride channels in the regulation of fibroblast physiology and its interactions with ET-1, BMP and melatonin signaling. These results are compatible with the notion that the participation of fibroblasts in the pathobiology of PAH or IPF is mediated by VRAC channels, which can be activated by ET-1 and inhibited by BMP’s or melatonin. The translational relevance of these findings will have to be investigated on fibroblasts from patients with PAH or IPF, or from animal models of pulmonary hypertension or lung fibrosis.<p><p><br>Doctorat en Sciences biomédicales et pharmaceutiques<br>info:eu-repo/semantics/nonPublished

Avermectins and milbemycins are believed to exert their anthelmintic effects by binding to glutamate-gated chloride channels (GluCls). Two GluCl subunits have been localized in the pharynx in Caenorhabditis elegans , and the pharynx has been implicated as a major target for avermectins in C. elegans. The HcGluCla gene encoding an alpha-type GluCl subunit has been cloned from Haemonchus contortus previously, however the localization of this gene has not been identified. To begin to investigate the expression site of this HcGluCla gene we have isolated a 1439bp 5'-flanking region and the entire genomic organization of this gene. The 1439bp 5'-flanking region and the first exon and intron and part of the second exon of the HcGluCla gene were fused to the green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. After microinjection of the construct into C. elegans, four stable transformed lines were established and assayed for GFP expression. The transformed animals exhibited fluorescence in the two pairs of MC and M2 pharyngeal neurons, but no expression was detected in the muscle cells. This result provides evidence that the pharynx is a major site for the mode of action of avermectins and milbemycins on parasitic nematodes, such as H. contortus.

The voltage-gated potassium channel Kv1.2 impacts membrane potential and therefore excitability of neurons. Expression of Kv1.2 at the plasma membrane (PM) is critical for channel function, and altering Kv1.2 at the PM is one way to affect membrane excitability. Such is the case in the cerebellum, a portion of the brain with dense Kv1.2 expression, where modulation of Kv1.2 at the PM can impact electrical activity of neurons and ultimately cerebellum-dependent learning. Modulation of Kv1.2 at the PM can occur through endocytic trafficking of the channel; however mechanisms behind this process in the brain remain to be defined. The goal of this dissertation was to identify and characterize modalities endogenous to the brain that influence the presence of Kv1.2 at the neuronal plasma membrane. Mass spectrometry (MS) was used to first identify interacting proteins and post-translational modifications (PTM) of Kv1.2 from cerebellar tissue, and the roles of these interactions and modifications on Kv1.2 function were evaluated in two studies: The first study investigated Trim32, a protein enzyme that catalyzes ubiquitylation, a PTM involved in protein degradation, but also in non-degradative events such as endocytic trafficking. Trim32 was demonstrated to associate and localize with Kv1.2 in cerebellar neurons by MS, immunoblotting (IB), and immunofluorescence (IF), and also demonstrated the ability to ubiquitylate Kv1.2 in vitro through purified recombinant proteins. Utilizing cultured cells through a combination of mutagenesis, biochemistry, and quantitative MS, a working model of Kv1.2 modulation was developed in which Trim32 influences Kv1.2 surface expression by two mechanisms that both involve cross-talk of ubiquitylation and phosphorylation sites of Kv1.2. The second study investigated WNK1, a chloride-sensitive kinase which regulates cellular homeostasis. Using MS, IB, and IF, WNK1 was demonstrated to associate and localize with Kv1.2 in the cerebellum, and a combination of mutagenesis and pharmacology in both wild-type and WNK1-knockout cultured cells produced a working model whereby WNK1 modulates surface Kv1.2. Activation of the downstream target SPAK kinase, also identified by MS to associate with Kv1.2 in the brain, by WNK1 was additionally found to influence the manner of WNK1 modulation of Kv1.2. In addition to providing new models of Kv1.2 modulation in the brain, these studies propose novel biological roles for Trim32 and WNK1 that may ultimately impact neuronal excitability.

Chloride channels play an essential role in the physiology of the respiratory system, the gastrointestinal tract, and secretory glands. Their dysregulation underlies debilitating pathologies such as cystic fibrosis, asthma, and certain cancers. The CLCA (Chloride Channel Accessory) gene family is thought to determine severity of these diseases by modulating an unidentified Calcium-activated Chloride Channel (CaCC). Recent evidence indicates Ano1 to be the mediator of strong quintessential calcium-activated chloride current in several cell types. Ano1 is highly expressed in airway epithelium and downregulated in cystic fibrosis patients. Human CLCA2 is also expressed in epithelium of airways and mammary glands, and there it promotes calcium-activated chloride current. Hence, we hypothesized that CLCA2 modulates the conductance of Ano1. We tested this by introducing Ano1 and CLCA2 together or separately into HEK293 cells, which express endogenous Ano1 at a low level. Using whole-cell voltage clamp, we found that CLCA2 enhanced the conductance of the endogenous CaCC. This current was inhibited by a specific inhibitor of Ano1, tannic acid. CLCA2 also increased both the amplitude and the onset rate of the Ano1-mediated current. To determine the mechanism by which CLCA2 amplifies Ano1 mediated current, we used co-immunoprecipitation with or without a protein cross-linking agent and to test whether the interaction if any, was stable or transient, respectively. Neither any interaction, nor any change in Ano1 multimerization was found. We next tested whether CLCA2 enhanced Ano1 conductance by increasing its stability or surface localization. Surface-labelling the cells expressing Ano1 alone or both proteins with biotin, no difference in Ano1 level or surface expression was detected. Ano1 has recently been shown to be activated by intracellular calcium released from endoplasmic reticulum (ER) stores and by subsequent store-operated calcium entry (SOCE). Therefore, we investigated whether CLCA2 could increase intracellular calcium levels. With Fluo-4 dye calcium imaging, we found that CLCA2 expression enhanced both ER calcium stores and SOCE upon exhaustion of intracellular stores, and the SOCE response could be abolished by a specific inhibitor of SOCE, BTP-2. This inhibitor also abolished CLCA2-induced chloride current, establishing that CLCA2 enhances CaCC via SOCE. Moreover, knockdown of CLCA2 in MCF10A cells, that naturally express both proteins, reduced both ER calcium stores and SOCE. Mutations that abolished the metalloprotease activity of CLCA2 or deleted the cytoplasmic tail had little effect on its enhancement of chloride current or intracellular calcium, suggesting that the uncleaved ectodomain was responsible for both effects of CLCA2. Since, the ectodomain is the most conserved region of the protein, we found that another member of the CLCA family, CLCA1, was also effective in enhancing intracellular calcium storage and SOCE. Co-immunoprecipitation studies further revealed that CLCA2 interacts in a ternary complex with mediators of SOCE, STIM1 and ORAI1. These results explain the CaCC-enhancing effects of CLCA family members and suggest a broader role in other calcium-dependent processes. Understanding the modulatory relationship between these molecules may lead to better therapies for airway diseases and Ano1-dependent cancers. Furthermore, the discovery that CLCA2 regulates intracellular calcium levels may explain its effects on cellular differentiation, stress response, and cell death.

The glutamate-gated chloride (GluCl) channel family of receptors are members of the ligand-gated ion channel (LGIC) family. In addition to being essential for physiological and behavioural aspects of an organism, they are also exploited as a target for the drug ivermectin. A novel GluCl subunit in C. elegans, GLC-4, was characterized in order to further understand the diversity of GluCls and their implications in biological and behavioural processes. Sequencing of cDNAs generated by RT-PCR indicate that GLC-4 possesses the typical features of a GluCl subunit. In addition, they also suggest possible alternative splicing of glc-4 resulting in a slightly truncated transcript. A glc-4 mutant, glc-4(ok212), was used to investigate the role of GLC-4 in C. elegans ivermectin sensitivity and behaviour. Glc-4(ok212) worms were found to be hypersensitive to low concentrations of ivermectin and experienced a slight hyperreversal behaviour. Genetic, molecular, and electrophysiological evidence is also provided suggesting an interaction between GLC-4 and AVR-14, another GluCl subunit in C. elegans. We hypothesize a direct association between GLC-4 and AVR-14 which together form a heteromeric channel in vivo .