Regulation of transient receptor potential melastatin 8 (TRPM8) channel activity by its short isoforms.

One important mechanism of membrane ion channels regulation involves their non-functional isoforms generated by alternative splicing. However, knowledge of such isoforms for the members of transient receptor potential (TRP) superfamily of ion channels remains quite limited. This study focuses on TRPM member, TRPM8, which functions as a cold receptor in sensory neurons, but is also expressed in tissues not exposed to ambient temperatures, as well as in cancer tissues. We report the cloning from prostate cancer cells of new short-splice variants of TRPM8, termed short TRPM8a (sM8a) and short TRPM8ß (sM8ß). Our results show that both variants are in a closed configuration with the C-terminal tail of the full-size TRPM8 chan-nel, resulting in stabilization of its closed state and thus reducing both its cold sensitivity and its activity. Our findings, therefore, uncover a new mode of the regulation of TRPM8 channel by its splice variants.

The Role of K+ and Cl- Channels in the Regulation of Retinal Arteriolar Tone and Blood Flow

Purpose: This study tested the role of K(+)- and Cl(-)-channels in retinal arteriolar smooth muscle in the regulation of retinal blood flow.

Methods: Studies were carried out in adult Male Hooded Lister rats. Selectivity of ion channel blockers was established using electrophysiological recordings from smooth muscle in isolated arterioles under voltage clamp conditions. Leukocyte velocity and retinal arteriolar diameters were measured in anesthetised animals using leukocyte fluorography and fluorescein angiography imaging with a confocal scanning laser ophthalmoscope. These values were used to estimate volumetric flow, which was compared between control conditions and following intravitreal injections of ion channel blockers, either alone or in combination with the vasoconstrictor potent Endothelin 1 (Et1).

Results: Voltage activated K(+)-current (IKv) was inhibited by correolide, large conductance (BK) Ca(2+)-activated K(+)-current (IKCa) by Penitrem A, and Ca(2+)-activated Cl(-)-current (IClCa) by disodium 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). Intravitreal injections (10µl) of DIDS (estimated intraocular concentration 10mM) increased flow by 22%, whereas the BK-blockers Penitrem A (1µM) and iberiotoxin (4µM), and the IKv-inhibitor correolide (40µM) all decreased resting flow by approximately 10%. Et1 (104nM) reduced flow by almost 65%. This effect was completely reversed by DIDS but was unaffected by Penitrem A, iberiotoxin or correolide.

Conclusions: These results suggest that Cl(-)-channels in retinal arteriolar smooth muscle limit resting blood flow and play an obligatory role in Et1 responses. K(+)-channel activity promotes basal flow but exerts little modifying effect on the Et1 response. Cl(-)-channels may be appropriate molecular targets in retinal pathologies characterised by increased Et1 activity and reduced blood flow.

Localization and regulation by GABA of anion transporter present in pollen grain protoplasts and tube of Arabidopsis thaliana

Tese de doutoramento, Biologia (Biologia do Desenvolvimento), Universidade de Lisboa, Faculdade de Ciências, 2015

Nedd4-2-dependent ubiquitylation and regulation of the cardiac potassium channel hERG1.

The voltage-gated cardiac potassium channel hERG1 (human ether-à-gogo-related gene 1) plays a key role in the repolarization phase of the cardiac action potential (AP). Mutations in its gene, KCNH2, can lead to defects in the biosynthesis and maturation of the channel, resulting in congenital long QT syndrome (LQTS). To identify the molecular mechanisms regulating the density of hERG1 channels at the plasma membrane, we investigated channel ubiquitylation by ubiquitin ligase Nedd4-2, a post-translational regulatory mechanism previously linked to other ion channels. We found that whole-cell hERG1 currents recorded in HEK293 cells were decreased upon neural precursor cell expressed developmentally down-regulated 4-2 (Nedd4-2) co-expression. The amount of hERG1 channels in total HEK293 lysates and at the cell surface, as assessed by Western blot and biotinylation assays, respectively, were concomitantly decreased. Nedd4-2 and hERG1 interact via a PY motif located in the C-terminus of hERG1. Finally, we determined that Nedd4-2 mediates ubiquitylation of hERG1 and that deletion of this motif affects Nedd4-2-dependent regulation. These results suggest that ubiquitylation of the hERG1 protein by Nedd4-2, and its subsequent down-regulation, could represent an important mechanism for modulation of the duration of the human cardiac action potential.

Expression analysis of the AtPHO1 gene family in Arabidopsis thaliana and the involvement of AtPHO1 in the regulation of stomatal movements

Résumé Le transfert du phosphate des racines vers les feuilles s'effectue par la voie du xylème. Il a été précédemment démontré que la protéine AtPHO1 était indispensable au transfert du phosphate dans les vaisseaux du xylème des racines chez la plante modèle Arabidopsis thaliana. Le séquençage et l'annotation du génome d'Arabidopsis ont permis d'identifier dix séquences présentant un niveau de similarité significatif avec le gène AtPHO1 et constituant une nouvelle famille de gène appelé la famille de AtPHO1. Basée sur une étude moléculaire et génétique, cette thèse apporte des éléments de réponse pour déterminer le rôle des membres de ia famille de AtPHO1 chez Arabidopsis, inconnue à ce jour. Dans un premier temps, une analyse bioinformatique des séquences protéiques des membres de la famille de AtPHO1 a révélé la présence dans leur région N-terminale d'un domaine nommé SPX. Ce dernier est conservé parmi de nombreuses protéines impliquées dans l'homéostasie du phosphate chez la levure, renforçant ainsi l'hypothèse que les membres de la famille de AtPHO1 auraient comme AtPHO1 un rôle dans l'équilibre du phosphate dans la plante. En parallèle, la localisation tissulaire de l'expression des gènes AtPHO dans Arabidopsis a été identifiée par l'analyse de plantes transgéniques exprimant le gène rapporteur uidA sous le contrôle des promoteurs respectifs des gènes AtPHO. Un profil d'expression de chaque gène AtPHO au cours du développement de la plante a été obtenu. Une expression prédominante au niveau des tissus vasculaires des racines, des feuilles, des tiges et des fleurs a été observée, suggérant que les gènes AtPHO pourraient avoir des fonctions redondantes au niveau du transfert de phosphate dans le cylindre vasculaire de ces différents organes. Toutefois, plusieurs régions promotrices des gènes AtPHO contrôlent également un profil d'expression GUS non-vasculaire, indiquant un rôle putatif des gènes AtPHO dans l'acquisition ou le recyclage de phosphate dans la plante. Dans un deuxième temps, l'analyse de l'expression des gènes AtPHO durant une carence en phosphate a établi que seule l'expression des gènes AtPHO1, AtPHO1; H1 et AtPHO1; H10 est régulée par cette carence. Une étude approfondie de leur expression en réponse à des traitements affectant l'homéostasie du phosphate dans la plante a ensuite démontré leur régulation par différentes voies de signalisation. Ensuite, une analyse détaillée de la régulation de l'expression du gène AtPHO1; H1O dans des feuilles d'Arabidopsis blessées ou déshydratées a révélé que ce gène constitue le premìer gène marqueur d'une nouvelle voie de signalisation induite par l'OPDA, pas par le JA et dépendante de la protéine COI1. Ces résultats démontrent pour la première fois que l'OPDA et le JA peuvent activer différents gènes via des voies de signalisation dépendantes de COI1. Enfin, cette thèse révèle l'identification d'un nouveau rôle de la protéine AtPHO1 dans la régulation de l'action de l'ABA au cours des processus de fermeture stomatique et de germination des graines chez Arabidopsis. Bien que les fonctions exactes des protéines AtPHO restent à être déterminées, ce travail de thèse suggère leur implication dans la propagation de différents signaux dans la plante via la modulation du potentiel membranaire et/ou l'affectation de la composition en ions des cellules comme le font de nombreux transporteurs ou régulateur du transport d'ions. Summary Phosphate is transferred from the roots to the shoot via the xylem. The requirement for AtPHO1 protein to transfer phosphate to the xylem vessels of the root has been previously demonstrated in Arabidopsis thaliana. The sequencing and the annotation of the Arabidopsis genome had allowed the identification of ten sequences that show a significant level of similarity with the AtPHO1 gene. These 10 genes, of unknown functions, constitute a new gene family called the AtPHO1 gene family. Based on a molecular and genetics study, this thesis reveals some information needed to understand the role of the AtPHO1 family members in the plant Arabidopsis. First, a bioinformatics study revealed that the AtPHO sequences contained, in the N-terminal hydrophilic region, a motif called SPX and conserved among multiple proteins involved in phosphate homeostasis in yeast. This finding reinforces the hypothesis that all AtPHO1 family members have, as AtPHO1, a role in phosphate homeostasis. In parallel, we identified the pattern of expression of AtPHO genes in Arabidopsis via analysis of transgenic plants expressing the uidA reporter gene under the control of respective AtPHO promoter regions. The results exhibit a predominant expression of AtPHO genes in vascular tissues of all organs of the plant, implying that these AtPHO genes could have redundant functions in the transfer of phosphate to the vascular cylinder of various organs. The GUS expression pattern for several AtPHO promoter regions was also detected in non-vascular tissue indicating a broad role of AtPHO genes in the acquisition or in the recycling of phosphate in the plant. In a second step, the analysis of the expression of AtPHO genes during phosphate starvation established that only the expression of the AtPHO1, AtPHO1; H1 and AtPHO1; H10 genes were regulated by Pi starvation. Interestingly, different signalling pathways appeared to regulate these three genes during various treatments affecting Pi homeostasis in the plant. The third chapter presents a detailed analysis of the signalling pathways regulating the expression of the AtPHO1; H10 gene in Arabidopsis leaves during wound and dehydrated stresses. Surprisingly, the expression of AtPHO1; H10 was found to be regulated by OPDA (the precursor of JA) but not by JA itself and via the COI1 protein (the central regulator of the JA signalling pathway). These results demonstrated for the first time that OPDA and JA could activate distinct genes via COI1-dependent pathways. Finally, this thesis presents the identification of a novel role of the AtPHO1 protein in the regulation of ABA action in Arabidopsis guard cells and during seed germination. Although the exact role and function of AtPHO1 still need to be determined, these last findings suggest that AtPHO1 and by extension other AtPHO proteins could mediate the propagation of various signals in the plant by modulating the membrane potential and/or by affecting cellular ion composition, as it is the case for many ion transporters or regulators of ion transport.

Airway surface liquid volume regulation determines different airway phenotypes in liddle compared with betaENaC-overexpressing mice.

Studies in cystic fibrosis patients and mice overexpressing the epithelial Na(+) channel beta-subunit (betaENaC-Tg) suggest that raised airway Na(+) transport and airway surface liquid (ASL) depletion are central to the pathogenesis of cystic fibrosis lung disease. However, patients or mice with Liddle gain-of-function betaENaC mutations exhibit hypertension but no lung disease. To investigate this apparent paradox, we compared the airway phenotype (nasal versus tracheal) of Liddle with CFTR-null, betaENaC-Tg, and double mutant mice. In mouse nasal epithelium, the region that functionally mimics human airways, high levels of CFTR expression inhibited Liddle epithelial Nat channel (ENaC) hyperfunction. Conversely, in mouse trachea, low levels of CFTR failed to suppress Liddle ENaC hyperfunction. Indeed, Na(+) transport measured in Ussing chambers ("flooded" conditions) was raised in both Liddle and betaENaC-Tg mice. Because enhanced Na(+) transport did not correlate with lung disease in these mutant mice, measurements in tracheal cultures under physiologic "thin film" conditions and in vivo were performed. Regulation of ASL volume and ENaC-mediated Na(+) absorption were intact in Liddle but defective in betaENaC-Tg mice. We conclude that the capacity to regulate Na(+) transport and ASL volume, not absolute Na(+) transport rates in Ussing chambers, is the key physiologic function protecting airways from dehydration-induced lung disease.

Influence of temperature and photoperiod upon ionic regulation in rainbow trout Salmo gairdneri

Interactions of photoperiod and temperature upon waterelectrolyte balance were examined in rainbow trout acclimated to six combinations of two photoperiods {18h light: 6h dark, o 6h light: l8h dark) and three temperatures (2, 10 and 18 C). The influence of temperature and photoperiod upon plasma, skeletal muscle, cardiac muscle and liver levels of sodium, potassium, magnesi.um, calcium, chloride, water content, water distribution and cellular ion concentrations was determined by a one way analysis of variance. Significant (p < 0.05 or better) temperature effects at common photoperiods were observed in 70% of the analyses performed, showing no bias toward either photoperiod. Significant photoperiod effects occured in 57% of the analyses performed at common temperatures. The influence of photoperiod was most prevalent at reduced temperatures. Potassium and magnesium appeared to be particularly thermosensitive, while sodium and calcium were the most photosensitive of the electrolytes. The ionic composition of all tissues studied were relatively thermosensitive, with liver apparently being the most sensitive. On the other hand; the ionic composition of skeletal and cardiac muscle appear to be the mos.t photosensitive of the tissues examined. Water content and distribution in skeletal muscle and liver were significantly influenced by temperature in 50% of the analyses performed showing a very strong bias toward UwinterU animals. Photoperiod effects were significant in 56% of the water parameters measured with a strong bias toward the two lower temperatures. Body weight was of significant influence in 16% of the 174 analyses performed. These data are discussed in terms of the effect of temperature upon ionregulatory mechanisms and the possible impact of photoperiod variations on endocrine systems influencing water-electrolyte metabolism.

Ionic regulation in goldfish, Carassius auratus, acclimated to constant and diurnally-cycling temperature conditions

The effects of a diurnal sine-wave temperature cycle (250 +- 5° C) on the wa terI-e etc r o1 yt est a t us 0 f gol df1' Sh , Carassius auratus, was assessed through determination of Na+, K+, Mg2+, Ca2+, Cl- and water content in plasma, Red blood cells and muscle tissue. Animals were also acclimated to o 0 0 static temperatures (20 C, 25 c, 30 C) corresponding to the high, low and mid-ooint temperatures of the cycle. All groups were sampled at 03:00, 09:00, 15:00 and 21:00 hr. Hemoglobin content and packed cell volume, as well as electrolyte and 'water levels were determined for each animal and red cell ion concentrations and ion : hemoglobin ratios estimated. Cycled animals were distinct from those at constant temperatures in several respects. Hematological parameters were elevated above those of animals at constant temperature and were, on a diurnal basis, more stable. Red blood cell electrolyte levels varied in an adaptively appropriate fashion to cycle temperatures. This was not the case in the constant temperature groups_ Under the cycling regime, plasma ion levels were more diurnally stable than those of constant temperature fish. Although muscle parameters in cycled fish exhibited more fluctuation than was observed in plasma, these also tended to be relatively more stable than was the caseErythrocytic data are discussed in terms of their effects on hemoglobin-oxygen affinity while plasma and muscle observations were considered from the standpoint of overall water-electrolyte balance. In general, cycled fish appeared to be capable of stabilizing overall body fluid composition, while simultaneously effecting adaptively-appropriate modifications in the erythrocytic ionic microenvironment of hemoglobin. The sometimes marked diurnal variability of water-electrolyte status in animals held at constant temperature as opposed to the conservation of cycled fish suggests that this species is, in some fashion, programmed for regulation in a thermally-fluctuating environment. If this interpretation is valid and a phenomenon of general occurrence, some earlier studies involving constant acclimation of eurythermal species normally occupying habitats which vary in temperature on a daily basis may require reconsideration. at constant temperature.

The production of 4-adminobutyrate in response to treatments reducing cytosolic pH and the regulation of intracellular pH

GABA (4-aminobutyrate) is synthesized through the decarboxylation of LGlu- (L-Glu-+ H+ ---> GABA + C02), and compared to many free amino acids is present in high concentrations in plant cells. GABA levels rise rapidly and dramatically in response to varied stress conditions including anaerobiosis. Recent papers suggest that GABA production and associated H+ consumption are parts of a metabolic pH-stat mechanism which ameliorates the intracellular pH decline associated with anaerobiosis or other treatments. To test this hypothesis GABA production and efflux have been measured in isolated Asparagus sprengeri cells in response to three treatments which potentially cause intracellular acidification. Acid loads were imposed using 60 min of (i) anaerobiosis, (ii) H+/LGlu- cotransport, and (iii) treatment with permeant weak acids (butyric, acetic and propionic). Both intra- and extracellular GABA concentrations increased more than 100% after anaerobiosis, almost 1000% after H+/L-Glu- cotransport (light or dark) and almost 5000/0 after addition of 5 mM butyric acid at pH 5.0. HPLC analysis of amino acids indicates that as GABA concentrations increased in response to butyric acid addition, glutamate concentrations decreased. Time-course studies demonstrated that added butyric acid stimulates GABA production by 2800/0 within 15 seconds. A fluorescent determination of cytosolic pH indicates that addition of butyric or other weak acids resulted in a rapid reduction in cytosolic pH of 0.6 pH units. The half time for the response to butyric acid addition is 2.1 seconds, indicating that the decline in cytosolic pH is rapid enough to account for the rapid stimulation of GABA production. The acid load in response to butyric acid addition was assayed by measurements of 14C-butyric acid uptake. Calculations indicate that GABA production accounted for 45% of the imposed acid load. The biological significance of GABA efflux is not yet understood. The results support the original hypothesis suggesting a role for GABA production in cellular pH regulation.

The regulation of oat coleoptile phosphoenolpyruvate carboxylase and malic enzyme by Hâ ½ and metabolites : kinetic evidence for and against a cytosolic pH-stat

Phosphoenolpyruvate carboxylase (PEPC) and malic enzyme activities in soluble protein extracts of Avena coleoptiles were investigated to determine whether their kinetics were consistent with a role in cytosol pH regulation. Malic enzyme activity was specific for NADP+ and Mn2+. Maximal labelled product formation from [14C]-substrates required the presence of all coenzymes, cofactors and substrates. Plots of rate versus malate concentration, and linear transformations there- 2 of, indicated typical Michaelis-Menten kinetics at non-saturating malate levels and substrate inhibition at higher malate levels. pH increases between 6.5 and 7.25 increased near-optimal activity, decreased the degree of substrate inhibition and the Kmapp(Mn2+) but did not affect the Vmax or Kmapp(malate). Transformed data of PEPC activity demonstrated non-linear plots indicative of non-Michaelian kinetics. pH increases between 7.0 and 7.6 increased the Vmax and decreased the Km app (Mg2+) but did not affect the Kmapp(PEP). Various carboxylic acids and phosphorylated sugars inhibited PEPC and malic enzyme activities, and these effects decreased with pH increases. Metabolite inhibited malic enzyme activity was non-competitive and resulted mainly from Mn2+ chelation. In contrast, metabolite inhibited PEPC activity was unique for each compound tested, being variously dependent on the PEP concentration and the pH employed. These results indicate that fluctuations in pH and metabolite levels affect PEPC and malic enzyme activities similarly and that 3 the in vitro properties of PEPC are consistent with its proposed role in a pH-stat, whereas the in vitro properties of the malic enzyme cannot be interpreted in terms of a role in pH regulation.

Modulation of ion channels by hydrogen sulfide

Increasing current awareness and understanding of the roles and mechanisms of action of ion channel regulation by H(2)S will open opportunities for therapeutic intervention with clear clinical benefits, and inform future therapies. In addition, more sensitive methods for detecting relevant physiological concentrations of H(2)S will allow for clarification of specific ion channel regulation with reference to physiological or pathophysiological settings.

Modulation of potassium ion channel proteins utilising antibodies

The application of antibodies to living cells has the potential to modulate the function of specific proteins by virtue of their high specificity. This specificity has proven effective in determining the involvement of many proteins in neuronal function where specific agonists and antagonists do not exist, e.g. ion channel subunits. We discuss a way to utilise subunit specific antibodies to target individual channel subunits in electrophysiological experiments to determine functional roles within native neurones. Utilising this approach, we have investigated the role of the voltage-gated potassium channel Kv3.1b subunit within a region of the brainstem important in the regulation of autonomic function. We provide some useful control experiments in order to help validate this method. We conclude that antibodies can be extremely valuable in determining the functions of specific proteins in living neurones in neuroscience research.

Oxygen-dependent hydroxylation by Factor Inhibiting HIF (FIH) regulates the TRPV3 ion channel

Factor Inhibiting HIF (FIH) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains 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 ankyrin repeat domain. 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 oxygendependent asparaginyl hydroxylation is likely to extend to other ion channels.

Helium ion microscopy visualizes lipid nanodomains in mammalian cells

Cell membranes are composed of two-dimensional bilayers of amphipathic lipids, which allow a lateral movement of the respective membrane components. These components are arranged in an inhomogeneous manner as transient micro- and nanodomains, which are believed to be crucially involved in the regulation of signal transduction pathways in mammalian cells. Because of their small size (diameter 10-200 nm), membrane nanodomains cannot be directly imaged using conventional light microscopy. Here, we present direct visualization of cell membrane nanodomains by helium ion microscopy (HIM). We show that HIM is capable to image biological specimens without any conductive coating, and that HIM images clearly allow the identification of nanodomains in the ultrastructure of membranes with 1.5 nm resolution. The shape of these nanodomains is preserved by fixation of the surrounding unsaturated fatty acids while saturated fatty acids inside the nanodomains are selectively removed. Atomic force microscopy, fluorescence microscopy, 3D structured illumination microscopy and direct stochastic optical reconstruction microscopy provide additional evidence that the structures in the HIM images of cell membranes originate from membrane nanodomains. The nanodomains observed by HIM have an average diameter of 20 nm and are densely arranged with a minimal nearest neighbor distance of ~15 nm.

Regulation of cell migration in cancer: investigation into the regulation of cancer cell blebbing in the extracellular matrix

Cell migration is a highly coordinated process and any aberration in the regulatory mechanisms could result in pathological conditions such as cancer. The ability of cancer cells to disseminate to distant sites within the body has made it difficult to treat. Cancer cells also exhibit plasticity that makes them able to interconvert from an elongated, mesenchymal morphology to an amoeboid blebbing form under different physiological conditions. Blebs are spherical membrane protrusions formed by actomyosin-mediated contractility of cortical actin resulting in increased hydrostatic pressure and subsequent detachment of the membrane from the cortex. Tumour cells use blebbing as an alternative mode of migration by squeezing through preexisting gaps in the ECM, and bleb formation is believed to be mediated by the Rho-ROCK signaling pathway. However, the involvement of transmembrane water and ion channels in cell blebbing has not been examined. In the present study, the role of the transmembrane water channels, aquaporins, transmembrane ion transporters and lipid signaling enzymes in the regulation of blebbing was investigated. Using 3D matrigel matrix as an in vitro model to mimic normal extracellular matrix, and a combination of confocal and time-lapse microscopy, it was found that AQP1 knockdown by siRNA ablated blebbing of HT1080 and ACHN cells, and overexpression of AQP1-GFP not only significantly increased bleb size with a corresponding decrease in bleb numbers, but also induced bleb formation in non-blebbing cell lines. Importantly, AQP1 overexpression reduces bleb lifespan due to faster bleb retraction. This novel finding of AQP1-facilitated bleb retraction requires the activity of the Na+/H+ pump as inhibition of the ion transporter, which was found localized to intracellular vesicles, blocked bleb retraction in both cell lines. This study also demonstrated that a differential regulation of cell blebbing by AQP isoforms exists as knockdown of AQP5 had no effect on bleb formation. Data from this study also demonstrates that the lipid signaling PLD2 signals through PA in the LPA-LPAR-Rho-ROCK axis to positively regulate bleb formation in both cell lines. Taken together, this work provides a novel role of AQP1 and Na+/H+ pump in regulation of cell blebbing, and this could be exploited in the development of new therapy to treat cancer.