Mecanismes d'alliberació d'ATP a travès de la membrana plasmàtica: paper de la CD39 i les connexines.

[cat] L'ATP és una molècula molt carregada elèctricament important per a l'obtenció de l'energia necessària per l'activitat cel·lular. A més a més, també actua com a molècula de senyalització cel·lular. Normalment, la secreció controlada d'ATP té lloc a través de l'exocitosi de grànuls o vesícules. Tot i això, en algunes cèl·lules, altres mecanismes poden controlar l'alliberació d'ATP. En aquesta tesi s'han estudiat dos d'aquests mecanismes: l'alliberació d'ATP a través de la proteïna CD39 i l'alliberació d'ATP a través de connexines.La CD39, o NTPDasa 1, és una proteïna que hidrolitza nucleòtids trifosfat i difosfat fins a nucleòtids monofosfat. Resultats anteriors del nostre laboratori suggerien que, aplicant polsos hiperpolaritzants en la membrana dels oòcits de Xenopus, l'ectoenzim NTPDasa1 podia esdevenir permeable per l'ATP. Continuant amb l'estudi d'aquesta proteïna, es va voler determinar també si estava relacionada amb l'alliberació d'ATP induïda per estrès hipertònic descrita en oòcits. Per portar a terme aquests estudis, era necessari aconseguir un oligonucleòtid antisentit contra la CD39 dels oòcits de Xenopus. Per tant, el nostre primer objectiu va ser sintetitzar i validar aquest oligonucleòtid, per tal de poder-lo utilitzar posteriorment a l'hora de fer els experiments. Mitjançant el disseny de dos oligonucleòtids antisentit per dita proteïna es va mesurar l'alliberació d'ATP en grups d'oòcits injectats amb els oligonucleòtids i en grups control, després de ser sotmesos a un xoc hiperosmòtic. Al mateix temps, per a comprovar l'efectivitat dels oligonucleòtids injectats, es va mesurar l'activitat ATPàsica en aquests oòcits. Es va observar com l'antisentit per la CD39 reduïa de manera significativa l'alliberació d'ATP i el corrent iònic després de 13 minuts d'exposició hipertònica.Els hemicanals, formats per connexines, és un altre dels possibles mecanismes de regulació de l'alliberació d'ATP. En les gap junctions, les connexines uneixen el citoplasma de dues cèl·lules adjacents establint un canal intercel·lular que permet el pas d'ions i molècules més petites d'1 KDa. Es va estudiar aquest mecanisme en oòcits de Xenopus, estudiant la Cx38, un tipus específic de connexina endògena dels oòcits, i la Cx32, expressada en els oòcits de manera heteròloga. En els experiments amb la Cx38, vam observar que solucions lliures d'ions divalents activaven un corrent d'entrada en els oòcits que era inhibit per octanol i àcid flufenàmic, dos inhibidors de les gap junctions. Aquest corrent sensible a calci, depenia de l'expressió de Cx38: disminuïa en oòcits injectats amb un oligonucleòtid antisentit per la Cx38 (ASCx38) i augmentava en oòcits que sobrexpressaven la Cx38. A més, l'activació dels hemicanals de Cx38 induïa l'alliberació d'ATP, la qual era inhibida per l'àcid flufenàmic, l'octanol i l'ASCx38 i augmentada en la sobrexpressió de Cx38. Aquests resultats suggerien que l'activació dels hemicanals era responsable de l'alliberació d'ATP en els oòcits de Xenopus.D'altra banda, vam investigar l'alliberació d'ATP a través dels hemicanals formats per Cx32 expressada en oòcits. Aquesta proteïna s'expressa en la majoria d'òrgans humans, però, particularment, mutacions de la Cx32 en les cèl·lules de Schwann produeixen la malaltia de Charcot-Marie-Tooth lligada al cromosoma X (CMTX). En oòcits que expressen la Cx32, vam combinar l'ús de tècniques de fixació de voltatge per registrar els corrents iònics generats pels hemicanals i la reacció bioluminiscent de la luciferina-luciferasa per mesurar l'alliberació d'ATP. Els hemicanals formats per la Cx32 eren estimulats mitjançant polsos despolaritzants generant un corrent iònic de sortida. Després de l'estimulació, en el període de repolarització, hi havia un corrent de cua que coincidia amb el moment d'alliberació d'ATP. L'àrea d'aquest corrent tenia una relació lineal amb la quantitat d'ATP alliberada. El conjunt de resultats d'aquesta tesi ens suggereixen que, tant les connexines com la CD39, participen en l'alliberació d'ATP a través de la membrana plasmàtica.[eng] ATP is an electrically charged molecule required to obtain the energy necessary for cellular activity; in addition, it is an intercellular signaling molecule. Usually, the controlled secretion of ATP occurs through the exocytosis of granules and vesicles. However, in some cells, other mechanisms control ATP release. Two of these mechanisms have been studied in this thesis: ATP release through CD39 and ATP release through connexin hemichannels. CD39, or NTPDasa 1, is a protein that hydrolyze nucleotide triphosphates and nucleotide diphosphates to nucleotide monophosfates. To study its relationship with ATP release we have synthetized an antisense oligonucleotide against Xenopus oocyte CD39. Our results indicate that the antisense against CD39 significanty reduce ATP release and also the ionic current due to the activation of CD39 by hypertonic solution. It has been suggested that hemichannels, formed by connexins, may be an alternative pathway for ATP release. In gap junctions, connexins link the cytoplasm of two adjacent cells by establishing an intercellular channel. We have investigated the release of ATP from Xenopus oocytes through hemichannels formed by connexin 38 (Cx38), an endogenous, specific type of connexin, and by connexin32 (Cx32) heterologously expressed in Xenopus oocytes. In Xenopus oocytes, calcium-free solution reversibly activates an inward current that is inhibited by octanol and flufenamic acid. This calcium-sensitive current depends on Cx38 expression: it's decreased in oocytes injected with an antisense oligonucleotide against Cx38 (ASCx38) and is increased in oocytes overexpressing Cx38. Moreover, the activation of Cx38 also induce the release of ATP, which is inhibited by flufenamic acid, octanol and ASCx38 and increased by Cx38 overexpression. Connexin 32 is expressed in most of human organs but, particularly, Cx32 mutations present in Schwann cells produce X-linked Charcot-Marie-Tooth disease. In oocytes expressing Cx32, application of depolarizing pulses to positive potentials induce outward hemichannel currents that become inward during the repolarization phase. The release of ATP occurred during the repolarization period and the amount of ATP released correlate with the area of the tail current

Morphine- and CaMKII-dependent enhancement of GIRK channel signaling in hippocampal neurons

G-protein-gated inwardly rectifying potassium (GIRK) channels, which help control neuronal excitability, are important for the response to drugs of abuse. Here, we describe a novel pathway for morphine-dependent enhancement of GIRK channel signaling in hippocampal neurons. Morphine treatment for ∼20 h increased the colocalization of GIRK2 with PSD95, a dendritic spine marker. Western blot analysis and quantitative immunoelectron microscopy revealed an increase in GIRK2 protein and targeting to dendritic spines. In vivo administration of morphine also produced an upregulation of GIRK2 protein in the hippocampus. The mechanism engaged by morphine required elevated intracellular Ca(2+) and was insensitive to pertussis toxin, implicating opioid receptors that may couple to Gq G-proteins. Met-enkephalin, but not the μ-selective (DAMGO) and δ-selective (DPDPE) opioid receptor agonists, mimicked the effect of morphine, suggesting involvement of a heterodimeric opioid receptor complex. Peptide (KN-93) inhibition of CaMKII prevented the morphine-dependent change in GIRK localization, whereas expression of a constitutively activated form of CaMKII mimicked the effects of morphine. Coincident with an increase in GIRK2 surface expression, functional analyses revealed that morphine treatment increased the size of serotonin-activated GIRK currents and Ba(2+)-sensitive basal K(+) currents in neurons. These results demonstrate plasticity in neuronal GIRK signaling that may contribute to the abusive effects of morphine

Release of ATP induced by hypertonic solutions in Xenopus oocytes

ATP mediates intercellular communication. Mechanical stress and changes in cell volume induce ATP release from various cell types, both secretory and non-secretory. In the present study, we stressed Xenopus oocytes with a hypertonic solution enriched in mannitol (300 mm). We measured simultaneously ATP release and ionic currents from a single oocyte. A decrease in cell volume, the activation of an inward current and ATP release were coincident. We found two components of ATP release: the first was associated with granule or vesicle exocytosis, because it was inhibited by tetanus neurotoxin, and the second was related to the inward current. A single exponential described the correlation between ATP release and the hypertonic-activated current. Gadolinium ions, which block mechanically activated ionic channels, inhibited the ATP release and the inward current but did not affect the decrease in volume. Oocytes expressing CFTR (cystic fibrosis transmembrane regulator) released ATP under hypertonic shock, but ATP release was significantly inhibited in the first component: that related to granule exocytosis. Since the ATP measured is the balance between ATP release and ATP degradation by ecto-enzymes, we measured the nucleoside triphosphate diphosphohydrolase (NTPDase) activity of the oocyte surface during osmotic stress, as the calcium-dependent hydrolysis of ATP, which was inhibited by more than 50 % in hypertonic conditions. The best-characterized membrane protein showing NTPDase activity is CD39. Oocytes injected with an antisense oligonucleotide complementary to CD39 mRNA released less ATP and showed a lower amplitude in the inward current than those oocytes injected with water

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability

Ras-association domain of sorting nexin 27 is critical for regulating expression of GIRK potassium channels

G protein-gated inwardly rectifying potassium (GIRK) channels play an important role in regulating neuronal excitability. Sorting nexin 27b (SNX27b), which reduces surface expression of GIRK channels through a PDZ domain interaction, contains a putative Ras-association (RA) domain with unknown function. Deleting the RA domain in SNX27b (SNX27b-DRA) prevents the down-regulation of GIRK2c/GIRK3 channels. Similarly, a point mutation (K305A) in the RA domain disrupts regulation of GIRK2c/GIRK3 channels and reduces H-Ras binding in vitro. Finally, the dominant-negative H-Ras (S17N) occludes the SNX27b-dependent decrease in surface expression of GIRK2c/GIRK3 channels. Thus, the presence of a functional RA domain and the interaction with Ras-like G proteins comprise a novel mechanism for modulating SNX27b control of GIRK channel surface expression and cellular excitability

Deletion of RA domain in SNX27b impairs functional regulation of GIRK channels.

<p><b>A</b>, Cartoon shows model of GIRK channels regulation by SNX27b. GIRK channels recycle through endosomal compartments. SNX27b associating with GIRK2c/3 channels in the early endosome (EE) reduces plasma membrane expression (PM) by targeting some channels to the late endosome (LE). <b>B</b>, SNX27 contains three functional domains; PDZ, PX and RA. GIRK2c and GIRK3 contain a C-terminal PDZ binding motif (-E(S/N)ESKV). <b>C,</b> Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293T cells transfected with cDNA for GABA_B1a/B2 receptors, GIRK2c/GIRK3 and either control vector, SNX27b or SNX27b-ΔRA. Agonist-independent basal currents are revealed by inhibition with 1 mM Ba²⁺. <b>D</b>, Average baclofen-induced current densities (I_Baclofen) for control (–41.3±5.2 pA⋅pF⁻¹, n = 8), SNX27b (–11.0±3.6 pA⋅pF⁻¹, n = 8) and SNX27b- ΔRA (–55.9±8.2 pA⋅pF⁻¹, n = 13) with GIRK2c/GIRK3. <b>E</b>, Average I_Baclofen for control (–15.7±3.6 pA⋅pF⁻¹, n = 6) and SNX27b-ΔRA (–18.6±4.9 pA⋅pF⁻¹, n = 5) with GIRK1/GIRK3 (**P<0.05, one way ANOVA followed by Bonferroni <i>post hoc</i> test; n.s. – not significant).</p

Dominant-negative H-Ras (H-Ras_S17NDN) prevents SNX27b-dependent down-regulation of GIRK channels.

<p><b>A</b>, Schematic shows GIRK2c and GIRK3 with PDZ-binding motif and GIRK2a and GIRK3-RR, which lack motifs that interact with SNX27-PDZ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Balana1" target="_blank">[7]</a>. <b>B</b>, Examples of baclofen-induced (100 µM) and Ba²⁺-sensitive (1 mM Ba²⁺) currents in HEK293 cells transfected with cDNA for GABA_B1a/B2, GIRK2c/GIRK3 and either empty vector (Control), H-Ras_S17NDN or H-Ras_S17NDN plus SNX27b. <b>C</b>, Bar graph shows average I_Baclofen for GIRK2c/GIRK3 alone (–47.7±8.5 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–22.0±3.8 pA⋅pF⁻¹, n = 19) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–23.7±4.7 pA⋅pF⁻¹, n = 16). <b>D</b>, Bar graph shows I_Baclofen for control (GIRK2a/GIRK3-RR alone) (–18.8±3.0 pA⋅pF⁻¹, n = 10) and GIRK2a/GIRK3-RR plus H-Ras_S17NDN (–18.6±2.0 pA⋅pF⁻¹, n = 11). <b>E</b>, Bar graph shows average I_Barium for GIRK2c/GIRK3 alone (–27.6±9.8 pA⋅pF⁻¹, n = 6), GIRK2c/GIRK3 and H-Ras_S17NDN (–7.3±2.2 pA⋅pF⁻¹, n = 18) or GIRK2c/GIRK3, SNX27b and H-Ras_S17NDN (–7.1±1.5 pA⋅pF⁻¹, n = 14). <b>F</b>, Bar graph shows average I_Barium for GIRK2a/GIRK3-RR alone (−2.49±0.9 pA⋅pF⁻¹, n = 10), GIRK2c/GIRK3 and H-Ras_S17NDN (−1.84±0.4 pA⋅pF⁻¹, n = 11). **P<0.05, one way ANOVA followed by Bonferroni post hoc test; n.s. – not significant.</p

Deletion of RA domain in SNX27b affects localization of GIRK2c/3 channels monitored with BiFC.

<p><b>A</b>, <i>Left</i>, Schematic shows placement of split YFP on GIRK2c and GIRK3. Note the C-terminal domains are free to interact with other proteins. <i>Right</i>, BiFC-tagged GIRK2c/3 channels are functional. Current-voltage plot is shown for ^CYGIRK2c/^NYGIRK3 channels. Baclofen (100 µM) activates and Ba²⁺ (1 mM) inhibits inwardly rectifying current. HEK293T cells were transfected with cDNA encoding GABA_B1a, GABA_B2 and ^CYGIRK2c/^NYGIRK3. Average baclofen-induced current densities were –13.2±6.0 pA⋅pF⁻¹ (n = 3) for ^CYGIRK2c/^NYGIRK3. <b>B</b>, HEK293 cells were co-transfected with ^CYGIRK2c, ^NYGIRK3 and either control cDNA (<i>i</i>), wild-type SNX27b (<i>ii</i>), SNX27b-ΔRA (deletion of Asp272-Trp358) (<i>iii</i>) or SNX27b-Y51L (a PDZ mutation) (<i>iv</i>). Green fluorescence in images represents molecular recombination of ^CYGIRK2c/^NYGIRK3 heterotetramers. Coexpression of wild-type SNX27b induced formation of puncta. By contrast, ^CYGIRK2c/^NYGIRK3 fluorescence was diffuse in the cytoplasm for SNX27b-ΔRA and for SNX27b-Y51L, similar to control. Inset shows zoom of boxed area. <b>C,</b> SNX27b-ΔRA exhibited a pattern of punctate expression similar that of wild-type SNX27b. YFP was fused to the C-terminus of SNX27b or SNX27b-ΔRA to directly visualize expression. HEK293T cells were transfected with cDNA for SNX27b-YFP and SNX27bΔRA-YFP. Scale bar: 10 µm.</p

K305A point mutation in SNX27b RA domain disrupts functional regulation of GIRK2c/GIRK3 channels.

<p><b>A</b>, Alignment of two different RA domains: a RID (Ras interacting domain) from RalGDS and a RA domain of RGL, with RA domain of SNX27. Residues implicated in Ras binding in Raf and RalGDS domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Block1" target="_blank">[28]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Vetter1" target="_blank">[30]</a> are highlighted in red. <b>B</b>, High-resolution structure shows R20, K32 and K52 at the binding interface of RID and Ras <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059800#pone.0059800-Huang1" target="_blank">[26]</a>. <b>C,</b> RA domain mutations impair the ability of SNX27b to induce formation of GIRK2c/3 puncta using BiFC. HEK293T cells were co-transfected with cDNA for ^CYGIRK2c/^NYGIRK3 and either wild-type SNX27b (<b>i</b>), SNX27b-R276A (<b>ii</b>), SNX27b-R288A/K291A (<b>iii</b>) or SNX27b-K305A (<b>iv</b>). Green fluorescence indicates molecular complementation of ^CYGIRK2c/^NYGIRK3. Scale bar: 10 µm. <b>D</b>, Colocalization of wild-type SNX27b (SNX27b-YFP; green) and SNX27b-K305A (anti-SNX27; green) with an early endosomal marker (anti-EEA1, green). Scale bar: 5 µm. <b>E</b>, Average I_Baclofen currents for control (–53.4±9.4 pA⋅pF⁻¹, n = 5), SNX27b (–8.05±3.44 pA⋅pF⁻¹, n = 6) and SNX27b-K305A (–52.9±8.8 pA⋅pF⁻¹, n = 10) with GIRK2c/GIRK3 channels.</p