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Gradogna, Antonella (2012) Biophysical analysis of the modulation of CLC-K channels by extracellular ligands. [Tesi di dottorato]

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Abstract (inglese)

CLC proteins form a family of voltage-gated homodimeric Cl- transporters comprising both Cl- channels (ClC-0, ClC-1, ClC-2, ClC-Ka, ClC-Kb) and Cl-/H+-exchangers (ClC-3 to ClC-7) (Accardi & Miller, 2004; Picollo & Pusch, 2005; Scheel et al, 2005). They are involved in important physiological processes, such as transepithelial transport, membrane excitability, cell volume regulation, and luminal acidification of the endosomal-lysosomal system (Zifarelli & Pusch, 2007). CLC proteins are found in all phyla; in particular in humans there are nine CLC genes. The role of these proteins is highlighted by the diseases associated with mutations in CLC protein encoding genes (myotonia, Dent’s disease, Bartter’s syndrome, osteopetrosis) (Zifarelli & Pusch, 2007; Jentsch, 2008). The human Cl- channels ClC-Ka and ClC-Kb, as their murine orthologues ClC-K1 and ClC-K2, are expressed in the kidney and in the inner ear where they are involved in NaCl reabsorption (Kieferle et al, 1994) and in the endolymph production (Estévez et al, 2001), respectively. CLC-K channels are associated with a small β-subunit called barttin (Birkenhäger et al, 2001; Estévez et al, 2001). Mutations in ClC-Kb and barttin cause Bartter’s syndrome, a kidney disease characterized by severe salt wasting and hypokalemia (Simon et al, 1997; Birkenhäger et al, 2001).
Because of their important physiological function, CLC-K channels are a potential pharmacological target. Among several chemical compounds that modulate CLC-K channels, niflumic acid (NFA), a known Cl- channel inhibitor, aroused our interest because of its unexpected effect on CLC-Ks. NFA blocks ClC-K1(Liantonio et al, 2004; Picollo et al, 2007), but surprisingly it increases ClC-Kb currents, and modulates ClC-Ka in a biphasic manner (Liantonio et al, 2006). To identify the residues responsible for the potentiating effect of NFA on ClC-Ka, we performed an extensive site-directed mutagenesis of ClC-Ka and voltage clamp measurements of the mutants expressed in Xenopus oocytes. The result of this work was the identification of three residues (L155, G345, and A349) which are involved in the potentiation by NFA (Zifarelli et al, 2010).
Extracellular calcium and protons modulate CLC-K channels in the physiological concentration range. In particular, the channels are activated by extracellular calcium and blocked by extracellular protons (Gradogna et al, 2010). Since the kidney has an important role in calcium reabsorption and in the maintenance of acid-base balance (Frick & Bushinsky, 2003; Jeck et al, 2005), calcium and proton regulation of CLC-K channels is potentially of physiological relevance. We performed a detailed biophysical analysis of Ca2+ and proton modulation of human CLC-K channels expressed in Xenopus oocytes. Experimental data showed an allosteric modulation of CLC-Ka for both, Ca2+ and protons. Modeling predicted a two state (blocked/unblocked) mechanism for proton modulation, while a four-state mechanism could properly describe Ca2+ modulation. An extensive mutagenic screen of ClC-Ka combined with voltage clamp measurements allowed us to identify two mutations, E261Q and D278N, which reduced calcium sensitivity of ClC-Ka. The proximity of the residues, E261 and D278, from different subunits suggested that they likely form an intersubunit Ca2+ binding site. In fact the double mutant E261D/D278N was completely Ca2+-insensitive. Moreover we identified a histidine, H497 responsible for the block of CLC-K channels at acidic pH (Gradogna & Pusch, 2010). We next decided to study the calcium effect on the other CLC-K channels. Human ClC-Kb displays a similar behaviour as ClC-Ka, while rat ClC-K1 is more calcium sensitive than the human CLC-Ks. The high sequence identity and the conserved calcium sensitivity suggested that the calcium binding site is a characteristic shared by all CLC-K channels. The double mutant E261Q/D278N of ClC-Kb and ClC-K1 completely abolished the calcium sensitivity supporting this hypothesis. To investigate the specificity of the Ca2+ binding site I studied the effect of various divalent cations (Zn2+, Mg2+, Ba2+, Sr2+, Mn2+) on ClC-Ka, ClC-K1, and ClC-Kb. Both WT ClC-Ka and the double mutant E261Q/D278N, were blocked by 5 mM Zn2+ suggesting that Zn2+ affects the channel interacting with a separate binding site. Mg2+ did not activate CLC-K channels at concentrations up to 50 mM. In contrast, CLC-Ka and ClC-Kb were activated by Ba2+, Sr2+, and Mn2+ with a rank order of potency Ca2+>Ba2+>Sr2+. Interestingly, ClC-K1 showed an altered rank order Ca2+> Sr2+>> Ba2+. Furthermore, the Ca2+ insensitive double mutant, E261/D278, was also insensitive to Ba2+ and Sr2+ (Gradogna et al, 2010; Gradogna & Pusch, 2010) demonstrating the specificity of the mechanism of activation of CLC-K channels by Ca2+ and the other divalent cations.
Finally, in collaboration with the group of Prof. Ildikò Szabò from the University of Padua, we tested the functional expression of the putative channel sll0993 from the cyanobacterium Synechocystis sp. PCC6803 (Kaneko et al, 1996). The coding sequence of the protein and that of a C-terminal EGFP-fusion protein were inserted in vectors optimized for the expression in Xenopus oocytes and tested for functional expression in oocytes. Oocytes injected with EGFP-fusion protein yielded a significant level of fluorescence demonstrating that the protein was produced. Unfortunately, for neither construct, we could detect any currents above those seen in non-injected oocytes using the voltage clamp technique.
In summary, we have provided an extensive biophysical and molecular characterization of the mechanisms of regulation of CLC-K chloride channels by extracellular ligands. All experiments have been executed at the Institute of Biophysics of the Italian National Research Council in Genoa, Italy in the group of Dr. Michael Pusch

Abstract (italiano)

Le proteine CLC sono una famiglia di trasportatori del cloruro voltaggio dipendenti, omodimerici comprendenti sia canali del cloruro (ClC-0, ClC-1, ClC-2, ClC-Ka, ClC-Kb) che Cl-/H+scambiatori (da ClC-3 a ClC-7) (Accardi & Miller, 2004; Picollo & Pusch, 2005; Scheel et al, 2005). Essi sono implicati in importanti processi fisiologici come il trasporto epiteliale, l’eccitabilità di membrana, la regolazione del volume cellulare e l’acidificazione del lume del sistema endosomale-lisosomiale (Zifarelli & Pusch, 2007). Le proteine CLC sono espresse in tutti i phyla, in particolare nell’uomo ci sono nove geni che codificano proteine CLC. Il ruolo di queste proteine è messo in luce dalle malattie associate con mutazioni in corrispondenza dei geni codificanti proteine CLC (miotonia, malattia di Dent, sindrome di Bartter, osteopetrosi) (Zifarelli & Pusch, 2007; Jentsch, 2008). I canali del cloruro ClC-Ka e ClC-Kb, come i loro ortologhi murini ClC-K1 e ClC-K2, sono espressi nel rene e nell’orecchio interno dove essi sono rispettivamente coinvolti nel riassorbimento di NaCl (Kieferle et al, 1994) e nella produzione di endolinfa (Estévez et al, 2001). I canali CLC-K sono associati con una piccola subunità β chiamata barttin (Birkenhäger et al, 2001; Estévez et al, 2001). Mutazioni in ClC-Kb e barttin determinano la sindrome di Bartter, una malattia caratterizzata da perdita di sali ed ipokalemia (Simon et al, 1997; Birkenhäger et al, 2001). A causa della loro importante funzione fisiologica, i canali CLC-K rappresentano un potenziale bersaglio farmacologico. Tra i molti composti chimici che modulano i CLC-K, l’acido niflumico, un noto inibitore dei canali del cloruro, destava il nostro interesse a causa del suo inaspettato effetto sui CLC-K. NFA blocca ClC-K1(Liantonio et al, 2004; Picollo et al, 2007), ma sorprendentemente esso attiva le correnti di CLC-Kb e modula in modo bifasico CLC-Ka (Liantonio et al, 2006). Per identificare i residui aminoacidici responsabili dell’effetto attivatorio di NFA su ClC-Ka, noi eseguivamo una estensiva mutagenesi sito specifica di ClC-Ka e misure di voltage clamp dei mutanti espressi in oociti di Xenopus. Il risultato di questo lavoro era l’identificazione di tre residui (L155, G345, and A349) che sono coinvolti nel potenziamento attraverso NFA (Zifarelli et al, 2010).
Calcio extracellulare e protoni modulano i canali CLC-K in un intervallo di concentrazione fisiologica. In particolare i canali sono attivati da aumento di calcio extracellulare e bloccati da pH acido extracellulare (Gradogna et al, 2010). Poichè il rene ha un ruolo importante nel riassorbimento di calcio e nel mantenimento dell’equilibrio acido-base (Frick & Bushinsky, 2003; Jeck et al, 2005), la regolazione da parte del calcio e dei protoni dei canali CLC-K è potenzialmente di rilevanza fisiologica. Noi eseguivamo una dettagliata analisi biofisica della modulazione da parte del Ca2+ e dei protoni dei canali CLC-K dell’uomo espressi in oociti di Xenopus. I dati sperimentali mostravano che sia Ca2+ che protoni modulano CLC-Ka in modo allosterico. Studi di modellistica indicavano un meccanismo a due stati (bloccato/non bloccato) per la modulazione dei protoni, mentre un modello comprendente quattro stati poteva descrivere in modo appropriato la modulazione da parte del calcio. Una estensiva analisi mutagenica combinata con misure di voltage clamp ci consentiva di identificare due mutazioni E261Q e D278N che riducevano la sensibilità al calcio di ClC-Ka. La vicinanza dei residui E261 e D278 appartenenti a differenti subunità suggeriva che probabilmente essi formano un sito di legame per il calcio intersubunità. Infatti il doppio mutante E261Q/D278N era completamente insensibile al calcio. Inoltre noi identificavamo una istidina, H497 responsabile per il blocco dei canali CLC-K causato dal pH acido (Gradogna & Pusch, 2010). Quindi decidevamo di studiare l’effetto del calcio sugli altri CLC-K. ClC-Kb presenta un comportamento simile a ClC-Ka, mentre ClC-K1 è più sensibile al calcio dei canali CLC-K dell’uomo. L’alta identità di sequenza e il fatto che la sensibilità al calcio è conservata tra i CLC-K suggeriva che il sito di legame per il calcio è una caratteristica condivisa da tutti i CLC-K. Il doppio mutante E261Q/D278N di ClC-Kb e ClC-K1 perdevano completamente la sensibilità al calcio confermando questa ipotesi. Per determinare la specificità del sito di legame del Ca2+ studiavo l’effetto di vari cationi divalenti (Zn2+, Mg2+, Ba2+, Sr2+, Mn2+) su ClC-Ka, ClC-K1e ClC-Kb. Sia ClC-Ka WT che il doppio mutante E261Q/D278N erano bloccati da 5 mM Zn2+ suggerendo che lo ione Zn2+ ha effetto sul canale interagendo con un sito di legame distinto. Lo ione Mg2+ non attivava i canali CLC-K a concentrazioni fino a 50 mM. Invece ClC-Ka e ClC-Kb erano attivati da Ba2+, Sr2+e Mn2+ con un ordine di potenziamento: Ca2+>Ba2+>Sr2+. E’ interessante osservare che ClC-K1 presentava un ordine di potenziamento modificato: Ca2+> Sr2+>> Ba2+. Inoltre il doppio mutante E261Q/D278N, insensibile al Ca2+, era anche insensibile al Ba2+ and Sr2+ (Gradogna et al, 2010; Gradogna & Pusch, 2010) dimostrando la specificità del meccanismo di attivazione dei canali CLC-K attraverso lo ione calcio e gli altri cationi divalenti.
Infine, in collaborazione con il gruppo della Prof. Ildikò Szabò dell’Università di Padova, noi provavamo l’espressione funzionale del canale putativo sll0993 del cianobatterio Synechocystis sp. PCC6803 (Kaneko et al, 1996). La sequenza codificante della proteina e quella della proteina con EGFP al C-terminale erano inserite in vettori ottimizzati per l’espressione in oociti di Xenopus e testati per quanto riguarda l’espressione funzionale. Oociti iniettati con la proteina fusa con EGFP presentavano un consistente livello di fluorescenza dimostrando che la proteina era prodotta. Sfortunatamente, per nessuno dei due costrutti, con la tecnica del voltage clamp, potevamo rilevare correnti superiori rispetto a quelle viste in oociti non iniettati.
Riassumendo, noi abbiamo effettuato una dettagliata caratterizzazione biofisica e molecolare dei meccanismi di regolazione dei canali del cloruro CLC-K attraverso ligandi extracellulari. Tutti gli esperimenti sono stati eseguiti presso l’Istituto di Biofisica del Consiglio Nazionale delle Ricerche a Genova (Italia) nel gruppo del Dott. Michael Pusch

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Tipo di EPrint:Tesi di dottorato
Relatore:Szabò, Ildikò
Correlatore:Pusch, Michael
Dottorato (corsi e scuole):Ciclo 24 > Scuole 24 > BIOSCIENZE E BIOTECNOLOGIE > BIOCHIMICA E BIOFISICA
Data di deposito della tesi:27 Gennaio 2012
Anno di Pubblicazione:27 Gennaio 2012
Parole chiave (italiano / inglese):canali CLC-K/CLC-K channels, sindrome di Bartter/Bartter's syndrome, acido niflumico/niflumic acid, calcio/calcium
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/09 Fisiologia
Struttura di riferimento:Dipartimenti > Dipartimento di Biologia
Codice ID:4658
Depositato il:26 Ott 2012 15:44
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