Dissertations / Theses on the topic 'Potassium channels'

This study uses the structural coordinates of the determined K+ channels to create comparative models of three diverse members of this family, with the aim of enabling a better understanding of the function of these channels. The K+ channel of primary interest is the hERG K+ channel. The pharmacology of this channel is of considerable interest as serendipitous block of K+ conduction pore may result in cardiac arrest. A set of known antagonists have been docked into novel comparative models of hERG to propose how these drugs interact with the channel. The models have also been subjected to molecular dynamics simulations to investigate the drug binding in more detail and to gain a structural understanding of two critical biophysical properties of this channel: activation and inactivation. Additionally, ancillary domains of the channel have been modelled to provide a tool for interpreting detailed structure-function relationships for the hERG channel. The second channel investigated is the TASK-1 channel. Comparative models of this channel have been created to evaluate mutations that alter selectivity and pH sensitivity. The final K+ channel studied is the Kir2.1 channel. A fundamental property of this channel is its block by polyamines, which prevents the efflux of K+. Comparative models have been created, with a series of polyamine analogues docked into the membrane and cytoplasmic pore regions of this channel. Overall, this study has illuminated the structural basis of several biophysical properties that are intrinsic to normal K+ channel function.

The pancreatic ATP-sensitive potassium (KATP) channels couple glucose metabolism to excitability of the pancreatic β-cells to regulate insulin secretion. The channel subunits, Kir6.2 and SUR1, are encoded by the KCNJ11 and ABCC8 genes respectively. Genetic polymorphisms in these genes, which reduce channel activity, cause congenital hyperinsulinism (CHI) characterized by insulin hyper-secretion and hypoglycemia. The hERG (human ether-a-go-go related gene) potassium channels,encoded by the KCNH2 gene, contribute to the rapidly activating delayed rectifier K+ current (IKr), which is responsible for rapid repolarisation of the cardiac action potential. Decreased hERG channel function causes the Long QT syndrome 2 (LQTS2) and life threatening cardiac arrhythmias. Several mutations in these two clinically important potassium ion channels alter their surface density leading to disease. Therefore, it is of fundamental importance to investigate the trafficking mechanisms that regulate the surface density of these channels. Techniques in cell biology, molecular biology and biochemistry were employed to identify the molecular basis of Sar1-GTPase dependent ER exit of the KATP and hERG channels in COPII vesicles. Blocking the cargo binding sites on the Sec24 protein of the COPII coat with membrane-permeable synthetic peptides prevented ER exit of both these channels. While the diacidic 280DLE282 sequence on the Kir6.2 subunit of KATP channels was found to be the ER exit motif required for entry of the channels into COPII vesicles at the ER exit sites, such a motif was found to be absent on hERG Cterminus. Further, endocytic trafficking mechanism of hERG channels was studied in recombinant (HEK MSRII and HeLa) and native (neonatal rat cardiac myocytes) systems using cell biological and pharmacological tools. hERG channels were found to be internalised by a dynamin-independent, raft-mediated, and ARF6-dependent pathway. A prolonged block of this pathway revealed that the channels could also undergo internalisation by an alternate dynamin-mediated pathway. Internalised hERG channels were found to recycle back to the cell surface and undergo lysosomal degradation. Degradation of the channels was enhanced when Rab11a-GTPase function was disrupted leading to reduced surface density indicating that recycling is crucial to maintain cell surface density of the channels. Thus this study investigated and compared the previously unknown mechanisms of biosynthetic and endosomal trafficking of the KATP and hERG potassium channels with a conclusion that these processes play an important role in maintaining surface density and thereby in the function of these channels in physiological and patho-physiological conditions.

Potassium channels control the conduction of K+ across cell membranes, down their electrochemical gradient. This rapid and highly selective movement of K+ is essential to many biological processes. K+ channels are largely alpha helical, tetrameric proteins that span the lipid bilayer. Diversity in K+ channels arises primarily in the mechanism of gating by various ligands or voltage; however, the basic structural elements, notably the selectivity filter are conserved within the family. Studies ~n this thesis focus on the single-channel behaviour of the K+ channels KcsA and Kcv. KcsA is a proton-activated channel from the bacterium Streptomyces lividans and its crystal structure was the first of a K+ channel to be solved. Kcv expressed by the Paramecium bursaria Chlorella virus is the smallest known K+ channel and thus represents the minimal structural entity necessary to form a functional and selective pore. Studies on the bacterial inward rectifying channels, KirBacs, have also been initiated. The KirBacs are a superfamily of prokaryotic channels homologous to eukaryotic Kir channels. In this work, KcsA and Kcv were found to form stable tetramers, which can be expressed by coupled in vitro transcription and translation and purified by polyacrylamide gel electrophoresis. The purified tetramers were reconstituted into planar lipid bilayers and studied at the single-channel level. Through single-channel recordings the ionic selectivity, gating behaviour, functional effects of site-directed mutagenesis and the interaction between Kcv and blockers have been studied. In addition, single-channel studies at elevated temperatures have revealed the remarkable thermostability of Kcv, as well as insight into the transport of ions through a narrow and selective pore. Through temperature studies, it has been possible to obtain the thermodynamic and kinetic parameters describing Kcv activity.

Limbic encephalitis (LE) is a central nervous system disorder that is characterised by memory impairments, confusion, agitation and seizures, and associated with hyperintense lesions of the medial temporal lobe, seen with MRI. Anti-voltage-gated potassium channel (VOKC) antibodies have been been detected in the plasma of a subset of LE patients using a 125I-a-dendrotoxin (a-DTX) radioimmunoprecipitation assay, suggesting that the likely antigens are VOKC Kv1.l, 1.2 and 1.6 subunits. Symptoms of the disease improve markedly with immunosuppression, correlating with similarly dramatic falls in the titre of anti-VOKC antibodies, thus implicating anti-VOKC antibodies in the pathogenesis of LE. Circumstantial evidence from studies of inherited channelopathies and animal models of reduced VOKC activity suggests that VOKC dysfunction may contribute to the pathogenesis of LE. This thesis addresses whether anti-VOKC antibodies (i) bind to a-DTX-sensitive subunits and (ii) affect VOKC function. Immunofluorescence data show that binding of LE patient IgO to the surface of primary neurons and Kvl-expressing HEK-293/HEKTSA cells could not be detected with indirect immunofluorescence. Comparison of intracellular labelling with patient and control IgO showed that no additional labelling could be detected with LE patient IgO. Electrophysiological data show first, that a-DTXsensitive currents could not be reliably isolated from primary cultured hippocampal neurons; second, that NMT or LE samples did not affect VOKCs expressed by neuroblastoma-l cells; third, that none of the LE samples affected potassium currents in Kvl-transfected HEK-293 cells. These data suggest that 'anti-VOKC' antibodies may not bind directly to Kv1.111.2/1.6 homomers, or to a range of Kv1.I/1.2/1.6 subunit-containing heteromers in transfected cells. The findings instead suggest that 'anti-VOKC' antibodies in LE patient plasma may bind to a Kvl-associated protein that contributes to a-DTX-sensitive complexes in the radioimmunoprecipitation assay, but is absent from Kv1.111.2/1.6-transfected cells. Future work to characterise whether another antigen is bound by LE patient anti-VOKC antibodies will be important in determining how these antibodies contribute to the pathogenesis of LE.

ATP-sensitive potassium (KATp) channels are known to play a vital role in the regulation of insulin secretion from pancreatic 0-cells. Changes in the ratio of [ATP]/[ADP] within the cell are known to regulate the activity of channels, but very little is known how the number of channels at the cell surface is regulated. The number of channels in the plasma membrane could be regulated in two ways; firstly by regulating the overall population of channels within the cells by increasing/decreasing the rates of channel synthesis or degradation, and secondly by regulating the insertion and removal of channels from the plasma membrane. The aim of the current study is to investigate the involvement of both of these mechanisms in regulating the cell surface density of KATp channels. It is shown that a sudden decrease in glucose concentration causes a rapid stimulation of KATp channel synthesis as shown by both immunocytochemistry and protein chemistry in both INS-le and isolated mouse pancreatic ß-cells. The intensity of fluorescence associated with Kir6.2 and SUR1 was - 2.5 fold greater in cells incubated with 3 mM compared to 25 mM glucose. This sudden increase in channel numbers is due to an increase in the rate of translation of pre-existing mRNA and may be mediated by the activation of AMP-activated protein kinase. Despite the - 2.5 fold increase in channel numbers only a small, but non significant, difference in cell surface density was observed as determined by patch-clamp. The internalisation of KATp channels with an extracellular HA-epitope was also investigated in stably transfected HEK293 cells. Channels were seen to internalise rapidly from the cell surface into a perinuclear compartment. The trafficking itinerary of these channels has been found to include the sorting endosome, late endosome and elements of the trans-Golgi network. Upon inhibition of protein kinase-C activity the internalised channels are redirected into a pathway which allows rapid recycling of the channels. Trafficking and function of KATP channels has also been shown to be disrupted by mutations of Kir6.2 known to cause congenital hyperinsulinism. In summary, it has been demonstrated that both regulated expression and trafficking are likely to be involved in determining the cell surface density of pancreatic KATP channels.

Regulation of inwardly-rectifying potassium (Kir) channels by intracellular ligands couples cell membrane excitability to important signaling cascades and metabolic pathways. Particularly, the ATP-sensitive potassium (KATP) channels, highly expressed in diverse excitable tissues, are the keystone components of glucose-stimulated insulin secretion in pancreatic β-cells. KATP channels are constituted by a canonical pore-forming transmembrane domain (TMD) and a large cytoplasmic domain (CTD) where ATP binds and inhibits the channel activity. A non-covalent interface domain in Kir channel structures is speculated as a mediator of coupling between the cytoplasmic (‘ligand-sensing’) and transmembrane (‘gating’) domains. The work described in this thesis aims to investigate the molecular mechanism that link ligand binding to the channel gating. A significant barrier is that many functionally important channel motifs are highly sensitive to mutagenesis, resulting in a loss-of-function (LOF) phenotype. We have developed a novel ‘forced gating’ approach by substituting a glutamate in the hydrophobic Kir channel bundle crossing (F168E), generating channels that open upon alkalization, likely due to mutual repulsion of the introduced glutamate side chain. This ‘forced gating’ approach is implemented in mutagenic scans of the Kir channel domain interface comprising numerous motifs, including the transverse ‘slide’ helix, the C-linker, the βC-βD loop, and the G-loop. Without exception, expression on the ‘forced gating’ background rescued all loss-of-function interfacial mutants in alkaline pH, and enabled functional characterization of all interfacial mutants on generation of conductive channels and ATP-dependent gating. Our findings highlight a small subset of ‘anchor residues’ at the Kir6.2 TMD-CTD interface, including D58 and T61 in the ‘slide helix’, R177 in the C-linker, T294 in the G-loop, and D204 in the βC-βD loop, which are required for both formation of conductive channels, and appropriate transmission of ligand binding to the channel gating. Disruption of these residues uncouples the TMD and CTD, causing loss-of-function phenotype combined with profound ligand insensitivity. Additionally, in contrast to a traditional ‘allosteric rescue’ approach (Kir6.2[C166S]) with an elevated intrinsic channel open probability, the ‘forced gating’ approach has a more general application due to its efficiency in rescuing all loss-of-function interfacial mutants without inherently perturbing ATP sensitivity in Kir6.2 channel.

The patch clamp technique was used to investigate the effect of mutations in the P-region on permeation characteristics and ionic selectivity in members of the Kir2.0 subfamily. Kir2.2 exhibits electrophysiological characteristics similar to those of Kir2.1 though there are differences in both unitary conductance and the kinetics of Ba2+ blockage. The P-region of these channels is virtually identical with the exception that leucine (L) at position 148 in Kir2.2 is replaced by phenylalanine (F) in Kir2.1. The effects of mutating L148 to phenylalanine in Kir2.2 and F147 to leucine in Kir2.1 on unitary conductance and channel sensitivity to Ba2+ were investigated. Neither mutation altered unitary conductance from that seen in the wild-type channel. However, mutation L148F in Kir2.2 reduced the association rate constant for Ba2+ blockage without affecting affinity. In contrast, mutation F147L in Kir2.1 increased channel affinity for Ba2+ without affecting the association rate constant. Thus, resides outside the P-region are responsible for the difference in unitary conductance and some of the differences in Ba2+ block in Kir2.2 and Kir2.1.;The effects on ionic selectivity of substituting single amino acid residues at position 143 in the P-region of Kir2.1 were also investigated. Substitution of isoleucine by hydrophobic residues such as valine (I143V) and leucine (I143L) raised the relative Rb+ permeability, whilst substitution of the more hydrophilic residue threonine (I143T) enhanced K+ selectivity. Two further mutants, I143C and I143S, failed to yield currents, but could be rescued by bathing cells in extracellular solution containing 10mM dithiothreitol (DTT). The permeability ratios were then similar to wild-type. The rescue of mutant channels by DTT suggests that the pore of Kir channels may undergo conformational changes. In vitro translation studies suggest that channel function is rescued through the disruption of an intra-subunit disulphide bond.

In this thesis, potassium channels human Kv2.1 and rat Kv2.1, along with calcium channels Ca, 1.2, Ca, 3.1, and chimeras have been studied. These channels were expressed in Xenopuso ocytesf or electrophysiological experiments using two electrode voltage clamp. For protein expression studies,DNA was expressed in BL21 or COS-7 cells and purified using glutathione or an ID4 affinity column. Firstly, the roles of the N- and C- terminal domains in the activation kinetics of rat and human forms of Kv2.1 were investigated. A mutant in the N- terminal domain and chimeras between the rat and human forms were constructed. All clones were expressed in Xenopuso ocytesa nd activation times obtained. The results suggested that key residues in the N- and C- terminal domains are involved in determining the activation kinetics of rat and human Kv2.1. Further experiments were carried out using GST fusion proteins, Biacore surface plasma resonance and FRET investigations. These confirmed that the N and C- terminal domains are important in determining activation kinetics, and that these regions interact. To determine the positions of both the N- and C- terminal domains in the folded channel, rat Kv2.1 and a C-terminal deleted protein were expressed purified, and shown to have correct protein folding. These samples were sent for electron microscopy experiments and a preliminary picture obtained. No studies of S4 movement in calcium channels have been reported previously. Cysteine residues were substituted into the domain I S4 of a calcium channel chimera (with domain I of Ca, 3.1 replaced by Caj. 2). Cysteine residues at positions 263,265,266,268,269 and 271 were characterized by electrophysiology. Cysteine mutants at residues 263,265,266 and 268 reacted when extracellular PCMBS was applied, but mutants at residues 269 and 271 did not. This suggests that under depolarising conditions the S4 segment is exposed to the extracellular environment up to and including residue 268, with residues 269 and 271 remaining buried. Further investigation of residue 263 indicated that this movement occurs at potentials more negative than the resting membrane potential of -80mV. The data suggests that under depolarisation, the S4 becomes exposed to the extracellular solution and this movement of the S4 occurs before the ionic flow.

Three distinct K$ sp+$ currents were identified in corn (Zea mays) protoplasts using the whole-cell patch-clamp technique. Inward-rectifying K$ sp+$ currents were evoked at membrane potentials more negative than $-$100 mV. The activation range was sensitive to external K$ sp+$ and shifted in the positive direction as the K$ sp+$ concentration was elevated. The second K$ sp+$ current was voltage-independent and contributed to the resting membrane conductance of the protoplast. Finally, a voltage- and Ca$ sp{2+}$-dependent K$ sp+$ current was observed at potentials positive to $-$60 mV. This current was inhibited by reagents which antagonize plasmalemma Ca$ sp{2+}$ influx (e.g. nitrendipine, verapamil). In contrast, currents were enhanced by increasing the cytosolic free Ca$ sp{2+}$ concentration from 40 to 400 nM. The Ca$ sp{2+}$-dependent K$ sp+$ current was inhibited by tetraethylammonium ions, Cs$ sp+$, Ba$ sp{2+}$, and charybdotoxin which suggested that the channel protein has structural similarities to the high conductance Ca$ sp{2+}$-dependent K$ sp+$ channel observed in animal systems.

Before Pre-Paleozoic, the Earth's atmosphere had a composition different from today; in fact the organisms are not able to live in aerobic condition. The advent of cyanobacteria brought significant innovations, in fact, these bacteria unlike other phototrophs existing at that time, had chlorophyll molecules and protein complexes that allowed the use of water as electron donor to produce oxygen gas. This innovation developed over millions of years to get the current atmosphere. All these changes led to an inevitable biochemical and metabolic evolution of organisms. In Proterozoic or in early Cambrian, cyanobacteria began to reside within certain eukaryote cells. According to the endosymbionthic theory, chloroplasts evolved from a small primitive cyanobacterium settled within eukaryotic cells. Today, cyanobacteria are found throughout the Earth's environment, from oceans to fresh water and soil in the arctic areas, deserts and hot springs. Our attention is focused on cyanobacterium Synechocystis sp. PCC 6803. This strain was isolated for the first time from fresh water in California and now is considered a good model for scientific studies. It is spontaneously transformable, is able to integrate foreign DNA into its genome by homologous recombination (allowing targeted gene replacement) and can grow in the absence of photosynthesis if a suitable fixed-carbon source such as glucose is provided. Moreover it is the first photosynthetic organism for which the complete genome was sequenced (Kaneko et al., 1996). In the proteome of Synechocystis several putative ion channels can be identified (Kuo et al., 2005). However, none of them have been characterized from the functional point of view and their physiological role is still unknown. Ion channels are ubiquitous membrane proteins that control the passage of ions through biological membranes. These proteins, in all prokaryotes and eukaryotes, allow the correct ion distribution necessary to cellular functions. The basic features of the channels are selectivity and gating, the first is the property that controls the kind of ion that flows across the membrane and the second is the process of opening and closing the ion pathway. In fact the passage through the pore is governed by a "gate," which may be opened or closed by chemical, mechanical or electrical signals (Hille, 2001). Potassium (K+) is the most abundant cation in organisms and plays a crucial role in the survival and development of cells, by regulating enzyme activity and tuning membrane potential. This is one of the reasons for which potassium channels are one of the most studied among classes of channels. The field of prokaryotic potassium channels underwent a rapid development over the past years thanks to the application of a combination of bioinformatics and molecular biology, beside electrophysiology and structural studies. Understanding of their structure and actual mechanism of ion conduction allow to obtain more information about the function of potassium channels in general. A bioinformatic screening of Synechocystis sp. PCC 6803 proteome identified two putative proteins on which we focused our attention. The first one was named SynK and it displays sequence homology with KvAP (a voltage gated potassium channel of A. pernix) (Jiang et al., 2003). The second one, SynCaK, displays sequence homology to MthK, a Ca2+-dependent potassium channel from M. thermoautotrophicum (Jiang et al., 2002). Our goal was to understand whether they actually function as ion channels and to reveal their roles in the physiology of cyanobacteria. The characteristics and function of these proteins were studied through an integrated approach involving molecular biology techniques, biochemistry, electrophysiology and microscopy. SynK was initially identified in the genome of Synechocystis sp. PCC 6803 using the selectivity filter amino acid sequence (TMTTVGYGD) as a query sequence. This protein of unknown function shows six membrane spanning segments (S1-S6) and a pore region between S5 and S6 helixes. Before starting my Ph.D, SynK gene has been cloned and expressed in mammalian cells (Chinese Hamster Ovary, CHO) in fusion with EGFP (a fluorescent protein). Subsequent Western-blotting analysis showed that the fusion protein was correctly expressed. Confocal microscopy studies demonstrated its membrane localization and patch-clamp analysis revealed an activity of voltage-gated outwardly rectifying potassium selective channel in CHO cells. In addition, the double location of SynK in plasma and thylakoid membrane of cyanobacteria was shown by immunogold electron microscopy and Western blot on isolated membrane fractions. During my P.h.D, I performed the construction of two different mutants of SynK channel. The first SynK mutant, corresponding to the protein with a single amminoacid mutation in the selectivity filter of the pore (mutation Y181A), was used for expression in CHO cells. In accordance to the literature, this mutant protein loses its potassium channel activity. I also produced a ∆SynK Synechocystis mutant strain. Its functional analysis allowed to understand the physiological role of SynK in cyanobacteria. In order to characterize the function of the SynK protein, we initially verified that the deletion mutant did not express Synk, using Western blot technique. To evaluate the physiological role of the SynK protein, we initially compared growth of the wild type (WT) and mutant strain in different conditions. Characterization of the mutant phenotype was investigated by comparing photosynthetic activity in WT and mutant strains. Using a similar approach we have identified in the genome of Synechocystis sp. PCC 6803 a second protein classified as a putative potassium channel that displays sequence homology with MthK, a calcium dependent potassium channel from the archeon Methanobacterium thermoautophicum. Using several structural prediction programs, we analyzed the primary sequence of the protein translated from sll0993 and we observed that this protein (that we called SynCaK), like MthK, is predicted to contain two membrane spanning segments, a recognizable K+ channel signature sequence, with only conservative substitutions, and a regulatory sequence for K+ conductance (RCK). Also in the case of sll0993, we cloned and expressed the protein in fusion with GFP in CHO cells and studied their activity by patch clamp. Moreover, in order to study the role of SynCaK in cyanobacteria physiology we produced a SynCaK-deficient Synechocystis mutant. To gain further information about the activity of the channel, we have expressed and started the purification of the protein in another heterologous system, E. coli. Purified recombinant channel proteins are often studied by incorporating them into an artificial planar bilayer system (Ruta et al., 2003). During my Ph.D, I also continued the work begun during my thesis in Biotecnology on the study of ion channels in mitochondria of Graminaceae. Classical bioenergetics techniques reveal activities compatible with the presence of a potassium channel in durum wheat mitochondria, but the study of channels in mitochondria of plant systems is a still unexplored field in the world. To this end, we started a study through the parallel use of different techniques, which allowed a more complete characterization of the activity of channels present in wheat mitochondria. In particular, we followed two approaches. First, biochemical studies on isolated mitochondria, through the use of SDS-PAGE and immunoblotting, allowed the evaluation of the sample used in terms of enrichment and purity (data completely absent in the literature to date). Second, preparations of mitochondria from roots of durum wheat were suitable for electrophysiological studies in particular patch clamp technique, applied for the first time on plant mitochondria. Finally, I was involved in collaboration with the laboratory of Professor Nobuyuki Uozumi at Tohoku University in Japan. This group obtained a mutant for Synechocystis aquaporin. Aquaporins are membrane proteins embedded in the cell membrane that regulate the flow of water. I contributed to the characterization of the acquaporin-less mutant by performing experiments measuring photosynthetic activity. In particular, we performed several experiments of oxygen evolution demonstrating that the photosyntetic efficiency is higher in the mutant with respect to the WT when the organisms are incubated in hyperosmotic medium. The next step is to clarify how exactly a hyperosmotic stress and the absence of aquaporin are correlated with the photosynthesis and what is the underlying mechanism.
Prima del Pre-Paleozoico, l'atmosfera terrestre aveva una composizione diversa rispetto a quella quella di oggi, infatti, gli organismi non erano in grado di vivere in condizioni aerobiche. L'avvento dei cianobatteri ha portato rilevanti innovazioni, infatti, questi a differenza di altri batteri fototrofi esistenti a quel tempo, presentavano molecole di clorofilla e complessi proteici che permisero di utilizzare l’acqua come donatore di elettroni per la produzione dell’ossigeno. Questa modificazione ha permesso, lungo milioni di anni, di ottenere l'attuale atmosfera. Tutti questi cambiamenti portarono ad una inevitabile evoluzione biochimica e metabolica degli organismi. Nel Proterozoico o agli inizi del Cambriano, i cianobatteri iniziarono a risiedere all'interno di alcune cellule eucariotiche. Secondo la teoria endosimbiotica, i cloroplasti evolsero da un piccolo cianobatterio primitivo presente all'interno delle cellule eucariotiche. Oggi, i cianobatteri si trovano in diversi ambienti terrestri, da oceani ad acque dolce, in terre artiche, in deserti ed in sorgenti termali. La nostra attenzione è focalizzata sul cianobatterio Synechocystis sp. PCC 6803. Questo ceppo è stato isolato per la prima volta da una sorgente di acqua dolce in California e ora è considerato un buon organismo modello per studi scientifici. È spontaneamente trasformabile, è in grado di integrare DNA estraneo nel suo genoma attraverso ricombinazione omologa (consentendo la sostituzione mirata dei geni) e può crescere in assenza di fotosintesi se viene fornita un'adeguata fonte di carbonio, come il glucosio. Inoltre è il primo organismo fotosintetico per il quale il genoma è stato sequenziato (Kaneko et al., 1996). Nel proteoma di Synechocystis sono stati identificati diversi putativi canali ionici (Kuo et al., 2005). Tuttavia, nessuno di essi è stato caratterizzato da un punto di vista funzionale e il loro ruolo fisiologico rimane ancora sconosciuto. I canali ionici sono proteine di membrana che controllano il passaggio degli ioni attraverso esse. Queste proteine, in tutti i procarioti e gli eucarioti, permettono la corretta distribuzione ionica necessaria per le funzioni cellulari. Le caratteristiche base dei canali sono la selettività ed il gating, la prima è la proprietà che controlla il tipo di ioni che attraversa la membrana, la seconda è il processo di apertura e chiusura del percorso degli ioni. In realtà il passaggio attraverso il poro è regolato da un gate, che può essere aperto o chiuso da segnali chimici, meccanici o elettrici (Hille, 2001). Il potassio (K+) è il catione più abbondante negli organismi viventi e svolge un ruolo cruciale per la sopravvivenza e lo sviluppo delle cellule, regolando l'attività enzimatica e il potenziale di membrana. Questo è uno dei motivi per i quali i canali del potassio sono una delle classi di canali più studiate. Il campo dei canali del potassio procariotici ha subito un rapido sviluppo negli ultimi anni grazie all'applicazione di una combinazione di tecniche di bioinformatica e biologia molecolare, affiancate a studi di elettrofisiologia e studi strutturali. La comprensione della loro struttura e del meccanismo di conduzione degli ioni permette di ottenere ulteriori informazioni sulla funzione dei canali di potassio in generale. Uno screening bioinformatico del proteoma di Synechocystis sp. PCC 6803 ha individuato due proteine putative su cui abbiamo concentrato la nostra attenzione. La prima è stata chiamata SynK e mostra omologia di sequenza con KvAP (un canale del potassio voltaggio di A. pernix) (Jiang et al., 2003). La seconda, SynCaK, mostra omologia di sequenza con MthK, un canale del potassio Ca2 +-dipendente di M. thermoautotrophicum (Jiang et al., 2002). Il nostro obiettivo era quello di capire se effettivamente queste proteine funzionano come canali ionici e di comprendere il loro ruolo nella fisiologia dei cianobatteri. Le caratteristiche e la funzione di queste proteine sono state studiate attraverso un approccio integrato comprendente tecniche di biologia molecolare, biochimica, elettrofisiologia e microscopia. Il gene SynK è stato inizialmente identificato nel genoma di Synechocystis sp. PCC 6803 utilizzando la sequenza amminoacidica del filtro di selettività (TMTTVGYGD) come sequenza query. Questa proteina di funzione sconosciuta mostra sei segmenti transmembrana (S1-S6) ed una regione del poro tra le eliche S5 e S6. Prima di iniziare il mio Dottorato, SynK è stato clonato ed espresso in cellule di mammifero (Chinese Hamster ovary, CHO) in fusione con la EGFP (una proteina fluorescente). Una successiva analisi western-blotting ha dimostrato che la proteina di fusione è correttamente espressa. Studi di microscopia confocale hanno dimostrato la sua localizzazione nella membrana di cellule CHO e l'analisi patch-clamp ha rivelato un'attività di canale outwardly rectifying selettivo per il potassio. Inoltre, è stata dimostrata per SynK, in frazioni di membrana isolate da cianobatteri, mediante microscopia elettronica (attraverso la tecnica dell’immunogold) e tecniche di western blot, una doppia localizzazione nella plasmamembrana e nelle membrane tilacoidi.Durante il mio Dottorato, è stata eseguita la costruzione di due diversi mutanti del canale SynK. Il primo mutante corrisponde alla proteina con una mutazione puntiforme nel filtro di selettività del poro (mutazione Y181A) e utilizzato per l'espressione in cellule CHO. In base alla letteratura, questa proteina mutante perde la sua attività di canale del potassio. Inoltre, è stato prodotto un ceppo mutante knock-out (ΔSynK) in Synechocystis. La sua analisi funzionale ha permesso di capire il ruolo fisiologico di SynK nei cianobatteri. Al fine di caratterizzare la funzione della proteina SynK, abbiamo inizialmente verificato, attraverso western blot, che il ceppo mutante effettivamente non esprimesse la proteina. Mentre per valutare il ruolo fisiologico della proteina SynK, abbiamo confrontato la crescita del ceppo wild-type (WT) e mutante in diverse condizioni. La caratterizzazione del fenotipo mutante è stata studiata confrontando l’attività fotosintetica nel WT e nel mutante. Utilizzando un approccio simile abbiamo identificato nel genoma di Synechocystis sp. PCC 6803 una seconda proteina classificata come putativo canale del potassio che mostra omologia di sequenza con MthK, un canale del potassio calcio dipendente di Methanobacterium thermoautophicum. Attraverso l’utilizzo di vari programmi di predizione strutturale, abbiamo analizzato la sequenza primaria della proteina tradotta e abbiamo osservato che questa (che abbiamo chiamato SynCaK), come MthK, contiene due segmenti transmembrana, un filtro di selettività tipico dei canali del K+, con sostituzioni conservative, e un dominio di regolazione della conduttanza del potassio (RCK domain). Anche in questo caso, abbiamo clonato ed espresso la proteina in fusione con EGFP in cellule CHO e studiato la loro attività tramite patch clamp. Inoltre, al fine di studiare il ruolo di SynCaK nella fisiologia dei cianobatteri abbiamo prodotto un mutante knock-out per SynCaK. Per ottenere ulteriori informazioni sull’attività del canale, abbiamo espresso e iniziato la purificazione della proteina in un altro sistema eterologo, E. coli. Le proteine canale-ricombinanti sono spesso studiate mediante la loro integrazione in doppi strati artificiali (Ruta et al., 2003). Durante il mio Dottorato, ho anche continuato il lavoro iniziato durante la mia tesi di laurea in Biotecnologie Industriali sullo studio dei canali ionici nei mitocondri delle Graminaceae. Tecniche classiche di bioenergetica hanno rivelato attività compatibili con la presenza di un canale di potassio nei mitocondri di grano duro, ma lo studio dei canali nei mitocondri di sistemi vegetali è un campo ancora inesplorato nel mondo. A tal fine, è stato iniziato uno studio attraverso l'utilizzo parallelo di diverse tecniche, che hanno consentito una caratterizzazione più completa delle attività dei canali presenti nei mitocondri di grano. In particolare, sono stati seguiti due approcci. In primo luogo, studi biochimici sui mitocondri isolati, attraverso l'uso di SDS-PAGE e immunoblotting, che hanno permesso la valutazione del campione utilizzato in termini di arricchimento e di purezza (dati del tutto assenti in letteratura fino ad oggi). In secondo luogo, sono state definite preparazioni di mitocondri da radici di grano duro adatte per studi elettrofisiologici. In particolare, per la prima volta è stata applicata la tecnica di patch clamp su mitocondri vegetali. Infine, ho svolto una collaborazione con il laboratorio del Professor Nobuyuki Uozumi presso la Tohoku University in Giappone. Questo gruppo ha ottenuto un mutante per l’acquaporina di Synechocystis. Le acquaporine sono proteine di membrana incorporate nelle membrane cellulari che regolano il flusso dell'acqua. Ho contribuito alla caratterizzazione del mutant-less acquaporin attraverso esperimenti di misura dell'attività fotosintetica. In particolare, sono stati eseguiti diversi esperimenti di evoluzione di ossigeno che dimostrano che l'efficienza fotosintetica è più alta nel mutante rispetto al WT quando gli organismi vengono incubati in un mezzo iperosmotico. Il passo successivo sarà quello di chiarire esattamente come uno stress iperosmotico e l'assenza di acquaporina sono correlati con la fotosintesi e quindi il meccanismo sottostante.

My Ph.D. project has focused on the characterization of TPK3, a putative channel selective for potassium (K+) with a predicted chloroplast localization in higher plants, from biochemical, physiological and electrophysiological point of view. This protein belongs to the TPK channel family (from Tandem-Pore K+ channels) and displays amino acid sequence homology with another K+ channel studied in our laboratory, called SynK (Zanetti et al., 2010). SynK shows thylakoid localization in Cyanobacteria. The SynK channel has been shown to be critical for photosynthetic performances in Cyanobacteria, given the photosensitive phenotype displayed by the mutants lacking the SynK protein. Given the homology, we hypothesized that similarly, TPK3 might be involved in the regulation of photosynthetic processes in higher plants. So far, no information is available about the properties of TPK3, nor about its physiological roles, neither about its possible involvement in photosynthesis; the work presented in this thesis had the aim of clarifying some important aspects of the functions of TPK3. Following subcellular localization studies carried out using biochemistry and confocal microscopy techniques, the TPK3 channel was expressed in E. coli cells for subsequent electrophysiological characterization in a planar lipid bilayer setup in order to prove its function as K+ channel. The unavailability of commercial mutants for tpk3 gene required setting up of a silencing procedure via RNA interference of the messenger for the protein, in order to analyze the possible physiological roles of TPK3. The resulting silenced plants have been studied under different growth conditions to determine changes in physiology of the plants including their photosynthetic parameters. In parallel with the TPK3 project, the most important part of my Ph.D., I also followed two other major areas of research: one concerning the study of the functions of two members of plant Glutamate Receptors (GluRs) and the other one concerning the characterization of the plant homologous of the recently identified MCU (Mitochondrial Calcium Uniporter) of mammals. This thesis also includes a manuscript (Checchetto et al., 2012) to which I contributed with the heterologous expression of a calcium-activated K+ channel, SynCaK, of Cyanobacteria.
Il mio progetto di dottorato si è focalizzato sulla caratterizzazione, dal punto di vista biochimico ed elettrofisiologico, di una proteina denominata TPK3 che è predetta di funzionare come canale selettiva per il potassio (K+) ed essere localizzata nei cloroplasti nelle piante superiori,. Questa proteina appartiene alla famiglia dei canali TPK (da Tandem-Pore K+ channels) e mostra omologia di sequenza a un altro canale del K+ studiato nello stesso nostro laboratorio, denominato SynK (Zanetti et al., 2010), a localizzazione tilacoidale ed appartenente al phylum dei Cianobatteri. È stato dimostrato in più esperimenti che il canale SynK è fondamentale per la regolazione della fotosintesi nei Cianobatteri, in considerazione del fenotipo fotosensibile mostrato dai mutanti per il gene synk. Visto la localizzazione predetta del TPK3, è stato ipotizzato in partenza che TPK3 potesse svolgere un ruolo simile nelle piante superiori. Finora nulla si conosceva sulle proprietà di TPK3, ne sui suoi ruoli fisiologici, ne su di un suo eventuale coinvolgimento nella fotosintesi nelle piante superiori; il lavoro contenuto nel progetto presentato ha cercato di chiarire alcuni aspetti salienti delle funzioni di TPK3. Dopo studi di localizzazione subcellulare condotti con tecniche di biochimica e microscopia confocale, il canale TPK3 è stato espresso in E. coli per la successiva caratterizzazione elettrofisiologica in bilayer lipidico planare allo scopo di determinare la sua funzione come canale di K+. L’assenza di mutanti commerciali per il gene tpk3 ha necessitato la messa a punto del suo silenziamento tramite RNA interference del messaggero per la proteina suddetta, al fine di analizzarne i possibili ruoli fisiologici. Le piante silenziate risultanti, sottoposte a differenti condizioni di crescita, sono state studiate in vari esperimenti atti a determinarne vari parametri inclusi quelli fotosintetici. Contemporaneamente allo studio del TPK3, quello di maggior rilievo nel mio dottorato, ho seguito anche altri due filoni di ricerca principali, riguardanti l’uno l’approfondimento delle funzioni di due membri dei Recettori di Glutammato vegetali (GluRs) e l’altro la caratterizzazione degli omologhi del recentemente identificato MCU (Mitochondrial Calcium Uniporter) di Mammiferi. Nella presente tesi è inoltre incluso un manoscritto (Checchetto et al., 2012) per il quale ho collaborato nell’espressione eterologa del canale di K+ calcio-dipendente (SynCaK) di Cianobatteri.

Two kinetically and pharmacologically distinct outward K+ conductances were found to be simultaneously activated at the plasma membrane of TBY-2 suspension culture protoplasts using the whole-cell patch-clamp technique. We used a modified Hodgkin-Huxley model to quantify the activation kinetics of the outward current. Time constants for the two conductances derived from the model differed in magnitude by 5--10 fold over the voltage range from -10 mV to +50 mV, allowing their classification as either fast or slow. Deactivation kinetics were better fit by two exponential terms rather than one, yielding fast and slow deactivation time constants. The voltage dependence of time constants derived from these two independent two-channel models followed a bell-shaped distribution with mid-point potentials for both components at -20 mV with a standard 10-fold K+ gradient (10 K+o/100K+i).
Both components were highly K+-selective, however the tail current amplitudes of the slowly activating component at hyperpolarized potentials exhibited non-linear rectification whereas the tail current amplitudes of the fast activating component were linear. The ratio of inward tail current/activated outward current (envelope of tails test) was not constant during the depolarizing step; during the first 50--100 milliseconds the ratio was 6 times higher than at quasi-steady-state (i.e. after 0.3 second).
A pharmacological dissection of outward currents revealed that external Ba2+ in the range from 10 muM to 1 mM selectively inhibited a fast, sigmoidally activating, slowly inactivating current as revealed by examining difference currents. The more slowly activating component was inhibited by only 20% with 5 mM Ba2+. Conversely, nitrendipine or bepridil (5--100 muM) selectively inhibited the slower component of outward current. External TEA inhibited both the fast and slow components equally; tail current amplitudes of both components were inhibited by 40% with 2 mM TEA and the activation time courses in the presence of TEA conserved the same kinetic parameters as control currents.

The beta 3 subunit of voltage gated has been recently identified as a modulatory macromolecule of pannexin 1. Our interest has focused on the possible interaction between the alpha subunit of Kv1.1 channel and pannexin 1. Through voltage clamp studies we have analyzed the electrical activity of the single channels and their behavior when they were coexpressed. With our results we have demonstrated that pannexin 1 was less susceptible to its inhibitors, like probenecid and DTT, when it was coexpressed with Kv1.1 channel, on the contrary pannexin 1 did not seem to influence the activity of Kv1,1 channel. Through immunocytochemistry on HEK-hBK1 cells expressing in stable way Kv1.1 channel we have observed the colocalization of the two channels but through coimmunoprecipitation we proved the lack of a physical interaction between these proteins, therefore the interaction should be functional. Moreover previous studies has reported an involvement of pannexin 1 in apoptosis at elevated concentrations of extracellular potassium. So we wanted to estimate the cell death in presence and absence of pannexin 1 inhibitors, using like control SH-SY5Y cells, being a cell line that does not express Kv1.1 channel. With our results we have obtained a decreased in the cell death when HEK-hBK1 cells were treated with probenecid 1 mM in presence of KCl 140 mM suggesting that this behavior was the consequence of pannexin 1 inhibition, therefore in these conditions Kv1.1 channel did not influence in some way its activity; on the contrary the treatment with 10 mM DTT did not produce any benefical effects in HEK-hBK1 cells. These findings confirmed that Kv1,1 channel influenced the sensibility of pannexin 1 to its inhibitors only when redox potential was altered and then in presence of reducing agents, or when a depolarization of the membrane was induced like in oocytes, but this phenomenon didn t occur in other conditions. Probably this indirect interaction is mediated by other proteins, such as calmodulin and kinase proteins or by lipids of the membrane such as PIP2; it could represent a regulatory mechanism that replaces or enhances that exercised by beta 3 subunit on pannexin 1, in order to control the potassium buffering' and then the cell excitability and survival, both in pathological and physiological conditions.

The membrane potential of arterial smooth cells, and consequently arterial tone, is. regulated by the activity of plasmalemmal K+ selective ion channels. K2P channels are responsible for background K+ (KB) conductances which contribute to generation of the resting membrane potential. They have been identified in arterial smooth muscle cells but their significance is poorly understood. A Ks conductance is expressed in rat femoral arterial smooth muscle cells (rFASMCs), and the aim of this project was to (i) determine the expression profile of K2P subunits in rat femoral artery and (ii) identify the molecular correlate of the KB conductance in rFASMCs. Using RT-PCR, we identified transcripts encoding TWIK-2, TASK-I, TASK-2, TREK-I, TREK-2 and THIK-I in total rat femoral artery RNA. TWIK-2, TASK-I, TASK-2 and TREK-I were further identified at the protein level in isolated FASMCs by antibody staining. Only TWIK-2 was detected at the cell surface, while TASK-I, TASK-2 and TREK-I had a predominantly intracellular localisation. Immunohistochemistry of femoral artery sections may also support the expression of TWIK-2, TASK-I and TASK-2 (but not TREK-I) in the endothelium. To allow comparison between cloned and native conductances, the open reading frames of rTWIK-2, rTASK-2 and rTREK-I were cloned by PCR amplification. The sequence encoding rTASK-2 was cloned de novo and submitted to the EBI gene data base (Accession no: AM229406). Functional expression ofrTWIK-2 and rTASK-2 was investigated in Xenopus 'laevis oocytes. rTWIK-2 did not exhibit strong expression in the Xenopus laevis expression system. Conversely, recombinant rTASK-2 channels formed K+ selective, extracellular pH sensitive ion channels. The basic pharmacological properties of cloned rTASK-2 channels were also investigated. Currents were insensitive to extracellular TEA, 4-AP and Cs+, but were sensitive to inhibition by Ba2+, Zn2+and quinidine. Inhibition by extracellular Ba2+was strongly time and voltage-dependent. Ba2+ inhibition of rTASK-2 was characterised by steady-state analysis and voltage pulses. Although we were unable to describe the functional correlate of the KB conductance in rFASMCs, this thesis has established the groundwork for further correlative studies. The information provided will assist in the identification of native TASK-2 conductances.

Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed July 10, 2008). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references.