Ion channels are fundamental molecules in the nervous system that catalyze the flux of ions across the cell membrane. There are mounting evidences suggesting that the kinetic properties of ion channels undergo activity-dependent changes in various pathophysiological conditions. Here such activity-dependent changes were studied in case of two different ion channels; the rat brain derived voltage-gated Na+ channel, rNav1.2 and the human background leak K+ channel, hTREK1 using the single channel patch-clamp technique. Our results on the voltage-gated Na+ channel (Chapter III) illustrated that sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, induced a defined non-linear variation in the unitary conductance, activation, inactivation and recovery kinetic properties of the channel. Signal processing tools attributed a pseudo-oscillatory nature to the non-linearity observed in the channel properties. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clustering of dwell time events and strong autocorrelation in the dwell time series. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Similar plastic changes were observed in case of the hTREK1 channel in presence of saturating concentrations of agonist, trichloroethanol (TCE) (Chapter IV). TREK1 channel behaves similar to single enzyme molecules with a single binding site for the substrate K+ ion whereas TCE acts as an allosteric activator of the channel. We observed that with increasing concentration of TCE (10 M to 10 mM) the catalytic turnover rate exhibited progressive departure from monoexponential to multi-exponential distribution suggesting the presence of ‘dynamic disorder’ analogous to single enzyme molecules. In addition, we observed the induction of strong correlation in successive waiting times and flux intensities, exemplified by distinct mode switching between high and low flux activity, which implied the induction of memory in single ion channel. Our observation of such molecular memory in two different ion channels in different experimental conditions highlights the importance and generality of the phenomenon which is normally hidden under the ensemble behaviour of ion channels. In the final part of the work (chapter V) we observed strong negative cooperativity and half-of-sites saturation kinetics in the interaction of local anesthetic, lidocaine with hTREK1 channel. We also mapped the specific anesthetic binding site in the c-terminal domain of the channel. Further, single channel analysis and the heterodimer studies enabled us to propose a model for this interaction and provide a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Οι νικοτινικοί υποδοχείς της ακετυλοχολίνης (nAChRs) ανήκουν στην υπερ-οικογένεια των ιοντικών καναλιών που ενεργοποιούνται από τη δέσμευση ενός προσδέτη (LGICs) και αποτελούνται από πέντε ομόλογες υπομονάδες. Κάθε μονομερής υπομονάδα αποτελείται από μία Ν-τελική εξωκυττάρια περιοχή (ΕΚΠ), από τέσσερεις διαμεμβρανικές α-έλικες και από μία κυτταροπλασματική περιοχή. Στην ΕΚΠ βρίσκεται η χαρακτηριστική Cys-θηλιά της υπερ-οικογένειας, καθώς και οι θέσεις πρόσδεσης αγωνιστών και ανταγωνιστών του υποδοχέα. Οι γνώσεις μας γύρω από τη δομή των nAChRs προέρχονται κυρίως από κρυσταλλογραφικές δομές ομολόγων πρωτεϊνών δέσμευσης της ACh (AChBP) μαλακίων, από μια δομή του nAChR από ιχθείς του γένους Torpedo που προέρχεται από ηλεκτρονική μικροσκοπία, από την κρυσταλλογραφική δομή της α1-ΕΚΠ ποντικού σε σύμπλοκο με α-μπουγκαροτοξίνη (α-Btx) και από κρυσταλλογραφικές δομές δύο προκαρυωτικών LGICs. Παρά τη μεγάλη πρόοδο που πραγματοποιήθηκε με τα παραπάνω επιτεύγματα, ακόμη δεν έχει επιλυθεί πειραματικά η δομή ανθρώπινου υποδοχέα. Επίσης λίγα είναι γνωστά για την επίδραση της γλυκοζυλίωσης των ΕΚΠ στη λειτουργία του nAChR. Χρησιμοποιώντας ως εκμαγείο την κρυσταλλογραφική δομή του συμπλόκου α1-ΕΚΠ ποντικού/α-Btx δημιουργήθηκαν υπολογιστικά μοντέλα της ανθρώπινης α1-ΕΚΠ προσδεμένης στις τοξίνες α-μπουγκαροτοξίνη (α-Btx), α-κομπρατοξίνη (α-Cbtx), α-κωνοτοξίνη (α-Ctx) ImI και α-κωνοτοξίνη GI. Στα σύμπλοκα με α-Btx και α-Cbtx προστέθηκε η υδατανθρακική αλυσίδα, συνδεδεμένη με το κατάλοιπο Asn141, που συγκρυσταλλώθηκε μαζί με την α1-ΕΚΠ ποντικού. Για να μελετηθεί η δυναμική συμπεριφορά της αλληλεπίδρασης υποδοχέα-τοξίνης καθώς και η συνεισφορά των σακχάρων σε αυτήν πραγματοποιήθηκαν προσομοιώσεις Μοριακής Δυναμικής σε υδατικό περιβάλλον. Με τη χρήση υπολογιστικών εργαλείων για τη μελέτη των συμπλόκων προσδιορίστηκαν σε ατομικό επίπεδο οι αλληλεπιδράσεις που καθοδηγούν την πρόσδεση τοξινών στην α1-ΕΚΠ. Βρέθηκε ότι η υδατανθρακική αλυσίδα συμμετέχει δυναμικά στη δέσμευση της τοξίνης στον υποδοχέα. Τα σάκχαρα συγκλίνουν προς την προσδεμένη τοξίνη στηριζόμενα στα κατάλοιπα Ser187 και Trp184 της α1 υπομονάδας. Η τοξίνη επίσης μετακινείται φέρνοντας τη θηλιά Ι σε επαφή με τα σάκχαρα. Αναγνωρίστηκαν σημαντικές αλληλεπιδράσεις των σακχάρων με τα τοξινικά κατάλοιπα Thr6, Ser9, και Th15 της α-Btx και Thr6 και Pro7 της α-Cbtx. Επίσης επιβεβαιώθηκε η ύπαρξη μιας υδρόφιλης κοιλότητας στο εσωτερικό του υδρόφοβου πυρήνα της α1-ΕΚΠ, η οποία πιθανόν εμπλέκεται στο άνοιγμα του ιοντικού καναλιού του nAChR. Τα αποτελέσματα αυτά παρέχουν σημαντικά δεδομένα για την κατανόηση της επίδρασης της υδατανθρακικής αλυσίδας στη λειτουργία του υποδοχέα, η οποία μπορεί να αξιοποιηθεί στην αντιμετώπιση των πολλών παθολογικών καταστάσεων στις οποίες εμπλέκονται οι nAChRs. / Nicotinic acetylcholine receptors (nAChRs) belong to the superfamily of ligand-gated ion channels (LGICs). LGICs form homo- or hetero-pentamers of related subunits, and each of them consists of a N-terminal extracellular ligand-binding domain (ECD), four transmembrane α-helixes and an intracellular region. The characteristic Cys-loop of the superfamily is found in the ECD of each subunit. The ECD also contains binding sites for agonists and competitive antagonists. Our knowledge regarding the nAChR structure mainly derives from the X-ray crystal structures of the molluscan ACh-binding proteins (AChBPs), the electron microscopy structure of the Torpedo nAChR, the X-ray crystal structure of the mouse nAChR α1-ECD bound to α-bungarotoxin (α-Btx), and the X-ray crystal structures of two prokaryotic LGICs. Despite the progress made by these achievements, the determination of any human nAChR structure has not yet been accomplished. Furthermore, the effect of glycosylation on nAChR function has not yet been explored. Based on the crystal structure of the extracellular domain of the mouse nAChR α1 subunit bound to α-Btx we have generated in silico models of the human nAChR α1-ECD bound to the toxins α-bungarotoxin (α-Btx), α-cobratoxin (α-Cbtx), α-conotoxin (α-Ctx) ImI and α-conotoxin GI. In the case of the α1-ECD/α-Btx and α-Cbtx complexes, a Asn141-linked carbohydrate chain was modeled, its coordinates taken from the crystal structure of the mouse α1-ECD. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. The use of computational methods allowed the monitoring of the interactions that govern toxin binding. The MD simulations revealed the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain. A shift in the position of the sugars towards the bound toxin was observed. Residues Ser187 and Trp184 of nAChR act as critical anchor points for the stabilization of the sugar chain in a close position to the toxin. Toxin Finger I shifts closer to the mannoses, forming important toxin-sugar interactions that implicate residues Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Additionally the MD simulations of the human α1 ECD–toxin complexes confirmed the possible accommodation of two water molecules into a hydration cavity inside the hydrophobic core of the subunit, which may contribute to the gating mechanism of the receptor. These findings provide additional structural data that are intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism of nAChR and also guide the development of novel therapeutic agents for the treatment of nAChR-associated diseases.

Pentameriska ligandstyrda jonkanaler (pLGICs) är avgörande för omvandlingen av kemisk till elektrisk signalöverföring i djurs nervsystem. Dysfunktion i dessa kanaler har visat sig vara kopplad till flera sjukdomar inklusive epilepsi, schizofreni, Alzheimers och autism, vilket gör dem till en måltavla för en mängd olika läkemedel. Att studera eukaryota kanaler är dock mycket utmanande, så upptäckten av prokaryota homologer, som är mycket lättare att studera, har därmed bidragit mycket till förståelsen för struktur och funktion hos proteiner i denna familj. I detta projekt producerades och renades en prokaryotisk pLGIC kallad DeCLIC från Escherichia coli. Strukturell bestämning av kanalen genomfördes med användning av kryo-elektronmikroskopi vid lågt pH och i närvaro av kalcium. En elektrontäthet med 3.4 Å upplösning uppnåddes och jämfördes med tidigare bestämda strukturer vid olika förhållanden i ett försök att bestämma hur proteinets struktur moduleras av kalcium och pH. Resultaten visar flera skillnader i kanalens konformation i närvaro och frånvaro av kalcium såväl som vid olika pH-värden. Dessutom antyder analys av den bestämda elektrontätheten ett möjligt intermediärt tillstånd vid lågt pH i närvaro av kalcium. / Pentameric ligand-gated ion channels (pLGICs) are crucial for the conversion of chemical to electrical signaling in the nervous system of mammals. Dysfunction in these channels has been found to be connected to several diseases including epilepsy, schizophrenia, Alzheimer’s, and autism, making them the target of a wide variety of therapeutic agents. However, studying eukaryotic channels is challenging so the discovery of prokaryotic homologs that are much easier to study has thus greatly helped in the understanding of the structure and function in this family of proteins. In this project, a prokaryotic pLGIC called DeCLIC was produced and purified from Escherichia coli. Structural determination of the channel was pursued using cryo-electron microscopy at a low pH and in the presence of calcium. An electron density at 3.4 Å resolution was achieved and compared to previously determined structures at different conditions in an attempt to determine the structural modulation of calcium and pH. Results show multiple differences in channel conformation in the presence and absence of calcium as well as in different pH conditions. Furthermore, analysis of the determined electron density suggests a possible intermediate state at low pH in the presence of calcium.

Membrane protein dynamics is of great importance for living organisms. The precise localization of proteins composing a synapse on the membrane facing a nerve terminus is essential for proper functioning of the nervous system. In muscle fibers, the nicotinic acetylcholine is densely packed under the motor nerve termini. A receptor associated protein, rapsyn, acts as a linker between the receptor and the other components of the synaptic suramolecular assembly. Advances in fluorescence microscopy have allowed to measure the behavior of a single receptor in the cell membrane. In this work single-molecule microscopy was used to track the motion of ionotropic acetylcholine (nAChR) and serotonin (5HT3R) receptors in the plasma membrane of cells. We present methods for measuring single-molecule diffusion and their analysis. Single molecule tracking has shown a high dependence of acetylcholine receptors diffusion on its associated protein rapsyn. Comparing muscle cells that either express rapsyn or are devoid of it, we found that rapsyn plays an important role on receptor immobilization. A three-fold increase of receptor mobility was observed in muscle cells devoid of rapsyn. However, in these cells, a certain fraction of immobilized receptors was also found immobile. Furthermore, nAChR were strongly confined in membrane domains of few tens of nanometers. This showed that membrane composition and membrane associated proteins influence on receptor localization. During muscle cell differentiation, the fraction of immobile nAChR diminished along with the decreasing nAChR and stable rapsyn expression levels. The importance of rapsyn in nAChR immobilization has been further confirmed by measurements in HEK 293 cells, where co-expression of rapsyn increased immobilization of the receptor. nAChR is a ligand-gated ion-channel of the Cys-loop family. In mammals, members of this receptor family share general structural and functional features. They are homo- or hetero-pentamers and form a membrane-spanning ion channel. Subunits have three major regions, an extracellular ligand binding domain, a transmembrane channel and a large intracellular loop. 5HT3R was used as a model to study the effect of this loop on receptor mobility. Single-molecule tracking experiments on receptors with progressively larger deletions in the intracellular loop did not show a dependence of the size of the loop on the diffusion coefficient of mobile receptors. However, two regions were identified to play a role in receptor mobility by changing the fractions of immobile and directed receptors. Interestingly, a prokaryotic homologue of cys-loop receptors, ELIC, devoid of a large cytoplasmic loop was found to be immobile or to show directed diffusion similar as the wild-type 5HT3R. The scaffolding protein rapsyn stabilizes nAChR clusters in a concentration dependent manner. We have measured the density and self-interactions of rapsyn using FRET microscopy. Point-mutations of rapsyn, known to provoke myopathies, destabilized rapsyn self-interactions. Rapsyn-N88K, and R91L were found at high concentration in the cytoplasm suggesting that this modification disturbs membrane association of rapsyn. A25V was found to accumulate in the endoplasmic reticulum. Fluorescent tools to measure intracellular concentration of calcium ions are of great value to study the function of neurons. Rapsyn is highly abundant at the neuromuscular junction and thus is a genuine synaptic marker. A fusion protein of rapsyn with a genetically encoded ratiometric calcium sensor has been made to probe synapse activity. This thesis has shown that the combined use of biologically relevant system and modern fluorescence microscopy techniques deliver important information on pLGIC behaviour in the cell membrane. / <p>QC 20151217</p>