Tesi sul tema "Sodium channels"

Mammalian sodium (NaV) channels are membrane proteins with potential therapeutic applications. Lack of crystal structures is the main bottleneck for studying these channels. Constructing a model of NaV channels using computational methods is an alternative way to study NaV channels and would be valuable in structure-based drug design. I constructed a homology model for NaV1.4 based on the crystal structure of bacterial counterparts. The extensive functional data for the binding of µ–conotoxin GIIIA to NaV1.4 were used to validate the model. The predictions of the binding were in good agreement with mutagenesis data. The standard binding free energy of GIIIA was also calculated from its potential of mean force (PMF) and was consistent with the experimental value. I then used the validated model to study binding of other µ-conotoxins, including KIIIA, PIIIA, and BuIIIB. The results indicated that there is a common motif for binding of µ–conotoxins to NaV1 which is useful in understanding experimental results and designing new analogues. The NaV1.4 model was also used to study the ion permeation. Linking of the residues at the selectivity filter (DEKA) with residues in the neighbouring domain was found to be important for keeping the permeation pathway open. The results revealed that there was a Na+ ion binding site inside the DEKA locus, and 1-2 Na+ ions could occupy the vestibule near the EEDD ring. These sites are separated by a low free energy barrier, suggesting inward conduction occurs when a Na+ ion in the vestibule goes over the free energy barrier and pushes the Na+ ion in the filter to the intracellular cavity, consistent with the classical knock-on mechanism. The model also provides a good description of the observed Na+/K+ selectivity. In summary, the validated model of NaV1.4 provides a reasonable platform for future studies of mammalian NaV channels and design selective analogues with therapeutic applications.

Voltage-gated sodium (Nav) channels are therapeutic targets for several disorders affecting humans, including epilepsy, neurodegeneration and neuropathic pain. Typically, drugs treating these conditions exert a use- and voltage-dependent inhibition of Na currents, an action attributed to the stabilisation of the slow inactivated state which is formed during prolonged depolarisation. The binding site has been suggested to reside in the channel pore at a site only accessible from the intracellular environment. What gives different chemicals having this action in common selectivity for certain disorders (e.g. neuroprotection versus epilepsy) remains a mystery. Several channel subtypes exist, with types Nav1.2 and Nav1.6 being major isoforms found in the brain, raising the possibility that different subtypes exhibit differential drug sensitivity. To investigate this issue and explore the site of drug action further, the action of several Nav channel modulators, including lidocaine (a local anaesthetic) and sipatrigine (a neuroprotective agent), were studied on Na currents generated by Nav1.2a and Nav1.6 channels stably expressed in cell lines, and by acutely dissociated native cells, using electrophysiological techniques. The findings indicated that different drugs have some selectivity for particular channel subtypes. In addition, to study the slow inactivated state more selectively, fast inactivation was inhibited chemically. Inhibition of this mechanism altered both normal channel function and drug action. Drug effect during external and internal application to cells was also compared. Seemingly contrary to the current hypothesis of an internal site of action, drugs were more potent following external application, suggesting that their site of action may be different from the putative intracellularly-accessible “local anaesthetic receptor”. These experimental results were tested using a mathematical simulation of drug diffusion during whole-cell voltage-clamped electrophysiological recordings, to determine whether variations in the physicochemical properties of these drugs could explain their different potencies on internal and external application.

The voltage-gated sodium channel, Nav 1.8, is known to play an important role in pain signalling. In this thesis, the functional properties and drug binding sites of wild type and mutant Nav 1.8 sodium channel currents were studied in mammalian sensory neuron-derived ND7/23 cells using whole-cell patch clamp. While the voltage-dependence of activation was similar for wild type human and rat Nay 1.8 channels, the voltage-dependence of steady-state inactivation was more hyperpolarised for hNav 1.8 compared to rNav 1.8. Furthermore, as a consequence of the different time course for inactivation between human and rat channels, inhibition during frequent stimulation was less pronounced for hNav 1.8 than for rNav 1.8. Thus, this would imply that the human channel is more inactivated at normal resting potentials, and can support higher firing frequencies than the rat channel. The action of tetracaine, ralfmamide, 227c89, Vl02862, and Nav1. 8-selective compound A-803467 on wild type hNav 1.8 and rNav 1.8 channels was studied. All compounds showed preferential block of inactivated channels rather than resting channels. Compound A-803467 showed greater affinity for inactivated hNav 1.8 channels than for inactivated rNav 1.8 channels. Unexpectedly, an increase in current was observed for V102862 and A-803467 during recovery from inactivation, likely due to "disinhibition" of resting block. For A-803467, rather than usedependent inhibition, this disinhibition increased the current during frequent stimulation, while for VI 02862 it led to the absence of inhibition during low frequency stimulation. Thus while both V 102862 and A-803467 are potent inhibitors ofNav 1.8, V102862, rather than A-803467 might be a more useful blocker where physiological firing frequencies are higher. Alanine mutations at residues 1381, N390, L14l0, V14l4, Il706, F1710 and Y1717 were made in the pore-lining S6 segments of the hN av 1. 8 channel, and at the corresponding positions in the rNav 1.8 channel. Many of the mutations caused shifts in voltage-dependence of activation and inactivation, and gave a faster time course of inactivation, indicating that the native residues at these positions are important for both activation and inactivation in Nav 1.8 sodium channels. The affinity of tetracaine for the resting and inactivated channels was reduced by hNav1.8 mutations 138lA, F1710A and Y1717A (only inactivated state affinity was measured for the latter), and by mutation F17l0A for A-803467. For mutation L1410A both compounds caused complete resting block at very low concentrations; this block was removed by further stimulation. While tetracaine did not show disinhibition for wild type channels during recovery from inactivation, it was seen particularly for mutants L1410 and F1710A. All mutations increased the extent of disinhibition of A-803467. These results suggest that the Nav 1.8-selective compound A-803467 acts within the pore S6 segments with a differing but partially overlapping site to that of the local anaesthetic tetracaine.

The pedigree of voltage-gated sodium channels spans the millennia from eukaryotic members that initiate the action potential firing in excitable tissues to primordial ancestors that act as enviro-protective complexes in bacterial extremophiles. Eukaryotic sodium channels (eNavs) are central to electrical signaling throughout the cardiovascular and nervous systems in animals and are established clinical targets for the therapeutic management of epilepsy, cardiac arrhythmia and painful syndromes as they are inhibited by local anesthetic compounds. Alternatively, bacterial voltage-gated sodium channels (bNavs) likely regulate the survival response against extreme pH conditions, electrophiles and hypo-osmotic shock and may represent a founder of the voltage-gated cation channel family. Despite apparent differences between eNav and bNav channel physiology, gating and gene structure, the discovery that bNavs are amenable to crystallographic study opens the door for the possibility of structure-guided rational design of the next generation of therapeutics that target eNavs. Here I summarize the gating behavior of a bacterial channel NaChBac and discuss mechanisms of local anesthetic inhibition in light of the growing number of bNav structures. Also, an interesting novel observation on cross-lineage modulation of NaChBac by eNav beta subunit is reported. This auxiliary subunit modulation is isoform specific and I show the discrete effects of each isoforms on NaChBac, with functional and biochemical analysis. I also report a novel mutation that alters inactivation kinetic drastically and a possible mechanism of NaChBac inactivation is discussed.

Voltage gated sodium channels (VGSC) are integral membrane proteins that selectively transport sodium ions across cellular membranes in response to changes in membrane potential. In vertebrates sodium channels are composed of a pore forming alpha subunit and one or more auxiliary beta subunit. There are a total of nine different alpha subunit isoforms and four known beta subunits. In higher eukaryotes VGSCs are responsible for fast electrical signalling through the propagation of action potentials in excitable cells such as neurons and muscle. Voltage gated sodium channels due to their physiological importance are established drug targets for anticonvulsants, local anaesthetics and antiarrhythmics. They are also the target of a number of naturally produced neurotoxins that form a diverse group of compounds including polypeptides such as scorpion toxins, alkaloids such as veratridine and others such as heterocyclic guanidines. The work of this thesis has been to isolate and purify firstly a higher eukaryotic sodium channel sourced from the electroplax tissue of the eel Electrophorus electricus and secondly a prokaryotic channel (NaChBac) that was over expressed in an E. Coli cell system. Purified channels were biophysically characterised using circular dichroism spectroscopy to analyse secondary structure content. Purified NaChBac was chemically and thermally unfolded using SDS and monitored using CD. The drug mibefradil was able to bind NaChBac and increase its Tm by 4°C. Ligand binding to the Eel channel was also investigated using CD and tryptophan fluorescence to establish if secondary or tertiary structural changes could be observed upon ligand binding.

Developments in heterologous expression of ion channels and determination of toxin-channel complex structures have prompted searches for peptide drugs to treat diseases caused by dysfunctional channels. In this project, we build computational models of voltage-gated sodium (NaV) channels using the latest experimental results for the human NaV1.2 channel and analyze the binding modes, which are then applied to the NaV1.7 channel with the aim of developing selective channel blockers. Our main focus is NaV1.2 in the central nervous system and NaV1.7 in the peripheral nervous system. NaV1.7 is involved in transmission of pain signals, so drugs that inhibit its action could be used in treatment of chronic pain. However, there are no known toxin peptides that selectively block NaV1.7. Therefore, in order to use toxin peptides to treat chronic pain, we need to improve their selectivity and affinity profiles for NaV1.7, which could be achieved most rationally using computational methods and validating the results in the lab. Using the experimental structure of NaV1.2–KIIIA complex as a guide, we have designed a KIIIA analogue with enhanced binding affinity, which could be used in treatment of epilepsy. We then constructed a homology model for NaV1.7 and docked KIIIA using HADDOCK. The NaV1.7–KIIIA model was refined in molecular dynamics (MD) simulations and validated by comparing the binding mode with alanine mutation experiments. Comparison of the binding modes of NaV1.7–KIIIA and NaV1.2–KIIIA complexes provided valuable information on how to reduce its affinity for NaV1.2 while increasing that for NaV1.7. We predicted several mutations on KIIIA that could improve the NaV1.7/NaV1.2 selectivity and confirmed the stability of the KIIIA analogues in complex with NaV1.7 in MD simulations. Our integrative protocol will be useful for rational design of novel peptide-based drugs targeting specific NaV channels.

All voluntary movements in mammals are accomplished by muscles which are controlled by the brain through a specialized synapse called neuromuscular junction (NMJ). To have full control over the movement disorders caused due to dysfunctions of neuromuscular junction (e.g., Myasthenia Gravis, Lambert-Eaton Myasthenic Syndrome) and for the engineering of rehabilitation neuroprosthetic devices, a clear knowledge about the neuromuscular junction transmission mechanism during the communication between the neuron and the muscle is essential. The NMJ is the place where the axon terminal of a motor neuron and the motor endplate of a muscle fiber meet creating a synapse. The pulse carried by the presynaptic neuron when reaches the synapse causes release of Acetylcholine (ACh) into the synaptic cleft which binds to the AChR at the motor end plate causing the sodium ions to flow in and potassium ions to flow out of the muscle's cytosol producing a local depolarization of the motor end plate called end-plate synaptic potential. The extracellular potential in the narrow extracellular space between nerve cell and muscle fiber (cleft) is affected by the current flow generated by the opening of sodium channels; consequently, voltage-dependent processes may be affected in the adjacent membranes. This phenomenon of current passing through weakly opened sodium channels and decreasing the potential of the extracellular space by boosting channels activation and by a positive feedback is termed “self-gating”. According to this hypothesis applied to NMJ, the extracellular voltage drop in the cleft may modify the voltages across the muscle fiber membrane, thus increasing the probability of opening the voltage-gated ion channels. In the last few decades some mathematical models of NMJ have been derived considering different aspects. But in the existing models the effect of voltage in the cleft is left unnoticed. The proposed model incorporates the voltage of the synaptic cleft and self-gating effect of the sodium channel at the NMJ to study for the NMJ properties of rat and frog. In modeling the NMJ a barrel shaped muscle is considered (50 µm diameter × 500 µm long). To form a synapse, an axon terminal is connected to the highly-excitable muscle fiber with a synaptic cleft (distance: 50 nm). The potential at the cleft (Vj) is calculated using the sodium conductance of the endplate motoneuron (global conductance) and the resistance of the cleft. The Acetylcholine Receptor current (AChR) together with the endplate sodium current (EPINa) act as stimulus current for the Hodgkin-Huxley based membrane potential (Vm) calculation at the muscle fiber. The conductance associated to the voltage-gated sodium channels depends on the voltage drop between the potentials of the junction and the membrane. The junction potential calculated for different membrane potentials shows that the voltage-dependent sodium conductance at the NMJ demonstrates self-gating behavior. This is mainly due to the drop of junction potential in the synaptic cleft caused by depolarization of the muscle membrane. We have studied the activation, inactivation, voltage and time dependency of recovery from inactivation of sodium channel and voltage and time dependent Hodgkin-Huxley model parameters under voltage clamp condition for both self-gating and non-self-gating states to check out the effect of junction potential drop in the muscle membrane. Some action potential generation based performance i.e., Overshoot, Maximum Rate of Rise have been evaluated. Implications for the firing frequency have been addressed. A Radius-Conductance factor has been proposed to get a clear idea about the relation among the size of the radius of the motorneuron, maximum sodium conductance and the AChR based stimulus current. The proposed transmission model considering the contribution of sodium channels at the muscle membrane infolds and extracellular voltages in the cleft detect the ability of neuromuscular transmission to remain effective under various physiological conditions and stresses (safety-factor) of the NMJ around 15%. During the self-gating condition a switching behavior was noticed in the sodium channels conductance: from low to high during depolarization and from high to low during repolarization of membrane potential. At this time bistability and hysteresis of sodium channels occurred which explains the transmission efficiency of the NMJ under self-gating condition. The remedies of several NMJ diseases (i. e., Myasthenia Gravis, Lembert-Eaton Myasthenic Syndrome) are also hampered by limited understanding of the transmission process at the NMJ. The proposed model explores the transmission mechanism at the NMJ and may be used to plan therapy for such diseases. However, more experimental validations have to be performed before implementing it at clinical level. Also, the model may contribute to the engineering of bio-inspired neuroprosthetic devices for rehabilitation and therapy in patients with motor impairments.
I movimenti volontari nel muscolo di mammifero sono controllati dal cervello tramite una sinapsi specializzata: la giunzione neuro-muscolare (NMJ). Al fine di comprendere nel dettaglio le origini fisiopatologiche di disfunzioni di questa giunzione (ad es. Miastenia Gravis, Sindrome miastenica di Lambert-Eaton) e per favorire l’ingegnerizzazione di raffinate neuroprotesi a scopo riabilititativo, è necessario approfondire le nostre conoscenze sull’esatto meccanismo di comunicazione tra neurone e muscolo a livello dell NMJ. La NMJ è la sede in cui il terminale assonico di un motoneurone e la membrana post-sinaptica della fibra muscolare si giustappongono per creare una sinapsi. L’impulso inviato dal neurone pre-sinaptico causa un rilascio del neurotrasmettitore, l’acetilcolina, nella fessura sinaptica che, a sua volta, induce un potenziale intracellulare post-sinaptico nella fibra muscolare. Il potenziale extracellulare nello spazio sinaptico è influenzato dalla corrente ionica generata dall’apertura di canali del sodio voltaggio-dipendenti localizzati nelle pieghe post-giunzionali della NMJ (v. dopo). Di conseguenza, il gating voltaggio-dipendente degli stessi canali può essere influenzato dal voltaggio extracellulare. Questo fenomeno in cui correnti che passano attraverso canali del sodio diminuendo il potenziale extracellulare e attivando i canali con meccanismo a feed-back positivo, viene chaimato “self-gating”. Secondo questa ipotesi, applicata alla NMJ, la caduta del voltaggio extracellulare potrebbe modificare il potenziale transmemebrana a livello della fibra e aumentare la probabilità di apertura dei canali del sodio. Negli ultimi decenni sono stati proposti alcuni modelli matematici della NMJ, ma nessuno ha considerato il potenziale ruolo del potenziale extracellulare nello spazio sinaptico della giunzione. Il modello proposato in questa tesi prende in considerazione questo effetto, simulando il comportamento della NMJ nel caso del ratto e della rana. Nel simulare la NMJ, si è presa in considerazione una fibra di forma tubulare (diametro 50 µm e longhezza 500 µm). La sinapsi è costituita da un terminale assonico che si affaccia sulla membrana della fibra muscolare con una distanza di 50 nm. Il potenziale della giunzione (Vj) è calcolato usando la conduttanza del sodio della membrana post-sinaptica (conduttanza globale) e la resitenza della fessura sinaptica. La corrente del sodio nella membrana post-sinaptica (EPINa) è calcolata usando il ptenziale della giunzione e la conduttanza globale. Il recettore per l’acetilcolina (AChR) assieme con la EPINa agisce come corrente di stimolo per la fibra che viene simulata, da un punto di vista elettrico, mediante un modello di tipo Hodgkin-Huxley. La conduttanza associata ai canali voltaggio dipendenti dipende dalla caduta di potenziale tra l’esterno e l’interno della membrana post-sinaptica. Il potenziale nello spazio sinaptico della NMJ calcolato per diversi potenziali di membrana dimostra che la conduttanza dei canali voltaggio dipendenti del sodio ha un comportamento compatibile con un effetto di self-gating. Abbiamo studiato l’attivazione, l’inattivazione e il recupero dall’inattivazione (e dei parametri Hodgkin-Huxley ad essi legati) in condizioni normali (di self-gating) e assumendo uno spazio sinaptico con distanza infinita (condizioni di non self-gating). In tal modo, abbiamo verificato l’effetto del potenziale extracellulare sul gating dei canali e sulla trasmissione sinaptica focalizzandoci su parametri del potenziale d’azione generato nella fibra muscolare quali l’overshoot, la massima velocità di salita e la frequenza di firing. E’ stato anche introdotto un fattore Raggio-Conduttanza per rendere più chiara e immediata la relazione tra la dimensione del raggio del terminale assonico del motoneurone, la conduttanza massima del sodio e la corrente di stimolo dovuta agli AChR. La dipendenza dal raggio della bistabilità dei canali è stata simulata. Il modello di trasmissione proposto, considerando il contributo dei canali del sodio nelle pieghe post-giunzionali e il potenziale extracellulare nella fessura sinaptica, dimostra la capacità della trasmissione neuromuscolare di rimanere efficace in varie condizioni fisiologiche e di stress, con un fattore di sicurezza (safety-factor) del 15%. In condizioni di self-gating si può notare un comportamento di switching della conduttanza del sodio: da bassa a alta, durante la depolarizzazione e da alta a bassa durante la ripolarizzazione della fibra. Durante questo processo, si evidenziano bistabilità ed isteresi dei canali del sodio. Le terapie per diverse patologie della NMJ (ad es. Miastenia Gravis, sindorme miastenica di Lambert-Eaton) sono limitate da incompleta caratterizzazione dei meccanismi che governano la trasmissione tra motoneurone e muscolo. Il modello qui proposto esplora il meccanismo di trasmissione nella NMJ e potrebbe essere usato in futuro per migliorare le terapie di queste patologie. Tuttavia, ulteriori validazioni sperimentali dovranno essere condotte. Inoltre, il modello potrebbe ispirare nuove e più efficienti neuroprotesi per pazienti con disabilità motorie.

The epithelial sodium channel (ENaC) is a key regulator of salt homeostasis. The classic ENaC consists of three subunits: α, β and γ, which are highly expressed in the kidney and colon where they mediate electrogenic Na⁺ influx into cells under the tight hormonal regulation of aldosterone. A fourth ENaC subunit named [delta]ENaC also generates Na⁺ influx with the β- and γENaC subunits in Xenopus oocytes. However [delta]ENaC differs to the other subunits in its channel properties and tissue distribution, suggesting that [delta]ENaC may possess a physiological role other than salt regulation. A copper-toxicosis related protein called COMMD1/Murr1 was previously identified to directly interact with [delta]ENaC and downregulate [delta]ENaC activity. COMMD1 is linked with multiple ubiquitination pathways, therefore we hypothesised that COMMD1 directly interacts with [delta]ENaC through novel protein-protein interaction motifs and promotes internalisation of [delta]ENaC from the cell surface through enhanced ubiquitination. With the use of GST pulldown assays and coimmunoprecipitation, it was found that the binding of COMMD1 to [delta]ENaC is mediated by the COMM domain of COMMD1, primarily through amino acids 120-150 of COMMD1. Immunocytochemical studies showed that the intracellular interaction between [delta]ENaC and COMMD1 predominantly occurred in the early and recycling endosomes, suggesting that COMMD1 may promote the retrieval of [delta]ENaC from the cell surface to the intracellular pool. COMMD1 mediated a decrease in the [delta]ENaC cell surface population, as shown by a biotinylation surface labelling assay. This may be driven by an ubiquitin-regulated endocytosis, as COMMD1 increased ubiquitination, but not proteasomal/lysosomal degradation, of [delta]ENaC. COMMD1 may promote [delta]ENaC ubiquitination through the action of the ubiquitin ligase Nedd4-2 as coexpression with Nedd4-2 enhanced the COMMD1-mediated decrease in surface [delta]ENaC expression. This is abolished by the addition of the Nedd4-2 downregulator kinase sgk1, suggesting that COMMD1 may downregulate [delta]ENaC through the Nedd4-2/sgk1 pathway. Surface levels of [delta]ENaC may also be affected by XIAP, a RING domain ubiquitin ligase which is able to decrease the levels of COMMD1. Coimmunoprecipitation of endogenous [delta]ENaC and COMMD1 proteins, and the enhanced colocalisation of endogenous [delta]ENaC in the recycling endosomes with transfected COMMD1, indicate that interaction between transfected [delta]ENaC and COMMD1 reflect the intracellular interactions of the endogenous proteins. Taken together, these findings suggest that COMMD1 downregulates [delta]ENaC activity by promoting the internalisation of surface [delta]ENaC into early and recycling endosomes and this may be mediated by enhanced [delta]ENaC ubiquitination via the ubiquitin ligase Nedd4-2.

Chronic pain is a highly unmet clinical need; often treatment is only partially effective, and is frequently accompanied by unpleasant side effects. The voltage-gated sodium channel Nav1. 7 has emerged as an attractive target in the treatment of pain. Expressed preferentially in nociceptive DRG neurons, Nav1.7 functions at the plasma membrane to transmit peripherally generated pain signals towards the central nervous system. Manipulating Nav1.7 trafficking mechanisms to reduce the cell surface density of the channel is thus a promising approach to reduce the sensation of pain, yet there are no previous reports on Nay1.7 trafficking mechanisms. To allow investigation of human Nav1.7 trafficking mechanisms, epitope tags were inserted into the extracellular domains of the channel. Tagged channels expressed within the cells, but lacked surface expression when assayed by immunostaining. chemiluminescence assays and electrophysiology. Studies then focussed on the role of individual cytosolic domains of Nav1.7 - which harbour a number of consensus trafficking motifs- in the trafficking of the channel. Peptide domains of Nav1.7 were employed in a peptide competition assay to screen the domains for a dominant negative effect on the trafficking of the channel. Results from these assays were inconclusive. Internalisation of the full-length channel was demonstrated using a surface biotinylation assay. Chimeras of C04 and three of the cytosolic domains of Na,,1.7 were constructed to investigate the underlying mechanism of channel endocytosis. Using immunostaining assays, all three domains were shown to promote internalisation of C04, consistent with the presence of consensus endocytosis motifs in each. Furthermore, the Na,,1.7-C-terminal-CD4 chimera was shown to co-Iocalise with markers of early endosomes following endocytosis. These data provide the first evidence of endocytosis of Na.,.1.7 and have implicated a role for the cytosolic domains of the channel in regulating this process. Further elucidation of these mechanisms could reveal novel targets for the treatment of pain.

COORDENAÇÃO DE APERFEIÇOAMENTO DO PESSOAL DE ENSINO SUPERIOR
PROGRAMA DE SUPORTE À PÓS-GRADUAÇÃO DE INSTS. DE ENSINO
O presente trabalho apresenta uma revisão sobre os Canais Iônicos de Sódio e de Potássio fazendo uma relacão com os aspectos biofísicos, computacionais e matemáticos. Estudamos os canais iônicos, sob o ponto de vista da Mecânica Estatìstica, mais precisamente, considerando os canais iônicos como um processo markoviano. O objetivo principal deste trabalho é representar a dinâmica dos canais iônicos utilizando a caminhada aleatória e elaborar programas computacionais que simulem o funcionamento dos mesmos. Para tanto, estudamos a partida de íons do início do canal (fonte) e a sua chegada em um sítio final (sorvedouro) a uma distância D. Para a simulacão dos Canais de Sódio e de Potássio foram consideradas as massas atômicas dos referentes elementos químicos, não foram levados em consideracão, a dinâmica de ativacão e inativacão dos canais, assim como, a estrutura celular. A importância do presente trabalho está em generalizar o estudo dos canais iônicos e assim, buscar parâmetros que possam diferenciálos. Consideramos também o parâmetro do tempo, pois entendemos a sua importãncia para a compreensão do comportamento dos sistemas biológicos. Por fim, a dissertacão é encerrada com alguns resultados da simulacão do Canal de Sódio e de Potássio além de propor ao leitor alguns trabalhos futuros.
This dissertation work presents an overview of ion channels for sodium and potassium for making a relationship with the biophysical, computational and mathematical. Studied in a qualitative way ion channels from the point of view of statistical mechanics, more precisely, considering the ion channel as a Markov process. The main objective of this work is to represent the dynamics of ion channels using the random walk and develop computer programs that simulate the operation thereof. We studied the departure of ions from the beginning of the channel (source) and its final arrival at a site (sink) a distance D. For the simulation of the channels for sodium and potassium were considered for the atomic masses of related chemicals, were not taken into account, the dynamic activation and inactivation of the channels, as well as the cellular structure. The importance of this work is to generalize the study of ion channels and thus seek parameters that could differentiate them. We also consider the importance of time, because we understand that real-time analysis is an important tool for understanding the behavior of biological systems. Finally, the essay ends with some simulation results of the channel for sodium and potassium to the reader and propose some future work.

Luminal fluid homeostasis in the respiratory system is crucial to maintain the gas- blood exchange in normal lungs and mucociliary clearance in the airways. Epithelial sodium channels (ENaC) govern ~70% of alveolar fluid clearance. Four ENaC subunits have been cloned, namely, α, β, γ, and δ ENaC subunits in mammalian cells. This critical review focuses on the expression and function of ENaC in human and murine lungs, and the post-translational regulation by fibrinolysins. Nebulized urokinase was intratracheally delivered for clinical models of lung injury with unknown mechanisms. The central hypothesis is that proteolytically cleaved ENaC channels composed of four subunits are essential pathways to maintain fluid homeostasis in the airspaces, and that fibrinolysins are potential pharmaceutical ENaC activators to resolve edema fluid. This hypothesis is strongly supported by our following observations: 1) δ ENaC is expressed in the apical membrane of human lung epithelial cells; 2) δ ENaC physically interacts with the other three ENaC counterparts; 3) the features of αβγ ENaC channels are conferred by δ ENaC; 4) urokinase activates ENaC activity; 5) urokinase deficiency is associated with a markedly distressed pulmonary ENaC function in vivo; 6) γ ENaC is proteolytically cleaved by urokinase; 7) urokinase augments the density of opening channels at the cell surface; and 8) urokinase extends opening time of ENaC channels to the most extent. Our integrated publications laid the groundwork for an innovative concept of pulmonary transepithelial fluid clearance in both normal and diseased lungs.

The epithelial sodium channel (ENaC) is the central component of the sodium absorption pathway in epithelia. It is critical for sodium homeostasis and blood pressure control, which is demonstrated by rare genetic disorders such as Liddle�s syndrome and pseudohypoaldosteronism type I, that are associated with hyper- and hypotension, respectively. ENaC is mainly regulated by mechanisms that control the expression of active channels at the cell surface. Ubiquitin ligases of the Nedd4-like family, such as Nedd4 and Nedd4-2 decrease epithelial sodium absorption by binding to and targeting ENaC for endocytosis and degradation. This is most likely achieved by catalyzing the ubiquitination of ENaC. Conversely the serum- and glucocorticoid regulated kinase (SGK) increases ENaC activity. This effect is partly mediated by the interaction of SGK with the ubiquitin ligases Nedd4 and Nedd4-2. SGK is able to bind to both Nedd4 and Nedd4-2, however only Nedd4-2 is phosphorylated by SGK. The phosphorylation of Nedd4-2 inhibits its interaction with ENaC, thus reducing ENaC ubiquitination, thereby increasing surface expression and sodium absorption. Nedd4-like proteins interact with ENaC via their WW-domains. These domains bind PY-motifs (PPXY) present in ENaC subunits. Nedd4 and Nedd4-2 both have four highly similar WW-domains. Previous studies have shown that interaction between Nedd4 and ENaC is mainly mediated by WW-domain 3. SGK also has a PY-motif; therefore it was analyzed whether the WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK. Here, it is shown that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that, despite their high similarity, different WW-domains of Nedd4 and Nedd4-2 are involved. These data also suggest that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a concerted manner, and that in vitro the phosphorylation of SGK at serine residue 422 increases its affinity for the WW-domains of Nedd4-2. The stimulatory effect of SGK on ENaC activity is partly mediated via Nedd4-2 and will decrease if competition between Nedd4 and Nedd4-2 for binding to SGK occurs. Here it is shown that Nedd4 and Nedd4-2 are located in the same subcellular compartment and that they compete for binding to SGK. Besides its function in the proteasomal degradation pathway ubiquitination is involved in the regulation of membrane protein trafficking, including their endocytosis. ENaC was shown previously to be ubiquitinated. Here, we provide evidence that ENaC can be ubiquitinated differentially depending on its cellular location. Channels residing in the plasma membrane are multiubiquitinated and we suggest that this serves as an internalization signal for ENaC and a control for further trafficking. Cytosolic ENaC is mainly polyubiquitinated, and therefore probably targeted for proteasomal degradation. However, mono- and multiubiquitination of ENaC located within the cytosol is very likely to occur as well. In addition, it is shown that both proteasomal and lysosomal pathways are involved in the regulation of ENaC.

Thesis (Ph. D.)--Harvard University--Massachusetts Institute of Technology Division of Health Sciences and Technology, Program in Medical Engineering and Medical Physics, 1989.
On t.p. "a" is subscript.
Includes bibliographical references (leaves 165-175).
by Daniel Michael Chernoff.
Ph.D.

Book Summary: Computational models of neural networks have proven insufficient to accurately model brain function, mainly as a result of simplifications that ignore the physical reality of neuronal structure in favor of mathematically tractable algorithms and rules. Even the more biologically based "integrate and fire" and "compartmental" styles of modeling suffer from oversimplification in the former case and excessive discretization in the second. This book introduces an integrative approach to modeling neurons and neuronal circuits that retains the integrity of the biological units at all hierarchical levels. With contributions from more than 40 renowned experts, Modeling in the Neurosciences, Second Edition is essential for those interested in constructing more structured and integrative models with greater biological insight. Focusing on new mathematical and computer models, techniques, and methods, this book represents a cohesive and comprehensive treatment of various aspects of the neurosciences from the molecular to the network level. Many state-of-the-art examples illustrate how mathematical and computer modeling can contribute to the understanding of mechanisms and systems in the neurosciences. Each chapter also includes suggestions of possible refinements for future modeling in this rapidly changing and expanding field. This book will benefit and inspire the advanced modeler, and will give the beginner sufficient confidence to model a wide selection of neuronal systems at the molecular, cellular, and network levels.

Nutrient recognition is one of the main physiological roles of the gustatory system. In mammals, it is well established that the taste of sodium salts is primarily mediated by sodium influx through the epithelial sodium channel. The epithelial sodium channel is a sodium-specific ion channel that is expressed across a wide range of transporting epithelia such as colon, kidney, and taste. In addition to its role as a salt taste receptor, sodium influx through the epithelial sodium channel is important systemically for maintaining sodium balance and blood pressure. Following our earlier work on the endocrine regulation of salt taste at the level of the epithelial sodium channel, we hypothesize that the epithelial sodium channel expressed in mouse taste receptor cells plays a central role in the restoration of salt and water balance. Using a multidisciplinary approach that includes patch clamp recording, functional sodium imaging, molecular biology, Western blotting, and behavioral assays, we have begun to investigate different mechanisms of the epithelial sodium channel regulation in the taste system. In the present study, we have demonstrated a number of mechanisms that regulate the epithelial sodium channel by both ions and/or hormones in mouse taste cells. In general, three new mechanisms of the epithelial sodium channel regulation were identified: (1) regulation of the epithelial sodium channel by chloride ions, (2) regulation of the epithelial sodium channel by insulin, and (3) alterations of the epithelial sodium channel function in diabetic taste cells. To test the relevance of one or more of these regulatory mechanisms in the animals' behavior, we used a variety of short-term behavioral assays. Interestingly, the results suggested that insulin regulates salt intake in rodents, which dovetails nicely with our functional and molecular findings. Consistent with insulin's physiological role in salt taste transduction, we investigated the modification of the epithelial sodium channel function during the onset of diabetes. Diabetic rodents displayed alterations in salt taste transduction via epithelial sodium channel from the gene level to the animals' behavior. These results are an example of how regulatory cues, like hormones, act on specific transduction elements to modulate the peripheral gustatory system.

Voltage-gated sodium channels auxiliary subunits evolutionary emerged nearly 500 million years ago during the Cambrian explosion. These subunits alter one the most important ion channels to electrical signaling, the voltage-gated sodium channels support the propagation of electric impulses in animals. The mechanism for the auxiliary subunits effects on the channels is poorly understand, as is the stoichiometry between the auxiliary subunit and the channel. The focus of my thesis is to generate assays and to use these approaches to understand the interactions different types of voltage-gated channels and their auxiliary subunits. A biochemical approach was taken to identify novel interactions between the eukaryotic sodium channel auxiliary subunits and a prokaryotic voltage-gated sodium channel, a protein that diverged from the eukaryotic voltage-gated sodium channels billions of years ago. These interactions between the auxiliary subunits and channels were probed with chemical and photochemical crosslinkers in search of interaction surfaces and similarity to explain the mechanisms of interaction. The work in this thesis identified novel interactions between the voltage-gated sodium channel auxiliary subunits and voltage-gated channels that are distantly related to the voltage-gated sodium channels principally thought to be modulated by the auxiliary subunits. From this work a rudimentary concept can be theorized that the voltage-gated sodium channel β-subunits and not only β1 have a more primary role in electrophysiology by associating with multiple different types of ion channels.

The nodes of Ranvier are short, periodical interruptions in the myelin sheath of myelinated axons, at which voltage-gated sodium channels are highly concentrated. The correct targeting of sodium channels to the nodes of Ranvier permits rapid propagation of action potentials in myelinated axons. The nodes of Ranvier contain a unique set of ion channels, cell-adhesion molecules, and cytoplasmic adaptor proteins. Neurofascins are cell adhesion molecules of the immunoglobulin superfamily and previous work has shown they are involved in the assembly of the node of Ranvier. The Neurofascin (Nfasc) gene is subject to extensive alternative splicing. RT-PCR studies have suggested that there were several different Neurofascin (Nfasc) transcripts. Thus far, research on the Neurofascins has concentrated on two isoforms, Nfasc186 and Nfasc155, which are expressed in neurons and glia respectively. A third Neurofascin isoform, Nfasc140, lacking the Mucin domain and two of the fibronectin repeats was originally identified in the laboratory of V. Bennett. However, neither the location nor function of this protein was known. By RT-PCR I successfully cloned the Nfasc140 cDNA and determined its domain composition, which was confirmed by a series of Western blots using domain-specific antibodies. The developmental expression of Nfasc140 revealed that it is the predominate isoform of Neurofascin during the embryonic stage. Using cell-type-specific conditional Neurofascin knock-out mice, I have also found that Nfasc140 is a neuronal isoform, like Nfasc186. I have used transgenic mouse lines to characterize the location and function of Nfasc140. Like Nfasc186, Nfasc140 is targeted to the nodes of Ranvier and axonal initial segment. Also Nfasc140 alone can reconstitute the nodal complex in Neurofascin knock-out mice in CNS and PNS in the absence of Nfasc186 and Nfasc155. It can also partially restore the electrophysiological function of PNS nerves. In order to address the role of the paranodes in sodium channel clustering, I generated a new neuronal-Cre-expressing transgenic line which, when bred with floxed Nfasc mice, generated early neuronal Neurofascin knock-out mice. Using those animals I have shown that after the ablation of all neuronal Neurofascins, when only glial Nfasc155 is presented, sodium channels can still target to the nodes of Ranvier in both PNS and CNS. These conditional knock-out mice have a longer life span than pan-Neurofascin knock-out mice. This indicates the importance of paranodal junctions, in addition to nodal neuronal Neurofascins, in clustering sodium channels at the node.

Book Summary: With contributions from more than 40 renowned experts, Modeling in the Neurosciences: From Ionic Channels to Neural Networks is essential for those interested in neuronal modeling and quantitative neiroscience. Focusing on new mathematical and computer models, techniques and methods, this monograph represents a cohesive and comprehensive treatment of various aspects of the neurosciences from the biophysical, cellular and netwrok levels. Many state-of-the-art examples are presented as to how mathematical and computer modeling can contribute to the understanding of mechanisms and systems in the neurosciences. Each chapter also includes suggestions of possible refinements for future modeling in this rapidly changing and expanding field. This book will benefit and inspire the advanced modeler, and give the beginner sufficient confidence to model a wide selection of neuronal systems at the biophysical, cellular and network levels.

La contraction des cellules musculaires est due à la propagation d'un stimulus électrique appelé potentiel d'action (PA). Il est produit par l'ouverture séquentielle de plusieurs canaux ioniques générant des courants à travers les membranes cellulaires. Les canaux sodiques dépendant du voltage (Nav) déclenchent la première phase du PA. Les variants Nav1. 4 et Nav1. 5 sont exprimés respectivement dans le muscle et le coeur. Des mutations de leurs gènes (SCN4A et SCN5A) provoquent respectivement des pathologies neuromusculaires et des arythmies cardiaques. Mon travail de thèse a consisté à étudier les canalopathies. J'ai ainsi examiné les conséquences de deux mutations sur les propriétés biophysiques des canaux en les exprimant dans des cellules HEK293. La mutation du gène SCN4A engendrait des modifications importantes de la fonction de Nav1. 4 expliquant certains aspects de la myotonie observée chez les patients. Le patient de la deuxième étude, atteint d'un syndrome de Brugada, présentait une mutation du gène SCN5A provoquant une perte de fonction des canaux Nav1. 5 à 37°C. Enfin, nous avons identifié une nouvelle protéine impliquée dans la régulation de Nav1. 5. Les trois derniers acides aminés du C-terminus de Nav1. 5 (SIV) interagissent avec la protéine SAP97. Une réduction de l'expression de SAP97 ainsi que l'expression de canaux tronqués (sans SIV) induisent une baisse importante du courant sodique. Ces résultats suggèrent que SAP97 est impliquée dans la régulation du canal Nav1. 5. Ainsi, certaines maladies musculaires et cardiaques peuvent être la conséquence directe de mutations de canaux ioniques, mais l'action de protéines auxiliaires peut aussi affecter leur fonction
The contraction of muscles cells is due to the propagation of an electrical influx called action potential (AP). It results from the sequential opening of ion channels generating currents through the cell membranes. The voltage-gated sodium channels (Nav) are responsible for the rising phase of the AP. Nav1. 4 and Nav1. 5 are respectively the main skeletal muscle and cardiac sodium channels. Mutations in their encoding genes (SCN4A and SCN5A) lead respectively to neuromuscular disorders or cardiac arrhythmias. The general aim of my PhD has been to study channelopathies. I first characterized the effects of two mutations. I used HEK293 cells to express wild-type or mutant channels and study their biophysical properties. We found that the SCN4A mutation produced complex alterations of Nav1. 4 function, explaining the myotonic phenotype described in patients carrying the mutation. In the second study, the index case carried a SCN5A mutation that leads to a "loss of function" of the channel. The decreased sodium current (lNa) measured at 37°C with mutated Nav1. 5 channels could explain the observed Brugada syndrome. The last project aimed at identifying a new potential protein interacting with the cardiac sodium channel. We found that SAP97 binds the three last amino-acids (SIV) of the C-terminus of Nav1. 5. Silencing the expression of SAP97 as well as the truncating the SIV motif, decreased 1Na. These preliminary results suggest that SAP97 is implicated in the regulation of sodium channel directly or indirectly. These studies reveal that cardiac and muscle diseases may result from ion channel mutations but also from regulatory proteins affecting their regulation

Thesis (Ph.D.)--Indiana University, 2010.
Title from screen (viewed on April 1, 2010). Department of Pharmacology and Toxicology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Theodore R. Cummins, Grant D. Nicol, Gerry S. Oxford, Andy Hudmon, John H. Schild. Includes vitae. Includes bibliographical references (leaves 232-266).

Hyperkalemic periodic paralysis (HyperKPP) is an autosomal dominant human skeletal muscle channelopathy that causes periods of myotonic discharge and periodic paralysis due to defective Nav1.4 sodium channels. Patients are asymptomatic at birth, attacks become short and frequent during childhood, and more severe during adolescence. Since the Nav1.4 content in the cell membrane is relatively constant during childhood, it was hypothesized that some symptoms start with the defective Nav1.4 channels, while other symptoms start after some changes occur in gene expression affecting other membrane channel content and/or activity. To test the hypothesis, the contractile characteristics of EDL and soleus muscles from HyperKPP mice from the age of 0.5 to 12 months were tested in vitro. For both EDL and soleus, contractile defects, including low force generation, instability and large unstimulated force were observed by two weeks of age. With aging, the defects did not worsen, but muscles actually showed some improvement. Considering that Nav1.4 protein content reaches maximum at three weeks of age, the data suggests that HyperKPP symptoms are solely due to the defective Nav1.4 channels.