Proton fluxes through plasma membranes are essential for regulating intracellular and extracellular pH and mediating co-transport of metabolites and ions. Although conventional electrical measurements are highly sensitive and precise for proton current detection, they provide limited specificity and spatial information. My thesis focuses on developing optical approaches to visualize proton fluxes from ion channels and transporters. It has been demonstrated that channel-mediated acid extrusion causes proton depletion at the inner surface of the plasma membrane. Yet, proton dynamics at the extracellular microenvironment are still unclear. In Chapter II, we developed an optical approach to directly measure pH change in this nanodomain by covalently attaching small-molecule, fluorescent proton sensors to the cell’s glycocalyx using glyco-engineering and copper free ‘click’ chemistry. The extracellularly facing sensors enable real-time detection of proton accumulation and depletion at the plasma membrane, providing an indirect readout of channel and transporter activity that correlated with whole-cell proton current. Moreover, the proton wavefront emanating from one cell was readily visible as it crossed over nearby cells. The transport of monocarboxylates, such as lactate and pyruvate is critical for energy metabolism and is mainly mediated by proton-coupled monocarboxylate transporters (MCT1-MCT4). Although pH electrodes and intracellular fluorescent pH sensors have been widely used for measuring the transport of proton-coupled MCTs, they are unable to monitor the subcellular activities and may underestimate the transport rate due to cell’s volume and intracellular buffering. In Chapter III, we used the Chapter II approach to visualize proton-coupled transport by MCT1-transfected HEK293T cells and observed proton depletion followed by a recovery upon extracellular perfusion of L-lactate or pyruvate. In addition, we identified a putative MCT, CG11665/Hrm that is essential for autophagy during cell death in Drosophila. The results demonstrate that Hrm is a bona fide proton-coupled monocarboxylate transporter that transports pyruvate faster than lactate. Although the approach developed in Chapter II enables visualization of proton fluxes from ion channels and transporters, it’s not applicable in some cell types which cannot incorporate unnatural sialic acid precursors into their glycocalyx, such as INS-1 cells and cardiomyocytes. To address this, in Chapter IV we developed a pH-sensitive, fluorescent WGA conjugate, WGA-pHRho that binds to endogenous glycocalyx. Compared to the results in Chapter II and III, cell surface-attached WGA-pHRho has similar fluorescent signals in response to proton fluxes from proton channel Hv1, omega mutant Shaker-IR R362H and MCT1. With WGA-pHRho, we were able to label the plasma membrane of INS-cells and cardiomyocytes and visualized the transport activity of MCT1 in these cells. Taken together, these findings provide news insights into proton dynamics at the extracellular environment and provide new optical tools to visualize proton fluxes from ion channels and transporters. Moreover, the modularity of the approaches makes them adaptable to study any transport events at the plasma membrane in cells, tissues, and organisms.

Understanding the nature of adaptive evolution has been the recent focus of research detailing the genetic basis of adaptation and theoretical work describing the mechanics of adaptive evolution. Nevertheless, key questions regarding the process of adaptive evolution remain. Ultimately, a detailed description of the ecological context, evolutionary history, and genetic basis of adaptations is required to advance our understanding of adaptive evolution. To address some of the contemporary issues surrounding adaptive evolution, I examine phenotypic and genotypic changes in a snake feeding adaptation. Adaptations can arise through fixation of novel mutations or recruitment of existing variation. Some populations of the garter snakes Thamnophis sirtalis, T. couchii, and T. atratus possess elevated resistance to tetrodotoxin (TTX), the lethal toxin of their newt prey. I show that TTX resistance has evolved independently through amino acid changes at critical sites in a voltage-gated sodium channel protein (Nav1.4) targeted by TTX. Thus, adaptive evolution has occurred multiple times in garter snakes via de novo acquisition of beneficial mutations. Detailing the genetic basis of adaptive variation in natural populations is the first step towards understanding the tempo and mode of adaptive evolution. I evaluate the contribution of Nav1.4 alleles to TTX resistance in two garter snake species from central coastal California. Allelic variation in Nav1.4 explains 29% and 98% of the variation in TTX resistance in T. atratus and T. sirtalis, respectively, demonstrating that Nav1.4 is a major effect locus. The simple genetic architecture of TTX resistance in garter snakes may significantly impact the dynamics of trait change and coevolution. Patterns of convergent evolution are cited as some of the most compelling examples of the strength of natural selection in shaping organismal diversity. Yet repeated patterns may tell us as much about the constraints that restrict evolution as about the importance of natural selection. I present data on convergent molecular adaptations in parallel arms races between diverse snakes and amphibians from across the globe. Six snake species that prey on TTX bearing amphibians have independently acquired amino acid changes in Nav1.4. The derived mutations are clustered in two portions of the gene, often involving the same sites and substitutions. While a number of amino acid changes can make Nav1.4 insensitive to TTX, most of these negatively impact or abolish the ion-conducting function of the protein. Thus, intramolecular pleiotropy likely prevents most replacements from becoming fixed and imposes limits on protein evolution.

Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (VGSCs) are dynamic membrane-spanning proteins crucial for determining the electrical excitability in nerve and muscle. VGSCs transition, or gate, between opened, closed, and inactivated states, in response to changes in transmembrane potential. Altered VGSC gating can affect electrical communication and is implicated in numerous channelopathies. Nav1.7, a VGSC isoform highly expressed in the peripheral nervous system, plays a unique role in pain perception as evidenced by single point missense mutations causing a spectrum of pain syndromes (inherited erythromelalgia; IEM and paroxysmal extreme pain disorder; PEPD) and nonsense mutations resulting in human insensitivity to pain (CIP). These studies indicate Nav1.7 is critical in pain transduction and, as such, structural perturbations to Nav1.7 affecting conformational stability and response to changes in transmembrane potential have the potential to cause pain. Therefore, the aims of this dissertation were to (1) examine the effects of PEPD mutations on the voltage-dependent properties Nav1.7; (2) investigate the effects Nav1.7 alternative splicing has on the impact of IEM and PEPD mutations; (3) evaluate the effects channelopathies, resulting from slowed inactivation, have on modulating an unusual type of sodium current that flows during membrane repolarization; and (4) determine the structural components involved in stabilizing Nav1.7 inactivation. Standard patch-clamp electrophysiology was used to study changes in channel properties. Results from this dissertation demonstrate that (1) PEPD mutations significantly shift the voltage-dependent properties of Nav1.7 channels, destabilize an inactivated state in a residue specific manner, and render nociceptive neurons hyperexcitable; (2) alternative splicing can functionally impact PEPD; (3) channelopathies, resulting from slowed inactivation in neuronal and muscle VGSC isoforms, increase an unusual sodium conductance that flows during repolarization; and (4) specific residues located in distinct regions of Nav1.7 serve as docking sites to stabilize inactivation at different membrane potentials. Overall, this dissertation answers key questions regarding the molecular mechanics required during inactivation and the biophysical consequences of Nav1.7 mutations implicated in painful disorders. The results of this dissertation are important for a more detailed understanding of pain perception and validate the applicability of studying Nav1.7 for discovery of therapeutic targets for treatment of pain. – Theodore R. Cummins, Chair

A well documented laboratory setup, leech preparation process, and bio-potential data recording process are needed. Repeatability and quality data recordings are essential and thus dictate the requirements of the laboratory setup and processes listed above. Advances in technology have both helped and hindered this development. While very precise equipment is required to record the low voltage bio-potentials, noisy electronic equipment and wires surrounding the work area provide high levels of interference. Proper laboratory setup and data recording processes, however, limit the unwanted interference. Quality data can only be recorded from a properly handled and prepared leech subject. Proper setup and procedures result in quality recordings which lend a clean signal for furthering the understanding of nerve functionality. The electrophysiology lab at California Polytechnic State University in San Luis Obispo is an example of a proven lab setup for high quality signal capture.

Pathogenic variants in KCNA2, encoding for the voltage-gated potassium channel Kv1.2, have been identified as the cause for an evolving spectrum of neurological disorders. Affected individuals show early-onset developmental and epileptic encephalopathy, intellectual disability, and movement disorders resulting from cerebellar dysfunction. In addition, individuals with a milder course of epilepsy, complicated hereditary spastic paraplegia, and episodic ataxia have been reported. By analyzing phenotypic, functional, and genetic data from published reports and novel cases, we refine and further delineate phenotypic as well as functional subgroups of KCNA2-associated disorders. Carriers of variants, leading to complex and mixed channel dysfunction that are associated with a gain- and loss-of-potassium conductance, more often show early developmental abnormalities and an earlier onset of epilepsy compared to individuals with variants resulting in loss- or gain-of- function. We describe seven additional individuals harboring three known and the novel KCNA2 variants p.(Pro407Ala) and p.(Tyr417Cys). The location of variants reported here highlights the importance of the proline(405)–valine(406)–proline(407) (PVP) motif in transmembrane domain S6 as a mutational hotspot. A novel case of self-limited infantile seizures suggests a continuous clinical spectrum of KCNA2-related disorders. Our study provides further insights into the clinical spectrum, genotype–phenotype correlation, variability, and predicted functional impact of KCNA2 variants.

No description available.

In this study, four anion transporter (AT) blockers, DIDS (4, 4′-diisothiocyanatostilbene-2, 2′-disulfonic acid), 9-AC (anthracene-9-carboxylic acid), NPPB (5-nitro-2-(3-phenylpropylamino) benzoic acid), and IAA-94 (indanyloxy acetic acid) were selected to evaluate ATs as potential target sites for insect and nematode control. All the AT blockers showed slowly developing toxicity against second-stage larvae of <i>Meloidogyne incognita</i> (Kofoid and White 1919) Chitwood 1949 and adults of <i>Caenorhabditis elegans</i> Maupas 1900 but not against third-stage larvae of <i>Heterorhabditis bacteriophora</i> Poinar 1975 even at 200 ppm. Symptoms of AT blocker toxicity observed in <i>C. elegans</i> adults were increased pharyngeal muscle contractions and decreased locomotion. Exposure of <i>C. elegans</i> as fourth-stage larvae to double-stranded RNA (dsRNA) of <i>ceclc-1</i> and <i>ceclc-2</i> (VGCC genes coding for CeClC-1 and CeClC-2, respectively) either alone or together for 24 h decreased their expression in F1 progeny in a time-dependent manner. Reduction in expression of <i>ceclc-2</i> alone or together with <i>ceclc-1</i> significantly increased pharyngeal contractions and decreased locomotion in significantly higher percentage of F1 progeny. The above findings suggested AT blockers nematicidal activity primarily comes from inhibition of CeClC-2 channels, while inhibition of CeClC-1 channels may enhance this activity. All the AT blockers showed slowly developing toxicity against adults of a susceptible strain (Oregon-R) of <i>Drosophila melanogaster</i> Meigen 1830, while DIDS, was equally toxic to dieldrin-resistant rdl flies. All AT blockers, except 9-AC, at 100 µM showed significant excitatory effect on desheathed central nervous system (CNS) of third-instar larvae of <i>Drosophila</i>, while DIDS showed a modest excitatory effect on ascending peripheral nerves. Feeding adult flies on 10% sugar solution mixed with 100 ppm of DIDS for 6 h decreased the midgut pH by 2 units approximately. All the AT blockers inhibited the growth of larvae (in weight), increased the developmental time, and decreased survival when <i>Ostrinia nubilalis</i> (Hübner 1796) second-instar larvae were fed for seven days. All the AT blockers decreased the midgut alkalinity and inhibited chloride ion transport from midgut lumen into epithelia in fifth-instar larvae when fed for 3 h on treated diet. Positive correlations observed among growth, midgut alkalinity, and midgut chloride transport in AT blocker-fed larvae suggested that inhibition of chloride/bicarbonate exchangers by AT blockers may have contributed to midgut alkalinity decrease affecting the digestion and resulting in observed lethal and sublethal effects. / Ph. D.

Cardiac arrhythmias are caused by a disruption of the normal initiation or propagation of electrical impulses in the heart. Hundreds of mutations in genes encoding ion channels or ion channel regulatory proteins are linked to congenital arrhythmia syndromes that increase the risk for sudden cardiac death. This dissertation focuses on how mutations in a gene (KCNQ1) that encodes a voltage-gated K+ ion channel (Kv7.1) can disrupt proper channel function and lead to abnormal repolarization of atrial and ventricular cardiomyocytes. In the heart, Kv7.1 coassembles with a regulatory protein to conduct the slowly activating delayed rectifier K+ current (IKs). Loss-of-function KCNQ1 mutations are linked to type 1 long QT syndrome (LQT1), and typically decrease IKs, which can lead to ventricular action potential (AP) prolongation. In patients, LQT1 is often characterized by an abnormally long corrected QT (QTc) interval on an electrocardiogram (ECG), and increases the risk for polymorphic ventricular tachycardias. KCNQ1 mutations are also linked to atrial fibrillation (AF), but cause a gain-of-function phenotype that increases IKs. Surprisingly, patients diagnosed with both LQT1 and AF are increasingly identified as genotype positive for a KCNQ1 mutation. The first aim of this dissertation was to determine a unique functional phenotype of KCNQ1 mutations linked to both arrhythmia syndromes by functional analyses via the whole-cell patch clamp technique in HEK293 cells. A proportion of patients with LQT1-linked KCNQ1 mutations do not have abnormal QTc prolongation known as latent LQT1. Interestingly, exercise can reveal abnormal QTc prolongation in these patients. During exercise, beta-adrenergic activation stimulates PKA to phosphorylate Kv7.1, causing an increase in IKs to prevent ventricular AP prolongation. Therefore, the second aim of this dissertation was to determine a molecular mechanism of latent LQT1 through functional analyses in HEK293 cells while incorporating pharmacological and phosphomimetic approaches to study PKA regulation of mutant Kv7.1 channels. The findings in this dissertation provide new insight into how KCNQ1 mutations disrupt the function of Kv7.1 in a basal condition or during beta-adrenergic activation. Also, this dissertation suggests these approaches will improve patient management by identifying mutation specific risk factors for patients with KCNQ1 mutations.

Les syndromes myotoniques congénitaux atypiques dus à des mutations du canal sodé voltage-dépendant Nav1.4 se distinguent des myotonies congénitales classiques (canal chlore ClC-1) par la présence de traits atypiques incluant des myotonies douloureuses aggravées au froid et à l’ingestion de potassium. La caractérisation clinique et moléculaire de plusieurs familles atteintes de ces conditions rares dans la région du Saguenay-Lac-St-Jean nous a permis de découvrir une nouvelle mutation SCN4A à effet fondateur causant un phénotype de myotonies douloureuses aggravées au froid, parfois accompagné de phénomènes dystrophiques ou paralytiques. L’ampleur de notre cohorte nous permet de commenter sur l’hétérogénité phénotypique observée, sur les traits caractéristiques des syndromes associés au gène SCN4A, sur les implications physiologiques probables d’une telle mutation ainsi que sur les facteurs modulant le phénotype observé. Enfin, notre étude nous permet de souligner l’importance du dépistage familial systématique afin de prévenir les complications anesthésiques potentielles associées à ces conditions. / Congenital myotonic syndromes due to mutations of the voltage-gated sodium channel Nav1.4 differ from those due to mutations of the chloride channel CLC-1 as they tend to present atypical traits including painful myotonias and aggravation of symptoms with cold and potassium ingestion. Indeed, after completing the clinical and molecular characterization of a large cohort of patients affected with these rare conditions in the Saguenay Lac-St-Jean area, we were able to describe a new founder SCN4A mutation presenting with painful cold-induced myotonias and occasional dystrophic and paralytic episodes. Our study illustrates the wide phenotypic variability and the typical traits of SCN4A mutations. In addition, we were able to speculate on the probable physiological consequences of such mutations. Finally, we conclude by stressing the importance of familial screening in order to reduce the incidence of anesthetic complications associated with these conditions.

Submitted by Ana Damasceno (ana.damasceno@unipampa.edu.br) on 2016-09-13T20:01:43Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Investigação bioquímica e eletrofisiológica da atividade neurotóxica da urease de Canavalia Ensiformis sobre o sistema nervoso de mamíferos.pdf: 4036702 bytes, checksum: 7c0fc1ca67b3af15c724790f838de2ed (MD5) / Made available in DSpace on 2016-09-13T20:01:43Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Investigação bioquímica e eletrofisiológica da atividade neurotóxica da urease de Canavalia Ensiformis sobre o sistema nervoso de mamíferos.pdf: 4036702 bytes, checksum: 7c0fc1ca67b3af15c724790f838de2ed (MD5) Previous issue date: 2016 / A urease de Canavalia ensiformis (Jack Bean Urease, JBU) possui uma cadeia polipeptídica de 90.770 kDa, com 840 resíduos de aminoácidos. Do ponto de vista biotecnológico, já foram caracterizadas suas ações fungicida e inseticida. Quando administrada na corrente sanguínea de mamíferos, esta, induz convulsões que culminam em morte dos animais, um efeito cujo mecanismo de ação ainda carece de investigação. O objetivo desse trabalho foi investigar os padrões eletrofisiológicos da JBU no sistema nervoso central e periférico de roedores in vivo e in vitro, e as correlações bioquímicas em termos de viabilidade celular e exocitose do L-glutamato. Nos ensaios bioquímicos, verificou-se aumento na liberação de L-glutamato em sinaptossomas corticais de ratos na concentração de 100 nM. Os ensaios de viabilidade celular por MTT em fatias de hipocampo de camundongos não demonstraram alterações. Nos ensaios eletrofisiológicos, verificou-se redução significativa na amplitude do potencial de ação composto (CAP) de nervo ciático de camundongo, nas doses de 1 e 10 nM. Nos ensaios eletroencefalográficos em ratos a injeção da urease (10nM) no hipocampo induziu um traçado de espícula-onda. A redução na amplitude do CAP provavelmente está relacionada a uma inibição dos canais de sódio voltagem-dependentes, já que a administração posterior de tetrodotoxina, não aumentou o nível de bloqueio da condução. Nossos resultados demonstram um efeito excitatório da JBU do tipo crise de ausência sobre o sistema nervoso central de mamíferos. Este resultado sugere o envolvimento dos canais de Ca2+ do tipo T nos efeitos excitatórios da JBU. O bloqueio da condução do nervo ciático de camundongos sugere o envolvimento de canais de Na+ voltagem dependentes, o qual corroboraria a atividade excitatória da urease sobre o sistema nervoso central de mamíferos. / The Canavalia ensiformis urease (Jack Bean Urease) has a 90,770 kDa, polypeptide containing 840 aminoacid residues. JBU is known to exhibit insecticidal and fungicidal activities. When administered endovenously in mammalians, it induces tonic clonic convulsions culminating in the death of the animals. This mechanisms involved in the excitatory activity of JBU has not been elucidated so far. In this work, we sought to investigate the central and peripheral electrophysiological patterns of Jack Bean Urease in rodents, in vivo and in vitro, as well as the biochemical correlation of cell viability and glutamate release. In the biochemical assays, JBU induced increase in L-Glutamate release in rat cortical sinaptossomes, with no alteration of mice hippocampal cell viability. The electrophysiological assays, showed that JBU induce a significant decrease on mice sciatic nerve compound action potentials (CAP), and spike-wave discharges (SWD) similar to “petit mal” seizures when injected directly in the hippocampus (10 nM). The decrease in CAP amplitude is related to a blockage of voltage-gated sodium channels, since it was not affected by the concomitant application of tetrodotoxin. Our results show that JBU exerts an effect of spike wave discharges-like activity over the mammalian central nervous system. This later result suggests an involvement of T-type voltage gated calcium channels in the excitatory activity of JBU. The blockade of mouse sciatic nerve compound action potential conduction corroborates the excitatory activity of the urease upon the mammalian central nervous system.