Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisors: Clifford Keil and Vincent D'Amico, Dept. of Entomology & Wildlife Ecology. Includes bibliographical references.

Orientador: Antônio Fernando Ribeiro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-23T16:58:35Z (GMT). No. of bitstreams: 1 Kmit_Arthur_M.pdf: 1478515 bytes, checksum: 34c7bf6fcce0ab6ab1b63b0927edcf98 (MD5) Previous issue date: 2013 / Resumo: O receptor purinérgico P2X7 é um canal iônico permeável a cátions pertencente da família P2X (P2X1-P2X7). Ele é ativado por altas concentrações (100?M - 1000?M) de ATP (Adenosine 5?-triphosphate), apresentando duas distintas respostas: 1) uma rápida ativação do canal, 2) uma segunda ativação, lenta e continua, que gera largos poros na membrana celular, permeáveis a grande moléculas (900 Da). O receptor P2X7 está envolvido em processos como morte celular, formação de células gigantes e secreção de citocinas como IL-1? e está predominantemente presente em células imunes. Neste estudo foi examinado como as altas correntes do P2X7 são geradas e qual o mecanismo necessário para serem ativadas em oócitos de Xenopus laevis. Os oócitos foram cirurgicamente retirados de uma rã adulta de Xenopus laevis e injetamos o cRNA do P2X7 para expressa-los na membrana celular. Medimos a condutância através da técnica de Voltage Clamp (TEVC). A incubação dos Oócitos superexpressos com P2X7 em BABTA-AM demonstrou que o Ca2+ extracelular, e não intracelular, é necessário para gerar altas correntes não seletivas através do P2X7, e a reposição de íons extracelular (Cl- e Na+) demonstrou regula-las. A mutação de truncamento da cauda C-terminal na proteína P2X7 gerou uma corrente menor após a estimulação com 1mM de ATP. E ainda três bloqueadores de canais o Ácido Tânico, o AO1 e o NPPB inibiram significativamente as correntes geradas pelo P2X7. Nós concluímos que (i) Os oócitos de Xenopus que expressam P2X7 produzem altas correntes não seletivas após estimulação com ATP, (ii) A ativação do P2X7 requer tanto o influxo de Ca2+ e a cauda C-terminal, e (iii) as correntes do P2X7 são regulados por cátions e ânions extracelulares / Abstract: The purinergic P2X7 receptor is an ion channel permeable to cations which belong to the P2X family (P2X1-P2X7). It is activated by high concentrations (100?M - 1000?M) of ATP (adenosine 5'-triphosphate), presenting two distinct responses: 1) a rapid activation of the channel, 2) a second activation, slow and continuous, which generates a large pore in the cell membrane permeable to large molecules (900 Dalton). The P2X7 receptor is involved in processes such as cell death, giant cell formation and secretion of cytokines such as IL-1? and is present predominantly on immune cells. In this study we examined how the P2X7 high currents are generated and what is the mechanism required to be activated in Xenopus laevis oocytes. Oocytes were surgically removed from an adult frog Xenopus laevis and injected with cRNA to express the P2X7 in the cell membrane. We measure the conductance through the Voltage Clamp technique (TEVC). Incubation of oocytes overexpressed with P2X7 receptors in BABTA-AM demonstrated that extracellular Ca2+, and do not intracellular, it is necessary to generate nonselective high currents through P2X7, and replacing extracellular ions (Cl- and Na+) showed regulate them. The truncation mutation in C-terminal tail of the P2X7 protein generated a smaller current after stimulation with 1 mM ATP. And three channel blockers Tannic Acid, AO1 and NPPB significantly inhibited the generated currents by P2X7. We conclude that (i) Xenopus oocytes expressing P2X7 produce a nonselective high currents after stimulation with ATP (ii) Activation of the P2X7 requires both the influx of Ca2+ and C-terminal tail, and (iii) the currents of the P2X7 are regulated by extracellular cations and anions / Mestrado / Saude da Criança e do Adolescente / Mestre em Ciências

Nematodes of the genus Ascaris are large gastrointestinal parasites. Ascaris lumbricoides infects ~1 billion people globally; causing malnutrition and general morbidity, and can block the gut or bile duct causing fatal complications. Ascaris suum is a parasite of pigs; in addition to its veterinary significance, it can occasionally be zoonotic, and is a good model of the human parasite. One of the main classes of drugs used to treat parasitic nematode infections are the cholinergic anthelmintics, such as levamisole and pyrantel, which act as agonists of nicotinic acetylcholine receptors at the nematode neuromuscular junction.

Pannexins are vertebrate proteins with limited sequence homology to the invertebrate gap junction proteins, the innexins. However, in contrast to innexins and the vertebrate connexins, pannexins do not form gap junction channels. Instead they appear to solely function as unpaired membrane channels allowing the flux of molecules, including ATP, across the plasma membrane. We provided additional evidence for their ATP release function by demonstrating that the connexin mimetic peptides, which were thought to inhibit ATP release through connexin channels, do not inhibit their host connexin channels but instead inhibit pannexin1 channels by a mechanism of steric block. Therefore, the inhibitory effects of mimetic peptides on ATP release may represent supporting evidence for a role of pannexin1 in ATP release. We also analyzed the pore structure of pannexin1 channels with the Substituted Cysteine Accessibility Method. The thiol reagents MBB and MTSET reacted with several positions in the external portion of the first transmembrane segment and the first extracellular loop. In addition, MTSET reactivity was found in the internal portion of TM3. These data suggest that portions of TM1, E1 and TM3 line the pore of pannexin1 channels. Thus, the pore structure of pannexin1 is similar to that of connexin channels.

Significant advancements in the field of membrane protein crystallography have provided in recent years invaluable images of transporter structures. These structures, however, are static and require complementary kinetic insight to understand how their mechanisms work. Electrophysiological studies of transporters permit the high quality kinetic measurements desired, but there are significant difficulties involved in analyzing and interpreting the data. Current methods allow a variety of kinetic parameters to be measured but there is a disconnect between these parameters and a fundamental understanding of the carrier. The intent of this research was to contribute new tools for studying the electrogenic kinetics of membrane transport proteins, to understand the link between these kinetics and the carrier, and to ultimately understand the mechanisms involved in transport. In this vein, two projects are explored covering two important kinetic time domains, transient and steady-state. The transient project studies the conformational changes of the unloaded carrier of SGLT1 through a multi-exponential analysis of the transient currents. Crystal structures have potentially identified a gated rocker-switch mechanism and the transient kinetics are used to support and study this kinetically. A protocol taking advantage of multiple holding potentials is used to measure the decay time constants and charge movements for voltage jumps from both hyperpolarizing and depolarizing directions. These directional measurements provide insight into the arrangement of the observed transitions through directional inequalities in charge movement, by considering the potential for a slow transition to hide a faster one. Ultimately, four carrier decays are observed that align with the gated rocker-switch mechanism and can be associated one-to-one with the movement of a gate and pore on each side of the membrane. The steady-state project considers a general theoretical model of transporter cycling. Recursive patterns are identified in the steady-state velocity equation that lead to a broad understanding of its geometric properties as a function of voltage and substrate concentration. This results in a simple phenomenological method for characterizing the I–V curves and for measuring the kinetics of rate limiting patterns in the loop, which we find are the basic structures revealed by the steady-state velocity.

Significant advancements in the field of membrane protein crystallography have provided in recent years invaluable images of transporter structures. These structures, however, are static and require complementary kinetic insight to understand how their mechanisms work. Electrophysiological studies of transporters permit the high quality kinetic measurements desired, but there are significant difficulties involved in analyzing and interpreting the data. Current methods allow a variety of kinetic parameters to be measured but there is a disconnect between these parameters and a fundamental understanding of the carrier. The intent of this research was to contribute new tools for studying the electrogenic kinetics of membrane transport proteins, to understand the link between these kinetics and the carrier, and to ultimately understand the mechanisms involved in transport. In this vein, two projects are explored covering two important kinetic time domains, transient and steady-state. The transient project studies the conformational changes of the unloaded carrier of SGLT1 through a multi-exponential analysis of the transient currents. Crystal structures have potentially identified a gated rocker-switch mechanism and the transient kinetics are used to support and study this kinetically. A protocol taking advantage of multiple holding potentials is used to measure the decay time constants and charge movements for voltage jumps from both hyperpolarizing and depolarizing directions. These directional measurements provide insight into the arrangement of the observed transitions through directional inequalities in charge movement, by considering the potential for a slow transition to hide a faster one. Ultimately, four carrier decays are observed that align with the gated rocker-switch mechanism and can be associated one-to-one with the movement of a gate and pore on each side of the membrane. The steady-state project considers a general theoretical model of transporter cycling. Recursive patterns are identified in the steady-state velocity equation that lead to a broad understanding of its geometric properties as a function of voltage and substrate concentration. This results in a simple phenomenological method for characterizing the I–V curves and for measuring the kinetics of rate limiting patterns in the loop, which we find are the basic structures revealed by the steady-state velocity.

This thesis presents a neuron emulator and headstage circuit for patch clamp setups and provides simulation, measurement and verification results. The circuit implemented on a printed circuit board (PCB) is battery powered and portable. The emulator provides both passive (resting potential) and active (action potential) electrical properties of a live neuron as seen from a single electrode by using the headstage circuit. It can be used to test electrophysiological equipment such as current-clamp, voltage-clamp or patch-clamp amplifiers. The action potentials (APs) are generated with a voltage-dependent frequency controlled by a microcontroller implementing a firing range from -60 mV to -30 mV and firing frequency from 1 Hz to10 Hz. The charge released by firing the neuron is initially stored on a 110 pC capacitor. Compared to directly using a current or voltage source, this design results in a more realistic simulation of the APs generated by ionic currents in a live neuron. The measured results from a prototype demonstrate that the neuron emulator meets the design specifications and it is capable of performing voltage clamp and rate responsive current clamp functionality. Measured results using a commercial clamp amplifier are provided to confirm the emulator operation in a practical recording environment.

In the heart, electrical signaling is responsible for its rhythmicity and is necessary to initiate muscle contraction. The net electrical activity in a cardiac myocyte during a contraction cycle is observed as the action potential (AP), which describes a change in membrane potential as a function of time. In ventricular cardiac myocytes, voltage-gated sodium channels (Nav) and voltage-gated potassium channels (Kv) play antagonistic roles in shaping the AP with the former initiating membrane depolarization and the latter repolarizing it. Functional changes in the primary cardiac Nav isoform, Nav 1.5, or any one of the many Kv isoforms expressed in the ventricle, as evidenced by those characterized in various congenital and/or acquired etiologies, can lead to severe cardiac pathologies. Nav and Kv are large transmembrane proteins that can be extensively post-translationally modified through processes that include glycosylation. The sequential glycosylation process typically ends with negatively charged sialic acid residues added through trans-Golgi sialyltransferase activity. Sialyltransferases belong to a much larger group of glycogene products that number in the hundreds and are responsible for creating a complex and variable glycan profile (glycome) unique to different cell types and tissues. Sialic acids impact Nav and Kv function likely by contributing to the extracellular surface potential and thereby causing channels to gate following smaller depolarizations. Additionally, developmentally regulated sialylation contributes to cardiac myocyte excitability in the neonatal mouse atria. However, little is understood concerning how the glycosylation machinery (glycogene products) influences cell and tissue electrical signaling. The sialytransferase Β-galactoside α-2,3-sialyltransferase 4 (ST3Gal4) adds sialic acids to galactose residues of N- and O-linked glycans through α-2,3-linkgages. ST3Gal4 is uniformly expressed throughout the chambers and developmental stages of the heart and therefore is likely a useful target to question whether and how glycosylation impacts these events. Additionally, diseases of glycosylation often cause symptoms that are consistent with changes in excitability that include arrhythmias and seizures. Congenital disorders of glycosylation lead to variably reduced glycoprotein and glycolipid glycosylation. However, because sialic acids are typically the terminal residues added to glycan structures, disease-related reduced glycosylation often leads to fewer sialic acids being attached. In addition, Chagas disease, which results in pathological changes in cardiac electrical function, may reduce sialic acids directly. Because of this, the ST3Gal4-/- strain was also used to investigate the role of glycosylation in the pathological cardiac electrical remodeling often associated with these diseases. The methodologies included cellular, tissue and whole-animal electrophysiology as well as biochemical assays. The data indicate that deletion of ST3Gal4 significantly affects Nav sialylation and gating with no change in maximum current density or protein expression. ST3Gal4 deletion also depolarizes the activation gating of both voltage-dependent kinetic components of repolarization found in the mouse ventricle: Ito and IKslow; however unlike the effect on INa, ST3Gal4 gene deletion causes a reduction in the peak IK density. Protein expression of the putative Kv isoforms responsible for Ito and IKslow was variably affected by ST3Gal4 gene deletion with Kv1.5 and Kv4.2 demonstrating no differences in protein densities. Contrastingly, a small but significant reduction in Kv2.1 protein from ST3Gal4-/- ventricular tissue was observed. In addition to effects on Nav and Kv activity, ST3Gal4 expression is necessary for normal cellular electrical signaling as demonstrated by a reduction in cellular refractory period and alterations in AP waveforms that include a slowing of cellular conduction and an extension of AP duration in ventricular myocytes from ST3Gal4-/- mice. Concurrent with aberrant excitability at the cellular level, the ST3Gal4-/- left ventricular epicardium demonstrated a reduced refractory period and was more susceptible to arrhythmias as observed through optical mapping studies. Additionally, ECGs of ambulatory ST3Gal4-/- mice demonstrated that deletion of the gene causes modest aberrant conduction under basal conditions and, in preliminary studies, appears to increase susceptibility to arrhythmias following a cardiac challenge, in the form of a low dosage of the Β-adrenergic agonist isoproterenol, suggesting a reduction in repolarization reserve in ST3Gal4 hearts. Based on the data reported here, it is apparent that relatively minor perturbations in the cardiac glycome cause significant changes in cardiac electrical signaling. These data highlight the role of glycosylation in normal physiology and underscore it as an important mediator in diseases where it may be altered.

The interaural level difference (ILD) is a sound localization cue first computed in the lateral superior olive (LSO) by comparing the loudness of sounds between the two ears. In the auditory cortex, one class of neurons is excited by contralateral but not ipsilateral monaural sounds. These "EO" neurons prefer ILDs where contralateral sounds are louder than ipsilateral sounds. Another class, the "PB" neurons, are unresponsive to monaural sounds but respond predominantly to binaural ILDs, when both ears receive simultaneous sounds of roughly equal loudness (0 ILD). Behavioral studies show that ILD sensitivity is invariant to increasing sound levels. However, in the LSO, ILD response functions shift towards the excitatory ear as sound level increases, indicating level-dependence. Thus, changes in firing rate can indicate either a change in sound location or sound level, or both. This suggests a transformation in level-sensitivity between the LSO and the perception of sound sources, yet the location of this transformation remains unknown. I performed recordings in the auditory cortex of the rat to test whether neurons were invariant to overall sound level. I found that with increasing sound levels, ILD responses were level-dependent, suggesting that level invariance of ILD sensitivity is not present in the rat auditory cortex. In general, neurons follow one of two processing strategies. The tuning of cortical cells typically follows the "inheritance strategy", such that the spiking output of the cell matches that of the excitatory synaptic input. However, cortical tuning can be modified by inhibition in the "local processing strategy". In this case, neurons are prevented from spiking at non-preferred stimuli by inhibition that overwhelms excitation. The tuning strategy of cortical neurons to ILD remains unknown. I performed whole-cell recordings in the anesthetized rat and compared the spiking output with synaptic inputs to ILDs within the same neurons. I found that the PB neurons showed evidence of the local processing strategy, which is a novel role for cortical inhibition, whereas the EO neurons utilized the inheritance strategy. This result suggests that an auditory cortical circuit computes sensitivity for midline ILDs. This dissertation includes previously published/unpublished co-authored material.

Sodium ion (Na+) channels play an important role in excitable cells, as they are responsible for the initiation of action potentials. Understanding the electrical characteristics of sodium channels is essential in predicting their behavior under different physiological conditions. We investigated several Markov models for the human cardiac sodium channel (NaV1.5) to derive a minimal mathematical model that can describe the reported experimental data obtained using major voltage-clamp protocols. We obtained simulation results for current-voltage relationships, steady-state inactivation, the voltage dependence of normalized ion channel conductance; activation and deactivation, fast and slow inactivation and recovery from inactivation kinetics. Good agreement with the experimental data provides us with the mechanisms of the fast and slow inactivation of the human sodium channel and the coupling of its inactivation states to the closed and open states in the activation pathway.