Acid-sensing ion channels (ASICs) are proton-gated cation channels expressed throughout the nervous system. These channels are activated by acidic pH conditions within an attainable physiologic range. The specific function of these channels has proven to be elusive, but it is clear that they are involved in various neuronal processes, both in the central nervous system as well as in the periphery.In order to further study the functions of these channels in an animal model system, transgenic animals were generated that overexpress individual ASIC subunits: ASIC2a and ASIC3. Transgenic proteins were detectable in brain and peripheral nervous tissue, and each had differential effects on acid-gated current properties in cultured neurons.Transgenes included N-terminal epitope tags to distinguish from endogenous ASICs, and expression was driven by a pan-neuronal promoter. Mechanical thermal sensory behaviors were tested in the transgenic mice. However, no effect was observed in these behaviors. The most interesting effect of overexpressing ASIC3 was the resulting impairment of conditioned fear behaviors in the transgenic animals without effect on unconditioned fear. ASIC3 transgenic behave like ASIC1a knockout mice in conditioned fear behaviors. Transgenic ASIC3 interacts with endogenous ASIC1, and is likely altering subunit composition of ASIC channels in the brain without abolishing proton-gated currenst like in the ASIC1a knockout. Overexpressing these two ASIC subunits in transgenic animals has produced tools that may be used to further study the functions of these channels. While this still is an artificial setting for studying ASIC functions, it nonetheless provides an in vivo method to study the effects of altering subunit composition in a whole animal and its behavioral effects, as well as in vivo expression of transgenes that can be studies biochemically. It is hopeful that studying localization in the transgenic mice will afford a better understanding of the localization and function of endogenous channels without the limitations of generating antibodies against endogenous mouse ASIC proteins, which is still in progress.

The variability of the postsynaptic response following a presynaptic action potential arises from: i) the neurotransmitter release being probabilistic and ii) channels in the postsynaptic cell involved in the response to neurotransmitter release, having stochastic properties. Spike timing dependent plasticity (STDP) is a form of plasticity that exhibits LTP or LTD depending on the precise order and timing of the firing of the synaptic cells. STDP plays a role in fundamental tasks such as learning and memory, thus understanding and characterizing the effect variability in synaptic transmission has on STDP is essential. To that end a model incorporating both forms of variability was constructed. It was shown that ion channel stochasticity increased the magnitude of maximal potentiation, increased the window of potentiation and severely reduced the post-LTP associated LTD in the STDP curves. The variability due to short term plasticity decreased the magnitude of maximal potentiation.

The variability of the postsynaptic response following a presynaptic action potential arises from: i) the neurotransmitter release being probabilistic and ii) channels in the postsynaptic cell involved in the response to neurotransmitter release, having stochastic properties. Spike timing dependent plasticity (STDP) is a form of plasticity that exhibits LTP or LTD depending on the precise order and timing of the firing of the synaptic cells. STDP plays a role in fundamental tasks such as learning and memory, thus understanding and characterizing the effect variability in synaptic transmission has on STDP is essential. To that end a model incorporating both forms of variability was constructed. It was shown that ion channel stochasticity increased the magnitude of maximal potentiation, increased the window of potentiation and severely reduced the post-LTP associated LTD in the STDP curves. The variability due to short term plasticity decreased the magnitude of maximal potentiation.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 108-119).

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 68-82).

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.

Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate. Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors. Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ion channel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions. / text

Capillary electrophoresis (CE) is an excellent analytical separation method with promising features such as small sample volumes (µL to pL), fast analysis times (s), high selectivity and efficiency, and excellent compatibility with biological samples. However, the inability of conventional CE detectors to sense biologically active compounds that are optically and electrochemically inactive limits their use for biosensing and drug screening. We have developed a highly stable electrophysiological detection platform consisting of ion channel (IC) reconstituted in synthetic bilayer membrane also known as black lipid membranes (BLM) suspended across a functionalized microaperture to be coupled to a high resolution capillary separation channel. Low energy surface modifiers were used to drastically improve the electrical, mechanical, and temporal stability of BLMs. Glass microapertures modified using tridecafluoro 1, 1, 2, 2-tetrahydrodimethylchlorosilane facilitated the rapid formation of highly stable BLMs due to the amphiphobic property (H₂O/oil repellency). Furthermore, a combination of chemically modified aperture surfaces and chemical cross-linking within the lipid membrane were used to dramatically improve BLM stability. Partial cross-linking within the bilayer maintained fluidity which allowed reconstitution of ion channel proteins while maintaining the stability of BLM-IC platform. The stable BLM-IC across glass pipette aperture was coupled to microchip electrophoresis and was shown to withstand field strength (>250 V/cm) from separation channel. Additionally, planar microapertures fabricated in SU8 were used for the formation of stable BLM-IC platform towards the construction of an integrated device. The chemical properties of the SU8 supported the formation and cross-linking within polymerizable lipid or lipid bilayer doped with polymerizable methacrylate monomers. Additionally, we expressed ion channel coupled receptor fusion protein in HEK 293 cells towards the development of ion channel sensors for wide range of ligand detection in BLM sensor platforms. The pharmacology of IC functionalized with muscarinic acetyl choline (M2-K) receptor using cell based assay by patch clamp electrophysiology showed activation by acetylcholine and inhibition by atropine. Thus this platform holds a great promise as the next-generation integrated analysis system for rapid screening of biologically active compounds (eg. glucagon) in complex matrix such as whole blood and urine for the diagnosis and management of chronic disease such as diabetes.

No description available.

No description available.