In the absence of X-ray structure of L-type Ca²⁺ channel (LCC), we have built a homology model of LCC based on the crystal structure of KcsA channel and have performed a series 0° docking simulations. The search for lowest-energy conformations was performed by the Monte Carlo energy-minimization method. To obtain the conformation with the lowest energy where dihydropyridine (DHP) ligand forms optimal contacts with the DHP-sensing residues of the channel, we have tested different sequence alignments between KcsA and LCC, and have docked the ligand inside the pore of the channel as well as into the interface between repeats IIIS5-IIIS6-IVS6. The LCC ligand tetrandrine was used during the studies of the selectivity filter of the channel. Conformational studies of the drug and its interaction with Ca²⁺ ions in a non-polar solution were performed by NMR spectroscopy. These experiments have demonstrated the binding of Ca²⁺ ions to the ligand. In the model based on the alignment proposed by Lipkind and Fozzard (2000), the DHP ligand nifedipine fits inside the pore and forms favorable contacts with several hydrophobic DHP-sensing residues, and forms hydrogen bonds with conserved tyrosines in repeats HI and IV. These interactions stabilize the portside-down docking mode of nifedipine, in which this blocker exposes its hydrophobic methoxy group to the bracelet of hydrophobic residues forming the gate of the channel near the crossing of the bundle of helices. The stabilizes the closed state of the channel. In contrast, the agonist has the hydrophilic group at its portside. The favorable interaction of this group with hydrated Ca²⁺ ion facilitates its permeation through hydrophobic gate. We have simulated the passage of the hydrated ion along the pore with the agonist bound inside and determined several residues crucial for this passage. The role of these residues can be tested experimentally. / Thesis / Master of Science (MSc)

Dissertation (Ph.D.)--University of Toledo, 2007. / "In partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biomedical Sciences." Title from title page of PDF document. Bibliography: p. 70-78, p. 93, p. 132-140, p. 164-168, p. 194-221.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 85-100).

There is an increasing demand for power converters with small size, light weight, high conversion efficiency and higher power density. Also, in many applications, there is a need for dc-to-dc converters to accept dc input voltage and provide regulated and/or isolated dc output voltage at a desired voltage level including telecommunications equipment, process control systems, and in industry applications. This thesis presents the analysis, design, simulation and experimental results of three-phase high-frequency transformer isolated resonant converters. The first converter presented is a three-phase LCC-type dc-dc resonant converter with capacitor output filter including the effect of the magnetizing inductance of the three-phase HF transformer. The equivalent ac load resistance is derived and the converter is analyzed by using approximation analysis approach. Base on this analysis, design curves have been obtained and a design example is given. Intusoft simulation results for the designed converter are given for various input voltage and for different load conditions. The experimental verification of the designed converter performance was established by building a 300 W rated power converter and the experimental results have been given. It is shown that the converter works in zero-voltage switching (ZVS) at various input voltage and different load conditions. A three-phase (LC)(L)-type dc-dc series-resonant converter with capacitive output filter has been proposed. Operation of the converter has been presented using the operating waveforms and equivalent circuit diagrams during different intervals. An approximate analysis approach is used to analyze the converter operation, and design procedure is presented with a design example. Intusoft simulation results for the designed converter are given for input voltage and load variations. Experimental results obtained in a 300 W converter are presented. Major advantages of this converter are the leakage and magnetizing inductances of the high-frequency transformer are used as part of resonant circuit and the output rectifier voltage is clamped to the output voltage. The converter operates in soft-switching for the inverter switches for the wide variations in supply voltage and load and it requires narrow switching frequency variation (compared to LCC-type) to regulate the output voltage. A three-phase high-frequency transformer isolated interleaved (LC)(L)-type dc-dc series-resonant converter with capacitive output filter using fixed frequency control is proposed. The converter operation for different modes is presented using the operating waveforms and equivalent circuit diagrams during different intervals. This converter is modeled and then analyzed using the approximate complex ac circuit analysis approach. Based on the analysis, design curves were obtained and the design procedure is presented with a design example. The designed converter is simulated using PSIM software to predict the performance of the converter for variations in supply voltage and load conditions. The converter operates in ZVS for the inverter switches with minimum input voltage and loses ZVS for two switches in each bridge for higher input voltages. / Graduate

No description available.

Tyrosine nitration results in altered function of smooth muscle voltage-gated L-type calcium channel. We explored the possibility that smooth muscle contains denitrase activity to allow functional recovery of the calcium channel without requiring synthesis of new channel proteins. Following peroxynitrite treatment of mouse colonic smooth muscle strips, CaCl2 (1 mM)-induced smooth muscle contraction was significantly reduced by 67% (P ≤ 0.05), which reversed by approximately 86% upon periodic washing within 2 hr period (P ≤ 0.001). The effect of the c-Src kinase inhibitor, PP2, on muscle contraction was also restored after 2 hr post-peroxynitrite treatment consistent with the thesis that recovery from tyrosine nitration allows for tyrosine phosphorylation of the calcium channel. In addition, sodium orthovanadate prevented nitration-induced inhibition of muscle contraction by approximately 90%. Moreover, denitration of nitrated proteins was observed by western blots in smooth muscle cells over 2 hr. Since nitrotyrosine formation interferes with tyrosine kinase pathways involved in cell signaling, the presence of denitrase activity in smooth muscle cells may have profound and important effects in restoring the function of nitrated proteins involved in cell signaling processes.

We study the activity-dependent refinement of sensory systems by using the mouse retinogeniculate system as a model. Spontaneous retinal waves lead to robust excitatory post-synaptic activity in developing relay cells in the dorsal lateral geniculate nucleus (dLGN) of the thalamus and are reportedly needed to help guide the segregation of retinal inputs into eye-specific domains as well as for the pruning of extraneous retinal inputs onto single dLGN relay cells. The composition of retinally evoked post-synaptic activity activated by these retinal waves in dLGN is largely unknown, but based on our in vitro recordings, such activity seems well suited to activate large, long-lasting, high-amplitude depolarizations mediated by L-type Ca2+ channel activation, plateau potentials. Plateau activity prevails early in life, at the peak of retinogeniculate refinement, however, little is known about the factors that contribute to the activation of these events, or the potential role of plateau potentials in mediating activity-dependent remodeling. In this thesis, we examined the factors and stimulus conditions that lead to the activation of plateau activity. We found that many aspects of developing retinogeniculatecircuitry (e.g., the high degree of retinal convergence, the temporal summation of excitatory post-synaptic potentials, and the lack of inhibitory connections) seem to favor their activation at early postnatal ages. We then tested whether such activity is necessary for the refinement of retinal projections, as well as their functional connections onto dLGN cells. To address this, we took a loss-of-function approach and made use of a transgenic mouse that lacks the β3 subunit of the L-type Ca2+ channel. These mutants have far fewer membrane-bound L-type Ca2+ channels and greatly attenuated L-type activity. In β3 nulls, L-type plateau potentials are rarely observed in the dLGN, even at young ages or when repetitive pulses of electrical stimulation are applied to the optic tract. Although these mice have normal stage II and III spontaneous retinal waves, the retinogeniculate projections of β3 null mice fail to segregate properly. In addition, the degree of retinal pruning is impaired. These results suggest that post-synaptic L-type Ca2+ channel activity is necessary to implement the activity-dependent refinement of the retinogeniculate pathway.

Brain function implies complex information processing in neuronal circuits, critically dependent on the molecular machinery that enables signal transmission across synaptic contacts between neurons. The types of ion channels and receptors in the neuronal membranes vary with neuron types and brain regions and determine whether neuronal responses will be excitatory or inhibitory and often allow for functional synaptic plasticity which is thought to be the basis for much of the adaptability of the nervous system and for our ability to learn and store memories. The present thesis is a study of synaptic transmission in the medial preoptic nucleus (MPN), a regulatory center for several homeostatic functions but with most clearly established roles in reproductive behaviour. The latter behaviour typically shows several distinct phases with dramatically varying neuronal impulse activity and is also subject to experience-dependent modifications. It seems likely that the synapses in the MPN contribute to the behaviour by means of activity-dependent functional plasticity. Synaptic transmission in the MPN, however, has not been extensively studied and is not well understood. The present work was initiated to clarify the synaptic properties in the MPN. The aim was to achieve a better understanding of the functional properties of the MPN, but also to obtain information on the functional roles of ion channel types for neurotransmission and its plastic properties in general. The studies were carried out using a brain slice preparation from rat as well as acutely isolated neurons with adhering nerve terminals. Presynaptic nerve fibres were stimulated electrically or, in a few cases, by raised external K+ concentration, and postsynaptic responses were recorded by tight-seal perforated-patch techniques, often combined with voltage-clamp control of the post-synaptic membrane potential. Glutamate receptors of α-amino-3-hydroxy-5-methyl-4-izoxazole propionic acid (AMPA) and N-methyl-D-aspartate (NMDA) types were identified as mediating the main excitatory synaptic signals and γ-aminobutyric acid (GABA)A receptors as mediating the main inhibitory signals. Both types of signals were suppressed by serotonin. The efficacy of AMPA-receptor-mediated transmission displayed several types of short-term plasticity, including paired-pulse potentiation and paired-pulse depression, depending on the stimulus rate and pattern. The observed plasticity was attributed to mainly presynaptic mechanisms. To clarify some of the presynaptic factors controlling synaptic efficacy, the role of presynaptic L-type Ca2+ channels, usually assumed not to directly control transmitter release, was investigated. The analysis showed that (i) L-type channels are present in GABA-containing presynaptic terminals on MPN neurons, (ii) that these channels provide a means for differential control of spontaneous and impulse-evoked GABA release and (iii) that this differential control is prominent during short-term synaptic plasticity. A model where Ca2+ influx through L-type channels may lead to reduced GABA release via effects on Ca2+-activated K+ channels, membrane potential and other Ca2+-channel types explains the observed findings. In addition, massive Ca2+ influx through L-type channels during high-frequency stimulation may contribute to increased GABA release during post-tetanic potentiation. In conclusion, the findings obtained in the present study indicate that complex neurotransmission mechanisms and different forms of synaptic plasticity contribute to the specific functional properties of the MPN.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.

The magnocellular neurosecretory cells (MNCs) of the hypothalamus are able to change their firing rate and pattern in response to small changes in external osmolality due to the involvement of osmosensitive ion channels. The firing rate and pattern determine the release of vasopressin (VP), a primary hormone regulating osmolality by controlling water excretion from the kidney. Both VP- and oxytocin (OT)-MNCs display irregular and infrequent fire when plasma osmolality is near normal, and they progressively increase the frequency of firing to fast continuous firing with increases in osmolality. VP-MNCs also respond to osmotic stimulation by adopting a phasic pattern of firing, which maximizes neuropeptide secretion. Sustained dehydration also causes structural and functional adaptations in MNCs.<p> Voltage-dependent Ca2+ channels play many important roles not only in the regulation of cell excitability but also in intracellular signal transduction, and L-type Ca2+ channel-mediated Ca2+ signals initiate intracellular signal transduction events that activate long-lasting changes in brain function and behavior. Our electrophysiological and immunocytochemical studies demonstrate that 16-24 h of water deprivation causes a significant increase in the amplitude of L-type Ca2+ current (from 55.5 ± 6.2 to 99.1 ± 10.0 pA) but not in other types of Ca2+ current. This increase occurred in both VP- and OT-MNCs. Such an increase in L-type Ca2+ current may contribute to modulation of firing rate and pattern, regulation of vasopressin release, structural adaptation in MNCs during sustained dehydration.<p> The mechanisms underlying the transition of the electrical behaviour are not completely understood. Ion channels, especially osmosensitive ion channels, play key roles in the modulation of MNC firing. A voltage-gated, 4-AP- and TEA-insensitive slowly activating outward current displayed a significant increase in about 66% of MNCs when the osmolality of the external solution was acutely increased from 295 to 325 mosmol kg-1. The responding cells showed an increase in net outward current from 12.3 ± 1.3 pA/pF to 21.4 ± 1.8 pA/pF. The reversal potential of this current was near the equilibrium for K+ and shifted with changes of K+ concentrations in external solution, suggesting that this current is a K+-selective current. The KCNQ/M current selective blockers linopirdine (150 µM) and XE991 (5 µM) suppressed this current. The IC50 of XE991 blockade was 3.9 ìM. The KCNQ/M channel openers retigabine (10 µM) and flupirtine (10 µM) significantly increased the current and shifted its activation curve toward more negative potentials. E4031, a specific blocker of ERG K+ channels, did not significantly block this current. The results from immunocytochemistry suggest that MNCs express KCNQ2, KCNQ3, KCNQ4, and KCNQ5, but not KCNQ1. These data suggest that this osmosensitive current could be a KCNQ/M current. Studies using single unit extracellular recording in hypothalamic explants showed that 10 µM XE991 increased MNC firing rate and that 20 µM retigabine decreased firing rate or caused a cessation of firing. These data suggest that a KCNQ/M current contributes to the regulation of MNC firing. KCNQ/M channels play key roles in regulating neuronal excitability in many types of central neurons. Slow activation of this current during firing might suppress activity by hyperpolarizing the cells and thus contribute to a transition between fast continuous and burst firing.<p> Our studies will be beneficial to understand the mechanisms that control VP and OT in response to acute changes in osmolality and also the mechanisms underlying MNC adaptation during sustained dehydration.