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Excitable cells display dynamically regulated changes in the properties of ion currents during development. These changes are crucial for the proper maturation of cellular excitability, and therefore have the potential to affect more sophisticated functions, including neural circuits, movements, and behaviors. Zebrafish skeletal muscle is an excellent model for studying the development of ion channels and their contributions to excitability. They possess distinguishable populations of red and white muscle fibers, whose biological functions are well understood. The main objectives of this thesis were: (1) To characterize the development of muscle excitability by examining properties of voltage-gated sodium and potassium currents expressed in embryonic and larval zebrafish during the first week of development. (2) To elucidate some of the mechanisms by which ion current development might be controlled, beginning with activity-dependent and phosphorylation-dependent mechanisms. These objectives were approached using whole-cell electrophysiological techniques to examine the voltage-dependent and kinetic properties of voltage-gated sodium and potassium currents in intact zebrafish skeletal muscle preparations. Mutant sofa potato zebrafish, which lack functional nicotinic acetylcholine receptors, were then utilized to determine whether synaptic activity at the neuromuscular junction is required for proper ion current development. Finally, protein kinases were activated pharmacologically in order to determine whether they were able to modulate ion currents during development. The results revealed that properties of ion currents undergo a developmental progression, including increased current density, accelerated kinetics, and shifts in voltage-dependence; these developments correlated well with the maturation of muscle action potentials and the movements and behaviors they mediate. Sofa potato mutants were found to be deficient in certain aspects of ion current development, but other aspects appeared to be unaffected by a lack of synaptic activity. Protein kinase A demonstrated the ability to drastically reduce potassium current density; however the effects of PKA were similar at all developmental stages. Overall, these findings provide novel insight into the roles played by voltage-gated currents during the development of excitability in zebrafish skeletal muscle, and expand the rapidly growing body of knowledge about ion channel function in general. / Physiology, Cell & Developmental Biology

Diss. (sammanfattning) Linköping : Linköpings universitet, 2007. / Härtill 4 uppsatser.

The primary goal of this work was to examine the role of voltage-dependent Ca2+ channels in regulating the output of larval Drosophila motoneurons functioning within an intact network. To accomplish this goal, two major aims were addressed: 1. To determine whether larval Drosophila motoneurons express voltage-dependent Ca2+ channels in their central processes, and further, to determine the genes responsible. 2. To determine the role of centrally expressed voltage-dependent Ca2+ channels in the regulation of motoneuron output as motoneurons receive behaviorally relevant input from the locomotor network. To address these goals, genetic tools available in Drosophila were used along side in situ patch clamp techniques from larval motoneurons.Using whole cell voltage-clamp techniques in situ, we have shown that two identified motoneurons, aCC and RP-2, carry voltage-dependent currents recorded from the soma. Dmca1D, the L-type like channel in Drosophila, is primarily responsible for this current. Expressing Dmca1D RNAi in aCC and RP-2, as the preparation displayed fictive bouts of locomotion, caused an increase in burst duration in both RP-2 and aCC as well as an increase in the number of action potentials fired per burst. Additionally, the afterhyperpolarization between spikes was greatly reduced and spiking became less regular. This work indicates a role for Dmca1D in the processing of synaptic information in Drosophila motoneurons aCC and RP-2.

Thesis (Ph. D.)--Cornell University, May, 2009. / Vita. Includes bibliographical references (leaves 163-186).

Hyperkalemic periodic paralysis, HyperKPP, is an inherited progressive disorder of the muscles caused by mutations in the voltage gated sodium channel (NaV1.4). The objectives of this thesis were to develop a technique for measurement symptoms in vivo using electromyography (EMG) and to determine the mechanism by which Ca2+ alleviates HyperKPP symptoms, since this is unknown. Increasing extracellular [Ca2+] ([Ca2+]e) from 1.3 to 4 mM did not result in any increases in45Ca2+ influx suggesting no increase in intracellular [Ca2+] ([Ca2+]i) acting on an intracellular signaling pathway or on an ion channel such as the Ca2+sensitive K+ channels. HyperKPP muscles have larger TTX-sensitive22Na+ influx than wild type muscles because of the defective NaV1.4 channels. When [Ca2+] was increased from 1.3 to 4 mM, the abnormal 22Na+ influx was completely abolished. Thus, one mechanism by which Ca2+alleviates HyperKPP symptoms is by reducing the abnormal Na+ influx caused by the mutation in the NaV1.4 channel.

Activation of the intrinsic aqueous water-wire proton conductance (GAQ) in Hv1 channels is controlled by changes in membrane potential and the transmembrane pH gradient (ΔpH). The mechanism by which changes in ΔpH affect the apparent voltage dependence of GAQ activation is not understood. In order to measure voltage sensor (VS) activation in Hv1, we mutated a conserved Arg residue in the fourth helical segment (S4) to His and measured H+ currents under whole-cell voltage clamp in transfected HEK-293 cells. Consistent with previous studies in VS domain containing proteins, we find that Hv1 R205H mediates a robust resting-state H+ ‘shuttle’ conductance (GSH) at negative membrane potentials. Voltage-dependent GSH gating is measured at more negative voltages than the activation GAQ, indicating that VS activation is thermodynamically distinct from opening of the intrinsic H+ permeation pathway. A hallmark biophysical feature of Hv1 channels is a ~-40 mV/pH unit shift in the apparent voltage dependence of GAQ gating. We show here that changes pHO are sufficient to cause similar shifts in GSH gating, indicating that GAQ inherits its pH dependence from an early step in the Hv1 activation pathway. Furthermore, we show for the first time that Hv1 channels manifest a form of electromechanical coupling VS activation and GAQ pore opening. Second-site mutations of D185 markedly alter GAQ gating without affecting GSH gating, indicating that D185 is required for a late step in the activation pathway that controls opening of the aqueous H+ permeation pathway. In summary, this work demonstrates that the Hv1 activation pathway contains multiple transitions with distinct voltage and pH dependencies that have not been previously identified. The results reported here novel insight into the mechanism of VS activation in Hv1 and raise fundamental questions about the nature of pH-dependent gating and electromechanical coupling in related VS domain-containing ion channels and phosphatases.

It is clear that the voltage-gated proton channel HVCN1 plays an essential role in a range of cell types, in particular immune cells. Previous published work has confirmed the existence of proton channels in both murine and human macrophages. However, the role of HVCN1 in macrophages has not been investigated. Given that the current literature on voltage-gated proton channels in immune cells has found HVCN1 to be involved in several cellular processes (such as the respiratory burst and signalling events) it is important to establish its functional role in macrophages, which are a crucial constituent of the immune system. The aim of my thesis was to investigate the function of voltage-gated proton channels in macrophages with the use of mice with a disrupting mutation within the Hvcn1 gene, which results in HVCN1 loss. In particular, I wanted to address how Hvcn1-/- macrophages responded to LPS activation. I hypothesised that HVCN1 regulates the respiratory burst of macrophages and that it potentially modulates mitochondrial ROS production, and in doing so, may affect several functional aspects of macrophage biology.

There is a complex relationship between genetic mutations and their phenotypic expression. Two patients who carry the exactly same disease-causing mutation can have drastically different severity in disease symptoms. Such phenotypic variations may be due to environmental factors, such as diet, stress and temperature, as well as genetic variations, including single nucleotide polymorphisms and copy number variations in other loci. From a clinical point of view, the environmental and genetic factors that modify phenotypic severity are important because, even when we cannot correct the original mutations, it may be able to reduce the burden of genetically inherited disorders by manipulating these modifiers. To study environmental and genetic modifiers, we used Shudderer (Shu), a Drosophila mutant for the voltage-gated sodium (Nav) channel gene, as an experimental model. Nav channels are essential for generation and propagation of action potentials in neurons. Reflecting their functional importance in neural function, mutations in the Nav channel genes are associated with a variety of human neurological disorders. Shu mutants display severe behavioral defects (e.g., spontaneous jerking and heat-induced seizures) and morphological abnormalities (e.g., indented thorax and down-turned wings). The goal of this study was to identify and characterize the environmental and genetic factors that modify behavioral and morphological phenotypes of Shu mutants. For environmental modifiers, we serendipitously discovered that a diet supplemented with milk dramatically reduces the Shu phenotypes. To identify genetic modifiers, we took two independent approaches, microarray analysis and unbiased forward genetic modifier screening. We found that reduction of Gadd45 or GstS1 activity leads to suppression of the Shu phenotypes to different degrees. Intriguingly, the effects of these genetic and environmental modifiers apparently converge into enhancement of the GABAergic inhibitory system. Because the molecular and cellular mechanisms underlying the basic neurobiological processes are highly conserved between flies and humans, our findings are expected to provide fundamental insights into genetic and environmental modifiers for human Nav channel gene mutations, leading to the future development of novel strategies for preventing and treating disorders caused by dysfunctional Navchannels.

Neurological disorders, such as epilepsy, often result from inherited or newly acquired genetic mutations. However, individuals possessing the exact same disease-causing mutations can exhibit dramatic differences in the severity of their symptoms. These differences can be explained in part by environmental factors, such as the microbes in our gut, that play an important role in the manifestation of disease symptoms. Within the last decade, microbes living in the gut have established themselves as an environmental factor with profound effects on our health and well-being. Of special interest is the relationship between the gut microbiota and neurological disease. The goal of my thesis was to: 1) characterize the gut microbiota composition and 2) understand how the gut microbiota modulates seizure-like behavior using Shudderer, a fruit fly (Drosophila melanogaster) model of epilepsy. Shudderer flies possess a mutation in the voltage-gated sodium channel gene and display seizure-like behavioral abnormalities including spontaneous tremors and heat-induced seizures. We identified differences in the microbial composition of the gut microbiota between Shudderer and control (healthy) flies. We also found that by removing the gut microbiota we could improve seizure-like behavior of Shudderer flies as well as another Drosophila mutant harboring a similar genetic mutation. Together, these findings provide evidence that a bidirectional interaction exists between the gut microbiota and neurological function. Since the molecular and cellular mechanisms controlling basic biological processes are highly conserved between fruit flies and humans, these findings are expected to be applicable to mammalian systems, including humans, and may lead to the future development of novel therapeutics to treat epilepsy and other neurological disorders.