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.

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.

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.

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.

<p>Phospho-MurNAc-pentapeptide translocase (MraY) is a conserved membrane-spanning enzyme involved in the biosynthesis of bacterial cell walls. MraY generates lipid I by transferring the phospho-MurNAc-pentapeptide to the lipid carrier undecaprenyl-phosphate. MraY is a primary target for antibiotic development because it is essential in peptidoglycan synthesis and targeted by 5 classes of natural product antibiotics. The structure of this enzyme will provide insight into the catalytic mechanism and a platform for future antibiotic development. MraY genes from 19 bacteria were cloned, expressed, purified and assayed for biochemical stability. After initial crystallization screening, I found that MraY from Aquifex aeolicus (MraYAA) produced diffracting crystals. Recombinant MraYAA is functional and shows inhibition by the natural inhibitor capuramycin. After extensive optimization of crystallization conditions, we extended the resolution limit of the crystal to 3.3 Å. The crystal structure, the first structure of the polyprenyl-phosphate N-acetyl hexosamine 1-phosphate transferase (PNPT) superfamily, reveals the architecture of MraYAA and together with functional studies, allow us to identify the location of Mg2+ at the active site and the putative binding sites of both substrates. My crystallographic studies provide insights into the mechanism of how MraY attaches a building block of peptidoglycan to the carrier lipid.</p><p>Voltage-gated Na+ (NaV) channels initiate action potentials in neurons and cardiac myocytes. NaV channels are composed of a transmembrane domain responsible for voltage-dependent Na+ conduction and a cytosolic C-terminal domain (CTD) that regulates channel function through interactions with many auxiliary proteins including members of the fibroblast growth factor homologous factor (FHF) family and calmodulin (CaM). Through the collaboration between our lab and Geoffrey Pitt's lab, we report the first crystal structure of the ternary complex of the human NaV1.5 CTD, FGF13, and Ca2+-free CaM at 2.2 Å. Combined with functional experiments based on structural insights, we present a platform to understand roles of these auxiliary proteins in NaV channel regulation and the molecular basis of mutations that lead to neuronal and cardiac diseases. Furthermore, we identify a critical interaction that contributes to the specificity between individual NaV CTD isoforms and distinctive FHFs.</p> / Dissertation

Voltage-gated sodium channels auxiliary subunits evolutionary emerged nearly 500 million years ago during the Cambrian explosion. These subunits alter one the most important ion channels to electrical signaling, the voltage-gated sodium channels support the propagation of electric impulses in animals. The mechanism for the auxiliary subunits effects on the channels is poorly understand, as is the stoichiometry between the auxiliary subunit and the channel. The focus of my thesis is to generate assays and to use these approaches to understand the interactions different types of voltage-gated channels and their auxiliary subunits. A biochemical approach was taken to identify novel interactions between the eukaryotic sodium channel auxiliary subunits and a prokaryotic voltage-gated sodium channel, a protein that diverged from the eukaryotic voltage-gated sodium channels billions of years ago. These interactions between the auxiliary subunits and channels were probed with chemical and photochemical crosslinkers in search of interaction surfaces and similarity to explain the mechanisms of interaction. The work in this thesis identified novel interactions between the voltage-gated sodium channel auxiliary subunits and voltage-gated channels that are distantly related to the voltage-gated sodium channels principally thought to be modulated by the auxiliary subunits. From this work a rudimentary concept can be theorized that the voltage-gated sodium channel β-subunits and not only β1 have a more primary role in electrophysiology by associating with multiple different types of ion channels.

Indiana University-Purdue University Indianapolis (IUPUI) / The  cardiac  voltage-­gated  sodium  channels  (Nav1.5)  play  a  specific  and   critical  role  in  regulating  cardiac  electrical  activity  by  initiating  and  propagating   action  potentials  in  the  heart.  The  association  between  Nav1.5  dysfunctions  and   generation  of  various  types  of  cardiac  arrhythmia  disease,  including  long-­QT3   and  Brugada  syndrome,  is  well  established.  Many  types  of  post-­translational   modifications  have  been  shown  to  regulate  Nav1.5  biophysical  properties,   including  phosphorylation,  glycosylation  and  ubiquitination.  However,  our   understanding  about  how  post-­translational  lipid  modification  affects  sodium   channel  function  and  cellular  excitability,  is  still  lacking.  The  goal  of  this   dissertation  is  to  characterize  Nav1.5  palmitoylation,  one  of  the  most  common   post-­translational  lipid  modification  and  its  role  in  regulating  Nav1.5  function  and   cardiac  excitability.     In  our  studies,  three  lines  of  biochemistry  evidence  were  shown  to  confirm   Nav1.5  palmitoylation  in  both  native  expression  background  and  heterologous   expression  system.  Moreover,  palmitoylation  of  Nav1.5  can  be  bidirectionally   regulated  using  2-­Br-­palmitate  and  palmitic  acid.  Our  results  also  demonstrated   that  enhanced  palmitoylation  in  both  cardiomyocytes  and  HEK293  cells   increases  sodium  channel  availability  and  late  sodium  current  activity,  leading  to   enhanced  cardiac  excitability  and  prolonged  action  potential  duration.  In  contrast,   blocking  palmitoylation  by  2-­Br-­palmitiate  increases  closed-­state  channel inactivation  and  reduces  myocyte  excitability.  Our  computer  simulation  results   confirmed  that  the  observed  modification  in  Nav1.5  gating  properties  by  protein   palmitoylation  are  adequate  for  the  alterations  in  cardiac  excitability.  Mutations  of   potential  palmitoylation  sites  predicted  by  CSS-­Palm  bioinformatics  tool  were   introduced  into  wild-­type  Nav1.5  constructs  using  site-­directed  mutagenesis.   Further  studies  revealed  four  cysteines  (C981,  C1176,  C1178,  C1179)  as   possible  Nav1.5  palmitoylation  sites.  In  particular,  a  mutation  of  one  of  these   sites(C981)  is  associated  with  cardiac  arrhythmia  disease.  Cysteine  to   phenylalanine  mutation  at  this  site  largely  enhances  of  channel  closed-­state   inactivation  and  ablates  sensitivity  to  depalmitoylation.  Therefore,  C981  might  be   the  most  important  site  that  regulates  Nav1.5  palmitoylation.  In  summary,  this   dissertation  research  identified  novel  post-­translational  modification  on  Nav1.5   and  revealed  important  details  behind  this  process.  Our  data  provides  new   insights  on  how  post-­translational  lipid  modification  alters  cardiomyocyte   excitability  and  its  potential  role  in  arrhythmogenesis.

Indiana University-Purdue University Indianapolis (IUPUI) / The voltage-gated sodium channels (VGSCs) are critical determinants of neuronal excitability. They set the threshold for action potential generation. The VGSC isoform Nav1.6 participates in various physiological processes and contributes to distinct pathological conditions, but how Nav1.6 function is differentially regulated in different cell types and subcellular locations is not clearly understood. Some VGSC isoforms are subject to S-palmitoylation and exhibit altered functional properties in different S-palmitoylation states. This dissertation investigates the role of S-palmitoylation in Nav1.6 regulation and explores the consequences of S-palmitoylation in modulating neuronal excitability in physiological and pathological conditions. The aims of this dissertation were to 1) provide biochemical and electrophysiological evidence of Nav1.6 regulation by S-palmitoylation and identify specific S-palmitoylation sites in Nav1.6 that are important for excitability modulation, 2) determine the biophysical consequences of epilepsy-associated mutations in Nav1.6 and employ computational models for excitability prediction and 3) test the modulating effects of S-palmitoylation on aberrant Nav1.6 activity incurred by epilepsy mutations. To address these aims, an acyl-biotin exchange assay was used for Spalmitoylation detection and whole-cell electrophysiology was used for channel characterization and excitability examination. The results demonstrate that 1) Nav1.6 is biochemically modified and functionally regulated by S-palmitoylation in an isoform- and site-specific manner and altered S-palmitoylation status of the channel results in substantial changes of neuronal excitability, 2) epilepsy associated Nav1.6 mutations affect different aspects of channel function, but may all converge to gain-of-function alterations with enhanced resurgent currents and increased neuronal excitability and 3) S-palmitoylation can target specific Nav1.6 properties and could possibly be used to rescue abnormal channel function in diseased conditions. Overall, this dissertation reveals S-palmitoylation as a new mechanism for Nav1.6 regulation. This knowledge is critical for understanding the potential role of S-palmitoylation in isoform-specific regulation for VGSCs and providing potential targets for the modulation of excitability disorders. / 2022-05-06

No description available.

Understanding how genetic changes ultimately affect morphology and physiology is essential for understanding and predicting how organisms will adapt to environmental changes. Although most traits are complex and involve the interplay of many different genetic loci, some exceptions exist. These include the convergent evolution of tetrodotoxin resistance in snakes, which has a simple genetic basis and can be used as a model system to investigate the genetic basis of adaptive evolution. Tetrodotoxin is a potent neurotoxin used as a chemical defense by various animals, including toxic newts. Snakes have evolved resistance through mutations in voltage-gated sodium channels, the protein targets of tetrodotoxin, sparking an evolutionary arms race between predator and prey. In this dissertation, I describe how genomic rearrangements have led to sex-linkage of four of the voltage-gated sodium channel genes in snakes and compare allele frequencies across populations and sexes to make inferences about how sex linkage has influenced the evolution of resistance in garter snakes. By measuring gene expression in different snake tissues, I show that three of these sex-linked sodium channel genes are dosage compensated in embryos, adult muscle, and adult brain. In contrast, two channels show sexual dimorphism in their expression levels in the heart, which may indicate differences in dosage compensation among tissues. I then use comparative genomics to track the evolutionary history of tetrodotoxin resistance across all nine sodium channel genes in squamate reptiles and show how historical changes have paved the way for full-body resistance in certain snakes. Finally, I use targeted sequence capture to obtain the sodium channel sequences of salamanders and show evidence that tetrodotoxin self-resistance in toxic newts was likely accelerated through gene conversion between resistant and non-resistant sodium channel paralogs. Together, these results illustrate parallelism in evolutionary mechanisms and processes contributing to the appearance of an extreme and complex trait that arose independently in two distinct taxa separated by hundreds of millions of years. / Doctor of Philosophy / Western North America is the site of an ongoing battle between highly toxic species of salamanders (toxic newts) and their garter snake predators. In certain regions, garter snakes have countered newt defenses by evolving resistance to their toxins, and the newts have become more toxic in response. This interaction has been the focus of scientists for decades because it teaches us about the ways in which animals can respond to changes in their environment. In living organisms, DNA is used a blueprint to determine the ultimate traits that are expressed (e.g., whether an organism will have five fingers or four, or whether it will be resistant or sensitive to a toxin). By comparing DNA sequences of different life forms, we are beginning to understand the rules that determine how these blueprints are read and how they can change over time. Because life is built upon the same basic building blocks (DNA, mRNA, and proteins), information about this snake-newt system can be used to understand the way that other systems, such as humans and pathogens, might interact. In my dissertation, I compare DNA sequences from snakes and lizards to identify the history of changes leading to the extreme toxin resistance in the garter snakes. I show that toxin resistance began hundreds of millions of years ago, with all lizards having a low baseline level of resistance, and that resistance built up slowly in the lineages leading to garter snakes. I also show that because of DNA rearrangements, female snakes have fewer copies of some of the genes involved in resistance, and this may have led to differences among the sexes. Lastly, I compare DNA sequences among salamanders, revealing a similar pattern to that in snakes and lizards. Specifically, newts have evolved self-resistance to their own toxin, and this has happened gradually over hundreds of millions of years, with all salamanders having some toxin resistance. I also show that an unusual process occurred within the DNA of toxic newts, resulting in a rapid change from toxin sensitivity to toxin resistance in some genes. Taken together, this work helps advance our understanding of the processes and limitations that determine how organisms can function and change over time.