Hyperkalemic periodic paralysis (HEPP) is an inherited, autosomal disorder characterized by myotonia and periodic paralysis in skeletal muscle. The hallmark of the disease is a severe sensitivity to the K+-induced force depression, the cause of the paralysis. Previous studies have provided evidence that the sensitivity to the K+-induced force depression can be alleviated when the Cl- conductance (GCl) is lowered. However, those studies were carried out at non-physiological temperatures (25°-30°C) and few stimulation frequencies. The overarching goal of this study was to examine whether manipulating GCl pharmacologically was a viable target for treating HEPP. This work sought to document the interactive effect of K+ and Cl- on force development in mouse skeletal muscle at 37°C, over a wide range of stimulation frequencies. Secondly, experiments were undertaken to determine if a reduction in GCl could protect against the severe K+ sensitivity in HEPP. The results show that in wildtype muscle, a reduction in GCl improved force generation at high [K+]e at stimulation frequencies that naturally occur in vivo for mouse EDL and soleus. While the effect in wildtype muscles was proof of principle that a reduction in GCl may be a potential approach to treat HEPP patients, the effects of reduced GCl at high [K+]e was quite variable in HEPP muscles. In a few cases, lowering GCl did improve force generation at high [K+]e. However, in most cases the decrease in GCl exacerbated the force depression at high [K+]e, suggesting that more studies will be necessary to understand the variability in the Cl- effect to conclude whether a decrease in GCl is a viable approach to treat HEPP patients.

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SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a multidomain scaffold protein that is highly augmented in the postsynaptic density (PSD) of excitatory glutamatergic synapses within the central and peripheral nervous systems. SHANK3 links neurotransmitter receptors, ion channels, and other critical membrane proteins to intracellular cytoskeleton and signal transduction pathways. Mutations in SHANK3 are linked with a number neuropsychiatric disorders including autism spectrum disorders (ASDs). Intellectual disability, impaired memory and learning, and epilepsy are some of the deficits commonly associated with ASDs that result from mutations in SHANK3. Interestingly, these symptoms show some clinical overlap with presentations of human neurological disorders involving hyperpolarization-activated cyclin nucleotide-gated (HCN) channels. In fact, it has recently been demonstrated in human neurons that SHANK3 haploinsufficiency causes Ih-channel dysfunction, and that SHANK3 has a physical interaction with HCN channels via its ANKYRIN repeat domain. These insights suggest that SHANK3 may play important roles in HCN channel expression and function, and put forward the idea that HCN channelopathies may actually encourage some of the symptoms observed in patients with SHANK-deficiency related ASDs. In this study, we provide preliminary data that suggests the ANK domain of SHANK3 interacts with COOH portion of HCN1. We also exploited the differences between two mouse models of autism to show that a subset of SHANK3 isoforms may be involved in the proper expression and function of HCN channels. We found that HCN2 expression is significantly decreased in a mouse model lacking all major isoforms of SHANK3 (exons 13-16 deleted; Δ13-16), while HCN2 expression is unaltered in a mouse model only lacking SHANK3a and SHANK3b (exons 4-9 deleted; Δ4-9). Surprisingly, we also found that HCN4 expression is altered in SHANK3Δ13-16, but not SHANK3Δ4-9. Taken together, our results show HCN channelopathy as a major downstream carrier of SHANK3 deficiency.

Hyperkalemic periodic paralysis (HyperKPP) is characterized by myotonic discharges and weakness/paralysis. It is a channelopathy that is caused by mutation in the SCN4A gene that encodes for the skeletal muscle Na+ channel isoform (Nav1.4) α-subunit. Limb muscles are severely affected while breathing musculature is rarely affected even though diaphragm expresses the Nav1.4 channel. The objective of this study was to investigate the mechanism(s) that render the HyperKPP diaphragm asymptomatic in order to find a novel long lasting therapeutic approach, to treat HyperKPP symptoms. A HyperKPP mouse model carrying the M1592V mutation was used because it has a similar phenotype to that of patients carrying the same mutation. HyperKPP diaphragm, the limb muscles soleus and EDL all had a higher tetrodotoxin (TTX) sensitive Na+ influx than wild type (WT), but only the soleus and EDL had a depolarized resting potential, lower force and greater K+-induced force loss when compared to WT. The lack of a membrane depolarization in HyperKPP diaphragm was because of greater electrogenic contribution of the Na+ K+ ATPase pump compared to WT while such increase was not observed in EDL and soleus. HyperKPP diaphragm also had greater action potential amplitude than EDL and soleus possibly because of higher Na+ K+ ATPase pump maintaining a low [Na+]i. An inhibition of PKA, but not of PKC, increased the sensitivity of the HyperKPP diaphragm to the K+-induced force depression. So, HyperKPP soleus was exposed to forskolin to increase cAMP levels in order to activate PKA to document whether greater activity of PKA will alleviate HyperKPP symptoms. At 4.7 mM K+, forskolin increased force production, but worsened the decrease in force at 8 and 11 mM K+. Forskolin also did not improve membrane excitability. Pinacidil a KATP channel opener, improved force production at all [K+]e by causing a hyperpolarization of resting EM which then allowed for greater action potential amplitude and more excitable fibers. It is concluded that the development of a better therapeutic approach to treat HyperKPP can include a mechanism which activates Na+ K+ ATPase pumps and KATP channels.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

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.

As the most widely consumed drug around the globe, there is a vast array of contradicting research available on caffeine. One of the most debated and researched topics on caffeine is its effect on the brain. Meanwhile, the data on the neurological condition of migraine has information scattered throughout countless research articles and experiments. Although neither migraine or caffeine are completely understood by the medical world, this analysis attempts to give a more coherent understanding of the relationship between the two. This is done by first understanding the known and theorized mechanisms of caffeine as well as the pathologies of migraine. Discussions on channelopathies, current migraine medications, and case studies will be presented. After much background research, we hypothesized that caffeine could excite neurons at physiological concentrations to the point of activation. This was tested by targeting the transcription factor cFos using immunocytochemistry in vitro. The protein cFos was identified due to its rapid translation—just 15 minutes after stimuli—to indicate activation. In addition to a control culture, three different caffeine concentrations were tested on the neurons: 50 micromoles— average plasma level after 1-2 cups of coffee consumption, 100 micromoles—average plasma level after 5-6 cups of coffee also believed to be the therapeutic amount to defend against neurological diseases such as Alzheimers Disease, and 250 micromoles—the average plasma level considered to be toxic in humans. Indeed, we saw a 53.8% increase in cFos expression in the neurons as 100 micromolar of caffeine was added and exposed to the cell cultures for 24 hours. In order to ensure the results obtained in this study were physiologically relevant in vivo, known toxic levels were tested for in vitro neurotoxicity. It was found in vitro that at the non toxic plasma concentrations of 50 micromolar and 100 micromolar of caffeine did not display cellular death as tested by Trypan Blue viability testing, Crystal Violet morphologies, and fleurojade immunochemistry that tests for degeneration. Each of these experiments identified a significant death increase as the toxic level of 250 micromoles of caffeine were utilized. This allowed us to theorize that the activation of neurons found in these experiments due to caffeine exposure would apply the same effect in vivo.

No description available.

Orientador: Alexandre Secorun Borges / Resumo: A principal causa de miotonia não distrófica hereditária ocorre devido à mutações no gene CLCN1, codificante para a proteína CLC1 que forma o canal iônico seletivo para o íon cloreto predominante no tecido muscular esquelético. Mutações no gene CLCN1 foram descritas como causadoras de miotomia hereditária em humanos e em várias espécies animais. Não existe descrição de miotonia hereditária na espécie suína. O objetivo deste estudo foi realizar a caracterização clínica e molecular de uma forma de miotonia hereditária em suínos. A hipótese desse estudo foi que animais com sinais clínicos compatíveis apresentavam a miotonia hereditária. Esses animais foram avaliados sob aspectos clínicos, eletromiográficos, histopatológicos e moleculares. Os sinais clínicos verificados foram hipertrofia e rigidez musculares, miotonia com startle response formação de dimples e fenômeno warm-up evidentes. Não foi constatada distrofia muscular ao exame histopatológico. Ao exame eletromiográfico foram demonstradas descargas miotônicas clássicas com formação de som característico diver bomb. A nível molecular foi verificada a ausência dos nucleotídeos referentes aos éxons 15 e 16 utilizando amostras de cDNA dos animais afetados. No DNA genômico foi encontrada uma grande deleção de 4165pb (g. NC_010460.4 del 6912538_6916702) na região do gene CLCN1. Análises de expressão relativa demonstraram níveis de expressão em tecido muscular de animais wild type para um transcrito associado a miotonia hereditári... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The major cause of hereditary non-dystrophic myotonia occurs due to mutations in the CLCN1 gene, coding for the CLC1 protein that forms the ionic channel selective for the predominant chloride ion in skeletal muscle tissue. The resulting hereditary disease is called congenital myotonia in human medicine. Mutations in the CLCN1 gene have been described as causing hereditary myotomy in several animal species, but in the swine species, no mutation in this gene has been described. The objective of this study was to perform the clinical and molecular characterization of hereditary myotonia in swine. The hypothesis of this study was that animals with compatible clinical signs had hereditary myotonia. These animals were evaluated under clinical, electromyographic, histopathological and molecular aspects. The clinical signs verifed were muscular hypertrophy and stifness, myotonia with startle response and formation of dimples. The phenomenon warm-up was evident. No muscular dystrophy was observed at the histopathological examination. Electromyographic examination showed classic myotonic discharges with characteristic sound. At the molecular level, the absence of nucleotides from exons 15 and 16 was verifed using cDNA samples of afected animals. In genomic DNA a large deletion of 4165bp (g NC_010460.4 del 6912538_6916702) was found in the region of the CLCN1 gene. Relative expression analyzes demonstrated expression levels of wild type animals for a transcript associated with heredita... (Complete abstract click electronic access below) / Doutor

L'unité de recherche EA4612 de l'Université Claude Bernard Lyon 1 s'intéresse à la physiopathologie des troubles du rythme cardiaque, en particulier d'origine héréditaire. Nous avons étudié des mutations de gène de canaux ioniques découvertes chez des patients hétérozygotes atteints d'un syndrome du QT long ou de bradycardie sinusale et de fibrillation atriale. La mutation R148W du gène hERG diminue le courant maximal de 29%. Dans un modèle mathématique, ceci allonge la durée du potentiel d'action ventriculaire, ce qui pourrait rendre compte du phénotype QT long des porteurs. La mutation F627L du gène hERG se situe au centre du motif de sélectivité ionique (GFG) de la protéine hERG. Elle cause une perte de la sélectivité ionique du courant, de la propriété d'inactivation et de la sensibilité aux bloqueurs spécifiques. Ainsi, la présence du groupement aromatique de la chaîne latérale semble essentielle au maintien des propriétés du canal. La mutation Q1476R du gène SCN5A provoque un gain de fonction du courant sodique persistant. Dans un modèle de cellule cardiaque ventriculaire humaine, nous montrons une surcharge sodique intracellulaire pouvant protéger de l'allongement de la durée du potentiel d'action ventriculaire. La mutation D600E du gène HCN4 accélère la désactivation, ce qui pourrait causer une bradycardie. Par ailleurs, la mutation abolit la réponse à la suppression de l'adénosine monophosphate cyclique (AMPc) intracellulaire. La mutation V501M du gène HCN4 cause une perte totale de courant à l'état homozygote. A l'état hétérozygote, l'amplitude moyenne du courant est inchangée par rapport au WT. Cependant, un décalage négatif de la courbe d'activation rendrait compte de la bradycardie des patients porteurs / The EA4612 unit of the University Lyon 1 focuses on the pathophysiology of heart rhythm disorders, especially hereditary. We studied ion channel gene mutations discovered in heterozygote patients with long QT syndrome or sinus bradycardia and atrial fibrillation.The R148W mutation of the hERG gene decreases the maximum current by 29%. In a mathematical model, this lengthens the duration of the ventricular action potential, which could account for long QT phenotype of the patients. The F627L mutation of the hERG gene is in the center of the ion selectivity filter (GFG) of the hERG protein. It causes a loss of the ionic selectivity of the current, the inactivating property and sensitivity to specific blockers. Thus, the presence of this aromatic group of the side chain seems to be essential to the maintenance of the channel properties. The mutation Q1476R in the SCN5A gene causes a gain-of-function of the persistent sodium current. In a model of human ventricular heart cells, we show an intracellular sodium overload that can protect against the lengthening of the duration of the ventricular action potential. The D600E mutation of the HCN4 gene accelerates deactivation, which could cause bradycardia. Moreover, the mutation abolishes the response to the suppression of intracellular cyclic adenosine monophosphate (cAMP). The V501M mutation of the HCN4 gene causes a total loss of current in the homozygous state. In the heterozygous state, the average amplitude of the current is unchanged from the WT. However, a negative shift of the activation curve would account for bradycardia in patients