Indiana University-Purdue University Indianapolis (IUPUI) / Voltage-gated sodium channels (Navs) undergo remarkably complex modes of modulation to fine tune membrane excitability and neuronal firing properties. In neurons, the isoform Nav1.6 is highly enriched at the axon initial segment and nodes, making it critical for the initiation and propagation of neuronal impulses. Thus, Nav1.6 modulation and dysfunction may profoundly impact the input-output properties of neurons in normal and pathological conditions. Phosphorylation is a powerful and reversible mechanism that exquisitely modulates ion channels. To this end, the multifunctional calcium/calmodulin-dependent protein kinase II (CaMKII) can transduce neuronal activity through phosphorylation of diverse substrates to serve as a master regulator of neuronal function. Because Nav1.6 and CaMKII are independently linked to excitability disorders, I sought to investigate modulation of Nav1.6 function by CaMKII signaling to reveal an important mechanism underlying neuronal excitability. Multiple biochemical approaches show Nav1.6 is a novel substrate for CaMKII and reveal multi-site phosphorylation within the L1 domain; a hotspot for post-translational regulation in other Nav isoforms. Consistent with these findings, pharmacological inhibition of CaMKII reduces transient and persistent sodium currents in Purkinje neurons. Because Nav1.6 is the predominant sodium current observed in Purkinje neurons, these data suggest that Nav1.6 may be modulated through CaMKII signaling. In support of this, my studies demonstrate that CaMKII inhibition significantly attenuates Nav1.6 transient and persistent sodium currents and shifts the voltage-dependence of activation to more depolarizing potentials in heterologous cells. Interestingly, I show that these functional effects are likely mediated by CaMKII phosphorylation of Nav1.6 at S561 and T642, and that each phosphorylation site regulates distinct biophysical characteristics of the channel. These findings are further extended to investigate CaMKII modulation of disease-linked mutant Nav1.6 channels. I show that different Nav1.6 mutants display distinct responses to CaMKII modulation and reveal that acute CaMKII inhibition attenuates gain-of-function effects produced by mutant channels. Importantly, computational simulations modeling the effects of CaMKII inhibition on WT and mutant Nav1.6 channels demonstrate dramatic reductions in neuronal excitability in Purkinje and cortical pyramidal cell models. Together, these findings suggest that CaMKII modulation of Nav1.6 may be a powerful mechanism to regulate physiological and pathological neuronal excitability. / 2022-02-02

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<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

12 month embargo; Available online 9 November 2016 / SCN8A encephalopathy is a severe, early-onset epilepsy disorder resulting from de novo gain-of-function mutations in the voltage-gated sodium channel Na(v)1.6. To identify the effects of this disorder on mRNA expression, RNA-seq was performed on brain tissue from a knock-in mouse expressing the patient mutation p.Asn1768Asp (N1768D). RNA was isolated from forebrain, cerebellum, and brainstem both before and after seizure onset, and from age-matched wildtype littermates. Altered transcript profiles were observed only in forebrain and only after seizures. The abundance of 50 transcripts increased more than 3-fold and 15 transcripts decreased more than 3 fold after seizures. The elevated transcripts included two anti-convulsant neuropeptides and more than a dozen genes involved in reactive astrocytosis and response to neuronal damage. There was no change in the level of transcripts encoding other voltage-gated sodium, potassium or calcium channels. Reactive astrocytosis was observed in the hippocampus of mutant mice after seizures. There is considerable overlap between the genes affected in this genetic model of epilepsy and those altered by chemically induced seizures, traumatic brain injury, ischemia, and inflammation. The data support the view that gain-of-function mutations of SCN8A lead to pathogenic alterations in brain function contributing to encephalopathy.

Traumatic axonal injury (TAI) contributes to the mortality and morbidity following diffuse traumatic brain injury (TBI). Previous work has shown that following TBI, alterations in the molecular domains of axons result in TAI. It is currently posited that injury induced ionic flux is responsible for activating deleterious proteolytic cascades, resulting in altered distributions of axonal components. However, the underlying mechanism of this progressive pathology remains elusive. This study further explores the hypothesis that altered molecular domains contributes to the progressive intra-axonal changes that characterize TAI. Using a rodent model of impact acceleration TBI we examined the expression of nodal and paranodal domains of myelinated axons in brainstem over a 24 h period post-injury. Western blot analysis was utilized to quantify changes in protein levels of Nav1.6, a prominent component at the node of Ranvier, and Caspr, a constituent of the paranodal tripartite complex. Here we report that diffuse TBI causes an up-regulation of Nav1.6 and a down-regulation of Caspr over a 24 h time-course post-injury. The results of this study support that alterations in the molecular components of the domains of injured axons contribute to the cellular mechanism of TAI and thus provides novel data in the field of TBI research.

Doctor of Philosophy / Department of Anatomy and Physiology / Bruce D. Schultz / The first aim of this project is to define the cellular mechanisms that account for the low Na[superscript]+ concentration in human milk. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short circuit current (I[subscript]sc), suggesting activity of the epithelial Na[superscript]+ channel, ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride sensitive I[subscript]sc at all time points tested, an effect that is not reduced with Ctx washout for 12 hours or by cytosolic pathways inhibitors. Ctx increases the abundance of both beta and gamma-ENaC in the apical membrane and increases its monoubiquitination but without changing total protein and mRNA levels. Additionally, Ctx increases the levels of both the phosphorylated and the nonphosphorylated forms of Nedd4-2, a ubiquitin-protein ligase that regulates ENaC degradation. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na[superscript]+ transport. The second project aim is to develop a protocol to isolate mammary gland epithelia for subsequent in vitro culture. Caprine (1[superscript]0CME) and bovine mammary epithelia (1[superscript]0BME) were isolated and cultured on permeable supports to study hormone- and neurotransmitter-sensitive ion transport. Both 1[superscript]0CME and 1[superscript]0BME cells were passed for multiple subcultures and all passages formed electrically tight barriers. 1[superscript]0CME were cultured in the presence of hydrocortisone and exhibited high electrical resistance and amiloride-sensitive I[subscript]sc, suggesting the presence of ENaC-mediated Na[superscript]+ transport. 1[superscript]0BME were grown in a complex media in the presence or absence of dexamethasone. In contrast to 1[superscript]0CME, 1[superscript]0BME exhibited no detectable amiloride-sensitive I[subscript]sc in either culture condition. However, 1[superscript]0BME monolayers responded to an adrenergic agonist, norepinephrine, and a cholinergic agonist, carbamylcholine, with rapid increases in I[subscript]sc. Thus, this protocol for isolation and primary cell culture can be used for future studies that focus on mammary epithelial cell regulation and functions. In conclusion, the results from these projects demonstrate that mammary epithelial cells form electrically tight monolayers and can exhibit neurotransmitter- and/or hormone-induced net ion transport. The mechanisms that regulate Na[superscript]+ transport across mammary gland may provide clues to prevent or treat mastitis.

O mosquito Aedes (Stegomyia) aegypti Linnaeus, 1762 é o principal vetor do vírus do dengue sorotipo 1-4 (DEN 1-4) e da febre amarela. Por não haver vacina disponível, a redução da transmissão da dengue só pode ser alcançada mediante o controle do vetor. Entre as medidas de controle, os órgãos responsáveis utilizam-se de compostos químicos, principalmente organofosforados. Além disso, devido ao grande incomodo causado pelo Aedes, a população faz uso de inseticidas domésticos, a base de piretróides, na tentativa de eliminar e ou repelir o mosquito. As repetidas aplicações destes inseticidas e seu uso contínuo possibilitam o desenvolvimento de resistência em populações de mosquitos, processo resultante do efeito seletivo de exposição a dosagens que matam os indivíduos suscetíveis, sobrevivendo os resistentes, que transferem essa capacidade a seus descendentes. Dentre os mecanismos de resistência, a redução da sensibilidade do sítio alvo é dada por mutações pontuais no sitio de ação dos inseticidas. Tais mutações podem levar a uma substituição de aminoácidos na molécula alvo e a uma diminuição da afinidade do inseticida com essa molécula. No caso dos piretróides a mudança estrutural na molécula formadora do canal de sódio (Nav), sítio alvo deste inseticida, é a causa da resistência tipo knockdown resistence (kdr), um mecanismo de resistência bastante conhecido. Em Aedes aegypti, a associação entre a presença da mutação V1016I e o fenótipo de resistência a piretróide já foi verificada em algumas regiões Brasileiras, utilizando primers alelo-específicos. A mutação I1011M também está associada à resistência a piretróides e já foi descrita no Brasil. O objetivo deste estudo foi determinar a frequência destas mutações que levam às substituições V1016I e I1011M no AaNav de populações de Aedes aegypti no estado de São Paulo e avaliar a evolução desta frequência no período de dez anos. Para isso, indivíduos coletados em 2001 e 2011 tiveram o DNA extraído e primers alelo específicos foram utilizados para a realização de PCR a fim de verificar a presença da mutação. Houve um aumento significativo do alelo 1016 Ile nas populações estudadas, comparando-se os anos 2001 e 2011. Entretanto para o alelo 1011 Met, somente a população de Santos apresentou essa diferença significativa. Este aumento na frequência destas mutações pode ter sido ocasionado pela utilização de inseticidas domésticos à base de piretróides, uma vez que os órgãos de controle interromperam a utilização desses compostos / The mosquito Aedes (Stegomyia) aegypti Linnaeus, 1762 is the main vector of the virus dengue serotypes 1-4 (DEN 1-4) and the yellow fever virus. As there is not vaccine available, the reduction of the transmission of dengue can only be achieved by controlling the vector. Among the control measures, the responsible agencies use chemical compounds, organophosphate mainly. Furthermore, due to the big nuisance caused by Aedes, the population makes use of domestic insecticide, pyrethroids based, trying to eliminate or repel the mosquito. The repeated applications of these insecticides and the continuous use of them enable the resistance development in the mosquitoes population. This process is the result of selective effect of exposure to dosages that can kill the susceptible individuals, and then the surviving individuals transfer these characteristics to their descendents. Among the mechanism of resistance, the reduction of sensibility of the target site is given by punctual mutation (SNIP) in the action site of the insecticides. This mutation can lead to a substitution of amino acids in the target molecule and a reduction of affinity of the insecticide with this molecule. In the case of pyrethroids, the structural change in the forming molecule of the sodium channel (Nav), target site of this insecticide, is the cause of knockdown resistence (kdr) type. In Aedes aegypti, the association between the presence of the mutation V1016I and the pyrethroid resistance phenotype has been previously verified in many Brazilian regions, using Allele Specific primers The mutation I1011M is also associated with the pyrethroids resistance and it has been described in Brazil. The objective of the this study was to determine the frequency of the mutations that leads to the substitutions V1016I e I1011M in AaNav of the Aedes aegypti population in the state of Sao Paulo and to evaluate the evolution of this frequency in the period of ten years. Therefore, collected individuals in 2001 and 2011 had their DNA extracted and specific primers were used for PCR, in order to verify the presence of the mutation. There was a significant raise of allele 1016 Ile in the studied population, comparing the years 2001 and 2011. Nevertheless for allele 1011 Met, only the population of Santos showed this significant difference. This raise in the frequency of these mutations may have been caused by the utilization of domestic insecticides pyrethroid based, whereas the control agencies interrupted the utilization of this compost

The mammalian suprachiasmatic nuclei (SCN) are located in the ventral part of the hypothalamus and orchestrate circadian rhythms in physiology and behaviour. The ~20.000 neurones of the murine SCN express key molecular clock components including the Cryptochrome (Cry1/2) and Period (Per1/2/3) genes and their protein products CRY1/2 and PER1/2/3. Using different mouse models, this work demonstrates that with disrupted expression of CRY in the after-hours (Afh/Afh) mouse, cells of the ventral part of the SCN (vSCN) have a propensity to desynchronise. They receive increased GABAergic inputs and are less excitable during the projected night but not during the day compared to congenic wildtype (+/+). The linkage between CRY protein expression and the reduced excitability at night is supported by recordings from SCN cells of Cry2 deficient mice (Cry2-/-), which exhibit similar electrophysiological behaviour. Luminometrical recordings of single cell Per2 expression confirms the involvement of GABAergic signalling in both, maintaining a coherent rhythm in synchronised SCN cells from +/+ controls and the propensity of Afh/Afh SCN cells to desynchronise. A mechanism by which neuronal excitability is regulated in mammals, is the modulation of activity of small-conductance Ca2+-activated K+ (SK) channels. Western blot analysis demonstrates the expression of SK2 and SK3 channel protein in SCN neurones. Functionally, we show with whole cell electrophysiology, calcium imaging and luminometry how SK channels regulate the levels of intracellular calcium ([Ca2+]i) from day to night. In the more hyperpolarised SCN network of the Afh/Afh genotype at night, SK channel activity is altered and contributes to the lower single cell excitability. Vasoactive intestinal polypeptide (VIP) and its cognate receptor, VPAC2, are synthesised by SCN neurones and this intercellular signal facilitates coordination of suprachiasmatic neuronal activity. How the loss of VPAC2 receptor signalling affects the electrophysiology of SCN neurones and their response to excitatory inputs is unclear. Here we made patch clamp recordings of SCN neurones in brain slices prepared from animals that do not express VPAC2 receptors (Vipr2-/- mice) as well as non-transgenic animals (Vipr2+/+ mice). While Vipr2+/+ SCN neurones exhibit coordinated day-night variation in their electrical state, Vipr2-/- neurones do not and instead manifest a range of states during both day and night. We find that Vipr2+/+ neurones vary the membrane threshold potential at which they start to fire actions potentials from day to night, while Vipr2-/- neurones lack this variation. This is due to Vipr2-/- neurones lacking a voltage-gated sodium current. Subsequently we determine that this aberrant temporal control of neuronal state and excitability alters appropriate neuronal responses to a neurochemical mimic of the light-input pathway to the SCN. Conclusively, these results highlight the critical role intercellular signalling plays in the activity of individual neuronal state and their response to neural input as well as ensemble activity and function of the suprachiasmatic neural network.

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.

Statement of the problem: The epithelial sodium channels (ENaC) play an important role in regulation of blood pressure (BP). Although the genes are identical in Dahl salt sensitive (S) and Dahl salt resistant (R) rats, expression of ENaC subunits is increased in kidneys of S rats on high salt diet. Intracerebroventricular (icv) infusion of ENaC blocker benzamil prevents Na+ induced hypertension. It was not known whether ENaC subunits are expressed in the brain and whether or not brain ENaC plays a role in regulation of [Na+] in CNS. Hypothesis: 1. Epithelial sodium channels are expressed in the brain. 2. Expression of ENaC is increased in the kidneys and brain of Dahl S rats on high salt diet. 3. ENaC in the brain contributes to regulation of [Na+] in the CSF and brain interstitium. Methods of investigation: We studied expression and distribution of the ENaC subunits and assessed the effects of icv infusion of Na+-rich aCSF in Wistar rats or high salt diet in Dahl S rats in different areas of the brain. Function of ENaC in the choroid plexus was evaluated by studying the effects of benzamil and ouabain on Na+ transport. Major findings: In Wistar rats, both mRNA and protein of all three ENaC subunits are expressed in brain epithelia and magnocellular neurons in the supraoptic (SON) and paraventricular (PVN) nucleus. ENaC abundance is higher on the apical versus basolateral membrane of choroid cells. Benzamil decreases Na+ influx into choroid cells by 20-30% and increases CSF [Na+] by ~8 mmol/L. Na+ rich aCSF increases apical membrane expression of βENaC in the choroid cells and of α and βENaC in basolateral membrane of ependymal cells, but has no effect on neuronal ENaC. Expression of ENaC is higher in choroid cells and SON of Dahl S versus R rats and the higher expression persists on a high salt diet. High salt attenuates the ouabain blockable efflux of Na+ from choroid cells and has no effect on CSF [Na+] in Dahl R rats. In contrast, high salt does not attenuate ouabain blockable efflux of 22Na+ and CSF [Na+] increases in Dahl S. Main Conclusion: ENaC in the brain contributes to Na+ transport into the choroid cells and appear to be involved in reabsorption of Na+ from the CSF. Aberrant regulation of Na+ transport and of Na+K+ATPase activity, might contribute to increases in CSF [Na+] in Dahl S rats on high-salt diet. ENaC in magnocellular neurons may contribute to enhanced secretion of mediators such as ‘ouabain’ leading to sympathetic hyperactivity in Dahl S rats.