Transient receptor potential (TRP) channels are expressed throughout the central nervous system and have a unique ability to detect a wide range of stimuli including changes in voltage, temperature, pH, lipid environment, small molecule agonists, and mechanical stress. While it is known that TRP channels contain the same six transmembrane helix (S1-S6), tetrameric architecture as voltage-gated channels, the degree to which functional and structural analogies are relevant remains poorly understood. This thesis describes a multidisciplinary approach toward understanding the structure and function of TRP channel transmembrane domains by focusing on the S1-S4 transmembrane helices of the TRPV1. This focus is inspired by the voltage-sensor domain (VSD) of the S1-S4 helices of voltage-gated channels, for which a range of studies show functional and structural independence. While some TRP channels are voltage-sensitive, their S4 helix does not contain the positive string of amino acids of canonical VSDs. However, the S1-S4 helices are functionally significant as the binding site of small molecule ligands in both TRPV1 and TRPM8 (for capsaicin and menthol, respectively). The question of TRP channel transmembrane domain modularity is addressed in this thesis by expression and purification trials as well as radioligand-binding assays. It is demonstrated that the S1-S4 and S1-S6 helices of TRPV1 can be properly inserted, overexpressed, and show signs of stability upon detergent-extraction from Saccharomyces cerevisiae membranes. However the TRPV1 S1-S4 and S1-S6 helices do not show wildtype (WT)-like binding in [³H]-RTX binding assays. These results indicate that the TRPV1 transmembrane domains are likely structural but not functional domains. The S. cerevisiae expression system remained promising for the overexpression of TRP transmembrane domains as well as the production of functional, though not stable upon detergent-extraction, WT TRPV1. This WT TRPV1 was subsequently found to functionally bind both RTX, used in ligand binding assays, as well as the double-knot toxin (DkTx), targeted to the pore domain (the S5-S6 helices). An effect of DkTx on RTX binding affinity demonstrates an allosteric interaction indicative of a possible tighter packing between the two transmembrane domains than is seen in voltage-gated channels containing the canonical VSD. Computational approaches additionally allowed for the investigation of the intramembrane capsaicin binding site in the TRPV1 S1-S4 helices, crucial to the initial motivations of this study. While the literature locates the capsaicin binding site to the TRPV1 S1-S4 helices, a `binding pocket' has yet to be defined, with regards to the orientation of bound capsaicin and its access route to the site via the bilayer. Using molecular dynamics (MD) simulations the preferred location of capsaicin within the bilayer is defined, as well as the elucidiation of capsaicin flip-flop between bilayer leaflets as a key event prior to TRPV1 binding. A transient binding was also observed between a homology model of the TRPV1 S1-S4 helices and capsaicin, possibly encouraging the idea that the S1-S4 helices still contain a partial binding site, though of too low affinity to be observed in the binding experiments performed here.

The Vestibulo-Ocular Reflex (VOR) produces compensatory eye movements in response to head and body rotations movements, over a wide range of frequencies and in a variety of dimensions. The individual components of the VOR are separated into parallel pathways, each dealing with rotations or movements in individual planes or axes. The Horizontal VOR (hVOR) compensates for eye movements in the Horizontal plane, and comprises a linear and non-linear pathway. The linear pathway of the hVOR provides fast and accurate compensation for rotations, the response being produced through 3-neuron arc, producing a direct translation of detected head velocity to compensatory eye velocity. However, single neurons involved in the middle stage of this 3-neuron arc cannot account for the wide frequency over which the reflex compensates, and the response is produced through the population response of the Medial Vestibular Nucleus (MVN) neurons involved. Population Heterogeneity likely plays a role in the production of high fidelity population response, especially for high frequency rotations. Here we present evidence that, in populations of bio-physical compartmental models of the MVN neurons involved, Heterogeneity across the population, in the form of diverse spontaneous firing rates, improves the response fidelity of the population over Homogeneous populations. Further, we show that the specific intrinsic membrane properties that give rise to this Heterogeneity may be the diversity of certain slow voltage activated Potassium conductances of the neurons. We show that Heterogeneous populations perform significantly better than Homogeneous populations, for a wide range of input amplitudes and frequencies, producing a much higher fidelity response. We propose that variance of Potassium conductances provides a plausible biological means by which Heterogeneity arises, and that the Heterogeneity plays an important functional role in MVN neuron population responses. We discuss our findings in relation to the specific mechanism of Desynchronisation through which the benfits of Heterogeneity may arise, and place those findings in the context of previous work on Heterogeneity both in general neural processing, and the VOR in particular. Interesting findings regarding the emergence of phase leads are also discussed, as well as suggestions for future work, looking further at Heterogeneity of MVN neuron populations.

Neurons are generated from unspecialized neural precursor cells (NPCs) in a process termed neurogenesis. This neuronal development continues throughout life in the ventricular zone (VZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus in the hippocampus. NPCs undergo a complex and highly regulated set of behaviours in order to ultimately integrate into the existing brain circuitry as fully functional neurons. Recently the pannexin (Panx) large-pore channel proteins were discovered. One family member, Panx1 is expressed in the nervous system in mature neurons, and acts as an ATP release channel in various cell types throughout the body. Post-natal NPCs are responsive to ATP via activation of purinergic receptors, which modulate a variety of NPC behaviours. I therefore investigated the hypothesis that Panx1 was expressed in post-natal VZ NPCs, where it functioned as an ATP release channel and regulated neuronal development. In the course of my studies, I found that Panx1 positively regulated NPC proliferation and migration, and negatively regulated neurite outgrowth in vitro. Using an NPC-specific Panx1 knock-out strategy, I showed that Panx1 expression was required for maintenance of a consistent population of VZ NPCs in vivo in both healthy and injured brain. Together these data indicated that Panx1 directed NPC behaviours associated with neuronal development both in vitro and in vivo. To further understand the molecular underpinnings of this regulation, I examined the Panx1 interactome, and uncovered a novel association with collapsin response mediator protein 2 (Crmp2). Functional studies suggested that this interaction likely was at least in part responsible for Panx1’s negative impact on neurite outgrowth. Overall, my results represent important novel findings that contribute to our understanding of post-natal neuronal development and the molecular function of Panx1 within the brain. / Graduate / 0317 / 0379 / leighws@uvic.ca

Transient receptor potential vanilloid 3 receptor channel (TRPV3) is a thermosensitive ion channel expressed in skin keratinocytes. There, in a molecular complex with the epidermal growth factor receptor (EGFR) contributes to proliferation and terminal differentiation of keratinocytes, temperature detection, pain and pruritus. TRPV3 is activated by a number of exogenous compounds, such as carvacrol from oregano, thymol from thyme and eugenol from clove. Its unique feature is sensitization, TRPV3 channel activity successively increases upon repeated stimulation. The molecular basis of this process is not yet understood. One of the considered possibility is a direct phosphorylation of TRPV3 protein through signaling pathways involving EGFR and mitogen-activated protein kinase MAPK1 / MAPK3 (also called ERK2 / ERK1). In this thesis we investigated whether sensitization of TRPV3 which is expressed in a human cell line immortalized keratinocytes could be influenced by mutations on the predicted consensual phosphorylation sites for MAPK1 / MAPK3. We used electrophysiological patch-clamp technique and tested eight mutants, in which was threonine or serine replaced with aspartic acid mimicking phosphorylation. We identified six residues where the mutations influenced at least one of the functional...

Ligand-gated and ligand-modulated ion channel (IC) sensors have received increased attention for their ability to transduce ligand-binding events into a readily measurable electrical signal. Ligand-binding to an IC modulates the ion flux properties of the channel in label-free manner, often with single-molecule sensitivity and selectivity. As a result, ICs are attractive sensing elements in biosensoring platforms, especially for ligands lacking optical (e.g. fluorescent) or electrochemical properties. Despite the growing number of available ligand-gated and ligand-modulated ICs and artificial lipid bilayer platforms for IC reconstitution, significant work remains in defining the analytical performance capabilities of IC sensors. Particularly, few studies have described platforms for making measurements with rapid temporal resolution and high sensitivity. In this work, we describe an artificial lipid bilayer platform which enables rapid measurement of ion channel activity, a key parameter for developing IC sensors suitable for studying biological events, e.g. single cell exocytosis (Chapter 2 and 3). Additionally, we developed expression, purification, and reconstitution protocols for Kir6.2, a model ligand-gated ion channel, for use in sensor development (Chapter 4). The final goal is to reconstitute ion channel-coupled receptors (ICCRs), G protein-coupled receptor-Kir6.2 fusion proteins, into artificial lipid bilayers to detect small molecules and hormones targeting GPCRs. Towards this goal, we characterized the expression and function of two ICCRs, M2-Kir and D2-Kir, in HEK293 cells (Chapter 5).

Doctor of Philosophy / Department of Biochemistry / John M. Tomich / Channel replacement therapy represents a new treatment modality that could augment existing therapies against cystic fibrosis. It is based on designing synthetic channel-forming peptides (CFPs) with desirable selectivity, high ion transport rates and overall ability to supersede defective endogenous chloride channels. We derived synthetic CFPs from a peptide initially reconstituted from the second transmembrane segment of the α-subunit of Glycine receptor (M2GlyR). Our best candidate peptide NK4-M2GlyR T19R, S22W (p22-T19R, S22W) is soluble in aqueous solutions, has the ability to deliver itself to the epithelial cell membranes without the use of a delivery system, is non-immunogenic, but when assembled into a pore, lacks the structural properties for anion selectivity. Previous findings suggested that threonine residues at positions 13, 17 and 20 line the pore of assembled p22-T19R, S22W and recent studies indicated that an introduction of positively charged 2, 3-diaminopropionic acid (Dap) at either T13 or T17 in the sequence increases transepithelial ion transport rates across the apical membranes of Madin-Darby canine kidney (MDCK) epithelial cells. This study focused on further structural modifications of the pore-lining interface of p22-T19R, S22W assembled pore. It was hypothesized that singly, doubly or triply introduced Dap residues modify the pore geometry and that their positively charged side chains impact discrimination for anions. Dap-substituted p22-T19R, S22W peptides retain the α-helical secondary structure characteristic for their parent p22-T19R, S22W. The sequences containing multiple Dap-substituted residues induce higher short circuit current across the epithelial MDCK cells compared to peptides with single Dap-substitutions or no Dap-substitutions. Whole-cell voltage clamp recordings using Xenopus oocytes indicate that Dap-substituted peptide assemblies induce higher levels of voltage-dependent but non-selective ion current relative to p22-T19R, S22W. Studies using the D-enantiomer of p22-T19R, S22W and shorter truncated sequences of a full length L-p22-T19R, S22W and L-Dap-substituted peptides provided evidence that peptide-induced ion transport rates can be attributed to formation of de novo pathways. Results of preliminary computer modeling studies indicate that Dap residues affect the pore geometry but not ion selectivity. Future studies focusing on modifying the existing electrostatic environment towards anion selectivity will focus on staggering the charged residues of Dap at various locations inside synthetic pores.

Pannexin 1 (Panx1) is a metabolite channel enriched in the brain and known to localize to the cell surface, where it is involved in a variety of neuronal processes including cell proliferation and differentiation. The mechanisms through which Panx1 is trafficked or stabilized at the surface, however, are not fully understood. The proximal Panx1 C-terminus (Panx1CT), upstream of a caspase-cleavage site has been demonstrated to be required for Panx1 cell-surface expression. We discovered a previously unreported putative leucine-rich repeat (LRR) motif within the proximal Panx1CT. I investigated the involvement of this putative LRR motif on Panx1 localization and oligomerization. Deletion of the putative LRR motif or uniquely the highly conserved segment of the putative LRR motif resulted in a significant loss of Panx1 cell surface expression. Finally, ectopic expression of Panx1-EGFP in HEK293T cells increased cell proliferation, which was not recapitulated by a Panx1 deletion mutant lacking the putative LRR motif. Overall the findings presented in this thesis provide new insights into the molecular determinants of Panx1 trafficking and oligomerization. / Graduate / 2020-02-14

Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.

Les canaux potassiques à deux domaines P (K2P) contrôlent l’excitabilité cellulaire et jouent un rôle central dans l’établissement et le maintien du potentiel de repos membranaire dans la majorité des cellules animales. Depuis leur identification dans les années 90, ces canaux ont été impliqués dans de nombreuses fonctions comme la modulation de l’activité neuronale, l’activité du muscle cardiaque ou encore la physiologie rénale. Malgré l’importance de ces canaux, peu de données existent sur les processus cellulaires qui contrôlent leur fonction in vivo. Au cours de ma thèse, j’ai utilisé des approches génétiques, d’imagerie et d’électrophysiologie pour comprendre comment l’expression, la distribution et l’activité du canal K2P UNC-58 sont contrôlés chez le nématode modèle C. elegans.Pour cela, j’ai effectué un crible suppresseur du phénotype locomoteur du mutant gain de fonction unc-58(e665). J’ai ainsi obtenu 133 mutants présentant une large gamme de niveaux de suppression, suggérant l’implication de plusieurs gènes dans les processus de régulation du canal. En utilisant les technologies de reséquençage complet de génome, j’ai pu cloner six nouveaux gènes requis pour la fonction d’unc 58.J’ai ensuite caractérisé en détail le rôle d’unc-44/ankyrine dans le contrôle du trafic d’unc 58. Ce travail a conduit à 4 conclusions majeures : (1) UNC-58, malgré sa structure de canal potassique, possède en réalité une sélectivité ionique altérée favorisant le passage des ions sodium, (2) l’addition à UNC 58 de protéine fluorescente par approche CRISPR/Cas9 nous a permis pour la première fois d’observer directement la distribution du canal UNC-58 in vivo, (3) l’ankyrine est nécessaire à l’adressage du canal UNC-58 à la surface des muscles et dans les axones des neurones mécanosenseurs ALM. Cette fonction fait intervenir une poche d’interaction lipidique localisée au sein du module Zu5N-Zu5C-UPA d’UNC-44, (4) ce mécanisme est hautement sélectif puisqu’il n’est pas requis pour l’adressage de 6 autres canaux potassiques musculaires. Mon crible a également identifié une interaction génétique entre unc-70/ß-spectrine et unc-44/ankyrine. Toutefois, la nature moléculaire de cette interaction reste encore à préciser / Two-pore potassium channels (K2P) control cell excitability and play a central role in the establishment and the maintenance of the resting membrane potential of almost all animal cells. Since their identification in the late 90s, these channels have been implicated in a large number of functions ranging from neuronal and cardiac activity to kidney physiology. Despite the crucial functions of these channels, comparatively little is known about the cellular processes controlling their function in vivo. During my PhD, I used a wide range of strategies including genetics, microscopy and electrophysiology to understand how the expression, the distribution and the activity of the K2P channel UNC-58 are controlled in the model nematode C. elegans. I have first performed a genetic suppressor screen targeting the locomotion phenotype of the gain of function mutant unc-58(e665). The screen yielded 133 mutants, displaying a wide range of suppression level, suggesting that several genes may be implicated in the channel regulation process. By using whole genome sequencing technologies, I’ve been able to clone six new genes required for the function of UNC-58.Then, I’ve characterized in detail the role of unc-44/ankyrin in the trafficking of UNC 58. This project led to 4 main conclusions : (1) UNC-58, despite its potassium channel structure, has an altered ionic selectivity, allowing preferably sodium ions to pass through the channel (2) the addition of a fluorescent protein to UNC-58 by CRISPR/Cas9 approaches allowed us for the first time to directly observe the addressing of the UNC-58 channel to the muscle surface and axons of ALM mechanosensory neurons. This function involves a lipid binding pocket located within the Zu5N-Zu5C-UPA module of UNC-44, (4) this mechanism is highly selective since it is not required for the addressing of 6 other muscular channels.My screen also identified a genetic interaction between unc-70/ß-spectrin and unc-44/ankyrin. However, the exact molecular nature of this interaction remains to be elucidated

Ca²⁺ influx is one of the critical events that trigger synaptic vesicular release, and the accumulation of residual free Ca²⁺ in synapses is also important for activity-dependent synaptic plasticity. Ca²⁺ imaging with fluorescence indicators (synthetic or genetically encoded) is a powerful approach to monitor Ca²⁺ levels in neurons and synapses. Although accumulating studies in vertebrate systems have been carried out to demonstrate the role of Ca²⁺ in synaptic transmission and plasticity, most of these studies rely on pharmacological methods to infer the molecular mechanism, with less emphasis on forward genetic analysis. The Drosophila neuromuscular junction (NMJ) is a powerful neurogenetic platform for studying synaptic transmission, because of the availability of many mutations. However, not many mutations have been analyzed with Ca²⁺ imaging. Besides, although Genetically Encoded Ca²⁺ Indicators (GECIs) including GCaMPs are increasingly popular as the tool to identify neuronal circuits activated by certain stimuli or mediating particular behaviors, the physiological and functional interpretation of neuronal Ca²⁺ transients reported by GECIs remain obscure. By expressing GCaMPs in NMJ synapses, I characterized a spectrum of genetic mutations including sodium channel alleles parats¹, parabss¹, potassium channel mutations Shaker (ShM, Sh¹²⁰), Shab³, ether-a-go-go (eag¹, eag⁴pm), and double mutant eag¹ Sh¹²⁰. Drosophila NMJs contain at least three different types of synapses, which include glutamatergic tonic motor synapse type Ib, phasic motor synapse type Is, and modulatory octopaminergic synapse type II. In this study, I found that the ion channel mutations did not uniformly alter the Ca²⁺ dynamics in type Ib, Is and II synapses. Based on genetic dissection and pharmacological analyses, I concluded that the excitability type I and type II synapses are differentially regulated by various ion channels, and that ion channels mainly influence the influx of Ca²⁺ upon membrane depolarization but not the subsequent clearance. I also attempted to interpret the significance of synaptic Ca²⁺ transients by correlating Ca²⁺ imaging with electrophysiological recordings. One important gap in the application of GCaMP indicators is its postsynaptic physiological relevance. Correlation of synaptic GCaMP Ca²⁺ transients with postsynaptic currents simultaneously recorded by focal extracellular recording indicated that Ca²⁺ transients reported by GCaMPs were slow, and did not reflect immediate synaptic transmission. Rather, the kinetics of synaptic Ca²⁺ transients was temporally correlated with short-term synaptic plasticity such as facilitation and depression. The hyperexcitable ion channel mutations Sh and parabss¹ enhanced the synaptic Ca²⁺ transient amplitudes as well as depression. Type Is synapses of hyperexcitable mutations such as eag¹ Sh¹²⁰ and parabss¹ often displayed single stimulus pulse-evoked Ca²⁺ transients, which were induced by high frequency repetitive firing of action potentials. Such Ca²⁺ transients were correlated with supernumerary peaks of postsynaptic currents. Based on the slow kinetics and the correlation with short-term plasticity, I conclude that GCaMP Ca²⁺ signals better reflect the accumulation of cytosolic residual Ca²⁺. The spontaneous Ca²⁺ waves in larval motor neurons were well correlated with high frequency nerve action potentials, suggesting that accumulation of residual Ca²⁺ occurs in larval crawling. Overall, this study provided important information about the different excitability control and Ca²⁺ clearance mechanisms in different synapses, and examined how membrane excitability controls the influx and accumulation of synaptic cytosolic residual Ca2+, as indicated by GCaMPs. Further, by correlating synaptic Ca²⁺ dynamics with electrophysiology, this study also investigated how to interpret GCaMP Ca²⁺ signals in the context of facilitation and depression, establishing a basis for an integrated approach of studying short-term synaptic plasticity from complementary physiological signals.