Relevant Thesis-Based Degree Programs
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Graduate Student Supervision
Doctoral Student Supervision
Dissertations completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest dissertations.
Seasonal and pandemic viral infections continue to pose severe challenges to human health. Antivirals that can effectually complement current vaccination strategies are vital in the battle against drug-resistant pandemic viral strains. Many viruses express small, predominantly hydrophobic and multi-functional proteins that can assemble into oligomeric ion channels capable of passing ions, called viroporins. These small proteins have historically been effective antiviral targets for drugs like amantadine that effectively inhibit the wild-type M2 ion channel of Influenza A. However, drug resistance has rendered adamantane-based antivirals ineffective against circulating strains of influenza A. Similarly, various studies have reported that hexamethylene amiloride (HMA), a previously used diuretic, is also capable of inhibiting the ion channel activity of influenza A M2 and SARS-CoV-1 and 2 Envelope (E) proteins. This dissertation elaborates on the functional properties of a panel of in-house designed and synthesized HMA derivatives and evaluates the activity of these amiloride-derived compounds against known viroporins of influenza A and SARS-CoV-2 through an iterative approach, employing synthetic organic chemistry, electrophysiological measurements and computational techniques and quantification of anti-viral activity through in vitro viral assays. Through studying the structure activity relationship of this chemical class, we report compounds that effectively inhibit the wild-type and adamantane-resistant forms of influenza A M2 ion channel. We found that tert-butyl 4'-(carbamimidoylcarbamoyl)-2',3-dinitro-[1,1'-biphenyl]-4-carboxylate exhibits dual inhibitory effect against both adamantane-sensitive as well as S31N mutant influenza A M2. This compound has an inhibitory effect against the replication of influenza viruses encoding wild-type M2 and M2(S31N) in vitro. Furthermore, by utilizing in silico techniques such as molecular modelling, docking simulations, molecular dynamics, and steered molecular dynamic, we report that these HMA derivatives are able to bind and effectively block the lumen of SARS-CoV-2 E protein with higher affinity as compared to HMA. Whilst, more work is still needed to utilize these HMA derivatives as therapeutic options, the findings presented in this thesis highlight the potential of amiloride-derived compounds as antivirals against drug-resistant strains of influenza A and important drug leads for the design and further development of inhibitors of SARS-CoV-2 E protein.
The IKs current, which is composed of KCNQ1 and KCNE1 subunits, increases in size as a result of beta-adrenergic stimulation. This response is a result of the KCNQ1 subunit being phosphorylated by protein kinase A (PKA). This is an important physiological response at high heart rates that allows the ventricles adequate time to fill. Mutations in either of these subunits can cause impaired cardiac repolarization and result in long and short QT syndromes, as well as familial atrial fibrillation. The mechanism by which the channel complex reacts to this stimulation has not been fully elucidated. To investigate the mechanism behind this, both total internal reflection florescence microscopy (TIRF) and single-channel recording were used. 8-CPT-cAMP, a membrane-permeant analog of cAMP, was used to induce PKA phosphorylation of KCNQ1. TIRF studies found no significant change in the number of channels at the cell surface. Single-channel recordings of IKs had a reduced first latency to opening showing that the channel opened more quickly in response to 8-CPT-cAMP. The IKs current has multiple open states and, in the presence of 8-CPT-cAMP, occupied the higher subconducting states more frequently. An increase in the open probability of the channel complex was also seen. In response to phosphorylation triggered by 8-CPT-cAMP, the first latency to opening of the channel is reduced and the channel opens more quickly, more often and passes more current by increasing the higher conducting open states. This results in an increase in IKs current at the macroscopic level. Using enhanced gating mutant KCNQ1 channels, it is shown that the effect of phosphorylation is likely through further activation of the voltage sensor. KCNE1 is required for a functional response to PKA phosphorylation. Both whole-cell and single-channel recordings show that as the number of KCNE1 subunits is reduced, a graded effect is seen in response to 8-CPT-cAMP; the less KCNE1 subunits, the smaller the response. However, in single-channel recordings, there was also a KCNE1-independent effect of phosphorylation as the first latencies for all KCNQ1-KCNE1 complexes were reduced in a non-graded manner. This suggests that phosphorylation may have both KCNE1-dependent and independent effects on the channel.
The IKs current has an important role in repolarizing the cardiac action potential. KCNQ1 subunits form a tetrameric voltage-gated potassium channel, with which an accessory beta-subunit, KCNE1, can interact. KCNE1 binding to KCNQ1 remarkably alters channel kinetics by delaying activation, increasing current density and removing inactivation. There has always been confusion about how many KCNE1 subunits can associate with the KCNQ1 tetramer. Several groups have reported a strict fixed stoichiometry of two KCNE1 subunits to four KCNQ1 subunits. However, others have shown that the ratio varies depending on the concentration of KCNE1 subunits available. Using whole cell and single channel patch clamp recordings of tethered fusion constructs with different ratios of KCNE1:KCNQ1, as well as photo-crosslinking experiments, we show that up to four KCNE1 subunits can associate with the complex. Therefore, in vitro, IKs can have a variable stoichiometry. In further photo-crosslinking experiments we show that two adjacent residues in KCNE1 interact with KCNQ1 in different channel states, open and closed. Additionally, we show that these interactions are likely taking place with the pore domain of the channel. We also confirm what other groups had proposed, that KCNE1 moves within the cleft of KCNQ1 during channel activation, since the rates of crosslinking change when the channels are held at more depolarized holding potentials. Finally, we investigate whether or not all four voltage sensors have to activate and the complex undergo a concerted step in order for the pore to conduct current. We employ a mutation, E160R, which applies electrostatic repulsion to the positive charges of the voltage sensor and keeps it in a resting conformation. By locking one, two, three and four voltage sensors down we show, via whole cell and single channel recordings, that the channel can conduct when only one voltage sensor is free to move. From these experiments, we provide additional evidence that the IKs channel gates in an allosteric fashion, where each additional voltage sensor movement results in a progressively higher open probability. Additionally, we propose that there is cooperativity between KCNQ1 subunits, where movement of one voltage sensor facilitates movement of a neighboring voltage sensor.
Ion channels are integral membrane proteins that form an aqueous pore through the cell lipid bilayer, and allow ions to traverse the membrane at rates approaching limits set by diffusion. Selectivity and gating differences amongst members of this protein family enable complex physiological processes such as action potentials. The diversity in ion channel selectivity and gating is endowed through structural permutations of protein structure that slightly alter factors such as the rate at which a channel activates or the width of the pore region and thus the type of ions it interacts with. This thesis investigates structural bases for the anomalous gating and drug interaction behaviour exhibited by the human ether-à-go-go related gene (hERG) voltage-gated potassium channel (VGKC). The unique gating kinetics of hERG allow it to fulfill its role as the rapid delayed rectifier potassium current of the cardiac action potential and the unique susceptibility to drug block can compromise this function. Chapter 2 describes how slow deactivation of hERG can be largely attributed to cytosolic domain interaction with channel gating, an interaction that serves to establish a mode shift of the channel gating charge, shifting the deactivation gating pathway to more hyperpolarized potentials. Chapter 3 demonstrates that an interaction between an acidic residue at the bottom of the S1 and a basic residue at the bottom of the S4 stabilizes the closed state of the channel and slows activation. Through gating currents and fluorescence experiments, we propose a model of hERG gating in which this unique interaction stabilizes an early closed state of the channel. Chapter 4 investigates the role of cation-π interactions in hERG drug block, testing the importance of the two most significant residues for drug interaction, Y652 and F656. Using unnatural amino acid mutagenesis, this final study shows that cation-π interactions do not appear to play a major role in drug interaction with the hERG pore.
Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the fibrillin-1 gene, with aortic aneurysm considered as the most life-threatening complication. Previous studies have shown that doxycycline, a general inhibitor of matrix metalloproteinases (MMPs), can improve aortic contractility and elastin structure in the mouse model of MFS. However, the longitudinal effects of MMPs inhibition on the gradual progression of aneurysm and aortic wall biophysical properties in a live animal have not yet been investigated. Therefore, we assessed the hypothesis that a long-term treatment with doxycycline would delay the progression of aortic aneurysm, improve aortic wall elasticity, and protect the ultrastructure of elastin in MFS mice.In this study, non-invasive and label-free multiphoton microscopy imaging was used to quantify fiber morphology and volumetric density of aortic and cutaneous elastin and collagen in MFS mice. We also utilized non-invasive high-resolution echocardiography to conduct a longitudinal in vivo study of the structural, functional, and biophysical properties of the aortic wall in control and MFS mice in the absence and presence of doxycycline treatment. The ultrastructure of aortic elastic fibers was also assessed by electron microscopy.Multiphoton imaging revealed significant elastin fragmentation and disorganization within the aortic wall of MFS mice, which was also associated with reduction in cutaneous volumetric density of elastin and collagen. Ultrasound imaging showed that aortic pulse wave velocity (PWV) was significantly elevated in MFS mice starting at the age of 6-month-old, which was associated with a distinct increase in aortic root dimeter in the regions of aortic annulus and sinus of Valsalva. Long-term treatment with doxycycline resulted in a significant improvement in elastin organization, reduction of aortic root growth and aortic PWV in MFS mice. These findings underscore the key role of MMPs in the pathogenesis, and provide new insights into the potential therapeutic value of doxycycline in blocking MFS-associated aneurysm.Furthermore, the use of multiphoton imaging to detect the signs of elastin degradation in the skin dermis may be considered as the first step towards the potential development of a non-invasive approach for monitoring the aneurysm progression in MFS patients.
Voltage-gated potassium (Kv) channels are essential membrane proteins in modulating membrane excitability and related cellular processes. Many details associated with the voltage response are unclear, particularly the complete role of the voltage sensing domain, and not just the densely charged S4 helix. Based on its crystal structure, the Kv1.2 channel represents an ideal model in which to study these questions. This thesis investigates Kv1.2 activation and deactivation gating, using the voltage clamp fluorimetry technique. This technique utilizes an environmentally sensitive fluorophore introduced at locations of interest in order to visualize conformational changes in protein structure. Labelling of Kv1.2 channels at the extracellular end of S4 reports a fast quenching of fluorescence emission upon depolarization that correlates extremely well with gating current measurements, suggesting it is a report of voltage-dependent S4 translocation. In addition, a slow quenching component is observed with a very negative voltage-dependence (V₁/₂ = -73.9 mV ± 1.4 mV), not seen in any other Kv channels studied to date, that involves regions of the voltage sensing domain in S1 and S2. This slow quenching is selectively removed from the fluorescence report with transfer of extracellular S1-S2 or S3-S4 linkers from the homologous Shaker potassium channel, suggesting that it arises from channel-specific interactions between the Kv1.2 linker segments. However, transfer of Kv1.2 linker segments into Shaker fail to recapitulate this quenching component, suggesting that these linker interactions likely underlie further differences in voltage sensor domain gating and/or structure. This slow quenching component correlates with deactivation of ionic current, and is prolonged with co-expression of the N-type inactivation-conferring Kvβ1.2 subunit. In the presence of the beta subunit, this likely reflects unbinding of the inactivation moiety from the pore domain, allowing deactivation and S4 return, but in the α-subunit alone we suggest that this may be a report of a voltage-dependent rearrangement in the voltage sensor domain that stabilizes the S4 in an activated conformation. Such interactions have been reported in other voltage-gated proteins, and provides further evidence that we must consider more than just S4 translocation when it comes to understanding the complete potassium channel voltage response, Kv1.2 or otherwise.
Master's Student Supervision
Theses completed in 2010 or later are listed below. Please note that there is a 6-12 month delay to add the latest theses.
No abstract available.
The slow potassium current, IKs, abbreviates the cardiac action potential by repolarizing the membrane to a resting state. Mutations in the pore-forming IKs subunit, KCNQ1, cause long QT syndrome type 1 (LQT1), which increases risk of fatal arrhythmia. Despite the physiological and clinical importance of IKs, little is known about the elementary events that underlie the unique biophysical properties of the channel, and how these elementary events are altered in the face of disease. This thesis investigates single channel recordings of IKs with and without mutations that cause LQT1 using patch clamp electrophysiology. Single channel IKs is described by slow and fast gating processes. The channel is slow to open, but flickers rapidly between open and closed states in non-deactivating bursts. Long latency periods to opening underlie the slow activation of IKs at depolarized potentials. Channel activity is cyclic with periods of high activity followed by quiescence, leading to an overall low open probability. The mean single channel conductance was determined to be 3.2 pS and long-lived subconductance levels coupled to activation were observed. Single channel properties of IKs with LQT1 mutations in the S3 helix of the voltage sensing domain in KCNQ1 were investigated to uncover pathogenic mechanisms at the single molecule level. Open probability was reduced in loss-of-function mutations (D202H, I204F and V205M) and increased in a unique gain-of-function mutation (S209F) that may cause LQTS from a reduced number of functional channels at the cell surface. The mean duration of open events correlated well with deactivation rate in all mutants and first latency to opening determined activation rate. From these results, we attributed the pathogenic mechanisms of LQT1 mutations to alterations in the stability of specific channel states.
Voltage-gated K⁺ channels (Kv channels) play important roles in the repolarization and the termination of electrical excitation in cardiomyocytes, but very little is known about how their surface expression and localization are regulated. In this thesis, I report the characterization of Kv1.5 localization as well as the trafficking pathways utilized by Kv4.2 in ventricular myocytes. We have developed a new gene gun transfection method that, for the first time, allows the ready and convenient transfection of acutely isolated adult rat cardiac myocytes. Using this system combined with electrophysiology and confocal imaging experiments, we unequivocally demonstrate that tagged Kv1.5 is efficiently localized to the intercalated disk in ventricular myocytes. Furthermore, Kv1.5 deletion mutations known to reduce the surface expression of the channel in heterologous cells do not affect the channel localization to this structure.The ventricular myocyte transfection system combined with electrophysiological and imaging techniques has also been used identify the small GTPases that regulate the trafficking of cardiac Kv4.2. We demonstrate that the small GTPases Sar1, Rab1, Rab5, Rab4, Rab11and Rab7 are involved in specific steps in the forward and retrograde trafficking of Kv4.2 in rat ventricular myocytes. This work has provided critical insights into the trafficking of cardiac potassium channels and allows us to better understand the modulation of their function in the heart.
Functional expression of voltage-gated potassium (Kv) channels in the plasmalemma is essential for repolarization phase of the cardiac action potential. Therefore, changes in Kv channel plasmalemmal expression can impact cardiac action potential duration and thereby result in arrythmias. The expression of these Kv channels is modulated by a number of different mechanisms, and among these, the most important and potent one is the regulation of the number of channels in the plasma membrane through modulation of channel trafficking. The trafficking process of Kv channel in cardiac background is poorly understood. The purpose of the studies presented in this thesis was to identify the specific kinesin isoform that is required for cardiac Kv1.5 channel forward trafficking and to investigate the roles of small GTPases in the trafficking of Kv4.2 channels in adult rat ventricular myocytes. Overexpression of wild type Kif5b increased the Kv1.5-conducted current and this increase was dependent on Golgi function; a 6 h treatment with Brefeldin A reduced Kv1.5 currents to control levels in Kif5b-overexpressing cells. Expression of dominant negative isoform of Kif5b prior to induction of Kv1.5 in a tetracycline inducible system blocked surface expression of the channel in both HEK293 cells and H9c2 cardiomyoblasts. These data confirmed the requirement for Kif5b for forward trafficking of newly synthesized Kv1.5 channels.The involvement of several Rab GTPases as well as Sar1 in the trafficking of an endogenous cardiomyocyte potassium channel has also been established. Kv4.2 traffics out of the cardiac endoplasmic reticulum via a conventional pathway involving Sar1 and Rab1 and its trafficking to the sarcolemma is enhanced by overexpression of wild type Rab11. Internalization of the channels is dependent upon Rab5 function and block of Rab4 somehow also inhibits that internalization. The internalized channels, if not recycle back into plasma membrane will be degraded via the proteasomal degradation pathway.Together, these studies enhance our knowledge of the players involved in the trafficking pathway of Kv channels in cardiomyocytes. In particular, the roles of kinesin and several small GTPases in regulating cardiac Kv channel plasmmalemmal expression through channel trafficking modulation.