Catherine C Pang

Epilepsy is among the most prevalent neurological disorders, affecting approximately 1% of the human population. There are many types of epilepsies, with diverse aetiologies, and many therapies that target ion channels as a means to combat neuronal hyper-excitability. However, most anti-epileptic drugs today target a limited number of ion channel types, mainly voltage-gated sodium and calcium channels. Retigabine is a recently approved anti-epileptic drug that operates through a novel mechanism of activating voltage-gated potassium channels. Previous research has established neuronal KCNQ channels as the therapeutic target of retigabine. However, detailed insights regarding the molecular mechanisms of retigabine action are lacking, such as its mode of binding, the factors underlying its ability to stabilize channel opening, and the stoichiometry of its action. A lack of such knowledge hampers the development of more potent and specific channel openers devoid of side effects associated with this first-generation drug.The work presented in this thesis utilizes various research techniques to investigate retigabine pharmacology at a molecular level. In the first objective, retigabine binding to KCNQ3 channels is investigated using unnatural amino-acid mutagenesis. The data pinpoint an essential hydrogen-bonding interaction that likely occurs between an S5 tryptophan residue and a carbonyl oxygen moiety present in most KCNQ activating drugs, providing the highest resolution understanding of the retigabine pharmacophore to date. In the second objective, voltage-clamp fluorometry is used to track conformational changes of the voltage-sensing domain of KCNQ3 channels. The data illustrate a network of interactions between the voltage-sensing and ion conducting regions of the channel protein that is dependent on the anionic membrane phospholipid PIP2; these interactions are not only essential for channel function, but also for retigabine binding to modulate channel voltage sensing downstream of binding in the pore domain. Finally, usingiiiconcatenated KCNQ3 constructs to express channels with variable stoichiometry of retigabine binding sites, we demonstrate that a minimum of one retigabine sensitive channel subunit is required for functional pharmacological effects. Overall, this work provides novel insights applicable to the development of retigabine derivatives with greater therapeutic impact, and improves our understanding of lipid and drug regulation of KCNQ channels.

View record

No abstract available.

Activation of inducible nitric oxide synthase (iNOS) and oxidative stress have been shown to be associated with compromised cardiovascular function in streptozotocin (STZ)-induced type 1 diabetes. The aim of the project is to investigate cardiovascular abnormalities in a rat model of type 2 diabetes (Zucker diabetes fatty or ZDF rats) and two models of metabolic syndrome (fructose-fed rats and Zucker obese rats), and to provide direct evidence linking iNOS and oxidative stress to abnormal cardiovascular function in these disorders. Blood pressure, cardiac contractility, cardiac index, regional flow, vascular resistance and venous tone were measured in diseased as well as normal rats. Biochemical analyses such as activities of iNOS, immunostaining of iNOS and western-blot analysis of iNOS in the heart tissue were carried out. The results showed that cardiac contractile response to dobutamine was compromised in the ZDF rats, and this was associated with increased myocardial protein expression as well as activity of iNOS. The formation of peroxynitrite was increased in the heart tissue of the ZDF rats. Selective inhibition of iNOS by 1400W (N-3-aminomethyl-benzyl-acetamidine) did not alter responses to dobutamine in the control rats, but augmented the contractile effects of dobutamine in the diabetic rats. The regional blood flow was altered in the ZDF rats, and iNOS played a negligible role in regulating regional flow in the ZDF rats. Although venous response to noradrenaline was also altered in the Zucker obese rats, NOS may not be involved in venous tone regulation. Anti-oxidative treatment with N-acetylcysteine inhibited the development of insulin resistance, blood pressure elevation and the increase of 8-isoprostane formation in the fructose-fed rats. We conclude that heart function is compromised and regional blood flow is altered in the ZDF rats. Activation of iNOS plays an important role in suppressing heart dysfunction but does not affect regional blood flow. In Zucker obese rats with metabolic syndrome, iNOS may not be involved in changes of venous function. Oxidative stress is associated with both abnormality of heart dysfunction in type 2 diabetes (by formation of peroxynitrite due to iNOS activation) and development of hypertension and insulin resistance in metabolic syndrome.

View record

Orthostatic intolerance following exposure to simulated or actual microgravity is observed following spaceflight and extended periods of bed rest, and is not always associated with simultaneous hypotension. Differential adaptation of cephalic and caudal arterial vasculatures (as a result of removal of the normal hydrostatic gradient) is proposed as a potential mechanism underlying this phenomenon. A potential role for changes to the L-arginine/nitric oxide pathway in such adaptations has been suggested, predominantly from previous in vitro studies; using an established model of simulated microgravity (head-down tilt; HDT). This thesis investigates whether findings in isolated vessels are reflected by in vivo measurements of cephalic and caudal vascular function.Using carotid or iliac artery flow normalized to mean arterial pressure as an index of cerebral or hind limb vascular conductance, autoregulatory cerebral vasodilatation in response to lower body negative pressure was found to be impaired following HDT. In addition, α¬1-adrenoceptor agonist-mediated vasoconstriction was decreased in the cerebral vasculature and increased in the peripheral and hind limb vasculature. Administration of acetylcholine or the non-selective nitric oxide synthase (NOS) inhibitor Nω-nitro-L-arginine methyl ester (L-NAME) demonstrated a decreased contribution of NOS to cerebrovascular tone, but an increased contribution of NOS to peripheral vascular resistance and tone of the hind limb vasculature. Together with a lack of difference in the response to the selective inducible NOS (iNOS) inhibitor 1400W, these results suggest that differential adaptation of eNOS may account for the observed differences between control and HDT animals. Further investigation of the changes to the L-arginine/nitric oxide pathway suggest that these changes are not associated with changes in eNOS expression, but may be related to altered activity of eNOS. Furthermore, the bioavailability (as measured by pharmacokinetic half life) or the vascular effector mechanisms (as measured by the haemodynamic response to exogenously administered nitric oxide) responsible for the effects of nitric oxide were also shown to be unaffected by HDT.These findings suggest that differential adaptation of the L-arginine/nitric oxide pathway may contribute to the inability to raise total peripheral resistance and impaired cerebral autoregulation following HDT, thereby representing a mechanism of orthostatic intolerance following exposure to microgravity.

View record