Voltage-gated potassium channels are primary determinants of cellular excitability in the mammalian nervous system. The localization of these channels to distinct cellular compartments influences components of neuronal function, including resting membrane potential, action potential characteristics and neurotransmitter release. Thus, understanding the mechanistic basis of ion channel localization can provide fundamental insight into human physiology. The overall goal of this dissertation was to elucidate the regulatory mechanisms governing localization and function of the Kv1.3 voltage-gated potassium channel. The sympathetic branch of the autonomic nervous system innervates many organ systems including the kidneys, heart and blood vessels and was used as a model to study endogenous Kv1.3. We found that postganglionic sympathetic neurons express Kv1.3 and that the channel exhibits a striking pattern of localization to the Golgi apparatus in the soma of these cells. Kv1.3 ionic current was also isolated from the soma of these neurons, indicating the channel is a determinant of the electrophysiological properties of sympathetic neurons. In addition, the specific inhibition of Kv1.3 with margatoxin was found to depolarize neuronal resting membrane potential, decrease the latency to action potential firing and increase nicotinic agonist-induced neurotransmitter release. Collectively, these findings demonstrate that Kv1.3 influences the function of postganglionic sympathetic neurons and led to the hypothesis that regulating channel localization may be a mechanism for modulating the activity of these cells. In this dissertation, we propose that the observed Golgi retention of Kv1.3 may be a trafficking-dependent mechanism of channel regulation. To test this hypothesis, we used HEK293 cells as our model system. Our data show that the degree of Kv1.3 Golgi localization is inversely correlated with the amount of channel at the plasma membrane. In addition, the amplitude of Kv1.3 ionic current measured in cells with low Kv1.3 Golgi localization was significantly greater than the current measured in cells with high Kv1.3 Golgi localization. One mechanism for localizing ion channels to the Golgi apparatus involves the Class I PDZ-binding motif (X-S/T-X-Φ). Deletion of the C-terminal PDZbinding motif of Kv1.3 decreased the intracellular Golgi localization of the channel and increased channel localization at the cell surface. Disrupting this canonical binding motif also increased the amplitude of Kv1.3 ionic current. These findings indicate that regulated subcellular distribution of the channel may be a determinant of Kv1.3 surface expression and function.

The divalent Ca2+ metal ion acts as a universal second messenger in virtually all eukaryotic cells from fungi to plants to mammals. In mammals, Ca2+ signaling is vital to a variety of physiological processes including fertilization, cell proliferation, secretion, and muscular contraction. In electrochemically non-excitable tissues, the release of Ca2+ from intracellular stores such as the endoplasmic reticulum is tightly regulated by the inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R Ca2+ release channel is activated by the binding of the small molecule inositol 1,4,5-trisphosphate (IP3) in response to extracellular stimuli such as hormones, growth factors, and neurotransmitters. The conformational changes accompanying IP3 binding were investigated using a biophysical approach. A specific focus of this work is to decipher how signals of ligand binding are transmitted from the N-terminal IP3-binding core to the C-terminal channel domain. To such end, biophysical studies of the ligand-induced conformational changes within the N-terminal domain of IP3R (a.a. 1 – 604) were performed. The results implicated the presence of two flexible linkers which join stably folded domains. This prompted the proposal of a model in which an equilibrium mixture of conformational substrates containing compact and more extended structures co-exist. Determinants within the N- and C-terminal regions of IP3R have previously been reported to be critical to channel function. Employing nuclear magnetic resonance (NMR) as well as biochemical methods, an intermolecular interaction between the S4-S5 linker, the cytoplasmic loop between the fourth and fifth transmembrane helices of IP3R, and the suppressor domain was identified. The determination of the crystal structure of the suppressor domain from isoform type 3 IP3R (IP3R3SUP) allowed us to map the residues involved in this interaction to one face of the molecule. The characterization of this interaction provides insight into the N- and C-terminal determinants essential to the IP3R channel gating mechanism.

The divalent Ca2+ metal ion acts as a universal second messenger in virtually all eukaryotic cells from fungi to plants to mammals. In mammals, Ca2+ signaling is vital to a variety of physiological processes including fertilization, cell proliferation, secretion, and muscular contraction. In electrochemically non-excitable tissues, the release of Ca2+ from intracellular stores such as the endoplasmic reticulum is tightly regulated by the inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R Ca2+ release channel is activated by the binding of the small molecule inositol 1,4,5-trisphosphate (IP3) in response to extracellular stimuli such as hormones, growth factors, and neurotransmitters. The conformational changes accompanying IP3 binding were investigated using a biophysical approach. A specific focus of this work is to decipher how signals of ligand binding are transmitted from the N-terminal IP3-binding core to the C-terminal channel domain. To such end, biophysical studies of the ligand-induced conformational changes within the N-terminal domain of IP3R (a.a. 1 – 604) were performed. The results implicated the presence of two flexible linkers which join stably folded domains. This prompted the proposal of a model in which an equilibrium mixture of conformational substrates containing compact and more extended structures co-exist. Determinants within the N- and C-terminal regions of IP3R have previously been reported to be critical to channel function. Employing nuclear magnetic resonance (NMR) as well as biochemical methods, an intermolecular interaction between the S4-S5 linker, the cytoplasmic loop between the fourth and fifth transmembrane helices of IP3R, and the suppressor domain was identified. The determination of the crystal structure of the suppressor domain from isoform type 3 IP3R (IP3R3SUP) allowed us to map the residues involved in this interaction to one face of the molecule. The characterization of this interaction provides insight into the N- and C-terminal determinants essential to the IP3R channel gating mechanism.

The transient receptor potential channels, ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), are non-selective cation-permeable channels that have retained their function as chemical sensors since their first appearance in metazoan species several hundred million years ago. In vertebrates, TRP channels have evolved multiple functions which make it difficult to understand exactly how they transmit signals to the brain that are interpreted very differently. For example, TRPA1 and TRPV1 are sensitive to various chemicals and activation of these channels produce sensations with opposing effects. Pain is felt when TRPV1 is activated by spider toxins, but activation by plant cannabidiol results in a pain-relieving sensation. Similarly, TRPA1 activation by delta-tetrahydrocannabinol is reported to relieve symptoms of pain, but TRPA1 activation by the active ingredient in wasabi results in a repulsive or noxious sensation. Much of what we know about TRPA1 and TRPV1 comes from the use of plant products or exposure to substances that cause or alleviate pain and inflammation. In this study, whole-cell voltage clamp recordings of heterologously expressed human TRPA1 and human TRPV1 were tested for sensitivity to a hallucinogenic plant compound, salvinorin A and an arthropod-defensive compound, para-benzoquinone. Neither compound has yet been reported to activate TRP channels but both are known to be involved in pain and inflammation signaling in humans. I show that the arthropod compound, para-benzoquinone, activates and desensitizes TRPA1 in a cysteine-dependent manner, but activation of TRPV1 is not dependent on cysteine reactivity. Although salvinorin A is known to be a potent agonist of the kappa-opioid and cannabinoid receptors, here I show that it also acts as a highly potent agonist of both TRPA1 and TRPV1. Its interaction with TRP channels may contribute to its antinociceptive effects in behavioral studies with animals that are reported to be independent of opioid signaling.

Influenza viral infection causes several hospitalizations and claims the lives of many people each year. The threat of epidemic and pandemic are more pressing than ever with newly mutated strains developing every year. Understanding the mechanism of infection of influenza can help identify new potential drug targets and help progress the development of antivirals. Currently there are two classes of FDA approved drugs, neuraminidase inhibitors and M2 ion channel inhibitors, to combat influenza infection. Unfortunately, viral resistance to M2 ion channel blockers has caused them to stop being used for treatment. This paper focuses on understanding influenzas ability to mutate and it mechanism of infection to develop new M2 ion channel blockers.

Previous studies on the rat P2X2 receptor demonstrated that structural modifications to amino acid side chains within the second transmembrane domain lead to receptor activation in the absence of exogenously applied ATP (Rassendren et al, 1997. Cao et al, 2007. Cao et al, 2009). Present work has been aimed towards the characterization of these apparently ATP-independent currents, to investigate the molecular mechanism underlying this phenomenon using a combination of computer modeling, amino acid substitution, heterologous expression in HEK293 cells, the real time modification of engineered cysteines by MTS compounds and electrophysiological techniques. A screen of cysteine substituted receptors at TM2 positions (from G323 to T354) with the membrane permeable MTS compound, MTSP, found that the compound evoked substantial currents from cells expressing P2X2[I328C] receptors, but not from cells expressing other TM2 cysteine substituted, nor wild type receptors. MTSP-evoked currents had similar properties to ATP currents in terms of rectification, NMDG+ permeability and unitary currents. Further investigation indicated that hydrophobic, unbranched modifications to the side chain at position 328 were the most effective. Overall, the results from this work demonstrate that increasing the length and hydrophobicity of an unbranched side-chain at position 328 leads to full receptor activation without the requirement for ATP. These results suggest that the highly conserved native Ile at position 328 stabilizes the closed of the receptor due to its branched nature.

Ion channel regulation is key to the control of excitability and behaviour. In the bag cell neurons of Aplysia californica, a voltage- and Ca2+-dependent nonselective cation channel drives a ~30-minute afterdischarge, culminating in the release of egg-laying hormone. Using excised, inside-out single channel patch-clamp, this study tested the hypothesis that inositol 1,4,5-trisphosphate (IP3), which is produced during the afterdischarge, and channel-associated protein kinase C (PKC), which is activated throughout the afterdischarge, cause a left-shift (enhancement) in both the voltage- and Ca2+-dependence of the cation channel. Kinetic analysis of bag cell neuron cation channel voltage-dependence revealed that, with depolarization, the channel remained open longer and reopened more often. A cation channel subconductance was also observed, and found to be 13 pS vs. the typical 23 pS full-conductance. The cytoplasmic face of cation channel-containing patches was exposed to 1 mM ATP, as a phosphate source for channel-associated PKC, and/or 5 uM IP3. Apparent PKC-dependent phosphorylation left-shifted voltage-dependence by -3 mV, although this effect was more prominent at negative voltages (between -90 and -30 mV). Conversely, IP3 right-shifted voltage-dependence (change in V1/2 of 6 mV). Cation channel Ca2+-dependence was similar to that previously reported, with a control EC50 of 3-5 uM. This was right-shifted by PKC (EC50 = 30 uM) and even more so by IP3 (apparent EC50 = 20 M). PKC largely rescued the Ca2+ responsiveness in the presence of IP3 (EC50 = 20 uM). Unexpectedly, IP3 plus ATP resulted in an increase in channel unitary conductance at more positive voltages. The multi-faceted regulation of the bag cell neuron cation channel suggests sophisticated modulatory control. Upregulation, such as depolarization and the left-shift in voltage-dependence with PKC, would drive the afterdischarge, while counteracting effects, such as IP3 right-shifting voltage-dependence, as well as PKC and IP3 suppressing Ca2+-dependence, would simultaneously or subsequently attenuate the channel, thus preventing an interminable afterdischarge. / Thesis (Master, Physiology) -- Queen's University, 2008-08-26 13:20:16.528

Based on preliminary reports, an extended series of bolamphiphilic oligoester compounds with structural symmetry were synthesized and then tested using a planar bilayer experiment with the voltage-clamp technique. The main structures of these compounds are identical, consisting of a mono or tri-aromatic core, two octamethylene chains and two benzoyl headgroups which are all connected through ester linkages. The structural variance was provided by the four differently functionalized benzoyl headgroups. The synthetic methods of three to five steps were mainly adapted from the previously reported method.1 The methods successfully produced eight compounds with overall yields of 20 to 30%. The voltage-clamp data suggested voltage-dependent behaviors occur at low concentrations while Ohmic behaviors require at high concentrations. The activity at low potentials showed relatively erratic behavior but the channels frequently switched between opening and closing states. The activity at high potential lasted longer as the channel maintained a longer state of opening. The exponential voltage-dependent behaviors were observed at higher potential while the voltage-independent Ohmic behaviors occur at low potential. These channel behaviors are highly time-dependent as there is no control over the stability and the aggregation level for the compounds forming active channels in the membrane. In some cases the current-voltage responses appear to be asymmetrical between the positive and the negative potentials. Mechanisms consistent with the observations are proposed. / Graduate / 0485 / 0490 / yzong@uvic.ca

Based on preliminary reports, an extended series of bolamphiphilic oligoester compounds with structural symmetry were synthesized and then tested using a planar bilayer experiment with the voltage-clamp technique. The main structures of these compounds are identical, consisting of a mono or tri-aromatic core, two octamethylene chains and two benzoyl headgroups which are all connected through ester linkages. The structural variance was provided by the four differently functionalized benzoyl headgroups. The synthetic methods of three to five steps were mainly adapted from the previously reported method.1 The methods successfully produced eight compounds with overall yields of 20 to 30%. The voltage-clamp data suggested voltage-dependent behaviors occur at low concentrations while Ohmic behaviors require at high concentrations. The activity at low potentials showed relatively erratic behavior but the channels frequently switched between opening and closing states. The activity at high potential lasted longer as the channel maintained a longer state of opening. The exponential voltage-dependent behaviors were observed at higher potential while the voltage-independent Ohmic behaviors occur at low potential. These channel behaviors are highly time-dependent as there is no control over the stability and the aggregation level for the compounds forming active channels in the membrane. In some cases the current-voltage responses appear to be asymmetrical between the positive and the negative potentials. Mechanisms consistent with the observations are proposed. / Graduate / 0485 / 0490 / yzong@uvic.ca

Abstract Photoreceptors convert the energy of light into an electric signal to be processed by the visual system. Photoreceptors of nocturnal insects are adapted for night vision by sacrificing spatial and temporal resolution for improved sensitivity. While the sensitivity-increasing optical adaptations and the temporal properties of light responses have been studied earlier, the intermediate biophysical mechanisms responsible for shaping the captured light into voltage responses were previously not known in detail in any nocturnal species. Using electrophysiological tools and computer simulations the photoreceptors of the nocturnal cockroach (Periplaneta americana) were studied by characterising 1) the electrical properties responsible for shaping the light responses, 2) the properties of light responses at different stages of light and dark adaptation and 3) properties of low-intensity light stimuli and how they are processed by the photoreceptors. The high input resistance and whole-cell capacitance were typical for a nocturnal insect, but the two voltage-dependent potassium conductances were closer to those found in diurnal species. The dominant sustained conductance typically associated with day-light vision activated during simulated light responses whereas the lesser transient conductance previously linked to low-light vision did not. Light responses were persistently slow regardless of the adapting light level and saturated at low intensities, indicating a strong adaptation to vision in dim light. Simulations showed that at such low light levels the physical noise caused by random photons determines the information rate and the biological noise, caused by random latency and amplitude of single photon responses, has only a minor effect. At higher intensities the latency variability degraded the information rates but the amplitude variability did not. Thus, photoreceptors of nocturnal animals can sacrifice phototransduction precision in their natural illumination without compromising their coding performance.