Imaging tools in cellular and molecular biology have long relied on organic fluorophores to observe microorganisms or various cell constituents. The advent of semiconductor nanoparticles known as quantum dots (QDs) has offered the possibility to use this new class of fluorescent probes with very advantageous optical properties in cell biology. The imaging of transmembrane potential and ionic currents is of significant importance for monitoring the activity of the cell. It remains possible with relatively complicated instruments and methods such as patch clamping. A complementary approach to view the dynamics of ion channels with modern and efficient fluorophores is therefore of great interest to the field of biology in general. / We developed a construct based on the FRET signal between QDs and organic fluorescent dyes to monitor the conformational changes of voltage gated sodium channels. The amino acid histidine was used as a "landing platform" for QDs and the bacterial sodium channel NaChBac was chosen for testing. This study focused on the preliminary steps of the project and aimed to characterize the electrophysiological behavior of the histidine-tagged channel. The whole-cell configuration of patch clamping was the tool we used to understand the differences between the wild-type and the histidine-tagged variants of the channels. We also explore the possibility to land QDs on the histidine tag.

Pacific hagfish (Eptatretus stoutii) slime mucin vesicles are released by holocrine secretion with membranes that remain intact until the vesicle contacts seawater and ruptures. This thesis is an investigation of the mechanisms that drive mucin vesicle rupture for mucin release. Using isolated mucin vesicles collected from the slime glands of the hagfish, I tested the effects of a variety of solutions and drugs on vesicle rupture. I found that there are two categories of mucin vesicle that differ in their sensitivity to calcium ions, and that calcium-dependent vesicle rupture was inhibited with anion channel inhibitors. I also found that vesicle swelling rate was reduced by the aquaporin inhibitor mercuric chloride. Together, these data suggest that mucin vesicle rupture is partially dependent on the movement of chloride ions from seawater through calcium-activated anion channels and the rapid influx of water through aquaporin-like proteins in the vesicle membrane. / NSERC Discovery Grant, NSERC CGSM scholarship, Canada Foundation for Innovation, Ontario Ministry of Research and Innovation

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

The Arabidopsis thaliana genome encodes twenty putative cyclic nucleotide-gated channel (CNGC) genes. Studies on A. thaliana CNGCs so far have revealed their ability to selectively transport cations that play a role in various stress responses and development, however, the regulation of plant CNGCs is not yet fully understood. Thus, in this study I have attempted to analyze the structure-function relationship of AtCNGCs, mainly by using suppressor mutants of the rare gain-of function mutant, cpr22. The A. thaliana mutant cpr22 resulted from an approximately 3kb deletion that fused the 5’ half and the 3’ half of two CNGC-encoding genes, AtCNGC11 and AtCNGC12, respectively. The expression of this chimeric CNGC, the AtCNGC11/12 gene confers easily detectable characteristics such as stunted morphology with curly leaves and hypersensitive response-like spontaneous lesion formation. Through a suppressor screen, twenty nine new alleles were identified in AtCNGC11/12. Since the cytosolic C-terminal region contains important regulatory domains, such as a cyclic-nucleotide binding domain, eleven cytosolic C-terminal mutants, S17, S35, S81, S83, S84, S100, S135, S136, S137, S140 and S144, were analyzed. A detailed analysis of two mutants, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), suggested that G459 and R381 are important for basic channel function rather than channel regulation. Site-directed mutagenesis and fast protein liquid chromatography (FPLC) showed that these two amino acids influence both intra- and inter-subunit interactions that are involved in stabilizing the tertiary structure of the channel. In addition, calmodulin binding domain(s) (CaMBD) and cyclic nucleotide binding domain(s) (CNBD) of some of AtCNGCs were studied using computational modeling and biophysical analyses. The data indicated that AtCNGC12 has two CaMBDs in both N- and C- cytosolic termini, whereas AtCNGC11 has only one CaMBD located in the N-terminal region of the channel. In addition, a thermal shift assay suggested that AtCNGC12 has higher affinity to bind cAMP over cGMP. Taken together, the current study contributes to identify key residues for channel function and provides new insights into CaMBD and CNBD in plant CNGCs.

Nicotine addiction is a complex behavior linked to the alteration of nicotinic receptor (nAChR) expression within the brain caused by chronic nicotine. A crucial factor when elucidating an accurate picture of the underlying causes of addiction is the route of administration. Oral self-administration of nicotine is a non-invasive method of drug administration, able to mimic the episodic nature of nicotine consumption seen in human smokers and also provide a choice – the key feature of an accurate addiction model. Mice with YFP-tagged α4 nicotinic receptors (α4YFP) were pretreated with chronic nicotine via osmotic pumps in order to maximally upregulate their nAChRs before being subjected to a two bottle-choice assay of nicotine self-administration. The paradigm consists of periods of choice interspersed with periods of nicotine abstinence to affect withdrawal and perpetuate nicotine self-selection. Spectral confocal microscopy of the endogenous α4YFP was used to investigate the expression levels of nAChRs following chronic nicotine priming with osmotic pumps. Imaging confirms that mice exposed to chronic nicotine prior to entering the self-administration paradigm have upregulated α4-containing (α4*) nAChRs in the medial perforant path of the hippocampus, on GABAergic somata of the ventral tegmental area and on GABAergic and glutamatergic somata of the medial prefrontal cortex, areas implicated in mediating addictive behavior. Compared to control mice with basal levels of nicotinic receptors, nicotine-primed mice ingest a larger daily dose of nicotine and allocate a greater percentage of their daily fluid intake to their nicotine-containing bottle. They also show signs of withdrawal, observed as post-abstinence binging. The control mice show no withdrawal, but progress towards dependence by adjusting the percentage drank from their nicotine bottle in order to maintain a constant daily dose. Conversely, nicotine primed mice decrease their daily dose of nicotine, suggesting that maximal receptor upregulation caused by osmotic pumps is outside the physiologically relevant level that can be obtained by nicotine self-administration in mice. Taken together, these results show that our model is sufficient to yield addictive behavior in mice and also implicates nAChR upregulation as a key factor influencing nicotine self-administration. / Graduate / 0306 / 0379 / 0317 / tony.j.renda@gmail.com

We present the first characterization of K+ optimization of N uptake and metabolism in an NH4+-tolerant species, tropical lowland rice (cv. IR-72). 13N radiotracing showed that increased K+ supply reduces futile NH4+ cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low-affinity NH4+ influx may be mediated by both K+ and non-selective cation channels. Suppression of NH4+ influx by K+ occurred within minutes of increasing K+ supply. Increased K+ reduced free [NH4+] in roots and shoots by 50-75%. Plant biomass was maximized on 10 mM NH4+ and 5 mM K+, with growth 160% higher than 10 mM NO3--grown plants, and 220% higher than plants grown at 10 mM NH4+ and 0.1 mM K+. Unlike in NH4+-sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH4+ cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase (GS) and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K+, mirroring plant growth and protein content. Improved plant performance through optimization of K+ and NH4+ is likely to be of substantial agronomic significance in the world’s foremost crop species.

We present the first characterization of K+ optimization of N uptake and metabolism in an NH4+-tolerant species, tropical lowland rice (cv. IR-72). 13N radiotracing showed that increased K+ supply reduces futile NH4+ cycling at the plasma membrane, diminishing the excessive rates of both unidirectional influx and efflux. Pharmacological testing showed that low-affinity NH4+ influx may be mediated by both K+ and non-selective cation channels. Suppression of NH4+ influx by K+ occurred within minutes of increasing K+ supply. Increased K+ reduced free [NH4+] in roots and shoots by 50-75%. Plant biomass was maximized on 10 mM NH4+ and 5 mM K+, with growth 160% higher than 10 mM NO3--grown plants, and 220% higher than plants grown at 10 mM NH4+ and 0.1 mM K+. Unlike in NH4+-sensitive barley, growth optimization was not attributed to a reduced energy cost of futile NH4+ cycling at the plasma membrane. Activities of the key enzymes glutamine synthetase (GS) and phosphoenolpyruvate carboxylase (PEPC) were strongly stimulated by elevated K+, mirroring plant growth and protein content. Improved plant performance through optimization of K+ and NH4+ is likely to be of substantial agronomic significance in the world’s foremost crop species.

Irritable Bowel Syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. Visceral hypersensitivity is the most commonly reported symptom of IBS, yet is the least adequately treated. Mechanosensitive information from the colon is relayed to the CNS by extrinsic colonic primary afferent nerves which have their cell bodies within dorsal root ganglia (DRG). This thesis aims to identify the contribution of several putatively mechanosensitive ion channels (ASIC1, 2 and 3, TRPV4 and TRPA1) toward detection of mechanical stimuli in the colon. This involvement is assessed by both molecular and functional means. The abundance of each of these channels was assessed by comparing expression within whole DRG against that in specifically colonic DRG neurons using an in situ hybridization methodology developed as part of this PhD. The functional role TRPV4 and TRPA1 impart toward colonic mechanosensation was investigated by recording responses to mechanical stimuli from colonic primary afferent fibres and comparing the results from mice genetically modified to lack either TRPV4 or TRPA1 with those of their intact littermates. The results from these studies indicate expression patterns within whole DRG do not provide accurate representation of the organ of interest, with abundances of each of the channels investigated differing between colonic DRG cells and the whole DRG. In particular ASIC3 and TRPV4 are preferentially expressed in colonic DRG neurons, unlike ASIC2 and TRPA1. Further, TRPV4 is functionally restricted to detection of noxious mechanical stimuli in the colon, while expression of TRPA1 is more widespread and functionally less restricted. Each of these channels are each potential targets for the treatment of IBS as each affects specific aspects of colonic mechanotransduction. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1347202 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Sciences, 2008

The glycine receptor (GlyR) mediates fast inhibitory neurotransmission in the central nervous system (CNS). Although GlyR α1 subunits are widely distributed, α3 subunits are found only on spinal cord pain sensory neurons where they mediate central inflammatory pain sensitization. Thus, the α3 subunit is a potential therapeutic target for anti-inflammatory analgesia. It is yet to be understood why α3 subunits are represented in these synapses. Thus, α3 subunit-specific modulators are required both as therapeutic leads and as pharmacological probes for basic research. The Thesis, which consists of three independent studies, investigated the molecular pharmacology of three classes of compounds at GlyRs, especially those containing the α3 subunit. The dihydropyridines (DHPs), nifedipine and nicardipine, modulate native GlyRs at micromolar concentrations. Nicardipine has a biphasic potentiating and inhibitory effect, whereas nifedipine causes inhibition only. The first study investigated the molecular mechanism by which these compounds inhibit recombinant GlyRs. The rate of onset of inhibition in the open state was accelerated by pre-application of DHP in the closed state, with the degree of acceleration proportional to the concentration of pre-applied DHP. This implies a non-inhibitory binding site close to the DHP inhibitory site. DHP inhibition was use-dependent and independent of glycine concentration, consistent with a pore-blocking mode of action. DHP sensitivity was abolished by the G2’A mutation, providing a strong case for DHP binding site in the pore. Nifedipine exhibited an approximately 10-fold higher inhibitory potency at α1-containing relative to α3-containing receptors, whereas nicardipine was only weakly selective for α1-containing GlyRs. The differential sensitivities of nifedipine and nicardipine for different GlyR isoforms suggest that DHPs may be a useful resource to screen as pharmacological tools for selectively inhibiting different synaptic GlyR isoforms. To date there are few compounds known to pharmacologically discriminate between α1 and α3 subunit-containing GlyRs. The second study stemmed from an observation that β-alanine and taurine act as weak partial agonists of α3 GlyRs but as strong partial agonists at α1 GlyRs. Using chimeras of α1 and α3 subunits, we identified the relatively structurally divergent M4 transmembrane domain and C-terminal tail as a specific determinant of the efficacy difference. As mutation of individual non-conserved M4 residues had little influence on agonist efficacies, the reduced efficacy of α3 GlyRs is most likely a distributed effect of all non-conserved M4 residues. Given the lack of contact between M4 and other transmembrane domains, the efficacy differences are probably mediated by differential interactions between the respective M4 domains and the surrounding lipid environment. The strong influence of M4 primary structure on partial agonist efficacy suggests that the relatively poorly conserved α3 GlyR M4 domain may be a promising domain to target in the search for α3 GlyR-specific modulators. β-carbolines have recently been shown to inhibit glycine receptors in a subunit-specific manner. The third study screened four structurally similar β-carbolines, harmane (HM), tryptoline (TP), norharmane (NHM) and 6-methoxyharmalan (MH) at recombinantly expressed α1, α1β, α2 and α3 glycine receptors. The four compounds exhibited only weak subunit-specificity, rendering them unsuitable as pharmacological probes. Because they displayed competitive antagonist activity, we investigated the roles of known glycine binding residues in coordinating the four compounds. The structural similarity of the compounds, coupled with the differential effects of C-loop mutations (T204A, F207Y) on compound potency, implied direct interactions between variable β-carboline groups and mutated residues. Mutant cycle analysis employing HM and NHM revealed a strong pairwise interaction between the HM methyl group and the C-loop in the region T204 and F207. These results, which define the orientation of the bound β-carbolines, were supported by molecular docking simulations. The information may also be relevant to understanding the mechanism of β-carboline binding to GABAAR where they are potent pharmacological probes. The identification of compounds that specifically abolish α3 GlyR-mediated currents should provide a useful means to investigate the physiological roles of this subunit. Drugs that potently and selectively enhance α3-subtype GlyR function may potentially serve as lead compounds since α3-subtype GlyRs have emerged as a potential therapeutic target for pain treatment. Results from studies forming the Thesis have identified several structural elements that might be useful for developing novel α3 subunit-specific drugs in the future.

Dissertation (Ph.D.)--University of South Florida, 2005. / Includes vita. Includes bibliographical references. Also available online as a PDF document.