ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

ATP-sensitive K+ channels (KATP) channels were first described in the cardiac muscles. KATP channels are a complex of regulatory sulphonylurea receptor subunits and pore-forming inward rectifier subunits such as Kir6.1. Nicotine, an exogenous substance, adversely affects cardiovascular function in humans. Acetylcholine (ACh) is well known as a key neurotransmitter of the parasympathetic nervous system. ACh effects are usually related to binding to muscarinic receptors and stimulating second messengers that relay and direct the extracellular signals to different intracellular destinations, resulting in modulated cellular activity. We hypothesize that nicotine and ACh may modulate Kir6.1 channels via different mechanisms. Using the whole cell patch-clamp technique, the interactions of nicotine and ACh with Kir6.1 subunit permanently expressed in Human Embryonic Kidney (HEK-293) cells as well as the underlying mechanisms were studied.<p> Non-transfected HEK-293 cells possess an endogenous K+ current with current density of 3.2 ± 1.4 pA/pF at 150 mV (n = 9). Stable expression of Kir6.1 subunits cloned from rat mesenteric artery in HEK-293 cells yielded a detectable inward rectifier KATP current (-23.9 ± 1.6 pA/pF at 150 mV, n = 6). In the presence of 0.3 mM ATP in the pipette solution, nicotine at 30 and 100 µM increased the expressed Kir6.1 currents by 42 ± 11.8 and 26.2 ± 14.6%, respectively (n = 4-6, p<0.05). In contrast, nicotine at 1-3 mM inhibited Kir6.1 currents (p<0.05). Nicotine at 100 µM increased the production of superoxide anion (O2.-) by 20.3 ± 5.7% whereas at 1 mM it significantly decreased the production of O2.- by 37.7 ± 4.3%. The hypoxanthine/xanthine oxidase (HX/XO) reaction was used as a source of O2.-. Co-application of HX and XO to the transfected HEK-293 cells resulted in a significant and reproducible increase in Kir6.1 currents. Tempol, a scavenger of O2.-, abolished the stimulatory effect of HX/XO on Kir6.1 currents. Tempol also abolished the stimulatory effect of 30 mM nicotine on Kir6.1 currents (-28.3 ± 6.1 pA/pF vs. -31.2 ± 7.3 pA/pF at -150 mV, n = 6-9 for each group, p>0.05). <p> In the presence of 0.3 mM ATP in the pipette solution, ACh concentration-dependently increased the expressed Kir6.1 currents. At 1 µM, ACh increased Kir6.1 currents from -19 ± 2.5 to 31.7 ± 2.1 pA/pF (n = 8, p < 0.05). Pretreatment of the transfected HEK-293 cells with either 2 or 20 µM atropine, 100 nM a-bungarotoxin, 100 µM mecamylamine, 2 µM prazosin, 1 µM propranolol, or 10 µM dihydro-b-erythroidine hydrobromide did not alter the stimulatory effect of ACh on Kir6.1 currents (n = 4 - 5 for each group, p<0.05). When intracellular ATP was increased to 5 mM, ACh at 10 µM still exhibited its stimulatory effect (-16.4 ± 2.3 to 25.5 ± 3.8 pA/pF, n = 8, p<0.05). For the first time, the present study provides an insight for the interactions of nicotine and ACh with Kir6.1 subunits. Our data demonstrate that micromolar concentration of nicotine and ACh stimulated Kir6.1 channels. Nicotine at millimolar concentrations inhibited Kir6.1 channels. The dual effect of nicotine, not mediated by nAChR, are mediated partially by O2.- levels in the cells. The ACh excitatory effect is mediated neither by an AChR-dependent mechanism, nor by alteration in ATP metabolism. This study challenges the traditional explanations for the receptor-mediated effects of nicotine and ACh on ion channels and opens a new door to understand the effects of nicotine and ACh on KATP channels in many cellular systems.

Protein complexes are critical to virtually every biological process performed by living organisms. The cellular “interactome”, or set of physical protein-protein interactions, is of particular interest, but no comprehensive study of human multi-protein complexes has yet been reported. In this Thesis, I describe the development of a novel high-throughput profiling method, which I term Fractionomic Profiling-Mass Spectrometry (or FP-MS), in which biochemical fractionation using non-denaturing high performance liquid chromatography (HPLC), as an alternative to affinity purification (e.g. TAP tagging) or immuno-precipitation, is coupled with tandem mass spectrometry-based protein identification for the global detection of stably-associated protein complexes in mammalian cells or tissues. Using a cell culture model system, I document proof-of-principle experiments confirming the suitability of this method for monitoring large numbers of soluble, stable protein complexes from either crude protein extracts or enriched sub-cellular compartments. Next, I document how, using orthogonal functional genomics information generated in collaboration with computational biology groups as filters, we applied FP-MS co-fractionation profiling to construct a high-quality map of 622 predicted unique soluble human protein complexes that could be biochemically enriched from HeLa and HEK293 nuclear and cytoplasmic extracts. Our network is enriched in assemblies consisting of human disease-linked proteins and contains hundreds of putative new components and novel complexes, many of which are broadly evolutionarily conserved. This study revealed unexpected biological associations, such as the GNL3, FTSJ3, and MKI67IP factors involved in 60S ribosome assembly. It is my expectation that this first systematic, experimentally-derived atlas of putative human protein complexes will constitute a starting point for more in depth, hypothesis-driven functional investigations of basic human molecular and cellular biology. I also note that my generic FP-MS screening approach can, and is currently, being applied by other members of the Emili laboratory to examine the global interactomes of other mammalian cell lines, tissues, sub-cellular compartments, and diverse model organisms, which should expand our understanding of proteome adaptations and association networks associated with cell physiology, animal development and molecular evolution.

Protein complexes are critical to virtually every biological process performed by living organisms. The cellular “interactome”, or set of physical protein-protein interactions, is of particular interest, but no comprehensive study of human multi-protein complexes has yet been reported. In this Thesis, I describe the development of a novel high-throughput profiling method, which I term Fractionomic Profiling-Mass Spectrometry (or FP-MS), in which biochemical fractionation using non-denaturing high performance liquid chromatography (HPLC), as an alternative to affinity purification (e.g. TAP tagging) or immuno-precipitation, is coupled with tandem mass spectrometry-based protein identification for the global detection of stably-associated protein complexes in mammalian cells or tissues. Using a cell culture model system, I document proof-of-principle experiments confirming the suitability of this method for monitoring large numbers of soluble, stable protein complexes from either crude protein extracts or enriched sub-cellular compartments. Next, I document how, using orthogonal functional genomics information generated in collaboration with computational biology groups as filters, we applied FP-MS co-fractionation profiling to construct a high-quality map of 622 predicted unique soluble human protein complexes that could be biochemically enriched from HeLa and HEK293 nuclear and cytoplasmic extracts. Our network is enriched in assemblies consisting of human disease-linked proteins and contains hundreds of putative new components and novel complexes, many of which are broadly evolutionarily conserved. This study revealed unexpected biological associations, such as the GNL3, FTSJ3, and MKI67IP factors involved in 60S ribosome assembly. It is my expectation that this first systematic, experimentally-derived atlas of putative human protein complexes will constitute a starting point for more in depth, hypothesis-driven functional investigations of basic human molecular and cellular biology. I also note that my generic FP-MS screening approach can, and is currently, being applied by other members of the Emili laboratory to examine the global interactomes of other mammalian cell lines, tissues, sub-cellular compartments, and diverse model organisms, which should expand our understanding of proteome adaptations and association networks associated with cell physiology, animal development and molecular evolution.

V této diplomové práci je popsána přechodná transfekce buněk 293 HEK adaptovaných na růst při suspenzní kultivaci bez přítomnosti séra za použití polyethyleniminů (PEI). Buňky byly transfekovány plasmidem pcDNA5/SEAP, který exprimuje sekretovanou formu lidské placentální alkalické fosfatázy. K porovnání účinnosti jednotlivých transfekcí byla měřena koncentrace exprimované fosfatázy v buněčném supernatantu. Cílem této práce bylo optimalizovat různé faktory ovlivňující účinnost transfekcí s důrazem na nalezení optimálního poměru DNA:PEI.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal