Le cotransporteur Na-K-CI rénal (NKCC2) couple le transport de 4 ions (1 Na+, 1 K+ ou 1 Rb +, et 2 Cr) à travers la membrane apicale de l'anse ascendante de Renle, contribuant à la réabsorption de l'ultrafiltrat glomérulaire. L'épissage alternatif de l'exon 4 engendre 2 variantes appelées ±A¿ et ±F¿ dont la séquence dans le deuxième segment transmem-branaire (tm2) et dans la première boucle intracellulaire (cs1) diffère. Il confère aussi des différences d'affinité ioniques, i.e., ± A ¿ a des Km beaucoup plus bas que ± F ¿ pour tous les ions grâce à des résidus spécifiques que nous avons identifiés par des études mutagéniques, i.e., les résidus 216 et 220 dans tm2 (qui moduleraient l'affinité pour le Rb et Cl, respectivement) et certains résidus de csl incluant 223, 229, 233 et peut être 225 (qui moduleraient l'affinité pour le Cl). À ce stade-ci, notre but principal est d'avancer notre recherche sur la compréhension des déterminants qui confèrent à ± A ¿ et ± F¿ leurs caractéristiques cinétiques propres. Nous savons que les résidus ± modificateurs d'affinité¿ identifiés opèrent en combinaison mais ignorons la séquence des cassettes en ~ant que tel et leur spécificité ionique. Nous avons donc créé des . chimères en interchangeant des paires de résidus entre ± A ¿ et ± F ¿, i.e. résidus 216 et 220 dans tm2, 223 et 225 dans cs1, 223 et 229 dans csl, et 220 et 225 dans tm2-cs1, pour ensuite exprimer les mutants dans des oocytes de Xenopus laevis et analyser leur dépendance à chacun des ions. Nous avons trouvé, de façon fort intéressante, que la cassette 220; 223; 225; 233 modulait l'affinité pour les ions Na+-Cr (incluant le site à haute affinité pour le Cl) et que la 'cassette 216; 225 modulait celle pour le Rb. Toutefois, les résultats obtenus pour l'une des chimères (± F ¿ chez qui les résidus 216 et 220 ont été remplacés par ceux de ± A ¿) avait un comportement inattendu puisque son affinité pour le Rb est demeurée identique à celle de ± F ¿. En conclusion, les résidus de tm2 et csl fonctionnent probablement en synergie pour assurer la coordination ionique par NKCC2. Par ailleurs, nos résultats suggèrent l'existence d'au moins 2 cassettes ± modificatrices d'affinité ¿, l'une pour le Na-Cl et l'autre pour le K-Cl.

Potassium (K) deficiency of soybean [Glycine max (Merr.) L.] is common throughout the Mid-Southern region of the United States. Visual symptoms of K deficiency may be absent or subtle during early and mid-reproductive growth stages. Correcting K deficiency in-season is problematic due to limited research. Information regarding soybean yield response to in-season fertilizer K application time is limited to growth stages prior to the onset of R1. Research is also limited on new higher yielding soybean cultivars. The objective of this study was to evaluate influences of in-season K application timing during reproductive growth stages on soybean yield. Field experiments were established in 2019, 2020, and 2021 to evaluate the effect of soybean potassium rate and timing during reproductive growth stages. Four independent experiments were established to: (1) determine the effect of applying K fertilizer to sufficient soils at different times in-season on yield and plant K status on two different soil textures, (2) determine the effects of K timing, K rate, and dicamba rate on yield and plant stress, and (3) determine the effects of K timing and desiccation on yield and plant stress. Results indicate that potassium application rate and timing during soybean reproductive growth stages do not increase yield on soils with adequate K present. Soybean trifoliate leaf tissue K analysis and petiole sap K analysis had no response during reproductive growth stages when K fertilization occurred on soils with ample K. Potassium fertilization did not aid soybean recovery after dicamba injury.

The purpose of this study was to relate femoral venous plasma potassium concentrations ([K⁺]) following a fatiguing submaximal isometric quadriceps contraction, to the excitability of the muscle cell membrane as assessed by the compound muscle action potential (M-Wave) . Ten healthy male volunteers (22. 0 ± . 5 yrs) performed a unilateral 3 minute (min) sustained isometric quadriceps contraction at 30% of their maximum voluntary contraction (MVC) . M-Waves, peak evoked twitch torque, plasma lactate concentration ([La⁻]), and plasma potassium concentration ([K⁺]) were measured before, and at predetermined times over the course of a 15 min recovery period following the fatigue paradigm. Immediately post-exercise, twitch torque decreased to 58% of baseline, femoral venous [La⁻] had risen to 10 ± 0.8 mmol/1, and [K⁺] was significantly increased from 4.0 ± 0.1 mmol/1 to 5.9 ± 0.2 mmol/1. M-Wave amplitude illustrated a trend for potentiation increasing 9.5%-from 13.9 ± 2.4 mV pre-exercise, to 15.3 ± 2.8 mV at 1 min 20 seconds post-exercise. M-Wave area exhibited a similar trend from baseline, but values showed no statistical significance during this time. These results suggest that in spite of increased extracellular [K⁺] following this type of fatiguing exercise, muscle membrane excitability is maintained, which is probably due to the electrogenic nature of the highly active Na⁺ /K⁺ pump. This study was supported by the Natural Sciences and Engineering Research Council of Canada. / Thesis / Master of Science (MSc)

No description available.

Effects of potassium supplementation were evaluated in four studies in endurance horses during races and treadmill exercise. In the first and second studies a potassium-free experimental formula was compared to potassium rich commercial formulas. The first study showed that supplementation increased plasma [K+], and that the extra sodium in the potassium-free experimental formulas helped to attenuate acidosis at the end of the ride. In the second study supplementation also increased plasma [K+], however speeds were lower and no increases were observed in plasma concentrations during the race. Supplementation of potassium during recovery helped to restore plasma [K+]. Higher plasma [Ca++] was found in horses supplied with experimental feeds, due to a lower dietary cation anion balance (DCAB). Three eliminated horses had heart rate arrhythmias and labile heart rates accompanied with higher plasma [K+] and lower [Ca++] than finishers. Also horses supplied with the experimental sodium-rich formula were less dehydrated than the ones receiving commercial formulas. The third study involved an 80 km endurance exercise test on the treadmill, and plasma [K+] was affected by potassium supplementation during exercise and recovery. The supply of potassium caused higher plasma [K+] helping to restore body stores. Also chloride supply in the electrolyte formulas maintained plasma [Cl-] levels during exercise and affected plasma concentrations during recovery. The fourth study showed that potassium supply affects plasma concentration, but also increases lactate production and glucose during sub-maximal exercise. A potassium-free electrolyte supply caused higher plasma [Ca++] during exercise. Higher sodium supply in the potassium-free electrolytes improved hydration during exercise. These studies show that potassium should supplemented after exercise and but not be done during exercise because of the risk of increased neuromuscular excitability. / Ph. D.

Ce mémoire de maîtrise porte sur la régulation du cotransporteur Na+-K+-Cl- de type 2 (NKCC2), de ses variantes d’épissage NKCC2A, NKCC2B et NKCC2F plus précisément, par les with no lysine kinases (WNKs) dont certains isoformes sont associés au pseudohypoaldostéronisme de type II, une maladie héréditaire causant de l’hypertension artérielle. Ces variantes sont exprimées uniquement dans l’anse ascendante de Henle où elles réabsorbent une partie importante du NaCl filtré, et elles sont identiques entre elles excepté pour un segment de 32 acides aminés codé par l’exon 4. Dans ce travail, j’ai utilisé le système d’expression de l’oocyte du Xenopus laevis pour déterminer si l’exon 4 de NKCC2 confère des différences de sensibilité à l’action des WNK parmi les variantes. J’ai démontré que tel était bien le cas, et que certains résidus dans l’exon 4 jouaient probablement un rôle important dans le recyclage endocytique de NKCC2 sous l’effet de L-WNK1 et de WNK3. / This thesis is concerned with the regulation of Na+-K+-Cl- cotransporter type 2 (NKCC2), of its splice variants NKCC2A, NKCC2B and NKCC2F more specifically, by the with no lysine kinases (WNKs) among which certain isoforms have been linked to pseudohypoaldosteronism type II, a hereditary disorder causing systemic hypertension. These variants are expressed exclusively in the thick ascending limb of Henle where they reabsorb 20 % of the filtrated NaCl load, and they are identical among each other except for a 23-amino acid segment that is encoded by exon 4. In this work, I have used the Xenopus laevis oocyte expression system to determine whether exon 4 in NKCC2 conveys variant-specific sensitivities to the action of WNKs. I have shown that this was indeed the case, and that certain residues in exon 4 probably play an important role in endocytic recycling of NKCC2 under the effect of L-WNK1 and WNK3.

Cette thèse de doctorat porte sur la régulation du cotransporteur K+-Cl- de type 4 (KCC4) par la « WNK lysine deficient protein kinase 4 » (WNK4), une sérine-thréonine kinase qui a été découverte en 2001. Le cotransporteur KCC4 fait partie de la famille des cotransporteurs cation-Cl- (CCC) qui compte neuf membres. Le cotransporteur KCC4, et les autres isoformes de type KCC, sont des protéines de surface qui couplent l’efflux du Cl- à celui du K+ de manière électroneutre. Ce faisant, ils régulent le Cl- intracellulaire (Cl-i), le volume cellulaire (Vi) et la réabsorption basolatérale du Cl- dans les épithélia. Bien qu’ils soient activés par une augmentation du Cl-i et du Vi, les intermédiaires signalétiques impliqués demeurent mal caractérisés à ce jour. À cet effet, WNK4 pourrait correspondre à l’un de ces intermédiaires puisqu’elle régule plusieurs systèmes de transport, dont les KCC. Les objectifs de cette thèse étaient donc de déterminer si WNK4 régule l’activité de KCC4 suite à des changements de Vi et de Cl-i, et si c’est le cas, par quels mécanismes. Pour répondre à ces objectifs, nous avons adopté une approche structure-fonction par laquelle des mutations ont été introduites dans les protéines KCC4 ou WNK4. L’effet de ces mutations a été analysé dans le système d’expression hétérologue des ovocytes de Xenopus laevis grâce à des études fonctionnelles, d’expression de surface, d’immunofluorescence et de phosphorylation. Nos travaux ont permis de montrer pour la première fois que WNK4 et PP1 sont bel et bien impliquées dans la régulation de KCC4 suite à une augmentation du Vi, mais que contrairement à des hypothèses avancées antérieurement, PP1 agirait en aval plutôt qu’en amont de WNK4, et qu’elle agirait aussi sur KCC4 par l’intermédiaire d’autres intervenants signalétiques. Nous avons aussi identifié deux résidus dans le domaine C-terminal de KCC4 qui soutiennent l’effet de WNK4 et de PP1. En somme, ces travaux pourraient permettre d’identifier de nouvelles voies de signalisation impliquées dans la régulation de KCC4 par WNK4 et PP1 et, par conséquent, d’offrir d’autres cibles thérapeutiques pour traiter des désordres électrolytiques. / This Ph.D. thesis focuses on the regulation of K+-Cl- cotransporter type 4 (KCC4) by WNK lysine deficient protein kinase 4 (WNK4), a serine threonine kinase that was discovered in 2001. The cotransporter KCC4 belongs to the cation-Cl- cotransporter (CCC) family that includes nine members. This transporter, along with the three other KCC isoforms, corresponds to cell surface proteins that couple elctroneutral efflux of Cl- with that of K+. In doing so, KCC family members regulate intracellular Cl- (Cl-i), cell volume (Vi) and basolateral reabsorption of Cl- across a variety of epithelia. Although they are activated by an increase in Cl-i and Vi, the signaling intermediates involved remain largely unknown to date. In this regard, WNK4 could correspond to one such intermediate given that it regulates several transport systems including KCC. On the basis of these premises, the main objectives of this thesis were to determine whether WNK4 regulates KCC4 activity during changes in Cl-i and Vi and if so, through which mechanisms. To address these objectives, I exploited a structure-function approach in which mutations were introduced in both KCC4 or WNK4 and in which the effect of such mutations were analyzed in the Xenopus laevis expression system through functional, expression, immunofluorescence studies and phosphorylation. My studies allowed to show for the first time that WNK4 et PP1 are indeed involved in KCC4 regulation during an increase in Vi, but that in contrast to previous assumptions, PP1 would act downstream instead of upstream of WNK4, and that it would also act on KCC4 through additional signaling intermediates. Lastly, we have identified two residues in the C-terminus of KCC4 that mediate the effect of WNK4 and PP1. Taken together, these results could help identifying new signaling pathways of KCC4 regulation by WNK4 and PP1, and thus offer other therapeutic targets to treat electrolyte disorders.

Satellite glial cells (SGC) are laminar cells that wrap completely around the sensory neuron and are responsible for buffering extracellular K+ after neuronal excitation. A decrease in the potassium buffering capacity of SGC has been associated with neuropathic pain (NP) behavior and apoptosis. This dissertation investigated the role of the potassium buffering capacity and apoptosis of trigeminal satellite glial cells (SGC) in the maintenance and development of orofacial NP in rats using in vivo and in vitro methodologies. In vivo endpoints were evaluated after performing chronic constriction injury (CCI) of the infraorbital nerve (IoN). NP signs and behavior were evaluated at 5, 10, 20 40 and 80 hours after injury. We evaluated the potassium buffering capacity of SGC by measuring the intracellular potassium concentration and protein levels and gene expression of the Kir4.1 and the SK3 potassium channels and gap junction protein connexin 43 (Cx43). We evaluated apoptosis endpoints including protein levels and gene expression of apoptotic related proteins bcl-2, caspase 9, caspase 3 and p53. Results indicate that NP signs developed as early as 5 hours after injury. After PNI, SGC responded by increasing their intracellular potassium concentration and by increasing protein levels of Kir4.1, SK3 and Cx43. Nonetheless, this increase in protein levels was not accompanied by an increase in gene expression. Apoptosis results revealed that SGC decreased protein levels and gene expression of anti-apoptotic protein Bcl-2. Using in vitro methodologies, we developed primary trigeminal SGC cultures and evaluated how a decrease in the intracellular potassium concentration modulates apoptosis induced by the mitochondrial and death receptor pathways. SGC depleted of potassium after hypoosmotic shock showed a significant increase in early apoptosis after incubation with mitochondrial pathway apoptotic inducer staurosporine when compared to SGC with normal intracellular concentration. This research has revealed that SGC respond early to PNI by increasing their potassium buffering capacity. We also determined that the mitochondrial apoptotic pathway might be involved in the trigeminal SGC response to PNI. From our in vitro experiments we have revealed that potassium is an important modulator of apoptosis induced by the mitochondrial pathway in cultured trigeminal SGC. / Ph. D.

The conclusion of the alfalfa fertility investigations at Glade Spring and at Culpeper may be summarized as follows; 1. Potassium was the main limiting factor in the growth, duration of stand, vigor, and color of alfalfa. 2. In general, yields were related directly to rate of potash fertilization. 3. Yield variations due to phosphorus fertilization were insignificant. 4. Sodium gave increased yields at Glade Spring and Location A at Culpeper, but had little or no effect on yields at Culpeper at Location B. 5. In general, yields were decreased by the application of minor elements. 6. In 1950 and 1951 at Glade Spring, more potassium was removed from the soil than was applied, the deficits ranging from only 8.4 pounds per acre of potassium where a 2-24-24 fertilizer was used, to 68.9 pounds per acre of potassium where e 2-12-0 fertilizer was used. 7. The potassium removed per ton of hay was closely correlated with the rate of potassium fertilization. 8. The phosphorus removed in the hay varied from 1/2 to 1/8 of the phosphorus applied in the fertilizer. 9. The phosphorus removed was nearly constant regardless of the rates of phosphorus fertilization. 10. Potassium decreased sodium uptake in the alfalfa; on the other hand, sodium had no effect on potassium uptake. 11. Alfalfa appears to be a sodium accumulating plant when sodium is used as a supplement with potassium. 12. Potassium depressed the absorption of calcium, magnesium, and sodium. 13. At Glade Spring, potassium slightly depressed phosphorus absorption but had no noticeable effect on phosphorus uptake at Culpeper. Potassium appeared to have no appreciable effect on nitrogen content. 14. The soil at Glade Spring had approximately 30 times as much exchangeable calcium as potassium. As a result, the calcium content of the alfalfa was often higher than the potassium content. 15. Available phosphorus in the topsoil at Glade Spring was about five times higher than in the subsoil; on the other hand, available phosphorus at Culpeper was about equal in topsoil and subsoil. / Master of Science

M.S.