The voltage-gated potassium channel Kv1.2 impacts membrane potential and therefore excitability of neurons. Expression of Kv1.2 at the plasma membrane (PM) is critical for channel function, and altering Kv1.2 at the PM is one way to affect membrane excitability. Such is the case in the cerebellum, a portion of the brain with dense Kv1.2 expression, where modulation of Kv1.2 at the PM can impact electrical activity of neurons and ultimately cerebellum-dependent learning. Modulation of Kv1.2 at the PM can occur through endocytic trafficking of the channel; however mechanisms behind this process in the brain remain to be defined. The goal of this dissertation was to identify and characterize modalities endogenous to the brain that influence the presence of Kv1.2 at the neuronal plasma membrane. Mass spectrometry (MS) was used to first identify interacting proteins and post-translational modifications (PTM) of Kv1.2 from cerebellar tissue, and the roles of these interactions and modifications on Kv1.2 function were evaluated in two studies: The first study investigated Trim32, a protein enzyme that catalyzes ubiquitylation, a PTM involved in protein degradation, but also in non-degradative events such as endocytic trafficking. Trim32 was demonstrated to associate and localize with Kv1.2 in cerebellar neurons by MS, immunoblotting (IB), and immunofluorescence (IF), and also demonstrated the ability to ubiquitylate Kv1.2 in vitro through purified recombinant proteins. Utilizing cultured cells through a combination of mutagenesis, biochemistry, and quantitative MS, a working model of Kv1.2 modulation was developed in which Trim32 influences Kv1.2 surface expression by two mechanisms that both involve cross-talk of ubiquitylation and phosphorylation sites of Kv1.2. The second study investigated WNK1, a chloride-sensitive kinase which regulates cellular homeostasis. Using MS, IB, and IF, WNK1 was demonstrated to associate and localize with Kv1.2 in the cerebellum, and a combination of mutagenesis and pharmacology in both wild-type and WNK1-knockout cultured cells produced a working model whereby WNK1 modulates surface Kv1.2. Activation of the downstream target SPAK kinase, also identified by MS to associate with Kv1.2 in the brain, by WNK1 was additionally found to influence the manner of WNK1 modulation of Kv1.2. In addition to providing new models of Kv1.2 modulation in the brain, these studies propose novel biological roles for Trim32 and WNK1 that may ultimately impact neuronal excitability.

L’Hypertension Hyperkaliémique Familiale (FHHt) est une forme rare d’hypertension artérielle associée à une hyperkaliémie et une acidose métabolique hyperchlorémique. Ces troubles sont corrigés par les diurétiques thiazidiques qui inhibent le co-transporteur Na+-Cl- NCC exprimé dans le néphron distal. Cette sensibilité aux diurétiques thiazidiques a laissé́ supposer que la FHHt est causée par une activation de NCC. En 2001, des mutations, de type gain-de-fonction, responsables de la FHHt ont été découvertes dans les gènes codant les sérine-thréonine kinases WNK1 et WNK4 [With No (K) lysine]. Des études in vitro ont montré que WNK1 et WNK4 stimulent NCC de façon indirecte, via la phosphorylation et l'activation de la kinase SPAK (Ste20 like Proline-Alanine rich Kinase). In vivo, l’activation de SPAK joue un rôle central dans le développement de la FHHt due aux mutations de WNK4. L'implication de SPAK n'a pas été définie dans le cas des mutations de WNK1. Nous avons donc croisé les souris WNK1+/FHHt, porteuses d'une mutation FHHt de WNK1, avec les souris SPAK243A/243A, porteuses d’une mutation abolissant l’activation de SPAK par les WNK. L’ensemble des phénotypes observés chez les souris WNK1+/FHHt est corrigé chez les souris WNK1+/FHHt:SPAK243A/243A démontrant ainsi le rôle central de SPAK dans la FHHt causée par les mutations WNK1. En 2012, de nouvelles mutations ont été identifiées dans les gènes codant CUL3 et KLHL3, deux composants d’un complexe ubiquitine ligase. Comme les mutations de WNK1 et WNK4, ces mutations entraînent une augmentation de l’expression de WNK1 et de WNK4. De façon inattendue, les patients FHHt porteurs d'une mutation CUL3 présentent un phénotype plus sévère que les autres. Des études suggèrent que ces mutations perturbent la fonction non seulement du néphron distal mais également des artères. Afin de vérifier cette hypothèse, nous avons généré et comparé deux modèles de souris : les souris pgk-Cul3Δ9, exprimant la mutation Cul3 de façon ubiquitaire, comme les patients, et les souris sm22-Cul3Δ9, exprimant la mutation uniquement dans les cellules musculaires vasculaires lisses. Les deux modèles sont hypertendus, mais les souris pgk-Cul3Δ9 le sont significativement plus que les souris sm22-Cul3Δ9, ce qui prouve que l’hypertension causée par les mutations Cul3 résulte du cumul d’une atteinte rénale et vasculaire. Récemment, de nouvelles mutations faux-sens d'un domaine dit acide de WNK1 ont été identifiées dans un petit nombre de patients atteints de FHHt. Ce domaine est nécessaire à la liaison à KLHL3 et donc à l’ubiquitination des WNK. Notre étude montre que, in vitro, ces mutations entraînent une accumulation d'une seule isoforme de WNK1. Chez la souris, la mutation du domaine acide provoque le même phénotype que les patients, ainsi qu’une augmentation de la phosphorylation de SPAK et du co-transporteur NCC. Cette étude a donc permis de démontrer le rôle essentiel de ce domaine dans la régulation de l'abondance de WNK1 dans le néphron distal in vivo. En conclusion, mon travail a permis une meilleure compréhension du rôle joué par SPAK, WNK1 et CUL3 dans le développement de la FHHt et, plus largement, de la physiopathologie de la FHHt. / Familial Hyperkalemic Hypertension (FHHt) is a rare form of hypertension associated with hyperkalemia and hyperchloremic metabolic acidosis. These disorders are all corrected by thiazide diuretics that inhibit the Na+-Cl- NCC cotransporter expressed in the distal nephron. The sensitivity of FHHt patients to thiazide diuretics strongly suggested that FHHt is caused by NCC activation. In 2001, gain-of function-mutations were identified in the genes encoding the serine-threonine kinases WNK1 and WNK4 [With No (K) lysine] in a subst of FHHt patients. In vitro studies demonstrate that WNK1 and WNK4 indirectly stimulate NCC, through the phosphorylation and activation of SPAK (Ste20 like Proline-Alanine rich Kinase). In vivo, SPAK activation plays a central role in the pathogenesis of FHHt caused by WNK4 mutations. However, the implication of SPAK has never been shown for the WNK1 mutations. Thus, we crossed WNK1+/FHHt mice, bearing the WNK1-FHHt mutation, with SPAK243A/243A mice bearing a mutation abolishing SPAK activation by the WNKs. All FHHt phenotypes observed in WNK1+/FHHt were corrected in WNK1+/FHHt:SPAK243A/243A mice demonstrating the central role of SPAK in FHHt caused by WNK1 mutations. In 2012, new mutations have been identified in CUL3 and KLHL3 genes. The products of these genes are both part of a ubiquitin ligase complex. As WNK1 and WNK4 mutations, these mutations lead to an increased expression of L-WNK1 and/or WNK4. Surprisingly, patients with CUL3 mutations display a more severe phenotype. Previous studies have suggested that CUL3 could be involved not only in the regulation of ion transport in the distal nephron but also in the regulation of vascular tone. To verify this hypothesis, we generated and compared two mouse models: pgk-Cul3Δ9 mouse model bearing, as patients, an ubiquitous Cul3 mutation, and sm22-Cul3Δ9 mouse model that express the Cul3 mutation only in vascular smooth muscle cells. Both models are hypertensive, but pgk-Cul3Δ9 mice display a more severe hypertension than sm22-Cul3Δ9 mice. It demonstrates that the hypertension caused by Cul3 mutations results from both renal and vascular disorders. Recently, new missense mutations have been identified in WNK1 acidic motif in a small number of FHHt patients. This acidic motif is necessary for the liaison to KLHL3 and therefore for WNK ubiquitination. Our study shows that, in vitro, these mutations lead to the accumulation of only one isoform of WNK1. In mice, the mutation of WNK1 acidic motif leads to an increased phosphorylation of SPAK and NCC. Therefore, it demonstrates the essential role of the acidic motif in the regulation of WNK1 abundance in vivo in the distal nephron. This work contributes to a better comprehension of the role played by SPAK, WNK1 and CUL3 in FHHt and more generally in the regulation of blood pressure and Na+/K+ homeostasis.

Der Typ I Transmembran-Rezeptor SORLA gehört zur VPS10p-Rezeptor Familie in Säugern. Der Rezeptor mit starker Homologie zu Endozytose- und Sorting-Rezeptoren ist am stärksten im zentralen Nervensystem (CNS) exprimiert. Außerhalb des CNS ist SORLA in einer Vielzahl von Geweben zu finden, unter anderem in der Niere. Das klare Expressionsmuster des Rezeptors im distalen Nephron lässt eine Rolle des Rezeptors in transepithelialen Transportprozessen vermuten. Um genau festzustellen, welche Prozesse von SORLA beeinflusst werden, wurde die Nierenfunktion von Mäusen mit einer vollständigen Defizienz des Sorla-Gens (Sorla-/-) untersucht. Diese Tiere zeigen Defekte in der renalen Ionenhomöostase: sie verlieren Na+, Cl-, K+, und Ca2+ (im Normalzustand und/oder nach Trinkwasserentzug). Eine Erniedrigung von Blutdruck und Herzfrequenz sowie eine fehlregulierte Sekretion von Aldosteron gehen mit dem Salzverlust-Phänotyp einher. Passend zu dieser Beobachtung konnte eine Expression von SORLA in der Nebenniere – speziell in der Zona glomerulosa, dem Ort der Aldosteron-Synthese – gezeigt werden. Des weiteren wurde eine signifikant verminderte Expression mehrerer Gene des Adrenalin-Synthesewegs in Sorla-/--Mäusen festgestellt, welcher in einer verringerten Menge des Hormons in den Nebennieren der Tiere resultiert. In der Niere bewirkt das Fehlen von SORLA insbesondere eine veränderte Phosphorylierung der beiden Kation-Chlorid-Cotransporter NCC und NKCC2 hervor, deren Aktivität durch Phosphorylierung reguliert wird. Es ist bekannt, dass die Signalkinase SPAK die Aktivität von NKCC2 und NCC reguliert. Die anormale Phosphorylierung fällt mit einer untypischen Verteilung der Kinase im TAL der SORLA-defizienten Mäuse zusammen. Dies deutet auf eine Funktion des Rezeptors beim Trafficking von SPAK hin. Durch die Identifizierung von Transportproteinen als putative Interaktionspartner SORLAs konnte diese Hypothese bekräftigt werden. / The type I transmembrane receptor SORLA is a member of the mammalian VPS10p-receptor family. The receptor, which is mainly expressed in the central nervous system (CNS), is characterized by high structural homology to endocytosis- and sorting-receptors. Outside the CNS, expression of SORLA can be found in a variety of tissues, including kidney. This distinct expression pattern in the distal nephron suggests a role for SORLA in transepithelial transport processes. To determine which processes the receptor might be involved in, the kidney function of mice, wich carry a complete deficiency of the Sorla gene, was analyzed. These animals show defects in ion handling: they are wasting Na+, Cl-, K+, and Ca2+ (under normal conditions and/or after water deprivation). The salt loss phenotype is accompanied by decreased mean arterial pressure and heart rate as well as mis-regulated secretion of aldosterone. In line with this observation, SORLA is also expressed in the adrenal gland, particularly in the zona glomerulosa, the place of aldosterone synthesis. Additionally, a significant down-regulation of several genes of the epinephrine synthesis pathway in mice lacking SORLA was found. This defect results in lower adrenal levels of the hormone. In the kidney, the lack of SORLA results especially in altered phosphorylation of the two cation-chloride cotransporters NCC and NKCC2, as their activity is regulated by phosphorylation. The signaling kinase SPAK has been reported to regulate the activity of NKCC2 and NCC. The transporters’ abnormal phosphorylation coincides with the atypical distribution of the kinase in TAL of Sorla-/- mice, suggesting a role of the receptor in establishing the localization of SPAK. This hypothesis was further substantiated by the identification of putative SORLA-interacting proteins involved in trafficking.

Dysregulation of the sodium-chloride cotransporter (NCC) is believed to significantly impact blood pressure. Recent studies have implicated overactivity of the sympathetic nervous system as a mechanism driving renal NCC dysregulation to evoke the development of salt-sensitive hypertension. It is proposed that the sympathetic nervous system accomplishes this by norepinephrine (NE)-mediated over-activation of the beta2-adrenergic receptors located in the distal tubules of the kidney. Beta2-adrenoreceptor activation is hypothesized to stimulate the protein kinases SPAK and OxSR1 to phosphorylate and thus activate NCC. This beta2-receptor-SPAK/OxSR1-NCC pathway was elucidated in studies that challenged salt-resistant mice with high-salt diets, bilateral adrenalectomies, and NE infusion. To expand the scope of these studies, we investigated the effects of elevated circulating NE on blood pressure, NCC activity, and expression of NCC proteins, SPAK, and OxSR1 in a different salt-resistant animal species (the Sprague-Dawley rat). In this study we implanted male Sprague-Dawley rats with osmotic minipumps delivering a subcutaneous infusion of either saline, NE, a 50:50 solution of DMSO/isotonic saline, a combination of NE and the NCC antagonist hydrochlorothiazide, or a combination of NE and the beta-adrenoreceptor antagonist propranolol. Following implantation of the pumps the rats were randomly assigned to either a standard diet (0.4% NaCl) or a high-salt diet (8% NaCl) for two weeks. After fourteen days all animals underwent acute femoral artery, vein, and bladder cannulation in order to monitor heart rate and blood pressure, administer drugs intravenously, and track renal function, respectively. Following surgical recovery, blood pressure and heart rate were measured continuously, and urine was collected in ten-minute intervals in order to assess peak natriuretic responses to amiloride and hydrochlorothiazide. Following this protocol the rats received an intravenous bolus of hexamethonium (30 mg/kg), and their peak drops in blood pressure were recorded. Afterwards both kidneys were harvested and frozen at -80 °C for measurement of NCC proteins, SPAK, and OxSR1 expression. This study demonstrates that increased circulating NE induces salt-sensitive hypertension in the naturally salt-resistant Sprague-Dawley rat. Chronic infusion of NE raised the blood pressure of the rats, and a high-salt diet exacerbated this effect. Furthermore, NE prevented salt-evoked suppression of NCC activity and NCC, SPAK, and OxSR1 protein expression. Co-infusion of hydrochlorothiazide with NE attenuated NE-mediated hypertension and caused no variance in the blood pressures between the standard salt and high-salt groups. This indicates that chronically antagonizing NCC eliminated the salt-sensitive component of NE-mediated hypertension. Beta-receptor antagonism combined with NE infusion completely eliminated the hypertensive influence of NE and downregulated the expression of NCC proteins, SPAK, and OxSR1. However, NCC activity still remained at a level comparable with that observed in the NE-infused rats, demonstrating dissociation between protein expression and function. These data, the first report in a rat model of an interaction of NE and a high salt intake that impairs NCC function, demonstrate that increased levels of NE in combination with a high dietary salt intake result in NCC dysregulation and the development of NE-mediated salt-sensitive hypertension. To an extent the data also support the proposition that NE activates the beta-adrenergic receptors to influence the activity of NCC and the expression of NCC proteins, SPAK, and OxSR1. Beta-antagonism combined with NE infusion attenuated the effects of NE on blood pressure and the expression of NCC proteins, SPAK, and OxSR1. However, the NE-mediated elevation of NCC activity still remained high. We propose that the beta-receptors are not the only adrenergic receptors that can influence NCC activity. The presence of alpha-adrenergic receptors in the distal tubules suggests that they may be able to keep NCC activity elevated through a pathway independent of the beta-receptors, SPAK, and OxSR1.

Study of Gordon (PHAII) and Gitelman (GS) syndromes revealed the importance of the WNK pathway and thiazide-sensitive Na-Cl Cotransporter (NCC) in the renal control of blood pressure. PHAII mutations lead to WNK accumulation resulting in the hyperphosphorylation of the downstream effector, SPAK, which overactivates NCC causing salt retention and hypertension. Mutations causing deletion of exon-9 in Cullin-3, which normally ubiquitylates WNKs for degradation, were recently discovered to cause the severest subtype of PHAII (PHA2E) with early onset salt-sensitive hypertension and hyperkalaemia. The reasons for this severity have remained elusive, however clues came from SPAK knock-out mice which recapitulate GS, the phenotypic mirror image of PHAII, typically caused by activation-inhibiting NCC phosphorylation site mutations resulting in salt-wasting and hypotension. As these mice were also discovered to have reduced vascular tone, it suggests the WNK pathway may have extra-renal roles in vascular smooth muscle function and highlights inhibition of SPAK function as a promising anti-hypertensive strategy with multiple sites of action. To address these possibilities the work aimed to phenotype: (1) heterozygous CUL3$^{WT/\Delta403-459}$ mice to investigate a possible vascular contribution to PHAII pathophysiology, (2) homozygous knock-out mice of MO25, a master regulator known to increase SPAK activity up to 100-fold independent of WNKs, and (3) homozygous SPAK$^{L502A/L502A}$ knock-ins, predicted to have disrupted SPAK binding to WNK/NCC, in order to validate SPAK signalling inhibition as a viable anti-hypertensive strategy. In mice, the CUL3$^{\Delta403-459}$ proteins are hyperflexible, hypermodified and ultimately have reduced WNK ubiquitylation. This lead to hypertension, hyperkalaemia, hyperchloraemia with compensated metabolic acidosis and growth retardation, which closely recapitulates human PHA2E. The discovery of increased vascular tone suggests an explanation for the severity of CUL3$^{\Delta}$$^{ex9}$PHAII. In mice, homozygous MO25$\alpha$ knock-out proved embryonically lethal, while homozygous MO25$\beta$ knock-out did not meaningfully alter blood pressure or electrolyte homeostasis. However, the SPAK$^{L502A}$ protein had a decreased ability to bind WNKs and cation-chloride cotransporters NCC and NKCC1/2, serving to reduce their activation. SPAK$^{L502A/L502A}$ mice showed typical features of GS with mild hypokalaemia, hypomagnesaemia, hypocalciuria and salt-wasting hypotension. The mice also presented with decreased markers of vascular tone potentially due to effects on cardiovascular and neuronal NKCC1. These results show that SPAK binding is crucial for blood pressure control and pharmacological inhibition of this binding is an attractive anti-hypertensive strategy.