Evaluation of renal sodium handling in heart failure with preserved ejection fraction: A pilot study.

The pathophysiology behind sodium retention in heart failure with preserved ejection fraction (HFpEF) remains poorly understood. We hypothesized that patients with HFpEF have impaired natriuresis and diuresis in response to volume expansion and diuretic challenge, which is associated with renal hypo-responsiveness to endogenous natriuretic peptides. Nine HFpEF patients and five controls received saline infusion (0.25 mL/kg/min for 60 min) followed by intravenous furosemide (20 mg or home dose) 2 h after the infusion. Blood and urine samples were collected at baseline, 2 h after saline infusion, and 2 h after furosemide administration; urinary volumes were recorded. The urinary cyclic guanosine monophosphate (ucGMP)/plasma B-type NP (BNP) ratio was calculated as a measure of renal response to endogenous BNP. Wilcoxon rank-sum test was used to compare the groups. Compared to controls, HFpEF patients had reduced urine output (2480 vs.3541 mL; p = 0.028), lower urinary sodium excretion over 2 h after saline infusion (the percentage of infused sodium excreted 12% vs. 47%; p = 0.003), and a lower baseline ucGMP/plasma BNP ratio (0.7 vs. 7.3 (pmol/mL)/(mg/dL)/(pg/mL); p = 0.014). Patients with HFpEF had impaired natriuretic response to intravenous saline and furosemide administration and lower baseline ucGMP/plasma BNP ratios indicating renal hypo-responsiveness to NPs.

Iterative assay for transposase-accessible chromatin by sequencing to isolate functionally relevant neuronal subtypes.

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Circadian Rhythm Regulation by Pacemaker Neuron Chloride Oscillation in Flies.

Circadian rhythms in physiology and behavior sync organisms to external environmental cycles. Here, circadian oscillation in intracellular chloride in central pacemaker neurons of the fly, Drosophila melanogaster, is reviewed. Intracellular chloride links SLC12 cation-coupled chloride transporter function with kinase signaling and the regulation of inwardly rectifying potassium channels.

Large genetic analysis of alcohol resistance and tolerance reveals an inverse correlation and suggests 'true' tolerance mutants.

Tolerance occurs when, following an initial experience with a substance, more of the substance is required subsequently to induce the same behavioral effects. Tolerance is historically not well-understood, and numerous researchers have turned to model organisms, particularly Drosophila melanogaster, to unravel its mechanisms. Flies have high translational relevance for human alcohol responses, and there is substantial overlap in disease-causing genes between flies and humans, including those associated with Alcohol Use Disorder. Numerous Drosophila tolerance mutants have been described; however, approaches used to identify and characterize these mutants have varied across time and between labs and have mostly disregarded any impact of initial resistance/sensitivity to ethanol on subsequent tolerance development. Here, we have analyzed a large amount of data - our own published and unpublished data and data published by other labs - to uncover an inverse correlation between initial ethanol resistance and tolerance phenotypes. This inverse correlation suggests that initial resistance phenotypes can explain many 'perceived' tolerance phenotypes. Additionally, we show that tolerance should be measured as a relative increase in time to sedation between an initial and second exposure rather than an absolute change in time to sedation. Finally, based on our analysis, we provide a method for using a linear regression equation to assess the residuals of potential tolerance mutants. We show that these residuals provide predictive insight into the likelihood of a mutant being a 'true' tolerance mutant, and we offer a framework for understanding the relationship between initial resistance and tolerance.

Unanticipated domain requirements for Drosophila Wnk kinase in vivo.

WNK (With no Lysine [K]) kinases have critical roles in the maintenance of ion homeostasis and the regulation of cell volume. Their overactivation leads to pseudohypoaldosteronism type II (Gordon syndrome) characterized by hyperkalemia and high blood pressure. More recently, WNK family members have been shown to be required for the development of the nervous system in mice, zebrafish, and flies, and the cardiovascular system of mice and fish. Furthermore, human WNK2 and Drosophila Wnk modulate canonical Wnt signaling. In addition to a well-conserved kinase domain, animal WNKs have a large, poorly conserved C-terminal domain whose function has been largely mysterious. In most but not all cases, WNKs bind and activate downstream kinases OSR1/SPAK, which in turn regulate the activity of various ion transporters and channels. Here, we show that Drosophila Wnk regulates Wnt signaling and cell size during the development of the wing in a manner dependent on Fray, the fly homolog of OSR1/SPAK. We show that the only canonical RF(X)V/I motif of Wnk, thought to be essential for WNK interactions with OSR1/SPAK, is required to interact with Fray in vitro. However, this motif is unexpectedly dispensable for Fray-dependent Wnk functions in vivo during fly development and fluid secretion in the Malpighian (renal) tubules. In contrast, a structure function analysis of Wnk revealed that the less-conserved C-terminus of Wnk, that recently has been shown to promote phase transitions in cell culture, is required for viability in vivo. Our data thus provide novel insights into unexpected in vivo roles of specific WNK domains.

Hydrostatic Pressure Sensing by WNK kinases.

Previous study has demonstrated that the WNK kinases 1 and 3 are direct osmosensors consistent with their established role in cell-volume control. WNK kinases may also be regulated by hydrostatic pressure. Hydrostatic pressure applied to cells in culture with N2 gas or to Drosophila Malpighian tubules by centrifugation induces phosphorylation of downstream effectors of endogenous WNKs. In vitro, the autophosphorylation and activity of the unphosphorylated kinase domain of WNK3 (uWNK3) is enhanced to a lesser extent than in cells by 190 kPa applied with N2 gas. Hydrostatic pressure measurably alters the structure of uWNK3. Data from size exclusion chromatography in line with multi-angle light scattering (SEC-MALS), SEC alone at different back pressures, analytical ultracentrifugation (AUC), NMR, and chemical crosslinking indicate a change in oligomeric structure in the presence of hydrostatic pressure from a WNK3 dimer to a monomer. The effects on the structure are related to those seen with osmolytes. Potential mechanisms of hydrostatic pressure activation of uWNK3 and the relationships of pressure activation to WNK osmosensing are discussed.

Intracellular Ion Control of WNK Signaling.

The with no lysine (K) (WNK) kinases are an evolutionarily ancient group of kinases with atypical placement of the catalytic lysine and diverse physiological roles. Recent studies have shown that WNKs are directly regulated by chloride, potassium, and osmotic pressure. Here, we review the discovery of WNKs as chloride-sensitive kinases and discuss physiological contexts in which chloride regulation of WNKs has been demonstrated. These include the kidney, pancreatic duct, neurons, and inflammatory cells. We discuss the interdependent relationship of osmotic pressure and intracellular chloride in cell volume regulation. We review the recent demonstration of potassium regulation of WNKs and speculate on possible physiological roles. Finally, structural and mechanistic aspects of intracellular ion and osmotic pressure regulation of WNKs are discussed.

Regulation of Distal Nephron Transport by Intracellular Chloride and Potassium.

BACKGROUND: Low potassium increases the phosphorylation and activity of the sodium chloride cotransporter (NCC) in the distal convoluted tubule of the nephron, which contributes to the hypertensive effect of the modern low potassium/high sodium diet. A central mediator of potassium regulation of NCC is the chloride-sensitive With No Lysine [K] (WNK) kinase. SUMMARY: Chloride directly inhibits WNKs by binding to the active site. The mechanisms underlying WNK regulation by extracellular potassium are reviewed, as well as the modulatory effect of kidney-specific-WNK1. WNK1, but not WNK1 kinase activity, is also required for the aldosterone-independent regulation of the epithelial sodium channel by potassium. Whether intracellular chloride could be involved in this process is discussed. Recent studies demonstrating direct regulation of WNKs by intracellular potassium are also reviewed, and the potential physiological relevance to renal epithelial ion transport is discussed. KEY MESSAGES: WNKs are sensors of the intracellular ionic milieu. In the nephron, changes in extracellular ion concentrations, resulting in changes in intracellular ion concentration, regulate WNK activity and downstream transporters and channels to maintain total body ion homeostasis.

Apolipoprotein L1 Genotypes and the Association of Urinary Potassium Excretion with CKD Progression.

BACKGROUND AND OBJECTIVES: Progressive CKD in Black individuals is strongly associated with polymorphisms in the APOL1 gene, but it is unknown whether dietary risk factors for CKD progression vary in high- versus low-risk APOL1 genotypes. We investigated if APOL1 genotypes modify associations of dietary potassium and sodium with CKD progression and death. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We analyzed 1399 self-identified Black participants enrolled in the Chronic Renal Insufficiency Cohort from April 2003 to September 2008. Exposures were calibrated 24-hour urine potassium and sodium excretion. The primary outcome was CKD progression defined as the time to 50% decline in eGFR or kidney failure. The secondary outcome was CKD progression or death. We tested for an interaction between urinary potassium and sodium excretion and APOL1 genotypes. RESULTS: Median 24-hour urinary sodium and potassium excretions in Black participants were 150 mmol (interquartile range, 118-188) and 43 mmol (interquartile range, 35-54), respectively. Individuals with high- and low-risk APOL1 genotypes numbered 276 (20%) and 1104 (79%), respectively. After a median follow-up of 5.23 years, CKD progression events equaled 605, and after 7.29 years, CKD progression and death events equaled 868. There was significant interaction between APOL1 genotypes and urinary potassium excretion with CKD progression and CKD progression or death (P=0.003 and P=0.03, respectively). In those with high-risk APOL1 genotypes, higher urinary potassium excretion was associated with a lower risk of CKD progression (quartiles 2-4 versus 1: hazard ratio, 0.83; 95% confidence interval, 0.50 to 1.39; hazard ratio, 0.54; 95% confidence interval, 0.31 to 0.93; and hazard ratio, 0.50; 95% confidence interval, 0.27 to 0.93, respectively). In the low-risk APOL1 genotypes, higher urinary potassium excretion was associated with a higher risk of CKD progression (quartiles 2-4 versus 1: hazard ratio, 1.01; 95% confidence interval, 0.75 to 1.36; hazard ratio, 1.23; 95% confidence interval, 0.91 to 1.66; and hazard ratio, 1.53; 95% confidence interval, 1.12 to 2.09, respectively). We found no interaction between APOL1 genotypes and urinary sodium excretion with CKD outcomes. CONCLUSIONS: Higher urinary potassium excretion was associated with lower versus higher risk of CKD progression in APOL1 high-risk and low-risk genotypes, respectively.

WNK kinases sense molecular crowding and rescue cell volume via phase separation.

When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.

Harnessing changes in open chromatin determined by ATAC-seq to generate insulin-responsive reporter constructs.

BACKGROUND: Gene regulation is critical for proper cellular function. Next-generation sequencing technology has revealed the presence of regulatory networks that regulate gene expression and essential cellular functions. Studies investigating the epigenome have begun to uncover the complex mechanisms regulating transcription. Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is quickly becoming the assay of choice for many epigenomic investigations. However, whether intervention-mediated changes in accessible chromatin determined by ATAC-seq can be harnessed to generate intervention-inducible reporter constructs has not been systematically assayed. RESULTS: We used the insulin signaling pathway as a model to investigate chromatin regions and gene expression changes using ATAC- and RNA-seq in insulin-treated Drosophila S2 cells. We found correlations between ATAC- and RNA-seq data, especially when stratifying differentially-accessible chromatin regions by annotated feature type. In particular, our data demonstrated a weak but significant correlation between chromatin regions annotated to enhancers (1-2 kb from the transcription start site) and downstream gene expression. We cloned candidate enhancer regions upstream of luciferase and demonstrate insulin-inducibility of several of these reporters. CONCLUSIONS: Insulin-induced chromatin accessibility determined by ATAC-seq reveals enhancer regions that drive insulin-inducible reporter gene expression.

Mitochondrial calcium uniporter stabilization preserves energetic homeostasis during Complex I impairment.

Calcium entering mitochondria potently stimulates ATP synthesis. Increases in calcium preserve energy synthesis in cardiomyopathies caused by mitochondrial dysfunction, and occur due to enhanced activity of the mitochondrial calcium uniporter channel. The signaling mechanism that mediates this compensatory increase remains unknown. Here, we find that increases in the uniporter are due to impairment in Complex I of the electron transport chain. In normal physiology, Complex I promotes uniporter degradation via an interaction with the uniporter pore-forming subunit, a process we term Complex I-induced protein turnover. When Complex I dysfunction ensues, contact with the uniporter is inhibited, preventing degradation, and leading to a build-up in functional channels. Preventing uniporter activity leads to early demise in Complex I-deficient animals. Conversely, enhancing uniporter stability rescues survival and function in Complex I deficiency. Taken together, our data identify a fundamental pathway producing compensatory increases in calcium influx during Complex I impairment.

Optimized assay for transposase-accessible chromatin by sequencing (ATAC-seq) library preparation from adult Drosophila melanogaster neurons.

Assay for transposase-accessible chromatin by sequencing (ATAC-seq) is rapidly becoming the assay of choice to investigate chromatin-mediated gene regulation, largely because of low input requirements, a fast workflow, and the ability to interrogate the entire genome in an untargeted manner. Many studies using ATAC-seq use mammalian or human-derived tissues, and established protocols work well in these systems. However, ATAC-seq is not yet widely used in Drosophila. Vinegar flies present several advantages over mammalian systems that make them an excellent model for ATAC-seq studies, including abundant genetic tools that allow straightforward targeting, transgene expression, and genetic manipulation that are not available in mammalian models. Because current ATAC-seq protocols are not optimized to use flies, we developed an optimized workflow that accounts for several complicating factors present in Drosophila. We examined parameters affecting nuclei isolation, including input size, freezing time, washing, and possible confounds from retinal pigments. Then, we optimized the enzymatic steps of library construction to account for the smaller Drosophila genome size. Finally, we used our optimized protocol to generate ATAC-seq libraries that meet ENCODE quality metrics. Our optimized protocol enables extensive ATAC-seq experiments in Drosophila, thereby leveraging the advantages of this powerful model system to understand chromatin-mediated gene regulation.

Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade.

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies.1,2 Circadian variation in the intracellular chloride concentration of mammalian pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels.3 However, results have been contradictory,4-9 and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response.10 Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila small ventral lateral (sLNv) pacemaker neurons. In control flies, intracellular chloride increases in sLNvs over the course of the morning. Chloride transport through sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations.11Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride 6 h after lights on, loss of morning anticipation, and a prolonged circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of WNK (with no lysine [K]) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila sLNv pacemakers restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

Role of collecting duct principal cell NOS1ß in sodium and potassium homeostasis.

The nitric oxide (NO)-generating enzyme, NO synthase-1ß (NOS1ß), is essential for sodium (Na+ ) homeostasis and blood pressure control. We previously showed that collecting duct principal cell NOS1ß is critical for inhibition of the epithelial sodium channel (ENaC) during high Na+ intake. Previous studies on freshly isolated cortical collecting ducts (CCD) demonstrated that exogenous NO promotes basolateral potassium (K+ ) conductance through basolateral channels, presumably Kir 4.1 (Kcnj10) and Kir 5.1 (Kcnj16). We, therefore, investigated the effects of NOS1ß knockout on Kir 4.1/Kir 5.1 channel activity. Indeed, in CHO cells overexpressing NOS1ß and Kir 4.1/Kir 5.1, the inhibition of NO signaling decreased channel activity. Male littermate control and principal cell NOS1ß knockout mice (CDNOS1KO) on a 7-day, 4% NaCl diet (HSD) were used to detect changes in basolateral K+ conductance. We previously demonstrated that CDNOS1KO mice have high circulating aldosterone despite a high-salt diet and appropriately suppressed renin. We observed greater Kir 4.1 cortical abundance and significantly greater Kir 4.1/Kir 5.1 single-channel activity in the principal cells from CDNOS1KO mice. Moreover, blocking aldosterone action with in vivo spironolactone treatment resulted in lower Kir 4.1 abundance and greater plasma K+ in the CDNOS1KO mice compared to controls. Lowering K+ content in the HSD prevented the high aldosterone and greater plasma Na+ of CDNOS1KO mice and normalized Kir 4.1 abundance. We conclude that during chronic HSD, lack of NOS1ß leads to increased plasma K+ , enhanced circulating aldosterone, and activation of ENaC and Kir 4.1/Kir 5.1 channels. Thus, principal cell NOS1ß is required for the regulation of both Na+ and K+ by the kidney.

Serine Protease HTRA1 as a Novel Target Antigen in Primary Membranous Nephropathy.

BACKGROUND: Identification of target antigens PLA2R, THSD7A, NELL1, or Semaphorin-3B can explain the majority of cases of primary membranous nephropathy (MN). However, target antigens remain unidentified in 15%-20% of patients. METHODS: A multipronged approach, using traditional and modern technologies, converged on a novel target antigen, and capitalized on the temporal variation in autoantibody titer for biomarker discovery. Immunoblotting of human glomerular proteins followed by differential immunoprecipitation and mass spectrometric analysis was complemented by laser-capture microdissection followed by mass spectrometry, elution of immune complexes from renal biopsy specimen tissue, and autoimmune profiling on a protein fragment microarray. RESULTS: These approaches identified serine protease HTRA1 as a novel podocyte antigen in a subset of patients with primary MN. Sera from two patients reacted by immunoblotting with a 51-kD protein within glomerular extract and with recombinant human HTRA1, under reducing and nonreducing conditions. Longitudinal serum samples from these patients seemed to correlate with clinical disease activity. As in PLA2R- and THSD7A- associated MN, anti-HTRA1 antibodies were predominantly IgG4, suggesting a primary etiology. Analysis of sera collected during active disease versus remission on protein fragment microarrays detected significantly higher titers of anti-HTRA1 antibody in active disease. HTRA1 was specifically detected within immune deposits of HTRA1-associated MN in 14 patients identified among three cohorts. Screening of 118 "quadruple-negative" (PLA2R-, THSD7A-, NELL1-, EXT2-negative) patients in a large repository of MN biopsy specimens revealed a prevalence of 4.2%. CONCLUSIONS: Conventional and more modern techniques converged to identify serine protease HTRA1 as a target antigen in MN.

Molecular basis for epithelial morphogenesis and ion transport in the Malpighian tubule.

During development, the insect Malpighian tubule undergoes several programmed morphogenetic events that give rise to the tubule's ability to transport ions and water at unparalleled speed. Studies in Diptera, in particular, have greatly increased our understanding of the molecular pathways underlying embryonic tubule development. In this review, we discuss recent work that has revealed new insights into the molecular players required for the development and maintenance of structurally and functionally intact adult Malpighian tubules. We highlight the contribution of the smooth septate junction (sSJ) proteins to the morphogenesis and transport function of the epithelial cells of the Drosophila melanogaster Malpighian tubule and also discuss new findings on the role of the GATAe transcription factor. We also consider the roles of sSJ proteins in the fly midgut, as compared to the Malpighian tubule, and the importance of cellular context for the functions of these proteins.

The fly liquid-food electroshock assay (FLEA) suggests opposite roles for neuropeptide F in avoidance of bitterness and shock.

BACKGROUND: Proper regulation of feeding is important for an organism's well-being and survival and involves a motivational component directing the search for food. Dissecting the molecular and neural mechanisms of motivated feeding behavior requires assays that allow quantification of both motivation and food intake. Measurements of motivated behavior usually involve assessing physical effort or overcoming an aversive stimulus. Food intake in Drosophila can be determined in a number of ways, including by measuring the time a fly's proboscis interacts with a food source associated with an electrical current in the fly liquid-food interaction counter (FLIC). Here, we show that electrical current flowing through flies during this interaction is aversive, and we describe a modified assay to measure motivation in Drosophila. RESULTS: Food intake is reduced during the interaction with FLIC when the electrical current is turned on, which provides a confounding variable in studies of motivated behavior. Based on the FLIC, we engineer a novel assay, the fly liquid-food electroshock assay (FLEA), which allows for current adjustments for each feeding well. Using the FLEA, we show that both external incentives and internal motivational state can serve as drivers for flies to overcome higher current (electric shock) to obtain superior food. Unlike similar assays in which bitterness is the aversive stimulus for the fly to overcome, we show that current perception is not discounted as flies become more food-deprived. Finally, we use genetically manipulated flies to show that neuropeptide F, an orthologue of mammalian NPY previously implicated in regulation of feeding motivation, is required for sensory processing of electrical current. CONCLUSION: The FLEA is therefore a novel assay to accurately measure incentive motivation in Drosophila. Using the FLEA, we also show that neuropeptide F is required for proper perception or processing of an electroshock, a novel function for this neuropeptide involved in the processing of external and internal stimuli.

WNKs are potassium-sensitive kinases.

With no lysine (K) (WNK) kinases regulate epithelial ion transport in the kidney to maintain homeostasis of electrolyte concentrations and blood pressure. Chloride ion directly binds WNK kinases to inhibit autophosphorylation and activation. Changes in extracellular potassium are thought to regulate WNKs through changes in intracellular chloride. Prior studies demonstrate that in some distal nephron epithelial cells, intracellular potassium changes with chronic low- or high-potassium diet. We, therefore, investigated whether potassium regulates WNK activity independent of chloride. We found decreased activity of Drosophila WNK and mammalian WNK3 and WNK4 in fly Malpighian (renal) tubules bathed in high extracellular potassium, even when intracellular chloride was kept constant at either ∼13 mM or 26 mM. High extracellular potassium also inhibited chloride-insensitive mutants of WNK3 and WNK4. High extracellular rubidium was also inhibitory and increased tubule rubidium. The Na+/K+-ATPase inhibitor, ouabain, which is expected to lower intracellular potassium, increased tubule Drosophila WNK activity. In vitro, potassium increased the melting temperature of Drosophila WNK, WNK1, and WNK3 kinase domains, indicating ion binding to the kinase. Potassium inhibited in vitro autophosphorylation of Drosophila WNK and WNK3, and also inhibited WNK3 and WNK4 phosphorylation of their substrate, Ste20-related proline/alanine-rich kinase (SPAK). The greatest sensitivity of WNK4 to potassium occurred in the range of 80-180 mM, encompassing physiological intracellular potassium concentrations. Together, these data indicate chloride-independent potassium inhibition of Drosophila and mammalian WNK kinases through direct effects of potassium ion on the kinase.