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.

Bicarbonate is the primary inducer of KCC3a expression in renal cortical B-type intercalated cells.

A primary function of intercalated cells in the distal tubule of the kidney is to maintain pH homeostasis. For example, type B intercalated cells secrete bicarbonate largely through the action of the apical Cl-/HCO3- exchanger, pendrin, which helps correct metabolic alkalosis. Since both the K-Cl cotransporter, KCC3a and pendrin colocalize to the apical region of type B and non-A, non-B intercalated cells and since both are upregulated in models of metabolic alkalosis, such as with dietary NaHCO3 loading, we raised the possibility that apical KCC3a facilitates pendrin-mediated bicarbonate secretion, such as through apical Cl- recycling. The purpose of this study was to determine if KCC3a abundance changes through intake of bicarbonate alone or through bicarbonate plus its accompanying cation, and if it requires a direct interaction with pendrin or the renin-angiotensin-aldosterone system. We observed that KCC3a protein abundance, but not mRNA, increases in a mouse model of metabolic alkalosis, achieved with dietary NaHCO3 or KHCO3 intake. Bicarbonate ion increases KCC3a abundance, both in vivo and in vitro, independently of the accompanying cation. Moreover, bicarbonate intake upregulates KCC3a independently of aldosterone or angiotensin II. Since NaHCO3 intake increased KCC3a abundance in wild-type as well as in pendrin knockout mice, this KCC3a upregulation by bicarbonate does not depend on a direct interaction with pendrin. We conclude that increased extracellular bicarbonate, as observed in models of metabolic alkalosis, directly raises KCC3a abundance independently of angiotensin II, aldosterone, or changes in KCC3a transcription and does not involve a direct interaction with pendrin.NEW & NOTEWORTHY KCC3a expression is stimulated in alkalemia. This paper shows that bicarbonate itself is mediating this effect through a posttranscriptional mechanism. The paper also shows that this phenomenon is not mediated by aldosterone or angiotensin II.

Osmotic Response of Dorsal Root Ganglion Neurons Expressing Wild-Type and Mutant KCC3 Transporters.

BACKGROUND/AIMS: Loss-of-Function (LOF) of the potassium chloride cotransporter 3 (KCC3) results in hereditary sensorimotor neuropathy with Agenesis of the Corpus Callosum (HSMN/ACC). Our KCC3 knockout mouse recapitulated axonal swelling and tissue vacuolization observed in autopsies of individuals with HSMN/ACC. We previously documented the first human case of a KCC3 gain-of-function (GOF) in which the patient also exhibited severe peripheral neuropathy. Furthermore, the GOF mouse model exhibited shrunken axons implicating the cotransporter in cell volume homeostasis. It is unclear how both KCC3 LOF and GOF lead to peripheral neuropathy. Thus, we sought to study differences in cell volume regulation of dorsal root ganglion neurons isolated from different mouse lines. METHODS: Using wide-field microscopy, we measured calcein fluorescence intensity through pinhole measurements at the center of cells and compared cell swelling and cell volume regulation/recovery of wild-type, LOF, and GOF dorsal root ganglia neurons, as well as wild-type neurons treated with a KCC-specific inhibitor. RESULTS: In contrast to control neurons that swell and volume regulate under a hypotonic challenge, neurons lacking KCC3 swell but fail to volume regulate. Similar data were observed in wild-type neurons treated with the KCC inhibitor. We also show that sensory neurons expressing a constitutively active KCC3 exhibited a blunted swelling phase compared to wild-type neurons, questioning the purely osmotic nature of the swelling phase. CONCLUSION: These findings demonstrate the integral role of KCC3 in cell volume homeostasis and support the idea that cell volume homeostasis is critical to the health of peripheral nerves.

Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells.

During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl--dependent Rb+ influx at different external Rb+ and Cl- ion concentrations, [Rb+]o and [Cl-]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl- and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl--dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl- correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl-]o were strikingly bimodal with 60-80 mM in early, ~20-30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.

Kinase-KCC2 coupling: Cl- rheostasis, disease susceptibility, therapeutic target.

The intracellular concentration of Cl(-) ([Cl(-)]i) in neurons is a highly regulated variable that is established and modulated by the finely tuned activity of the KCC2 cotransporter. Despite the importance of KCC2 for neurophysiology and its role in multiple neuropsychiatric diseases, our knowledge of the transporter's regulatory mechanisms is incomplete. Recent studies suggest that the phosphorylation state of KCC2 at specific residues in its cytoplasmic COOH terminus, such as Ser940 and Thr906/Thr1007, encodes discrete levels of transporter activity that elicit graded changes in neuronal Cl(-) extrusion to modulate the strength of synaptic inhibition via Cl(-)-permeable GABAA receptors. In this review, we propose that the functional and physical coupling of KCC2 to Cl(-)-sensitive kinase(s), such as the WNK1-SPAK kinase complex, constitutes a molecular "rheostat" that regulates [Cl(-)]i and thereby influences the functional plasticity of GABA. The rapid reversibility of (de)phosphorylation facilitates regulatory precision, and multisite phosphorylation allows for the control of KCC2 activity by different inputs via distinct or partially overlapping upstream signaling cascades that may become more or less important depending on the physiological context. While this adaptation mechanism is highly suited to maintaining homeostasis, its adjustable set points may render it vulnerable to perturbation and dysregulation. Finally, we suggest that pharmacological modulation of this kinase-KCC2 rheostat might be a particularly efficacious strategy to enhance Cl(-) extrusion and therapeutically restore GABA inhibition.

KCC3a, a Strong Candidate Pathway for K+ Loss in Alkalemia.

Loss-of-function mutations in the human potassium chloride cotransporter-3 (KCC3) cause a hereditary motor sensory neuropathy associated with agenesis of the corpus callosum. While recapitulating the neuropathy, KCC3-knockout mice also exhibit high blood pressure. This phenotype is believed to have neurogenic and/or vascular origins. The role of KCC3 in the kidney is poorly understood. KCC3 is encoded by two major isoforms originating from alternative promoters: KCC3a and KCC3b, with KCC3b being the predominant transcript in the kidney. Although the transporter has previously been localized to the proximal tubule, we show here the unique expression of the KCC3a isoform in the connecting tubule. Using a KCC3a-specific polyclonal antibody validated for both immunofluorescence and immunoblotting, we showed an intense KCC3a signal restricted to cortical intercalated cells. No overlap is detected between KCC3a and sodium chloride cotransporter (NCC), a distal convoluted tubule (DCT) marker; or between KCC3a and ENaC or calbindin, which are both principal cell markers. KCC3a signal was observed in cells expressing the apical V-ATPase and pendrin, establishing a unique expression pattern characteristic of intercalated cells of type-B or type-nonA/nonB. We further show that treatment of wild-type mice with hydrochlorothiazide, amiloride, or fed a K+-deficient diet up-regulates KCC3a level, suggesting that volume depletion increases KCC3a abundance. This hypothesis was confirmed by showing a higher abundance of KCC3a protein after 23-h water restriction or after placing the mice on a low-salt diet. More importantly, abundance of the Cl-/HCO3 - exchanger, pendrin, which is known to secrete bicarbonate in alkalotic conditions, was significantly diminished in KCC3-knockout mice. In addition, KCC3a abundance increased significantly alongside pendrin abundance in bicarbonate-treated alkalotic mice, providing a credible mechanism for K+ loss in metabolic alkalosis.

Sickle cell dehydration: Pathophysiology and therapeutic applications.

Cell dehydration is a distinguishing characteristic of sickle cell disease and an important contributor to disease pathophysiology. Due to the unique dependence of Hb S polymerization on cellular Hb S concentration, cell dehydration promotes polymerization and sickling. In double heterozygosis for Hb S and C (SC disease) dehydration is the determining factor in disease pathophysiology. Three major ion transport pathways are involved in sickle cell dehydration: the K-Cl cotransport (KCC), the Gardos channel (KCNN4) and Psickle, the polymerization induced membrane permeability, most likely mediated by the mechano-sensitive ion channel PIEZO1. Each of these pathways exhibit unique characteristics in regulation by oxygen tension, intracellular and extracellular environment, and functional expression in reticulocytes and mature red cells. The unique dependence of K-Cl cotransport on intracellular Mg and the abnormal reduction of erythrocyte Mg content in SS and SC cells had led to clinical studies assessing the effect of oral Mg supplementation. Inhibition of Gardos channel by clotrimazole and senicapoc has led to Phase 1,2,3 trials in patients with sickle cell disease. While none of these studies has resulted in the approval of a novel therapy for SS disease, they have highlighted the key role played by these pathways in disease pathophysiology.

Deletion of KCC3 in parvalbumin neurons leads to locomotor deficit in a conditional mouse model of peripheral neuropathy associated with agenesis of the corpus callosum.

Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC or ACCPN) is an autosomal recessive disease caused by the disruption of the SLC12A6 gene, which encodes the K-Cl cotransporter-3 (KCC3). A ubiquitous deletion of KCC3 in mice leads to severe locomotor deficits similar to ACCPN patients. However, the underlying pathological mechanism leading to the disease remains unclear. Even though a recent study suggests that the neuropathic features of ACCPN are mostly due to neuronal loss of KCC3, the specific cell type responsible for the disease is still unknown. Here we established four tissue specific KCC3 knockout mouse lines to explore the cell population origin of ACCPN. Our results showed that the loss of KCC3 in parvalbumin-positive neurons led to significant locomotor deficit, suggesting a crucial role of these neurons in the development of the locomotor deficit. Interestingly, mice in which KCC3 deletion was driven by the neuron-specific enolase (NSE) did not develop any phenotype. Furthermore, we demonstrated that nociceptive neurons targeted with Nav1.8-driven CRE and Schwann cells targeted with a desert hedgehog-driven CRE were not involved in the development of ACCPN. Together, these results establish that the parvalbumin-positive neuronal population is an important player in the pathogenic development of ACCPN.

Arachidonic Acid Activates K-Cl-cotransport in HepG2 Human Hepatoblastoma Cells.

K(+)-Cl(-)-cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A(2) (PLA(2))-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K(+) efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator-induced K(+) efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium-independent PLA(2) (iPLA(2)), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF(3)) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA(2) (cPLA(2)) and the secretory PLA(2) (sPLA(2)), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF(3) and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA(2)-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.

Arachidonic Acid Activates K+-Cl--cotransport in HepG2 Human Hepatoblastoma Cells

K+-Cl--cotransport (KCC) has been reported to have various cellular functions, including proliferation and apoptosis of human cancer cells. However, the signal transduction pathways that control the activity of KCC are currently not well understood. In this study we investigated the possible role of phospholipase A2 (PLA2)-arachidonic acid (AA) signal in the regulatory mechanism of KCC activity. Exogenous application of AA significantly induced K+ efflux in a dose-dependent manner, which was completely blocked by R-(+)-[2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl]oxy]acetic acid (DIOA), a specific KCC inhibitor. N-Ethylmaleimide (NEM), a KCC activator-induced K+ efflux was significantly suppressed by bromoenol lactone (BEL), an inhibitor of the calcium-independent PLA2 (iPLA2), whereas it was not significantly altered by arachidonyl trifluoromethylketone (AACOCF3) and p-bromophenacyl bromide (BPB), inhibitors of the calcium-dependent cytosolic PLA2 (cPLA2) and the secretory PLA2 (sPLA2), respectively. NEM increased AA liberation in a dose- and time-dependent manner, which was markedly prevented only by BEL. In addition, the NEM-induced ROS generation was significantly reduced by DPI and BEL, whereas AACOCF3 and BPB did not have an influence. The NEM-induced KCC activation and ROS production was not significantly affected by treatment with indomethacin (Indo) and nordihydroguaiaretic acid (NDGA), selective inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), respectively. Treatment with 5,8,11,14-eicosatetraynoic acid (ETYA), a non-metabolizable analogue of AA, markedly produced ROS and activated the KCC. Collectively, these results suggest that iPLA2-AA signal may be essentially involved in the mechanism of ROS-mediated KCC activation in HepG2 cells.