Deafness can result from a variety of gene defects. Some genes involved in the physiology of hearing encode membrane transporters that regulate the ionic composition of the fluid bathing the inner ear. The endolymph is an extracellular fluid with an atypical composition that resembles the intracellular milieu, high in K+ and low in Na+. Recent studies have emphasized the prominent role of K+ channels in endolymph secretion and mechanical transduction. Coupled electroneutral transport of Na+, K+ and Cl- is mediated by two isoforms of the Na-K-2Cl co-transporter: the absorptive isoform BSC1 (also called NKCC2, encoded by Slc12a1 in mouse) that is exclusively expressed in kidney; and BSC2/NKCC1 (encoded by Slc12a2 in mouse), the secretory isoform which has a wider pattern of expression including epithelia, muscle cells, neurons and red blood cells. These co-transporters share 57% homology at the amino acid level and are pharmacologically inhibited by loop diuretics. There is functional and histochemical evidence for the presence of the secretory isoform of the Na-K-2Cl co-transporter in gerbil, rat and rabbit inner ear. We disrupted mouse Slc12a2 and report here that Slc12a2-/- mice are deaf and exhibit classic shaker/waltzer behaviour, indicative of inner-ear defects. We localized the co-transporter to key secreting epithelia of the mouse inner ear and show that absence of functional co-transporter leads to structural damages in the inner ear consistent with a decrease in endolymph secretion.

In the present study, we have demonstrated functional interaction between Ste20-related proline-alanine-rich kinase (SPAK), WNK4 [with no lysine (K)], and the widely expressed Na+-K+-2Cl- cotransporter type 1 (NKCC1). NKCC1 function, which we measured in Xenopus laevis oocytes under both isosmotic (basal) and hyperosmotic (stimulated) conditions, was unaffected when SPAK and WNK4 were expressed alone. In contrast, expression of both kinases with NKCC1 resulted in a significant increase in cotransporter activity and an insensitivity to external osmolarity or cell volume. NKCC1 activation is dependent on the catalytic activity of SPAK and likely also of WNK4, because mutations in their catalytic domains result in an absence of cotransporter stimulation. The results of our yeast two-hybrid experiments suggest that WNK4 does not interact directly with NKCC1 but does interact with SPAK. Functional experiments demonstrated that the binding of SPAK to WNK4 was also required because a SPAK-interaction-deficient WNK4 mutant (Phe997Ala) did not increase NKCC1 activity. We also have shown that the transport function of K+-Cl- cotransporter type 2 (KCC2), a neuron-specific KCl cotransporter, was diminished by the expression of both kinases under both isosmotic and hyposmotic conditions. Our data are consistent with WNK4 interacting with SPAK, which in turn phosphorylates and activates NKCC1 and phosphorylates and deactivates KCC2.

Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is a severe sensorimotor neuropathy associated with mental retardation, dysmorphic features and complete or partial agenesis of the corpus callosum. ACCPN is transmitted in an autosomal recessive fashion and is found at a high frequency in the province of Quebec, Canada. ACCPN has been previously mapped to chromosome 15q. The gene SLC12A6 (solute carrier family 12, member 6), which encodes the K+-Cl- transporter KCC3 and maps within the ACCPN candidate region, was screened for mutations in individuals with ACCPN. Four distinct protein-truncating mutations were found: two in the French Canadian population and two in non-French Canadian families. The functional consequence of the predominant French Canadian mutation (2436delG, Thr813fsX813) was examined by heterologous expression of wildtype and mutant KCC3 in Xenopus laevis oocytes; the truncated mutant is appropriately glycosylated and expressed at the cellular membrane, where it is non-functional. Mice generated with a targeted deletion of Slc12a6 have a locomotor deficit, peripheral neuropathy and a sensorimotor gating deficit, similar to the human disease. Our findings identify mutations in SLC12A6 as the genetic lesion underlying ACCPN and suggest a critical role for SLC12A6 in the development and maintenance of the nervous system.

Four genes encode electroneutral, Na+-independent, K-Cl cotransporters. KCC2, is exclusively expressed in neurons where it is thought to drive intracellular Cl- to low concentrations and shift the reversal potential for Cl- conductances such as GABA(A) or glycine receptor channels, thus participating in the postnatal development of inhibitory mechanisms in the brain. Indeed, expression of the cotransporter is low at birth and increases postnatally, at a time when the intracellular Cl- concentration in neurons decreases and gamma-aminobutyric acid switches its effect from excitatory to inhibitory. To assert the significance of KCC2 in neuronal function, we disrupted the mouse gene encoding this neuronal-specific K-Cl cotransporter. We demonstrate that animals deficient in KCC2 exhibit frequent generalized seizures and die shortly after birth. We also show upregulation of Fos, the product of the immediate early gene c-fos, and the significant loss of parvalbumin-positive interneurons, both indicative of brain injury. The regions most affected are the hippocampus and temporal and entorhinal cortices. Extracellular field potential measurements in the CA1 hippocampus exhibited hyperexcitability. Application of picrotoxin, a blocker of the GABA(A) receptor, further increased hyperexcitability in homozygous hippocampal sections. Pharmacological treatment of pups showed that diazepam relieved the seizures while phenytoin prevented them between postnatal ages P4-P12. Finally, we demonstrate that adult heterozygote animals show increased susceptibility for epileptic seizure and increased resistance to the anticonvulsant effect of propofol. Taken together, these results indicate that KCC2 plays an important role in controlling CNS excitability during both postnatal development and adult life.

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related prolinealanine-rich kinase (SPAK). Mutant NKCC1 that lacks the ability to bind to this kinase showed K ؉ transport function identical to wild-type NKCC1. Thus, preventing the binding of the kinase to the cotransporter does not affect cotransporter function. In contrast, several experiments suggest a possible role for SPAK as a scaffolding protein. First, Western blot analysis revealed the presence, and in some tissues abundance, of truncated forms of SPAK and OSR1 in which the kinase domains are affected and thus lack kinase activity. Second, a yeast two-hybrid screen of proteins that interact with the regulatory (binding) domain of SPAK identified several proteins all involved in cellular stress pathways. Third, p38, one of the three major MAPKs, can be coimmunoprecipitated with SPAK and with NKCC1 in an activitydependent manner. The amount of p38 coimmunoprecipitated with the kinase and the cotransporter significantly decreases upon cellular stress, whereas the interaction of the kinase with NKCC1 remains unchanged. These findings suggest that cation-chloride cotransporters might act as "sensors" for cellular stress, and SPAK, by interacting with the cotransporter, serves as an intermediate in the response to cellular stress. Cation-chloride cotransporters, which mediate the tightly coupled, electroneutral movement of cations (Na ϩ and K ϩ ) together with Cl Ϫ , can be divided into Na ϩ -dependent transporters, such as Na-K-2Cl cotransporters (NKCC1-2) and the Na-Cl cotransporter (NCC), and Na ϩ -independent K-Cl cotransporters (KCC1-4). All of these transporters have a well conserved topology with large intracellular amino-terminal and carboxyl-terminal tails and 12 transmembrane spanning domains. The core protein shares some homology to amino acid permeases (for reviews see Refs. 1 and 2). A variety of stimuli regulates these transporters, including hormonal (3), cytokines (4, 5), cell volume (6, 7), oxidative stress (8), etc. There is also accumulating evidence that cation-chloride cotransporters participate in pathways leading to cell differentiation, growth and proliferation (9, 10), and apoptosis (11,12). At the molecular level, the activation-deactivation of these cotransporters mostly involves phosphorylation/dephosphorylation mechanisms, the details of which are still the subject of intense investigation.To identify proteins that directly interact and regulate cation-chloride cotransporters, we recently performed a yeast twohybrid screen using the cytosolic amino-terminal tail of KCC3. We identified two closely related kinases that bind to KCC3, NKCC1 and NKCC2 (13). These kinases belong to the group of Ste20 kinases that function as regulators of MAPK 1 cascades (14). The first kinase, SPAK (Ste-20 related proline-alaninerich kinase, or PASK, as the rat homologue), is highly expressed in epithelia and neurons (15). Its gene is located on human chromosome...

Our recent studies demonstrate that SPAK (Ste20p-related Proline Alanine-rich Kinase), in combination with WNK4 [With No lysine (K) kinase], phosphorylates and stimulates the Na-K-2Cl cotransporter (NKCC1), whereas catalytically inactive SPAK (K104R) fails to activate the cotransporter. The catalytic domain of SPAK contains an activation loop between the well-conserved DFG and APE motifs. We speculated that four threonine residues (T231, T236, T243, and T247) in the activation loop might be sites of phosphorylation and kinase activation; therefore, we mutated each residue into an alanine. In this report, we demonstrate that coexpression of SPAK (T243A) or SPAK (T247A) with WNK4 not only prevented, but robustly inhibited, cotransporter activity in NKCC1-injected Xenopus laevis oocytes. These activation loop mutations produced an effect similar to that of the SPAK (K104R) mutant. In vitro phosphorylation experiments demonstrate that both intramolecular autophosphorylation of SPAK and phosphorylation of NKCC1 are significantly stronger in the presence of Mn 2؉ rather than Mg 2؉ . We also show that SPAK activity is markedly inhibited by staurosporine and K252a, partially inhibited by N-ethylmaleimide and diamide, and unaffected by arsenite. OSR1, a kinase closely related to SPAK, exhibited similar kinase properties and similar functional activation of NKCC1 when coexpressed with WNK4.Cation-chloride cotransporters (e.g., Na-K-2Cl and K-Cl cotransporters) serve multiple fundamental functions in a wide variety of tissues and organs. These include influencing ion and fluid movements in secreting or reabsorbing epithelia, control of CNS excitability, and cell volume regulation, proliferation, and survival (for a review, see reference 19). On the basis of the observations that the cotransporters are phosphoproteins and that phosphatase inhibitors affect cotransport activity, it is generally agreed that cotransporter regulation is mainly mediated through phosphorylation-dephosphorylation mechanisms. Activity of NKCC1, for instance, correlates directly with the phosphorylation state of the protein (33). For the past 20 years, the identity of the kinase in question has remained elusive. We have recently shown direct interaction between cation-chloride cotransporter and two Ste20p-related serine/threonine kinases, SPAK and OSR1 (45).There are some 30 protein kinases identified in mammals related to the budding Saccharomyces cerevisiae sterile 20 protein kinase (Ste20p). In 1991, Dan et al. divided the mammalian Ste20p-like kinases into two subgroups: the p21-activated kinases (PAKs; two subfamilies), which are characterized by a carboxyl-terminal catalytic domain, and the germinal center kinases (GCKs; eight subfamilies), which have their catalytic domain located at the amino terminus (10). Most Ste20p-like kinases stimulate mitogen-activated protein kinase (MAPK) cascades and thus participate in the modulation of cell motility (7), cell growth (31), and apoptosis (23).The GCK6 subfamily comprises two kinases: SPAK/PASK (Ste...

AimThe primary objective was to compare apixaban to heparin/vitamin K antagonist (VKA) in patients with atrial fibrillation (AF) and ≤48 h anticoagulation prior to randomization undergoing cardioversion.MethodsOne thousand five hundred patients were randomized. The apixaban dose of 5 mg b.i.d. was reduced to 2.5 mg b.i.d. in patients with two of the following: age ≥ 80 years, weight ≤ 60 kg, or serum creatinine ≥ 133 µmol/L. To expedite cardioversion, at the discretion of the investigator, imaging and/or a loading dose of 10 mg (down-titrated to 5 mg) was allowed. The endpoints for efficacy were stroke, systemic embolism (SE), and death. The endpoints for safety were major bleeding and clinically relevant non-major (CRNM) bleeding.ResultsThere were 1038 active and 300 spontaneous cardioversions; 162 patients were not cardioverted. Imaging was performed in 855 patients, and 342 received a loading dose of apixaban. Comparing apixaban to heparin/VKA in the full analysis set, there were 0/753 vs. 6/747 strokes [relative risk (RR) 0; 95% confidence interval (95% CI) 0–0.64; nominal P = 0.015], no SE, and 2 vs. 1 deaths (RR 1.98; 95% CI 0.19–54.00; nominal P > 0.999). In the safety population, there were 3/735 vs. 6/721 major (RR 0.49; 95% CI 0.10–2.07; nominal P = 0.338) and 11 vs. 13 CRNM bleeding events (RR 0.83; 95% CI 0.34–1.89; nominal P = 0.685). On imaging, 60/61 with thrombi continued randomized treatment; all (61) were without outcome events.ConclusionsRates of strokes, systemic emboli, deaths, and bleeds were low for both apixaban and heparin/VKA treated AF patients undergoing cardioversion.Clinical Trials.gov numberNCT02100228

Background: SPAK (Ste20p-related proline alanine-rich kinase) phosphorylates and activates NKCC1 (Na-K-2Cl cotransporter) in the presence of another serine/threonine kinase WNK4 (With No lysine (K)). However, whether or not the docking of SPAK to NKCC1 is a requirement for cotransporter activation has not been fully resolved. Methods: We mutated both SPAK binding motifs in the amino-terminal tail of NKCC1 and tested the interaction between SPAK and NKCC1 using a semi in vivo yeast two-hybrid assay, ³²P-ATP in vitro phosphorylation assays, and ⁸⁶Rb⁺ uptake (a K⁺ congener) assays in heterologously expressed Xenopus laevis oocytes. We also used site-directed mutagenesis to identify the principle phospho-regulatory threonine residues in the amino-terminal tail of NKCC1. Results: A single SPAK binding motif is necessary for isotonic NKCC1 activation. Mutation of the phenylalanine (F) residue within the motif abrogates binding and function. Phosphorylation of the cotransporter is markedly reduced in the absence of SPAK docking to NKCC1. Truncations of internal regions of the amino-terminus of NKCC1 do not disrupt protein structure enough to affect cotransporter function. Threonine residues (T²⁰⁶ and T²¹¹) are both identified as phospho-regulatory sites of NKCC1 function. Conclusion: We demonstrate that physical docking of SPAK to NKCC1 is necessary for cotransporter activity under both baseline and hyperosmotic conditions. We identify T²⁰⁶ and T²¹¹ as major phospho-acceptor sites involved in cotransporter function, with T²⁰⁶ common to two separate regulatory pathways: one involving SPAK, the other involving a still unknown kinase that is responsive to forskolin/PKA stimulation.