A Trafficking-Deficient Mutant of KCC3 Reveals Dominant-Negative Effects on K–Cl Cotransport Function

The K–Cl cotransporter (KCC) functions in maintaining chloride and volume homeostasis in a variety of cells. In the process of cloning the mouse KCC3 cDNA, we came across a cloning mutation (E289G) that rendered the cotransporter inactive in functional assays in Xenopus laevis oocytes. Through biochemical studies, we demonstrate that the mutant E289G cotransporter is glycosylation-deficient, does not move beyond the endoplasmic reticulum or the early Golgi, and thus fails to reach the plasma membrane. We establish through co-immunoprecipitation experiments that both wild-type and mutant KCC3 with KCC2 results in the formation of hetero-dimers. We further demonstrate that formation of these hetero-dimers prevents the proper trafficking of the cotransporter to the plasma membrane, resulting in a significant decrease in cotransporter function. This effect is due to interaction between the K–Cl cotransporter isoforms, as this was not observed when KCC3-E289G was co-expressed with NKCC1. Our studies also reveal that the glutamic acid residue is essential to K–Cl cotransporter function, as the corresponding mutation in KCC2 also leads to an absence of function. Interestingly, mutation of this conserved glutamic acid residue in the Na⁺-dependent cation-chloride cotransporters had no effect on NKCC1 function in isosmotic conditions, but diminished cotransporter activity under hypertonicity. Together, our data show that the glutamic acid residue (E289) is essential for proper trafficking and function of KCCs and that expression of a non-functional but full-length K–Cl cotransporter might results in dominant-negative effects on other K–Cl cotransporters.     

Characterization of the interaction of the stress kinase SPAK with the Na+-K+-2Cl- cotransporter in the nervous system: evidence for a scaffolding role of the kinase

Activity of heterologously expressed NKCC1 was analyzed under basal and activated conditions in the presence and absence of binding of Ste20-related proline-alanine-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 activity-dependent 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 interact with the stress-related kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1)

Cells respond to stress stimuli by mounting specific responses. During osmotic and oxidative stress, cation chloride cotransporters, e.g. Na-K-2Cl and K-Cl cotransporters, are activated to maintain fluid/ion homeostasis. Here we report the interaction of the stress-related serine-threonine kinases Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1) with the cotransporters KCC3, NKCC1, and NKCC2 but not KCC1 and KCC4. The interaction was identified using yeast two-hybrid assays and confirmed via glutathione S-transferase pull-down experiments. Evidence for in vivo interaction was established by co-immunoprecipitation of SPAK from mouse brain with anti-NKCC1 antibody. The interacting region of both kinases comprises the last 100 amino acids of the protein. The SPAK/OSR1 binding motif on the cotransporters consists of nine residues, starting with an (R/K)FX(V/I) sequence followed by five additional residues that are essential for binding but for which no consensus was found. Immunohistochemical analysis of choroid plexus epithelium revealed co-expression of NKCC1 and SPAK on the apical membrane. In contrast, in choroid plexus epithelium from NKCC1 null mice, SPAK immunostaining was found in the cytoplasm. We conclude that several cation chloride co-transporters interact with SPAK and/or OSR1, and we hypothesize that this interaction might play a role during the initiation of the cellular stress response.     

Further optimization of the K-Cl cotransporter KCC2 antagonist ML077: development of a highly selective and more potent in vitro probe

Further chemical optimization of the MLSCN/MLPCN probe ML077 (KCC2 IC(50)=537 nM) proved to be challenging as the effort was characterized by steep SAR. However, a multi-dimensional iterative parallel synthesis approach proved productive. Herein we report the discovery and SAR of an improved novel antagonist (VU0463271) of the neuronal-specific potassium-chloride cotransporter 2 (KCC2), with an IC(50) of 61 nM and >100-fold selectivity versus the closely related Na-K-2Cl cotransporter 1 (NKCC1) and no activity in a larger panel of GPCRs, ion channels and transporters.     

SPAK and OSR1: STE20 kinases involved in the regulation of ion homoeostasis and volume control in mammalian cells

Since the discovery of an interaction between membrane transport proteins and the mammalian STE20 (sterile 20)-like kinases SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1), a significant body of work has been performed probing the molecular physiology of these two kinases. To date, the function of SPAK and OSR1 is probably the best known of all mammalian kinases of the STE20 family. As they regulate by direct phosphorylation key ion transport mechanisms involved in fluid and ion homoeostasis, SPAK and OSR1 constitute key end-of-pathway effectors. Their significance in such fundamental functions as ion homoeostasis and cell volume control is evidenced by the evolutionary pressure that resulted in the duplication of the OSR1 gene in higher vertebrates. This review examines the distribution of these two kinases in the animal kingdom and tissue expression within a single organism. It also describes the main molecular features of these two kinases with emphasis on the interacting domain located at their extreme C-terminus. A large portion of the present review is devoted to the extensive biochemical and physiological studies that have resulted in our current understanding of SPAK/OSR1 function. Finally, as our understanding is a work in progress, we also identify unresolved questions and controversies that warrant further investigation.     

Expression of the Na-K-2Cl cotransporter is developmentally regulated in postnatal rat brains: A possible mechanism underlying GABA's excitatory role in immature brain

An inhibitory neurotransmitter in mature brain, γ-aminobutyric acid (GABA) also appears to be excitatory early in development. The mechanisms underlying this shift are not well understood. In vitro studies have suggested that Na-K-Cl cotransport may have a role in modulating immature neuronal and oligodendrocyte responses to the neurotransmitter GABA. An in vivo developmental study would test this view. Therefore, we examined the expression of the BSC2 isoform of the Na-K-2Cl cotransporter in the postnatal developing rat brain. A comparison of sections from developing rat brains by in situ hybridization revealed a well-delineated temporal and spatial pattern of first increasing and then diminishing cotransporter expression. Na-K-2Cl mRNA expression in the cerebral cortex and hippocampus was highest in the first week of postnatal life and then diminished from postnatal day (PND) 14 to adult. Cotransporter signal in white-matter tracts of the cerebrum, cerebellum, peaked at PND 14. Expression was detected in cerebellar progenitor cells of the external granular layer, in internal granular layer cells at least as early as PND 7, and in Purkinje cells beginning at PND 14. Double-labeling immunofluorescence of brain sections with anti-BSC2 antibody and cell type-specific antibodies confirmed expression of the cotransporter gene product in neurons and oligodendrocytes in the white matter in a pattern similar to that determined by in situ hybridization. The temporal pattern of expression of the Na-K-2Cl cotransporter in the postnatal rat brain supports the hypothesis that the cotransporter is the mechanism of intracellular Cl⁻ accumulation in immature neurons and oligodendrocytes. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 781–795, 1997     

The Ste20 Kinases Ste20-related Proline-Alanine-rich Kinase and Oxidative-stress Response 1 Regulate NKCC1 Function in Sensory Neurons

NKCC1 is highly expressed in dorsal root ganglion neurons, where it is involved in gating sensory information. In a recent study, it was shown that peripheral nerve injury results in increased NKCC1 activity, not due to an increase in cotransporter expression, but to increased phosphorylation of the cotransporter (Pieraut, S., Matha, V., Sar, C., Hubert, T., Méchaly, I., Hilaire, C., Mersel, M., Delpire, E., Valmier, J., and Scamps, F. (2007) J. Neurosci. 27, 6751–6759). Our laboratory has also identified two Ste20-like kinases that bind and phosphorylate NKCC1: Ste20-related proline-alanine-rich kinase (SPAK) and oxidative-stress response 1 (OSR1). In this study, we show that both kinases are expressed at similar expression levels in spinal cord and dorsal root ganglion neurons, and that both kinases participate equally in the regulation of NKCC1. Using a novel fluorescence method to assay NKCC1 activity in single cells, we show a 50% reduction in NKCC1 activity in DRG neurons isolated from SPAK knockout mice, indicating that another kinase, e.g. OSR1, is present to phosphorylate and activate the cotransporter. Using a nociceptive dorsal root ganglion sensory neuronal cell line, which expresses the same cation-chloride cotransporters and kinases as native DRG neurons, and gene silencing via short hairpin RNA, we demonstrate a direct relationship between kinase expression and cotransporter activity. We show that inactivation of either kinase significantly affects NKCC1 activity, whereas inactivation of both kinases results in an additive effect. In summary, our study demonstrates redundancy of kinases in the regulation of NKCC1 in dorsal root ganglion neurons.The regulation of intracellular Cl^– in neurons is a critical determinant of inhibitory synaptic neurotransmission. Sensory or peripheral neurons express, in abundance, an inwardly poised Na⁺- and K⁺-dependent Cl^– transport mechanism, NKCC1,² whose activity drives the uphill accumulation of Cl^– ions (2, 3). High intracellular Cl^– concentration in dorsal root ganglion (DRG) neurons permits depolarizing γ-aminobutyric acid responses, which mediate presynaptic inhibition and filtration of sensory noise (4). Consequently, the knockout of NKCC1 exhibits a redistribution of internal Cl^– in DRG neurons and a pain perception phenotype (3, 5). In addition, peripheral inflammation or nerve injury results in increased NKCC1 function in primary afferents (6, 7). Using a phosphopeptide-specific NKCC1 antibody, it was recently shown that NKCC1 phosphorylation instead of expression level increased in DRG neurons upon nerve injury (1). This observation points to the importance of NKCC1 regulation in the neuropathic pain pathway.In recent work, our laboratory identified two Ste20-like kinases that directly bind to the cytosolic N-terminal tail of NKCC1 (8). The binding is a pre-requisite for NKCC1 phosphorylation and activation (9–13). The kinases, named SPAK (Ste20-related proline-alanine-rich kinase) and OSR1 (oxidative-stress response 1), share high homology in both their catalytic and regulatory domains. Expression of the two proteins has been examined in tissues using both Northern and Western blot analysis. These studies established that both kinases are widely expressed and that their expression pattern often overlaps (14–18). Co-expression of the kinases has been confirmed using Western blot analysis of well established cultured cell lines (17).Heterologous expression of SPAK and OSR1 in Xenopus laevis oocytes demonstrates that both kinases are able to bind and activate NKCC1 (10). Whether or not they equally participate to the regulation of NKCC1 in vivo under normal physiological conditions has not yet been determined. We chose to address the role of SPAK and OSR1 by using an established nociceptive dorsal root ganglion sensory neuronal cell line (19), as well as isolated mouse DRG neurons. We demonstrate that undifferentiated 50B11 cells express the same cotransporters and regulatory kinases as native DRG cells. To manipulate the kinases, we decreased their expression via shRNA knockdown and used a SPAK knockout mouse for the isolated DRG neurons. To measure NKCC1 activity, we used unidirectional ⁸⁶Rb-uptake with the established cell line and developed a novel fluorescence assay to assess cotransporter activity in single isolated DRG neurons. Our data demonstrate that both kinases are expressed to similar levels in both the cell line and isolated mouse DRG neurons, and that the kinases participate in concert to regulate NKCC1 function.