NKCC1 knockdown decreases neuron production through GABA(A)-regulated neural progenitor proliferation and delays dendrite development.

Signaling through GABA(A) receptors controls neural progenitor cell (NPC) development in vitro and is altered in schizophrenic and autistic individuals. However, the in vivo function of GABA(A) signaling on neural stem cell proliferation, and ultimately neurogenesis, remains unknown. To examine GABA(A) function in vivo, we electroporated plasmids encoding short-hairpin (sh) RNA against the Na-K-2Cl cotransporter NKCC1 (shNKCC1) in NPCs of the neonatal subventricular zone in mice to reduce GABA(A)-induced depolarization. Reduced GABA(A) depolarization identified by a loss of GABA(A)-induced calcium responses in most electroporated NPCs led to a 70% decrease in the number of proliferative Ki67(+) NPCs and a 60% reduction in newborn neuron density. Premature loss of GABA(A) depolarization in newborn neurons resulted in truncated dendritic arborization at the time of synaptic integration. However, by 6 weeks the dendritic tree had partially recovered and displayed a small, albeit significant, decrease in dendritic complexity but not total dendritic length. To further examine GABA(A) function on NPCs, we treated animals with a GABA(A) allosteric agonist, pentobarbital. Enhancement of GABA(A) activity in NPCs increased the number of proliferative NPCs by 60%. Combining shNKCC1 and pentobarbital prevented the shNKCC1 and the pentobarbital effects on NPC proliferation, suggesting that these manipulations affected NPCs through GABA(A) receptors. Thus, dysregulation in GABA(A) depolarizing activity delayed dendritic development and reduced NPC proliferation resulting in decreased neuronal density.

Protein aggregation in neurons following OGD: a role for Na+ and Ca2+ ionic dysregulation.

In this study, we investigated whether disruption of Na(+) and Ca(2+) homeostasis via activation of Na(+)-K(+)-Cl(-) cotransporter isoform 1 (NKCC1) and reversal of Na(+)/Ca(2+) exchange (NCX(rev)) affects protein aggregation and degradation following oxygen-glucose deprivation (OGD). Cultured cortical neurons were subjected to 2 h OGD and 1-24 h reoxygenation (REOX). Redistribution of ubiquitin and formation of ubiquitin-conjugated protein aggregates occurred in neurons as early as 2 h REOX. The protein aggregation progressed further by 8 h REOX. There was no significant recovery at 24 h REOX. Moreover, the proteasome activity in neurons was inhibited by 80-90% during 2-8 h REOX and recovered partially at 24 h REOX. Interestingly, pharmacological inhibition or genetic ablation of NKCC1 activity significantly decreased accumulation of ubiquitin-conjugated protein aggregates and improved proteasome activity. A similar protective effect was obtained by blocking NCX(rev) activity. Inhibition of NKCC1 activity also preserved intracellular ATP and Na(+) homeostasis during 0-24 h REOX. In a positive control study, disruption of endoplasmic reticulum Ca(2+) with thapsigargin triggered redistribution of free ubiquitin and protein aggregation. We conclude that overstimulation of NKCC1 and NCX(rev) following OGD/REOX partially contributes to protein aggregation and proteasome dysfunction as a result of ionic dysregulation.

Compensatory enhancement of intrinsic spiking upon NKCC1 disruption in neonatal hippocampus.

Depolarizing and excitatory GABA actions are thought to be important in cortical development. We show here that GABA has no excitatory action on CA3 pyramidal neurons in hippocampal slices from neonatal NKCC1(-/-) mice that lack the Na-K-2Cl cotransporter isoform 1. Strikingly, NKCC1(-/-) slices generated endogenous network events similar to giant depolarizing potentials (GDPs), but, unlike in wild-type slices, the GDPs were not facilitated by the GABA(A) agonist isoguvacine or blocked by the NKCC1 inhibitor bumetanide. The developmental upregulation of the K-Cl cotransporter 2 (KCC2) was unperturbed, whereas the pharmacologically isolated glutamatergic network activity and the intrinsic excitability of CA3 pyramidal neurons were enhanced in the NKCC1(-/-) hippocampus. Hence, developmental expression of KCC2, unsilencing of AMPA-type synapses, and early network events can take place in the absence of excitatory GABAergic signaling in the neonatal hippocampus. Furthermore, we show that genetic as well as pharmacologically induced loss of NKCC1-dependent excitatory actions of GABA results in a dramatic compensatory increase in the intrinsic excitability of glutamatergic neurons, pointing to powerful homeostatic regulation of neuronal activity in the developing hippocampal circuitry.

NKCC1-dependent GABAergic excitation drives synaptic network maturation during early hippocampal development.

A high intracellular chloride concentration in immature neurons leads to a depolarizing action of GABA that is thought to shape the developing neuronal network. We show that GABA-triggered depolarization and Ca2+ transients were attenuated in mice deficient for the Na-K-2Cl cotransporter NKCC1. Correlated Ca2+ transients and giant depolarizing potentials (GDPs) were drastically reduced and the maturation of the glutamatergic and GABAergic transmission in CA1 delayed. Brain morphology, synaptic density, and expression levels of certain developmental marker genes were unchanged. The expression of lynx1, a protein known to dampen network activity, was decreased. In mice deficient for the neuronal Cl(-)/HCO(3)(-) exchanger AE3, GDPs were also diminished. These data show that NKCC1-mediated Cl(-) accumulation contributes to GABAergic excitation and network activity during early postnatal development and thus facilitates the maturation of excitatory and inhibitory synapses.

Impaired cardiac contractility in mice lacking both the AE3 Cl-/HCO3- exchanger and the NKCC1 Na+-K+-2Cl- cotransporter: effects on Ca2+ handling and protein phosphatases.

To analyze the cardiac functions of AE3, we disrupted its gene (Slc4a3) in mice. Cl(-)/HCO3(-) exchange coupled with Na+-dependent acid extrusion can mediate pH-neutral Na+ uptake, potentially affecting Ca2+ handling via effects on Na+/Ca2+ exchange. AE3 null mice appeared normal, however, and AE3 ablation had no effect on ischemia-reperfusion injury in isolated hearts or cardiac performance in vivo. The NKCC1 Na+-K+-2Cl(-) cotransporter also mediates Na+ uptake, and loss of NKCC1 alone does not impair contractility. To further stress the AE3-deficient myocardium, we combined the AE3 and NKCC1 knock-outs. Double knock-outs had impaired contraction and relaxation both in vivo and in isolated ventricular myocytes. Ca2+ transients revealed an apparent increase in Ca2+ clearance in double null cells. This was unlikely to result from increased Ca2+ sequestration, since the ratio of phosphorylated phospholamban to total phospholamban was sharply reduced in all three mutant hearts. Instead, Na+/Ca2+ exchanger activity was found to be enhanced in double null cells. Systolic Ca2+ was unaltered, however, suggesting more direct effects on the contractile apparatus of double null myocytes. Expression of the catalytic subunit of protein phosphatase 1 was increased in all mutant hearts. There was also a dramatic reversal, between single null and double null hearts, in the carboxymethylation and localization to the myofibrillar fraction, of the catalytic subunit of protein phosphatase 2A, which corresponded to the loss of normal contractility in double null hearts. These data show that AE3 and NKCC1 affect Ca2+ handling, PLN regulation, and expression and localization of major cardiac phosphatases and that their combined loss impairs cardiac function.

Endoplasmic reticulum Ca2+ dysregulation and endoplasmic reticulum stress following in vitro neuronal ischemia: role of Na+-K+-Cl- cotransporter.

We investigated the role of Na(+)-K(+)-Cl(-) cotransporter (NKCC1) in conjunction with Na(+)/Ca(2+) exchanger (NCX) in disruption of endoplasmic reticulum (ER) Ca(2+) homeostasis and ER stress development in primary cortical neurons following in vitro ischemia. Oxygen-glucose deprivation (OGD) and reoxygenation (REOX) caused a rise in [Na(+)](cyt) which was accompanied by an elevation in [Ca(2+)](cyt). Inhibition of NKCC1 with its potent inhibitor bumetanide abolished the OGD/REOX-induced rise in [Na(+)](cyt) and [Ca(2+)](cyt). Moreover, OGD significantly increased Ca(2+)(ER) accumulation. Following REOX, a biphasic change in Ca(2+)(ER) occurred with an initial release of Ca(2+)(ER) which was sensitive to inositol 1,4,5-trisphosphate receptor (IP(3)R) inhibition and a subsequent refilling of Ca(2+)(ER) stores. Inhibition of NKCC1 activity with its inhibitor or genetic ablation prevented the release of Ca(2+)(ER). A similar result was obtained with inhibition of reversed mode operation of NCX (NCX(rev)). OGD/REOX also triggered a transient increase of glucose regulated protein 78 (GRP78), phospho-form of the alpha subunit of eukaryotic initiation factor 2 (p-eIF2alpha), and cleaved caspase 12 proteins. Pre-treatment of neurons with NKCC1 inhibitor bumetanide inhibited upregulation of GRP78 and attenuated the level of cleaved caspase 12 and p-eIF2alpha. Inhibition of NKCC1 reduced cytochrome C release and neuronal death. Taken together, these results suggest that NKCC1 and NCX(rev) may be involved in ischemic cell damage in part via disrupting ER Ca(2+) homeostasis and ER function.

Excitatory actions of GABA in the suprachiasmatic nucleus.

Neurons in the suprachiasmatic nucleus (SCN) are responsible for the generation of circadian oscillations, and understanding how these neurons communicate to form a functional circuit is a critical issue. The neurotransmitter GABA and its receptors are widely expressed in the SCN where they mediate cell-to-cell communication. Previous studies have raised the possibility that GABA can function as an excitatory transmitter in adult SCN neurons during the day, but this work is controversial. In the present study, we first tested the hypothesis that GABA can evoke excitatory responses during certain phases of the daily cycle by broadly sampling how SCN neurons respond to GABA using extracellular single-unit recording and gramicidin-perforated-patch recording techniques. We found that, although GABA inhibits most SCN neurons, some level of GABA-mediated excitation was present in both dorsal and ventral regions of the SCN, regardless of the time of day. These GABA-evoked excitatory responses were most common during the night in the dorsal SCN region. The Na(+)-K(+)-2Cl(-) cotransporter (NKCC) inhibitor, bumetanide, prevented these excitatory responses. In individual neurons, the application of bumetanide was sufficient to change GABA-evoked excitation to inhibition. Calcium-imaging experiments also indicated that GABA-elicited calcium transients in SCN cells are highly dependent on the NKCC isoform 1 (NKCC1). Finally, Western blot analysis indicated that NKCC1 expression in the dorsal SCN is higher in the night. Together, this work indicates that GABA can play an excitatory role in communication between adult SCN neurons and that this excitation is critically dependent on NKCC1.

GABA regulates excitatory synapse formation in the neocortex via NMDA receptor activation.

The development of a balance between excitatory and inhibitory synapses is a critical process in the generation and maturation of functional circuits. Accumulating evidence suggests that neuronal activity plays an important role in achieving such a balance in the developing cortex, but the mechanism that regulates this process is unknown. During development, GABA, the primary inhibitory neurotransmitter in adults, excites neurons as a result of high expression of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1). Using NKCC1 RNA interference knockdown in vivo, we show that GABA-induced depolarization is necessary for proper excitatory synapse formation and dendritic development of newborn cortical neurons. Blocking NKCC1 with the diuretic bumetanide during development leads to similar persistent changes in cortical circuitry in the adult. Interestingly, expression of a voltage-independent NMDA receptor rescues the failure of NKCC1 knockdown neurons to develop excitatory AMPA transmission, indicating that GABA depolarization cooperates with NMDA receptor activation to regulate excitatory synapse formation. Our study identifies an essential role for GABA in the synaptic integration of newborn cortical neurons and suggests an activity-dependent mechanism for achieving the balance between excitation and inhibition in the developing cortex.

Roles of basolateral solute uptake via NKCC1 and of myosin II in vasopressin-induced cell swelling in inner medullary collecting duct.

Collecting duct cells swell when exposed to arginine vasopressin (AVP) in the presence of a transepithelial osmolality gradient. We investigated the mechanisms of AVP-induced cell swelling in isolated, perfused rat inner medullary collecting ducts (IMCDs) using quantitative video microscopy and fluorescence-based measurements of transepithelial water transport. We tested the roles of transepithelial water flow, basolateral solute entry, and the cytoskeleton (actomyosin). When a transepithelial osmolality gradient was imposed by addition of NaCl to the bath, AVP significantly increased both water flux and cell height. When the osmolality gradient was imposed by addition of mannitol, AVP increased water flux but not cell height, suggesting that AVP-induced cell swelling requires a NaCl gradient and is not merely dependent on the associated water flux. Bumetanide (Na-K-2Cl cotransporter inhibitor) added to the bath markedly diminished the AVP-induced cell height increase. AVP-induced cell swelling was absent in IMCDs from NKCC1-knockout mice. In rat IMCDs, replacement of Na, K, or Cl in the peritubular bath caused significant cell shrinkage, consistent with a basolateral solute transport pathway dependent on all three ions. Immunocytochemistry using an antibody to NKCC1 confirmed basolateral expression in IMCD cells. The conventional nonmuscle myosin II inhibitor blebbistatin also diminished the AVP-induced cell height increase and cell shape change, consistent with a role for the actin cytoskeleton and myosin II. We conclude that the AVP-induced cell height increase is dependent on basolateral solute uptake via NKCC1 and changes in actin organization via myosin II, but is not dependent specifically on increased apical water entry.

NKCC1 phosphorylation stimulates neurite growth of injured adult sensory neurons.

Peripheral nerve section promotes regenerative, elongated neuritic growth of adult sensory neurons. Although the role of chloride homeostasis, through the regulation of ionotropic GABA receptors, in the growth status of immature neurons in the CNS begins to emerge, nothing is known of its role in the regenerative growth of injured adult neurons. To analyze the intracellular Cl- variation after a sciatic nerve section in vivo, gramicidin perforated-patch recordings were used to study muscimol-induced currents in mice dorsal root ganglion neurons isolated from control and axotomized neurons. We show that the reversal potential of muscimol-induced current, E(GABA-A), was shifted toward depolarized potentials in axotomized neurons. This was attributable to Cl- influx because removal of extracellular Cl- prevented this shift. Application of bumetanide, an inhibitor of NKCC1 cotransporter and E(GABA-A) recordings in sensory neurons from NKCC1-/- mice, identified NKCC1 as being responsible for the increase in intracellular Cl- in axotomized neurons. In addition, we demonstrate with a phospho-NKCC1 antibody that nerve injury induces an increase in the phosphorylated form of NKCC1 in dorsal root ganglia that could account for intracellular Cl- accumulation. Time-lapse recordings of the neuritic growth of axotomized neurons show a faster growth velocity compared with control. Bumetanide, the intrathecal injection of NKCC1 small interfering RNA, and the use of NKCC1-/- mice demonstrated that NKCC1 is involved in determining the velocity of elongated growth of axotomized neurons. Our results clearly show that NKCC1-induced increase in intracellular chloride concentration is a major event accompanying peripheral nerve regeneration.

NKCC1 does not accumulate chloride in developing retinal neurons.

GABA excites immature neurons due to their relatively high intracellular chloride concentration. This initial high concentration is commonly attributed to the ubiquitous chloride cotransporter NKCC1, which uses a sodium gradient to accumulate chloride. Here we tested this hypothesis in immature retinal amacrine and ganglion cells. Western blotting detected NKCC1 at birth and its expression first increased, then decreased to the adult level. Immunocytochemistry confirmed this early expression of NKCC1 and localized it to all nuclear layers. In the ganglion cell layer, staining peaked at P4 and then decreased with age, becoming undetectable in adult. In comparison, KCC2, the chloride extruder, steadily increased with age localizing primarily to the synaptic layers. For functional tests, we used calcium imaging with fura-2 and chloride imaging with 6-methoxy-N-ethylquinolinium iodide. If NKCC1 accumulates chloride in ganglion and amacrine cells, deleting or blocking it should abolish the GABA-evoked calcium rise. However, at P0-5 GABA consistently evoked a calcium rise that was not abolished in the NKCC1-null retinas, nor by applying high concentrations of bumetanide (NKCC blocker) for long periods. Furthermore, intracellular chloride concentration in amacrine and ganglion cells of the NKCC1-null retinas was approximately 30 mM, same as in wild type at this age. This concentration was not lowered by applying bumetanide or by decreasing extracellular sodium concentration. Costaining for NKCC1 and cellular markers suggested that at P3, NKCC1 is restricted to Müller cells. We conclude that NKCC1 does not serve to accumulate chloride in immature retinal neurons, but it may enable Müller cells to buffer extracellular chloride.

Renal function in mice with targeted disruption of the A isoform of the Na-K-2Cl co-transporter.

Three different full-length splice isoforms of the Na-K-2Cl co-transporter (NKCC2/BSC1) are expressed along the thick ascending limb of Henle (TAL), designated NKCC2A, NKCC2B, and NKCC2F. NKCC2F is expressed in the medullary, NKCC2B mainly in the cortical, and NKCC2A in medullary and cortical portions of the TAL. NKCC2B and NKCC2A were shown to be coexpressed in the macula densa (MD) segment of the mouse TAL. The functional consequences of the existence of three different isoforms of NKCC2 are unclear. For studying the specific role of NKCC2A in kidney function, NKCC2A-/- mice were generated by homologous recombination. NKCC2A-/- mice were viable and showed no gross abnormalities. Ambient urine osmolarity was reduced significantly in NKCC2A-/- compared with wild-type mice, but water deprivation elevated urine osmolarity to similar levels in both genotypes. Baseline plasma renin concentration and the effects of a high- and a low-salt diet on plasma renin concentration were similar in NKCC2A+/+ and -/- mice. However, suppression of renin secretion by acute intravenous saline loading (5% of body weight), a measure of MD-dependent inhibition of renin secretion, was reduced markedly in NKCC2A-/- mice compared with wild-type mice. Cl and water absorption along microperfused loops of Henle of NKCC2A-/- mice were unchanged at normal flow rates but significantly reduced at supranormal flow. Tubuloglomerular feedback function curve as determined by stop flow pressure measurements was left-shifted in NKCC2A-/- compared with wild-type mice, with maximum responses being significantly diminished. In summary, NKCC2A activity seems to be required for MD salt sensing in the high Cl concentration range. Coexpression of both high- and low-affinity isoforms of NKCC2 may permit transport and Cl-dependent tubuloglomerular feedback regulation to occur over a wider Cl concentration range.

Macula densa control of renin secretion and preglomerular resistance in mice with selective deletion of the B isoform of the Na,K,2Cl co-transporter.

Na,K,2Cl co-transporter (NKCC2), the primary NaCl uptake pathway in the thick ascending limb of Henle, is expressed in three different full-length splice variants, called NKCC2F, NKCC2A, and NKCC2B. These variants, derived by differential splicing of the variable exon 4, show a distinct distribution pattern along the loop of Henle, but the functional significance of this organization is unclear. By introduction of premature stop codons into exon 4B, specific for the B isoform, mice with an exclusive NKCC2B deficiency were generated. Relative expression levels and distribution patterns of NKCC2A and NKCC2F were not altered in the NKCC2B-deficient mice. NKCC2B-deficient mice did not display a salt-losing phenotype; basal plasma renin and aldosterone levels were not different from those of wild-type mice. Ambient urine osmolarities, however, were slightly but significantly reduced. Distal Cl concentration was significantly elevated and loop of Henle Cl absorption was reduced in microperfused superficial loops of Henle of NKCC2B-deficient mice. Because of the presence of NKCC2A in the macula densa, maximum tubuloglomerular feedback responses were normal, but tubuloglomerular feedback function curves were right-shifted, indicating reduced sensitivity in the subnormal flow range. Plasma renin concentration in NKCC2B-deficient mice was reduced under conditions of salt loading compared with that in wild-type mice. This study shows the feasibility of generating mice with specific deletions of single splice variants. The mild phenotype of mice that are deficient in the B isoform of NKCC2 indicates a limited role for NKCC2B for overall salt retrieval. Nevertheless, the high-affinity NKCC2B contributes to salt absorption and macula densa function in the low NaCl concentration range.

Multisensory plasticity in congenitally deaf mice: how are cortical areas functionally specified?

The neocortex of congenitally deaf mice was examined using electrophysiological recording techniques combined with cortical myeloarchitecture. Our results indicate that relative activity patterns across sensory systems during development contribute to modality assignment of cortical fields as well as the size of cortical fields. In congenitally deaf mice, "auditory cortex" contained neurons that responded to somatosensory, visual, or both somatosensory and visual stimulation; the primary visual area contained a larger proportion of neurons that responded to somatosensory stimulation than in normal animals, and the primary visual area had significantly increased in size. Thus, cortical architecture and functional specification were de-correlated. When results are considered in the light of molecular studies and studies in which peripheral activity is altered in development, it becomes clear that similar types of changes to the neocortex, such as alterations in cortical field size, can be achieved in more than one way in the developing and evolving neocortex.

Neuronal chloride accumulation in olfactory epithelium of mice lacking NKCC1.

When stimulated with odorants, olfactory receptor neurons (ORNs) produce a depolarizing receptor current. In isolated ORNs, much of this current is caused by an efflux of Cl-. This implies that the neurons have one or more mechanisms for accumulating cytoplasmic Cl- at rest. Whether odors activate an efflux of Cl- in intact olfactory epithelium, where the ionic environment is poorly characterized, has not been previously determined. In mouse olfactory epithelium, we found that >80% of the summated electrical response to odors is blocked by niflumic acid or flufenamic acid, each of which inhibits Ca2+-activated Cl- channels in ORNs. This indicates that ORNs accumulate Cl- in situ. Recent evidence has shown that NKCC1, a Na+-K+-2Cl- cotransporter, contributes to Cl- accumulation in mammalian ORNs. However, we find that the epithelial response to odors is only reduced by 39% in mice carrying a null mutation in Nkcc1. As in the wild-type, most of the response is blocked by niflumic acid or flufenamic acid, indicating that the underlying current is carried by Cl-. We conclude that ORNs effectively accumulate Cl- in situ even in the absence of NKCC1. The Cl- -transport mechanism underlying this accumulation has not yet been identified.

Expression and function of chloride transporters during development of inhibitory neurotransmission in the auditory brainstem.

Glycine and GABA, the dominant inhibitory neurotransmitters in the CNS, assume a depolarizing role in early development, leading to increased cytoplasmic Ca2+ levels and action potentials. The effect is thought to be of some significance for maturation. The depolarization is caused by Cl- efflux, and chloride transporters contribute to the phenomenon by raising the intracellular Cl- concentration ([Cl-]i) above equilibrium, thereby generating an outward-directed electrochemical gradient for Cl-. In mature neurons, the [Cl-]i is reduced below equilibrium, thus rendering glycine activity hyperpolarizing. Here, we investigated the temporal expression of the K-Cl cotransporter KCC2 and the Na-K-Cl cotransporter NKCC1 in the lateral superior olive (LSO) of rats and mice. The two cation cotransporters normally extrude and accumulate Cl-, respectively. As evidenced by several methods, KCC2 mRNA was present in LSO neurons during both the depolarizing and hyperpolarizing periods. Western blots confirmed a constant level of KCC2 in the brainstem, and immunohistochemistry showed that the protein is diffusely distributed within neonatal LSO neurons, becoming integrated into the plasma membrane only with increasing age. The glycine reversal potential in KCC2 knock-out mice differed significantly from that determined in wild-type controls at postnatal day 12 (P12) but not at P3, demonstrating that KCC2 is not active in neonates, despite its early presence. NKCC1 mRNA was not detected during the depolarizing phase in the LSO, implying that this transporter does not contribute to the high [Cl-]i. Our results reveal major differences in the development of [Cl-]i regulation mechanisms seen in brainstem versus forebrain regions.

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.