Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.

Xu, Jian; Lytle, Christian; Zhu, Tracey T.; Payne, John A.; Benz, Edward J.; Forbush, Bliss

doi:10.1073/pnas.91.6.2201

Cited by 374 publications

(294 citation statements)

References 23 publications

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“…NKCC1 is activated under conditions in which the cell is depleted of cell chloride, and we have routinely used low Cl Ϫ preincubation to stimulate transport in HEK-293 cells (5,19,20). As illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Darman

Forbush

2002

Journal of Biological Chemistry

215

217

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The secretory Na-K-Cl cotransporter NKCC1 is activated by secretagogues through a phosphorylationdependent mechanism. We found a phosphorylation stoichiometry of 3.0 ؎ 0.4 phosphorylated residues/ NKCC1 protein harvested from shark rectal gland tubules maximally stimulated with forskolin and calyculin A, showing that at least three sites on the cotransporter are phosphorylated upon stimulation. Three phosphoacceptor sites were identified in the N-terminal domain of the protein (at Thr 184 , Thr 189 , and Thr 202 ) using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry to analyze tryptic fragments of the radiolabeled cotransporter. None of these residues occurs in the context of strong consensus sites for known Ser/Thr kinases. The threonines and the surrounding amino acids are highly conserved between NKCC1 and NKCC2, and similarities are also present in the Na-Cl cotransporter NCC (or TSC). This strongly suggests that the phosphoregulatory mechanism is conserved among isoforms. The secretory Na-K-Cl cotransporter NKCC1 is a major pathway for net influx of Cl Ϫ in many cells (1, 2). Along with the absorptive Na-K-Cl cotransporter NKCC2 and the thiazidesensitive Na-Cl transporter NCC (or TSC), NKCC1 belongs to the sodium-coupled branch of the cation chloride cotransporter family; this family also includes the K-Cl transporters (KCCs) (3) and two other major branches of putative transporters, including human CIP (4) and SLC12-8 (GenBank TM /EBI accession number AAK94307), whose transport functions are unknown. These proteins are all predicted to have 12 transmembrane-spanning domains that are responsible for ion transport properties (5) and large cytoplasmic N and C termini that are candidates for regulatory domains. NKCC1 is expressed in many cell types and is involved in the regulation of both cell volume and intracellular Cl Ϫ concentration (2, 6). This isoform is also a major component of the basolateral membrane of secretory cells, mediating Cl Ϫ influx, the first step in the transepithelial movement of Cl Ϫ . In contrast, the NKCC2 isoform is limited to the apical membranes of absorptive epithelia such as the thick ascending limb of Henle's loop, where it is a major determinant of NaCl reabsorption from the tubular fluid.In order that the processes of regulatory volume increase, secretion, and absorption may be tightly controlled, the Na-K-Cl cotransporter is subject to strict regulation. The cotransporter is inactive in the basolateral membranes of secretory cells until the application of secretagogues or cell shrinkage causes a phosphorylation-dependent activation of the cotransporter (7-10). Studies on shark rectal gland secretory epithelia have shown an increase in phosphorylation at serines and threonines in response to forskolin or hypertonic stress, and the N terminus of NKCC1 has been shown to be the locus of at least some of the phosphoregulatory sites (7, 11). Although, in many cells, PKA 1 agonists effect an increase in Na-K-Cl co...

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Section: Resultsmentioning

confidence: 99%

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Darman

Forbush

2002

Journal of Biological Chemistry

215

217

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“…During embryonic development, NKCC1 is highly expressed in the membrane, and as a result GABA acts as an excitatory neurotransmitter (40,41). However, during the first few weeks of postnatal development, there is a significant increase in KCC2 expression that results in a hyperpolarizing shift in E GABA (4,5).…”

Section: Discussionmentioning

confidence: 99%

Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons

Ivakine

Acton

Mahadevan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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KCC2 is a neuron-specific K + -Cl − cotransporter that is essential for Cl − homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl − extrusion, resulting in decreased synaptic inhibition in hippocampal neurons. that makes fast GABA A Rmediated synaptic inhibition possible is maintained by the neuronspecific K + -Cl − cotransporter KCC2 (4), a member of the cationchloride cotransporter SLC12 gene family (5). KCC2 is essential for survival, as KCC2 knockout mice die immediately at birth due to respiratory failure (6). In the adult nervous system, decreased KCC2 expression correlates with neuropathic pain, spasticity following spinal cord injury, and epileptic seizures (5, 7-11).Based on the critical importance of KCC2 in the brain, an understanding of the mechanisms that promote and maintain KCC2 expression and efficacy is essential for designing therapeutic strategies to treat neurological disorders characterized by KCC2 dysfunction and the subsequent loss of inhibitory synaptic transmission. A major limitation in the development of these strategies is a lack of understanding regarding the cellular mechanisms regulating KCC2. In particular, KCC2 interacting proteins required for KCC2 transport and function in the mature CNS have not been identified, which represents a large gap in our fundamental knowledge regarding neuronal Cl − regulation and inhibitory synaptic transmission.Here, we identify neuropilin and tolloid like-2 (Neto2) as a KCC2 interacting protein in vivo and demonstrate that this interaction is required for normal neuronal Cl − homeostasis. Neto2 is a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein abundantly expressed in neurons (12), which is important for proper neurological function (13). CUB domains are evolutionarily conserved protein domains (14) that participate in protein-protein interactions (15, 16). In the present study, we performed an unbiased proteomic screen and discovered that Neto2 interacts with KCC2. We elucidated the role of this association and characterized its importance in the normal neurophysiological function of KCC2. Because Neto proteins were previously identified as auxiliary subunits of ionotropic glutamate receptors, including kainate and NMDA receptors (17-21), this study further extends the role of the Neto proteins to inhibitory synapses. ResultsNeto2 and KCC2 Interact in Vivo. Recent reports have demonstrated that Neto2 and its homologous protein Neto1 have important functions at excitatory synapses. Neto1 interacts with both the N-methyl D-aspartate receptor (NMDAR) and kainate receptor comp...

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“…Two isoforms of the Na-K-2Cl co-transporter (NKCC) protein are currently known (3). NKCC1, the basolateral isoform, is by far the most widely distributed (4,5). By contrast, NKCC2 is exclusively targeted to the apical cell surface and is exclusively found in the kidney (4,6).…”

Section: Cation-cl Co-transporters (Cccs)mentioning

confidence: 99%

“…NKCC1, the basolateral isoform, is by far the most widely distributed (4,5). By contrast, NKCC2 is exclusively targeted to the apical cell surface and is exclusively found in the kidney (4,6). Apical sodium entry in the thick ascending limb of the loop of Henle is mediated by NKCC2.…”

Section: Cation-cl Co-transporters (Cccs)mentioning

confidence: 99%

NKCC2 Surface Expression in Mammalian Cells

Benziane

Demaretz

Defontaine

et al. 2007

Journal of Biological Chemistry

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Apical bumetanide-sensitive Na؉ -K ؉ -2Cl ؊ co-transporter, termed NKCC2, is the major salt transport pathway in kidney thick ascending limb. NKCC2 surface expression is subject to regulation by intracellular protein trafficking. However, the protein partners involved in the intracellular trafficking of NKCC2 remain unknown. Moreover, studies aimed at understanding the post-translational regulation of NKCC2 have been hampered by the difficulty to express NKCC2 protein in mammalian cells. Here we were able to express NKCC2 protein in renal epithelial cells by tagging its N-terminal domain. To gain insights into the regulation of NKCC2 trafficking, we screened for interaction partners of NKCC2 with the yeast two-hybrid system, using the C-terminal tail of NKCC2 as bait. Aldolase B was identified as a dominant and novel interacting protein. Real time PCR on renal microdissected tubules demonstrated the expression of aldolase B in the thick ascending limb. Co-immunoprecipitation and co-immunolocalization experiments confirmed NKCC2-aldolase interaction in renal cells. Biotinylation assays showed that aldolase co-expression reduces NKCC2 surface expression. In the presence of aldolase substrate, fructose 1,6-bisphosphate, aldolase binding was disrupted, and aldolase co-expression had no further effect on the cell surface level of NKCC2. Finally, functional studies demonstrated that aldolase-induced down-regulation of NKCC2 at the plasma membrane was associated with a decrease in its transport activity. In summary, we identified aldolase B as a novel NKCC2 binding partner that plays a key role in the modulation of NKCC2 surface expression, thereby revealing a new regulatory mechanism governing the co-transporter intracellular trafficking. Furthermore, NKCC2 protein expression in mammalian cells and its regulation by protein-protein interactions, described here, may open new and important avenues in studying the cell biology and post-transcriptional regulation of the co-transporter.

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Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.

Cited by 374 publications

References 23 publications

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons

NKCC2 Surface Expression in Mammalian Cells

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Molecular cloning and functional expression of the bumetanide-sensitive Na-K-Cl cotransporter.

Cited by 374 publications

References 23 publications

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

A Regulatory Locus of Phosphorylation in the N Terminus of the Na-K-Cl Cotransporter, NKCC1

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

NKCC2 Surface Expression in Mammalian Cells

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Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons