Disease relevance of SLC12A2

• RECENT FINDINGS: Previously, three genes (SLC12A2, the sodium-potassium-chloride co-transporter; KCNJ1, the ROMK potassium ion channel; ClC-Kb, the basolateral chloride ion channel) had been identified as causing antenatal and 'classic' Bartter syndrome [1].
• Inhibition of NKCC1 activity significantly reduces infarct volume and cerebral edema following cerebral focal ischemia [2].
• The mRNA levels of NKCC1, an inwardly directed Na(+), K(+)-2Cl(-) cotransporter that facilitates the accumulation of intracellular Cl(-), and of KCC2, an outwardly directed K(+)-Cl(-) cotransporter that extrudes Cl(-), were studied in surgically resected brain specimens from drug-resistant temporal lobe (TL) epilepsy (TLE) patients [3].
• In ganglion cells the upregulation of KCC2 by itself cannot explain the relatively fast switch in GABA's action; additional events, possibly KCC2's integration into the plasma membrane and downregulation of NKCC, might also contribute [4].
• With respect to pathophysiology, the focus is on ischemia and severe hypoxia where the roles of NHE1 and NKCC1 have been widely studied yet remain controversial and incompletely elucidated [5].

Psychiatry related information on SLC12A2

• In conclusion, alterations of NKCC control and NK-derived cytokine release in DAT could be involved in the neuroinflammatory mechanism related to the progression of neurodegeneration and dementia [6].
• The objective of the survey was to investigate psychological factors related to NKCC and NK cell populations in elderly women [7].
• Adjusting for covariates regarding lifestyle, multiple logistic regression analysis was applied; consequently, significant associations were found between reduced NKCC and high depressive symptoms and between increased NK cell numbers and life satisfaction [7].

High impact information on SLC12A2

• Evidence that the absolute dependence on ATP of the NKCC is the result of regulatory phosphorylation/dephosphorylation mechanisms is decribed [8].
• Although the NKCC has been studied for approximately 20 years, we are only beginning to frame the broad outlines of the structure, function, and regulation of this ubiquitous ion transport mechanism [8].
• The NKCC may play an important role in the cell cycle [8].
• NKCC1 expression level versus expression of the Cl(-)-extruding transporter (KCC2) in human and rat cortex showed that Cl(-) transport in perinatal human cortex is as immature as in the rat [9].
• The NKCC1 blocker bumetanide shifted E(Cl) negative in immature neurons, suppressed epileptiform activity in hippocampal slices in vitro and attenuated electrographic seizures in neonatal rats in vivo [9].

Chemical compound and disease context of SLC12A2

• These results suggest that KCC2, rather than NKCC1, mainly contributes to modulating excitability of the dorsal spinal cord neurons in the initial stage of formalin-evoked hyperalgesia [10].
• Administration of the NKCC1 inhibitor bumetanide (15 mg/kg, I.V.) significantly attenuated the contusion volume (464.03+/-23.62 mm(3)) and brain edema (water content: 79.12+/-0.28%) after traumatic brain injury [11].
• We concluded that regulatory volume increase in human glioma cells occurs through the uptake of Na(+), K(+), and Cl(-) by NKCC1 and is modulated by the presence of glutamine [12].
• We investigated whether addition of bismuth subcitrate (BSC, 4 x 120 mg) to a two week therapy scheme of omeprazole (OME, 2 x 40 mg)/amoxicillin (AMO, 4 x 500 mg) increases Helicobacter pylori eradication [13].
• Furosemide, a blocker of Na(+)/K(+)/2Cl(-) cotransporter (NKCC), is often used as a diuretic to improve edema, ascites, and pleural effusion of patients with cancers [14].

Biological context of SLC12A2

• In mammals, these proteins are encoded by three distinct but related genes: SLC12A1, SLC12A2, SLC12A3, which are located on different chromosomes [15].
• Hydropathy analysis of KCC1 indicates structural homology to NKCC, including 12 transmembrane domains, a large extracellular loop with potential N-linked glycosylation sites, and cytoplasmic N- and C-terminal regions [16].
• Changes in intracellular [Cl] ([Cl]i) appear to be involved in this regulation of NKCC1, which is directly phosphorylated by an unknown protein kinase in response to various secretagogues as well as reductions in [Cl]i and cell volume [17].
• These studies implicate the Na-K-2Cl cotransporter NKCC1 in hearing, salivation, pain perception, spermatogenesis, and the control of extracellular fluid volume [18].
• The co-expression of NKCC1 and Na,K-ATPase in highly specialized subpopulations of cochlear and vestibular fibrocytes provides further evidence for their role in recycling K+ leaked or effluxed through hair cells into perilymph back to endolymph, as postulated in current models of inner ear ion homeostasis [19].

Anatomical context of SLC12A2

• To date, two Na-K-Cl cotransporter isoforms have been identified: NKCC1, which is present in a wide variety of secretory epithelia and non-epithelial cells; and NKCC2, which is present exclusively in the kidney, in the epithelial cells of the thick ascending limb of Henle's loop and of the macula densa [17].
• In (32)P phosphorylation experiments with rectal gland tubules, we show that the R5 antibody signal is proportional to the amount of (32)P incorporated into NKCC1; and in experiments with NKCC1-transfected HEK-293 cells, we demonstrate that R5-detected phosphorylation directly mirrors functional activation [20].
• NKCC1 and NHE1 are abundantly expressed in the basolateral plasma membrane of secretory coil cells in rat, mouse, and human sweat glands [21].
• Immunoperoxidase labeling revealed abundant expression of NKCC1 and NHE1 in the basolateral domain of secretory coils of rat, mouse, and human sweat glands and low expression was found in the coiled part of the ducts [21].
• Immunohistochemical localization of the Na-K-Cl co-transporter (NKCC1) in the gerbil inner ear [19].

Associations of SLC12A2 with chemical compounds

• The relative expression level of the neuron-specific SLC12A5 and the Na(+)-K(+)-2Cl(-) cotransporter SLC12A2 appears to determine whether neurons respond to GABA with a depolarizing, excitatory response or with a hyperpolarizing, inhibitory response [22].
• We additionally show the near complete restoration of NKCC1 activity in the presence of the protein phosphatase type 1 inhibitor calyculin A, demonstrating that DNPASK inhibition results from an alteration in kinase/phosphatase dynamics rather than from a decrease in functional cotransporter expression [23].
• The molecular identity of the basolateral transporter(s) responsible for Cl(-) entry in ADPKD cells is unknown, although pharmacological studies suggest that a bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (NKCC/BSC) is involved [24].
• The high conservation of the regulatory threonine residues among NKCC1, NKCC2, and NCC family members, together with the fact that tissues from divergent vertebrate species exhibit similar R5-binding profiles, lends further support to the role of this regulatory locus in vivo [20].
• Our recent studies suggest that NKCC1 activation leads to high [K(+)](o(-)) induced astrocyte swelling and glutamate release, as well as neuronal Na(+) , and Cl(-) influx during acute excitotoxicity [2].

Enzymatic interactions of SLC12A2

• By immunocytochemistry, SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK) were colocalized with NKCC at the basolateral membrane [25].

Regulatory relationships of SLC12A2

• The results further indicate that the presence of functional CFTR enhances the expression of NKCC1 [26].
• We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif [27].
• These results suggest that C11 cells express an undetermined type of UTP-sensitive P2-purinoceptors or a unique P2Y-purinoceptor-triggered signaling cascade that leads to inhibition of NKCC1 [28].
• In nontransporting epithelia, inhibition of myosin light chain kinase (MLCK) prevents cell-shrinkage-induced activation of NKCC1 [29].

Other interactions of SLC12A2

• Staining of serial sections showed that cysts positive for NKCC1 also stained for CFTR [24].
• From the intrastrial space, K+ is taken up across the basolateral membrane of strial marginal cells via the Na+/2Cl-/K+ cotransporter SLC12A2 and the Na+/K+-ATPase ATP1A1/ATP1B2 [30].
• Immunostaining located NKCC1 in one-third of ADPKD cysts, with a pattern of basolateral reactivity [24].
• Overexpression of wild type PASK causes a small (sNKCC1 22 +/- 8% p < 0.05, hNKCC1 12 +/- 3% p < 0.01) but significant increase in shark and human cotransporter activity in HEK cells [23].
• KCC3 is 40 and 33% identical to two Caenorhabditis elegans K-Cl cotransporters and approximately 20% identical to other members of the cation-chloride cotransporter family (CCC), two Na-K-Cl cotransporters (NKCC1, NKCC2), and the Na-Cl cotransporter (NCC) [31].

Analytical, diagnostic and therapeutic context of SLC12A2

• Immunoblotting and immunoprecipitation detected NKCC1 at ~170 kDa in ADPKD cells and kidney extracts [24].
• These results support a model in which apical NKCC2 activity is matched to basolateral Cl exit through changes in [Cl]i. Reverse transcriptase-polymerase chain reaction of HEK-293 cell mRNA is positive with NKCC1 primers and negative with NKCC2 primers [32].
• Immunoelectron microscopy demonstrated abundant NKCC1 and NHE1 labeling of the basolateral plasma membrane of mouse sweat glands, with no labeling of the apical plasma membranes or intracellular structures [21].
• Using NKCC1 and NKCC2 isoform-specific PCR primers based on mouse and human sequences, only transcripts for NKCC1 were detected in the gerbil inner ear [19].
• In additional experiments, we immunoprecipitated the 355 kDa cross-linked complex from biotinylated membranes, then cleaved the cross-linking bonds and analyzed the resulting components of the NKCC1 oligomer by avidin blotting, silver staining, and 2D electrophoresis [33].

References