Cation Chloride Cotransporter NKCC1 Operates through a Rocking-Bundle Mechanism.

The sodium, potassium, and chloride cotransporter 1 (NKCC1) plays a key role in tightly regulating ion shuttling across cell membranes. Lately, its aberrant expression and function have been linked to numerous neurological disorders and cancers, making it a novel and highly promising pharmacological target for therapeutic interventions. A better understanding of how NKCC1 dynamically operates would therefore have broad implications for ongoing efforts toward its exploitation as a therapeutic target through its modulation. Based on recent structural data on NKCC1, we reveal conformational motions that are key to its function. Using extensive deep-learning-guided atomistic simulations of NKCC1 models embedded into the membrane, we captured complex dynamical transitions between alternate open conformations of the inner and outer vestibules of the cotransporter and demonstrated that NKCC1 has water-permeable states. We found that these previously undefined conformational transitions occur via a rocking-bundle mechanism characterized by the cooperative angular motion of transmembrane helices (TM) 4 and 9, with the contribution of the extracellular tip of TM 10. We found these motions to be critical in modulating ion transportation and in regulating NKCC1's water transporting capabilities. Specifically, we identified interhelical dynamical contacts between TM 10 and TM 6, which we functionally validated through mutagenesis experiments of 4 new targeted NKCC1 mutants. We conclude showing that those 4 residues are highly conserved in most Na+-dependent cation chloride cotransporters (CCCs), which highlights their critical mechanistic implications, opening the way to new strategies for NKCC1's function modulation and thus to potential drug action on selected CCCs.

Cryo-EM structure of the human NKCC1 transporter reveals mechanisms of ion coupling and specificity.

The sodium-potassium-chloride transporter NKCC1 of the SLC12 family performs Na+ -dependent Cl- - and K+ -ion uptake across plasma membranes. NKCC1 is important for regulating cell volume, hearing, blood pressure, and regulation of hyperpolarizing GABAergic and glycinergic signaling in the central nervous system. Here, we present a 2.6 Å resolution cryo-electron microscopy structure of human NKCC1 in the substrate-loaded (Na+ , K+ , and 2 Cl- ) and occluded, inward-facing state that has also been observed for the SLC6-type transporters MhsT and LeuT. Cl- binding at the Cl1 site together with the nearby K+ ion provides a crucial bridge between the LeuT-fold scaffold and bundle domains. Cl- -ion binding at the Cl2 site seems to undertake a structural role similar to conserved glutamate of SLC6 transporters and may allow for Cl- -sensitive regulation of transport. Supported by functional studies in mammalian cells and computational simulations, we describe a putative Na+ release pathway along transmembrane helix 5 coupled to the Cl2 site. The results provide insight into the structure-function relationship of NKCC1 with broader implications for other SLC12 family members.

Further confirmation of the association of SLC12A2 with non-syndromic autosomal-dominant hearing impairment.

Congenital hearing impairment (HI) is genetically heterogeneous making its genetic diagnosis challenging. Investigation of novel HI genes and variants will enhance our understanding of the molecular mechanisms and to aid genetic diagnosis. We performed exome sequencing and analysis using DNA samples from affected members of two large families from Ghana and Pakistan, segregating autosomal-dominant (AD) non-syndromic HI (NSHI). Using in silico approaches, we modeled and evaluated the effect of the likely pathogenic variants on protein structure and function. We identified two likely pathogenic variants in SLC12A2, c.2935G>A:p.(E979K) and c.2939A>T:p.(E980V), which segregate with NSHI in a Ghanaian and Pakistani family, respectively. SLC12A2 encodes an ion transporter crucial in the homeostasis of the inner ear endolymph and has recently been reported to be implicated in syndromic and non-syndromic HI. Both variants were mapped to alternatively spliced exon 21 of the SLC12A2 gene. Exon 21 encodes for 17 residues in the cytoplasmatic tail of SLC12A2, is highly conserved between species, and preferentially expressed in cochlear tissues. A review of previous studies and our current data showed that out of ten families with either AD non-syndromic or syndromic HI, eight (80%) had variants within the 17 amino acid residue region of exon 21 (48 bp), suggesting that this alternate domain is critical to the transporter activity in the inner ear. The genotypic spectrum of SLC12A2 was expanded and the involvement of SLC12A2 in ADNSHI was confirmed. These results also demonstrate the role that SLC12A2 plays in ADNSHI in diverse populations including sub-Saharan Africans.

High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters.

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl- with K+ and/or Na+ in an electroneutral manner. They play important roles in myriad fundamental physiological processes--from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis--and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na+/K+/Cl- cotransporter NKCC1 and the K+/Cl- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.

The effects of temperature on the biophysical properties of optic nerve F-fibres.

In multiple sclerosis, exacerbation of symptoms with rising body temperature is associated with impulse conduction failure. The mechanism is not fully understood. Remarkably, normal optic nerve axons also show temperature dependent effects, with a fall in excitability with warming. Here we show two properties of optic nerve axons, accommodation and inward rectification (Ih), respond to temperature changes in a manner consistent with a temperature dependent membrane potential. As we could find no evidence for the functional expression of KV7.2 in the axons, using the K+ channel blocker tetraethylammonium ions, we suggest this may explain the membrane potential lability. In order to understand how the axonal membrane potential may show temperature dependence, we have developed a hypothesis involving the electroneutral movement of Na+ ions across the axon membrane, that increases with increasing temperature with an appropriate Q10. Part, but probably not all, of the electroneutral Na+ movement is eliminated by removing extracellular Cl- or exposure to bumetanide, consistent with the involvement of the transporter NKCC1. Numerical simulation suggests a change in membrane potential of - 15-20 mV mimics altering temperature between room and physiological in the largest axons.

Variants encoding a restricted carboxy-terminal domain of SLC12A2 cause hereditary hearing loss in humans.

Hereditary hearing loss is challenging to diagnose because of the heterogeneity of the causative genes. Further, some genes involved in hereditary hearing loss have yet to be identified. Using whole-exome analysis of three families with congenital, severe-to-profound hearing loss, we identified a missense variant of SLC12A2 in five affected members of one family showing a dominant inheritance mode, along with de novo splice-site and missense variants of SLC12A2 in two sporadic cases, as promising candidates associated with hearing loss. Furthermore, we detected another de novo missense variant of SLC12A2 in a sporadic case. SLC12A2 encodes Na+, K+, 2Cl- cotransporter (NKCC) 1 and plays critical roles in the homeostasis of K+-enriched endolymph. Slc12a2-deficient mice have congenital, profound deafness; however, no human variant of SLC12A2 has been reported as associated with hearing loss. All identified SLC12A2 variants mapped to exon 21 or its 3'-splice site. In vitro analysis indicated that the splice-site variant generates an exon 21-skipped SLC12A2 mRNA transcript expressed at much lower levels than the exon 21-included transcript in the cochlea, suggesting a tissue-specific role for the exon 21-encoded region in the carboy-terminal domain. In vitro functional analysis demonstrated that Cl- influx was significantly decreased in all SLC12A2 variants studied. Immunohistochemistry revealed that SLC12A2 is located on the plasma membrane of several types of cells in the cochlea, including the strial marginal cells, which are critical for endolymph homeostasis. Overall, this study suggests that variants affecting exon 21 of the SLC12A2 transcript are responsible for hereditary hearing loss in humans.

Structure of the human cation-chloride cotransporter NKCC1 determined by single-particle electron cryo-microscopy.

The secondary active cation-chloride cotransporters (CCCs) utilize the existing Na+ and/or K+ gradients to move Cl- into or out of cells. NKCC1 is an intensively studied member of the CCC family and plays fundamental roles in regulating trans-epithelial ion movement, cell volume, chloride homeostasis and neuronal excitability. Here, we report a cryo-EM structure of human NKCC1 captured in a partially loaded, inward-open state. NKCC1 assembles into a dimer, with the first ten transmembrane (TM) helices harboring the transport core and TM11-TM12 helices lining the dimer interface. TM1 and TM6 helices break α-helical geometry halfway across the lipid bilayer where ion binding sites are organized around these discontinuous regions. NKCC1 may harbor multiple extracellular entryways and intracellular exits, raising the possibility that K+, Na+, and Cl- ions may traverse along their own routes for translocation. NKCC1 structure provides a blueprint for further probing structure-function relationships of NKCC1 and other CCCs.

A mutation in the Na-K-2Cl cotransporter-1 leads to changes in cellular metabolism.

The Na-K-Cl cotransporter-1 (NKCC1), by mediating the electroneutral transport of Na+ , K+ , and Cl- plays an important role in cell volume regulation, epithelial transport, and the control of neuronal excitability. Recently, we reported the first known human mutation in SLC12A2, the gene encoding NKCC1. The 17-year old patient suffers from multiorgan failure. Laboratory tests conducted on muscle and liver biopsies of the patient showed abnormal increase in mitochondrial DNA copy number and increased glycogen levels, indicating the possibility that the transporter may play a role in energy metabolism. Here, we show that fibroblasts isolated from the patient demonstrate a significant increase in mitochondrial respiration, compared to fibroblasts isolated from healthy individuals. Similarly, Madin Darby canine kidney (MDCK) cells transfected with enhanced green fluorescent protein (EGFP)-tagged mutant NKCC1 DNA demonstrated increased mitochondrial respiration when compared to MDCK cells expressing EGFP-tagged wild-type (WT) cotransporter. Direct inhibition of the cotransporter through addition of bumetanide did not change the rate of basal respiration, but led to increased maximal mitochondrial respiration. Fibroblasts extracted from NKCC1WT/DFX and NKCC1DFX/DFX mice also demonstrated a significant elevation in mitochondrial respiration, compared to fibroblasts isolated from their WT littermates. Expression of the mutant protein was associated with an increase in hydrogen peroxide and peroxidase activity and a decrease in messenger RNA transcript levels for protein involved in the unfolded protein response. These data reveal that cells expressing the mutant cotransporter demonstrate increased mitochondrial respiration and behave like they are experiencing a state of starvation.

Cryo-EM structures of DrNKCC1 and hKCC1: a new milestone in the physiology of cation-chloride cotransporters.

New milestones have been reached in the field of cation-Cl- cotransporters with the recently released cryo-electron microscopy (EM) structures of the Danio rerio (zebrafish) Na+-K+-2Cl- cotransporter (DrNKCC1) and the human K+-Cl- cotransporter (hKCC1). In this review we provide a brief timeline that identifies the multiple breakthroughs in the field of solute carrier 12 transporters that led to the structure resolution of two of its key members. While cation-Cl- cotransporters share the overall architecture of carriers belonging to the amino acid-polyamine-organocation (APC) superfamily and some of their substrate binding sites, several new insights are gained from the two individual structures. A first major feature relates to the largest extracellular domain between transmembrane domain (TMD) 5 and TMD6 of KCC1, which stabilizes the dimer and forms a cap that likely participates in extracellular gating. A second feature is the conservation of the K+ and Cl- binding sites in both structures and evidence of an unexpected second Cl- coordination site in the KCC1 structure. Structural data are discussed in the context of previously published studies that examined the basic and kinetics properties of these cotransport mechanisms. A third characteristic is the evidence of an extracellular gate formed by conserved salt bridges between charged residues located toward the end of TMD3 and TMD4 in both transporters and the existence of an additional neighboring bridge in the hKCC1 structure. A fourth feature of these newly solved structures relates to the multiple points of contacts between the monomer forming the cotransporter homodimer units. These involve the TMDs, the COOH-terminal domains, and the large extracellular loop for hKCC1.

Structure and mechanism of the cation-chloride cotransporter NKCC1.

Cation-chloride cotransporters (CCCs) mediate the electroneutral transport of chloride, potassium and/or sodium across the membrane. They have critical roles in regulating cell volume, controlling ion absorption and secretion across epithelia, and maintaining intracellular chloride homeostasis. These transporters are primary targets for some of the most commonly prescribed drugs. Here we determined the cryo-electron microscopy structure of the Na-K-Cl cotransporter NKCC1, an extensively studied member of the CCC family, from Danio rerio. The structure defines the architecture of this protein family and reveals how cytosolic and transmembrane domains are strategically positioned for communication. Structural analyses, functional characterizations and computational studies reveal the ion-translocation pathway, ion-binding sites and key residues for transport activity. These results provide insights into ion selectivity, coupling and translocation, and establish a framework for understanding the physiological functions of CCCs and interpreting disease-related mutations.

Differential expression of putative sodium-dependent cation-chloride cotransporters in Aedes aegypti.

The yellow fever mosquito, Aedes aegypti, has three genes that code for proteins with sequence similarity to vertebrate Na+-K+-Cl- cotransporters (NKCCs) of the solute-linked carrier 12 superfamily of cation-chloride cotransporters (CCCs). We hypothesized that these mosquito NKCC orthologues have diverged to perform distinct roles in salt secretion and absorption. In phylogenetic analyses, one protein (aeNKCC1) groups with a Drosophila melanogaster NKCC that mediates salt secretion whereas two others (aeCCC2 and aeCCC3) group with a Drosophila transporter that is not functionally characterized. The aeCCC2 and aeCCC3 genes probably result from a tandem gene duplication in the mosquito lineage; they have similar exon structures and are consecutive in genomic DNA. Predicted aeCCC2 and aeCCC3 proteins differ from aeNKCC1 and vertebrate NKCCs in residues from the third transmembrane domain known to influence ion and inhibitor binding. Quantitative PCR revealed that aeNKCC1 and aeCCC2 were approximately equally expressed in larvae and adults, whereas aeCCC3 was approximately 100-fold more abundant in larvae than in adults. In larval tissues, aeCCC2 was approximately 2-fold more abundant in Malpighian tubules compared to anal papillae. In contrast, aeCCC3 was nearly 100-fold more abundant in larval anal papillae compared to Malpighian tubules, suggesting a role in absorption. Western blots with polyclonal antibodies against isoform-specific peptides revealed stronger aeCCC2 immunoreactivity in adults versus larvae, whereas aeCCC3 immunoreactivity was stronger in larvae versus adults. The differential expression pattern of aeCCC2 and aeCCC3, and their sequence divergence in transmembrane domains, suggests that they may have different roles in transepithelial salt transport.

Bumetanide, an Inhibitor of NKCC1 (Na-K-2Cl Cotransporter Isoform 1), Enhances Propofol-Induced Loss of Righting Reflex but Not Its Immobilizing Actions in Neonatal Rats.

Gamma-aminobutyric acid (GABA) has been shown to induce excitation on immature neurons due to increased expression of Na+-K+-2Cl- co-transporter isoform 1 (NKCC1), and the transition of GABAergic signaling from excitatory to inhibitory occurs before birth in the rat spinal cord and spreads rostrally according to the developmental changes in cation-chloride co-transporter expression. We previously showed that midazolam activates the hippocampal CA3 area and induces less sedation in neonatal rats compared with adolescent rats in an NKCC1-dependent manner. In the present study, we tested the hypothesis that propofol-induced loss of righting reflex (LORR) but not immobilizing actions are modulated by NKCC1-dependent mechanisms and reduced in neonatal rats compared with adolescent rats. We estimated neuronal activity in the cortex, hippocampus and thalamus after propofol administration with or without bumetanide, an NKCC1 inhibitor, by immunostaining of phosphorylated cyclic adenosine monophosphate-response element binding protein (pCREB). We studied effects of bumetanide on propofol-induced LORR and immobilizing actions in postnatal day 7 and 28 (P7 and P28) rats. The pCREB expression in the cortex (P = 0.001) and hippocampus (P = 0.01) was significantly greater in the rats receiving propofol only than in the rats receiving propofol plus bumetanide at P 7. Propofol-induced LORR or immobilizing effects did not differ significantly between P7 and P28. Bumetanide significantly enhanced propofol-induced LORR (P = 0.031) but not immobilization in P7 rats. These results are partially consistent with our hypothesis. They suggest that propofol may activate the rostral but not caudal central nervous system dependently on NKCC1, and these differential actions may underlie the different properties of sedative and immobilizing actions observed in neonatal rats.

Inhibition of NKCC1 attenuated hippocampal LTP formation and inhibitory avoidance in rat.

The loop diuretic bumetanide (Bumex) is thought to have antiepileptic properties via modulate GABAA mediated signaling through their antagonism of cation-chloride cotransporters. Given that loop diuretics may act as antiepileptic drugs that modulate GABAergic signaling, we sought to investigate whether they also affect hippocampal function. The current study was performed to evaluate the possible role of NKCC1 on the hippocampal function. Brain slice extracellular recording, inhibitory avoidance, and western blot were applied in this study. Results showed that hippocampal Long-term potentiation was attenuated by suprafusion of NKCC1 inhibitor bumetanide, in a dose dependent manner. Sequent experiment result showed that Intravenous injection of bumetanide (15.2 mg/kg) 30 min prior to the training session blocked inhibitory avoidance learning significantly. Subsequent control experiment's results excluded the possible non-specific effect of bumetanide on avoidance learning. We also found the phosphorylation of hippocampal MAPK was attenuated after bumetanide administration. These results suggested that hippocampal NKCC1 may via MAPK signaling cascade to possess its function.

Molecular motions involved in Na-K-Cl cotransporter-mediated ion transport and transporter activation revealed by internal cross-linking between transmembrane domains 10 and 11/12.

We examined the relationship between transmembrane domain (TM) 10 and TM11/12 in NKCC1, testing homology models based on the structure of AdiC in the same transporter superfamily. We hypothesized that introduced cysteine pairs would be close enough for disulfide formation and would alter transport function: indeed, evidence for cross-link formation with low micromolar concentrations of copper phenanthroline or iodine was found in 3 of 8 initially tested pairs and in 1 of 26 additionally tested pairs. Inhibition of transport was observed with copper phenanthroline and iodine treatment of P676C/A734C and I677C/A734C, consistent with the proximity of these residues and with movement of TM10 during the occlusion step of ion transport. We also found Cu(2+) inhibition of the single-cysteine mutant A675C, suggesting that this residue and Met(382) of TM3 are involved in a Cu(2+)-binding site. Surprisingly, cross-linking of P676C/I730C was found to prevent rapid deactivation of the transporter while not affecting the dephosphorylation rate, thus uncoupling the phosphorylation and activation steps. Consistent with this, (a) cross-linking of P676C/I730C was dependent on activation state, and (b) mutants lacking the phosphoregulatory domain could still be activated by cross-linking. These results suggest a model of NKCC activation that involves movement of TM12 relative to TM10, which is likely tied to movement of the large C terminus, a process somehow triggered by phosphorylation of the regulatory domain in the N terminus.

Lipoic acid protects C6 cells against ammonia exposure through Na⁺-K⁺-Cl⁻ co-transporter and PKC pathway.

Astrocytes play an essential role in the central nervous system (CNS) homeostasis. They providing metabolic support and protecting against oxidative stress and glutamatergic excitotoxicity. Glutamate uptake, an electrogenic function, is driven by cation gradients and the Na⁺-K⁺-Cl⁻ co-transporter (NKCC1) carries these ions into and out of the cell. Elevated concentrations of ammonia in the brain lead to cerebral dysfunction. Ammonia toxicity can be mediated by an excitotoxic mechanism, oxidative stress and ion discharged. Astrocytes also convert excess ammonia and glutamate into glutamine, via glutamine synthetase (GS). Lipoic acid (LA) is a modulator of the cellular redox status potentially beneficial in neurodegenerative diseases. In this study, we investigated the effect of LA on glial parameters, in C6 cells exposed to ammonia. Ammonia increased S100B secretion and decreased glutamate uptake, GS activity and glutathione (GSH) content. LA was able to prevent these effects. LA exerts its protective effect on glutamate uptake and S100B secretion via mechanisms dependent of NKCC1 and PKC. These findings show that LA is able to modulate glial function impairments by ammonia in vitro, indicating a potential therapeutic agent to improve glutamatergic metabolism and oxidative stress against hyperammonemia.