Pharmacological tools to target NKCC1 in brain disorders
Authors: Annalisa Savardi, Marco Borgogno, Marco De Vivo, Laura Cancedda
Journal: Trends in Pharmacological Sciences (2021)

A growing number of studies indicate a defective ratio of the chloride importer NKCC1 and the chloride exporter KCC2, and consequent impaired Cl intracellular regulation, in several brain conditions, from neurodevelopmental to neurological and neurodegenerative disorders.The FDA-approved diuretic bumetanide, a nonselective inhibitor of NKCC1, reverses core symptoms of several neurological disorders in rodent models and/or in clinical trials in patients.Bumetanide has low brain penetration, and its repurposing in neurological disorders brings several collateral issues and limitations due to excessive diuresis caused by NKCC2 inhibition in the kidney.Recent drug discovery efforts have evaluated novel NKCC1 inhibitors and modulators designed with diverse strategies to avoid the adverse effects of bumetanide. The chloride importer NKCC1 and the chloride exporter KCC2 are key regulators of neuronal chloride concentration. A defective NKCC1/KCC2 expression ratio is associated with several brain disorders. Preclinical/clinical studies have shown that NKCC1 inhibition by the United States FDA-approved diuretic bumetanide is a potential therapeutic strategy in preclinical/clinical studies of multiple neurological conditions. However, bumetanide has poor brain penetration and causes unwanted diuresis by inhibiting NKCC2 in the kidney. To overcome these issues, a growing number of studies have reported more brain-penetrating and/or selective bumetanide prodrugs, analogs, and new molecular entities. Here, we review the evidence for NKCC1 pharmacological inhibition as an effective strategy to manage neurological disorders. We also discuss the advantages and limitations of bumetanide repurposing and the benefits and risks of new NKCC1 inhibitors as therapeutic agents for brain disorders. The chloride importer NKCC1 and the chloride exporter KCC2 are key regulators of neuronal chloride concentration. A defective NKCC1/KCC2 expression ratio is associated with several brain disorders. Preclinical/clinical studies have shown that NKCC1 inhibition by the United States FDA-approved diuretic bumetanide is a potential therapeutic strategy in preclinical/clinical studies of multiple neurological conditions. However, bumetanide has poor brain penetration and causes unwanted diuresis by inhibiting NKCC2 in the kidney. To overcome these issues, a growing number of studies have reported more brain-penetrating and/or selective bumetanide prodrugs, analogs, and new molecular entities. Here, we review the evidence for NKCC1 pharmacological inhibition as an effective strategy to manage neurological disorders. We also discuss the advantages and limitations of bumetanide repurposing and the benefits and risks of new NKCC1 inhibitors as therapeutic agents for brain disorders. In neurons, the sodium (Na+)–potassium (K+)–Cl– transporter isoform 1 (NKCC1, SLC12A2) and the K+–Cl– transporter isoform 2 (KCC2, SLC12A5) [1.Watanabe M. Fukuda A. Development and regulation of chloride homeostasis in the central nervous system.Front. Cell. Neurosci. 2015; 9: 371Crossref PubMed Scopus (101) Google Scholar] are key regulators of intracellular chloride concentration ([Cl–]i). In the central nervous system (CNS), NKCC1 functions as a Cl– importer, and is highly expressed in immature neurons during early development [2.Kaila K. et al.Cation-chloride cotransporters in neuronal development, plasticity and disease.Nat. Rev. Neurosci. 2014; 15: 637-654Crossref PubMed Scopus (345) Google Scholar]. Conversely, KCC2 expression is relatively low in early development (resulting in a high NKCC1/KCC2 ratio), increases during the postnatal period, and is more highly expressed in mature neurons (resulting in a low NKCC1/KCC2 ratio; Box 1). The fine regulation of [Cl–]i by NKCC1 and KCC2 is essential for brain development, including cell proliferation and apoptosis, and neuronal migration and maturation [3.Peerboom C. Wierenga C.J. The postnatal GABA shift: a developmental perspective.Neurosci. Biobehav. Rev. 2021; 124: 179-192Crossref PubMed Scopus (1) Google Scholar]. Moreover, NKCC1 and KCC2 are key for neuronal synaptic plasticity (see Glossary) and for maintaining a proper excitatory/inhibitory balance, which is fundamental for brain functions [3.Peerboom C. Wierenga C.J. The postnatal GABA shift: a developmental perspective.Neurosci. Biobehav. Rev. 2021; 124: 179-192Crossref PubMed Scopus (1) Google Scholar].Box 1Chloride homeostasis and GABAAergic transmissionGamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the CNS and regulates neuronal excitability. It exerts its action by binding to the different GABA receptors, including the ionotropic GABAA receptor (GABAAR). GABAARs are ligand-gated ion channels that respond to GABA binding by allowing neuronal permeability of Cl– (and partially HCO3–) through the cell membrane. Depending on its concentration gradient across the cell membrane and the membrane resting potential of the neuron, Cl– can flow through the GABAAR in both directions. Therefore, maintaining and regulating [Cl–]i in neurons is crucial for the proper functioning of GABAergic signaling.The main regulators of Cl– homeostasis in neurons are the Cl– importer NKCC1 and Cl– exporter KCC2 [1.Watanabe M. Fukuda A. Development and regulation of chloride homeostasis in the central nervous system.Front. Cell. Neurosci. 2015; 9: 371Crossref PubMed Scopus (101) Google Scholar]. During early neurodevelopment, a high Cl– concentration is present inside the cell due to a higher NKCC1/KCC2 expression ratio. Thus, opening of the GABAAR causes a Cl– efflux outside the cell. Consequently, GABA activation results in membrane depolarization [2.Kaila K. et al.Cation-chloride cotransporters in neuronal development, plasticity and disease.Nat. Rev. Neurosci. 2014; 15: 637-654Crossref PubMed Scopus (345) Google Scholar] (Figure I, left). Depolarization leads to activation of voltage-gated calcium channels and removal of the Mg2+ block from NMDA receptors, causing further membrane depolarization and calcium influx into the cell [4.Schulte J.T. et al.Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions.Neurosci. Biobehav. Rev. 2018; 90: 260-271Crossref PubMed Scopus (44) Google Scholar]. This sequence of events is pivotal for neuronal excitability and the activation of second messengers that participate in neuronal migration, differentiation, synaptogenesis, and physiological brain development [3.Peerboom C. Wierenga C.J. The postnatal GABA shift: a developmental perspective.Neurosci. Biobehav. Rev. 2021; 124: 179-192Crossref PubMed Scopus (1) Google Scholar]. After physiological brain maturation, adult neurons in the CNS present lower levels of [Cl–]i due to a lower NKCC1/KCC2 expression ratio [2.Kaila K. et al.Cation-chloride cotransporters in neuronal development, plasticity and disease.Nat. Rev. Neurosci. 2014; 15: 637-654Crossref PubMed Scopus (345) Google Scholar]. In this condition, opening of GABAAR leads to an increased conductance of Cl– into the neuron, which generates the hyperpolarizing and inhibitory GABAAergic transmission (Figure I, center). Hyperpolarizing GABA transmission has a significant role in regulating neuronal-network function and plasticity to optimize the processing of sensorial information throughout life [3.Peerboom C. Wierenga C.J. The postnatal GABA shift: a developmental perspective.Neurosci. Biobehav. Rev. 2021; 124: 179-192Crossref PubMed Scopus (1) Google Scholar].However, several studies have demonstrated a depolarizing GABAergic transmission in a range of brain disorders [5.Ben-Ari Y. NKCC1 chloride importer antagonists attenuate many neurological and psychiatric disorders.Trends Neurosci. 2017; 40: 536-554Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. This is due to an altered NKCC1/KCC2 ratio, which is caused by high NKCC1 expression/function and/or low KCC2 expression/function resulting in high [Cl–]i [4.Schulte J.T. et al.Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions.Neurosci. Biobehav. Rev. 2018; 90: 260-271Crossref PubMed Scopus (44) Google Scholar] (Figure I, right). This alteration affects proper neuronal functioning and, thus, contributes to the development and severity of some of the symptoms of the several brain disorders characterized by impaired Cl– homeostasis.Three main strategies are suitable for restoring physiological [Cl–]i, independent of which of the two transporters is dysregulated (Figure I, right): (i) NKCC1 inhibition/downregulation is the most widely used approach, described in detail in the main text; (ii) KCC2 activation/upregulation is a promising, but less explored strategy [134.Delpire E. Advances in the development of novel compounds targeting cation-chloride cotransporter physiology.Am. J. Physiol. Cell Physiol. 2021; 320: C324-C340Crossref PubMed Scopus (0) Google Scholar]; and (iii) targeting of NKCC1/KCC2 upstream effectors was recently proposed, and it is briefly described in the main text. Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the CNS and regulates neuronal excitability. It exerts its action by binding to the different GABA receptors, including the ionotropic GABAA receptor (GABAAR). GABAARs are ligand-gated ion channels that respond to GABA binding by allowing neuronal permeability of Cl– (and partially HCO3–) through the cell membrane. Depending on its concentration gradient across the cell membrane and the membrane resting potential of the neuron, Cl– can flow through the GABAAR in both directions. Therefore, maintaining and regulating [Cl–]i in neurons is crucial for the proper functioning of GABAergic signaling. The main regulators of Cl– homeostasis in neurons are the Cl– importer NKCC1 and Cl– exporter KCC2 [1.Watanabe M. Fukuda A. Development and regulation of chloride homeostasis in the central nervous system.Front. Cell. Neurosci. 2015; 9: 371Crossref PubMed Scopus (101) Google Scholar]. During early neurodevelopment, a high Cl– concentration is present inside the cell due to a higher NKCC1/KCC2 expression ratio. Thus, opening of the GABAAR causes a Cl– efflux outside the cell. Consequently, GABA activation results in membrane depolarization [2.Kaila K. et al.Cation-chloride cotransporters in neuronal development, plasticity and disease.Nat. Rev. Neurosci. 2014; 15: 637-654Crossref PubMed Scopus (345) Google Scholar] (Figure I, left). Depolarization leads to activation of voltage-gated calcium channels and removal of the Mg2+ block from NMDA receptors, causing further membrane depolarization and calcium influx into the cell [4.Schulte J.T. et al.Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions.Neurosci. Biobehav. Rev. 2018; 90: 260-271Crossref PubMed Scopus (44) Google Scholar]. This sequence of events is pivotal for neuronal excitability and the activation of second messengers that participate in neuronal migration, differentiation, synaptogenesis, and physiological brain development [3.Peerboom C. Wierenga C.J. The postnatal GABA shift: a developmental perspective.Neurosci. Biobehav. Rev. 2021; 124: 179-192Crossref PubMed Scopus (1) Google Scholar]. After physiological brain maturation, adult neurons in the CNS present lower levels of [Cl–]i due to a lower NKCC1/KCC2 expression ratio [2.Kaila K. et al.Cation-chloride cotransporters in neuronal development, plasticity and disease.Nat. Rev. Neurosci. 2014; 15: 637-654Crossref PubMed Scopus (345) Google Scholar]. In this condition, opening of GABAAR leads to an increased conductance of Cl– into the neuron, which generates the hyperpolarizing and inhibitory GABAAergic transmission (Figure I, center). Hyperpolarizing GABA transmission has a significant role in regulating neuronal-network function and plasticity to optimize the processing of sensorial information throughout life [3.Peerboom C. Wierenga C.J. The postnatal GABA shift: a developmental perspective.Neurosci. Biobehav. Rev. 2021; 124: 179-192Crossref PubMed Scopus (1) Google Scholar]. However, several studies have demonstrated a depolarizing GABAergic transmission in a range of brain disorders [5.Ben-Ari Y. NKCC1 chloride importer antagonists attenuate many neurological and psychiatric disorders.Trends Neurosci. 2017; 40: 536-554Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar]. This is due to an altered NKCC1/KCC2 ratio, which is caused by high NKCC1 expression/function and/or low KCC2 expression/function resulting in high [Cl–]i [4.Schulte J.T. et al.Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions.Neurosci. Biobehav. Rev. 2018; 90: 260-271Crossref PubMed Scopus (44) Google Scholar] (Figure I, right). This alteration affects proper neuronal functioning and, thus, contributes to the development and severity of some of the symptoms of the several brain disorders characterized by impaired Cl– homeostasis. Three main strategies are suitable for restoring physiological [Cl–]i, independent of which of the two transporters is dysregulated (Figure I, right): (i) NKCC1 inhibition/downregulation is the most widely used approach, described in detail in the main text; (ii) KCC2 activation/upregulation is a promising, but less explored strategy [134.Delpire E. Advances in the development of novel compounds targeting cation-chloride cotransporter physiology.Am. J. Physiol. Cell Physiol. 2021; 320: C324-C340Crossref PubMed Scopus (0) Google Scholar]; and (iii) targeting of NKCC1/KCC2 upstream effectors was recently proposed, and it is briefly described in the main text. A defective NKCC1/KCC2 expression ratio is often associated with several neurological and psychiatric disorders [4.Schulte J.T. et al.Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions.Neurosci. Biobehav. Rev. 2018; 90: 260-271Crossref PubMed Scopus (44) Google Scholar]. In particular, a large and growing body of literature reporting preclinical and clinical studies has shown that upregulation of NKCC1 and/or downregulation of KCC2 (resulting in an increased NKCC1/KCC2 ratio) underlie neurodevelopmental, insult-induced neurological, and neurodegenerative disorders [4.Schulte J.T. et al.Chloride transporters and GABA polarity in developmental, neurological and psychiatric conditions.Neurosci. Biobehav. Rev. 2018; 90: 260-271Crossref PubMed Scopus (44) Google Scholar] (Box 1; see Outstanding questions). Restoring neuronal [Cl–]i by inhibiting NKCC1 with bumetanide (an unselective NKCC1 inhibitor) was the first proof of concept for NKCC1 as a valuable target for several neurological conditions in preclinical (animal models) and clinical studies (patients) [5.Ben-Ari Y. NKCC1 chloride importer antagonists attenuate many neurological and psychiatric disorders.Trends Neurosci. 2017; 40: 536-554Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar,6.Kharod S.C. et al.Off-label use of bumetanide for brain disorders: an overview.Front. Neurosci. 2019; 13: 310Crossref PubMed Scopus (34) Google Scholar] (see Outstanding questions). Bumetanide is an FDA-approved thick ascending loop (TAL) of Henle diuretic, which acts by inhibiting the kidney transporter NKCC2. Due to its potent diuretic effect, bumetanide is currently indicated only to treat edema and swelling caused by congestive heart failure, acute pulmonary congestion, and hepatic and renal diseases [7.Sidhu G. Puckett Y. Bumetanide. StatPearls, 2021Google Scholar]. Notably, downregulation of NKCC1 via RNAi in rodent models [e.g., of schizophrenia [8.Kim H.R. et al.Depolarizing GABAA current in the prefrontal cortex is linked with cognitive impairment in a mouse model relevant for schizophrenia.Sci. Adv. 2021; 7eaba5032Crossref PubMed Scopus (2) Google Scholar], Huntington's disease (HD) [9.Hsu Y.T. et al.Enhanced Na(+) -K(+) -2Cl(-) cotransporter 1 underlies motor dysfunction in huntington's disease.Mov. Disord. 2019; 34: 845-857Crossref PubMed Scopus (0) Google Scholar], and Down syndrome (DS) [10.Parrini M. et al.Restoring neuronal chloride homeostasis with anti-NKCC1 gene therapy rescues cognitive deficits in a mouse model of Down syndrome.Mol. Ther. 2021; 29: 3072-3092Abstract Full Text Full Text PDF Scopus (0) Google Scholar]] and glioma cells [11.Ma H. et al.NKCC1 promotes EMT-like process in GBM via RhoA and Rac1 signaling pathways.J. Cell. Physiol. 2019; 234: 1630-1642Crossref PubMed Scopus (17) Google Scholar,12.Sun H. et al.NKCC1 involvement in the epithelial-to-mesenchymal transition is a prognostic biomarker in gliomas.PeerJ. 2020; 8e8787Crossref PubMed Scopus (2) Google Scholar] offered a more direct proof of NKCC1 as a target for some brain disorders. Here, we summarize studies demonstrating the involvement of altered NKCC1 and/or KCC2 expression in the pathogenesis of brain disorders (Table 1), contextualizing the multiple indications for which NKCC1 inhibitors are being developed.Table 1Summary of NKCC1 and/or KCC2 dysregulation in neurological disorders in preclinical and clinical studiesaAbbreviations: CBC, cerebellar cortex; CMECs, cerebral microvascular endothelial cells; CP, choroid plexus; CSF, cerebrospinal fluid; CTX, cerebral cortex; DRG, dorsal root ganglia; E, embryonic day; GW, gestational week; HP, hippocampus; iPSCs, induced pluripotent stem cells; MN, motor neurons; P, postnatal day; Ph, phosphorylation; PBMCs, peripheral blood mononuclear cells; Prot, protein; SC, spinal cord; SDH, spinal dorsal horn; STNC, spinal trigeminal nucleus caudalis; TG, trigeminal ganglion; yr, years.DisordersNKCC1 upregulationKCC2 downregulationNKCC1 upregulation and KCC2 downregulationRefsh'S' refers to references in the supplemental information online.RodentHumanRodentHumanRodentHumanRodentHumanAutismProt, CTX- HP-CBC (P0–P60)Prot, CTX (17–45 yr)[17.Tyzio R. et al.Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring.Science. 2014; 343: 675-679Crossref PubMed Scopus (340) Google Scholar, 18.Li Y. et al.Reduced protein expressions of cytomembrane GABAARbeta3 at different postnatal developmental stages of rats exposed prenatally to valproic acid.Brain Res. 2017; 1671: 33-42Crossref PubMed Scopus (9) Google Scholar, 19.Savardi A. et al.Discovery of a small molecule drug candidate for selective NKCC1 inhibition in brain disorders.Chem. 2020; 6: 2073-2096Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar,22.Eftekhari S. et al.Response to comment on 'Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring'.Science. 2014; 346: 176Crossref PubMed Google Scholar][19.Savardi A. et al.Discovery of a small molecule drug candidate for selective NKCC1 inhibition in brain disorders.Chem. 2020; 6: 2073-2096Abstract Full Text Full Text PDF PubMed Scopus (11) Google Scholar]Rett syndromeProt, CTX (P20–P25)Prot-mRNA, CTX CSF-iPSCs (2–22 yr)[20.Banerjee A. et al.Jointly reduced inhibition and excitation underlies circuit-wide changes in cortical processing in Rett syndrome.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: E7287-E7296Crossref PubMed Scopus (0) Google Scholar][25.Hinz L. et al.KCC2 expression levels are reduced in post mortem brain tissue of Rett syndrome patients.Acta Neuropathol. Commun. 2019; 7: 196Crossref PubMed Scopus (11) Google Scholar, 26.Duarte S.T. et al.Abnormal expression of cerebrospinal fluid cation chloride cotransporters in patients with Rett syndrome.PLoS ONE. 2013; 8e68851Crossref PubMed Scopus (45) Google Scholar, 27.Tang X. et al.KCC2 rescues functional deficits in human neurons derived from patients with Rett syndrome.Proc. Natl. Acad. Sci. U. S. A. 2016; 113: 751-756Crossref PubMed Scopus (103) Google Scholar]Fragile X syndromeProt, CTX (P10)Prot, HP (P15, P30)[17.Tyzio R. et al.Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring.Science. 2014; 343: 675-679Crossref PubMed Scopus (340) Google Scholar,21.He Q. et al.The developmental switch in GABA polarity is delayed in fragile X mice.J. Neurosci. 2014; 34: 446-450Crossref PubMed Scopus (123) Google Scholar,22.Eftekhari S. et al.Response to comment on 'Oxytocin-mediated GABA inhibition during delivery attenuates autism pathogenesis in rodent offspring'.Science. 2014; 346: 176Crossref PubMed Google Scholar]DiGeorge syndromeProt-mRNA, HP primary neurons[28.Amin H. et al.Developmental excitatory-to-inhibitory GABA-polarity switch is disrupted in 22q11.2 deletion syndrome: a potential target for clinical therapeutics.Sci. Rep. 2017; 7: 15752Crossref PubMed Scopus (22) Google Scholar]TSCProt-mRNA, cortical tubers (15 GW–31 yr)[29.Talos D.M. et al.Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia.Ann. Neurol. 2012; 71: 539-551Crossref PubMed Scopus (100) Google Scholar,30.Ruffolo G. et al.Functional aspects of early brain development are preserved in tuberous sclerosis complex (TSC) epileptogenic lesions.Neurobiol. Dis. 2016; 95: 93-101Crossref PubMed Scopus (19) Google Scholar]Down syndromeProt, CTX-HP (adult)Prot, CTX-HP (adult)[39.Deidda G. et al.Reversing excitatory GABAAR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome.Nat. Med. 2015; 21: 318-326Crossref PubMed Scopus (133) Google Scholar][39.Deidda G. et al.Reversing excitatory GABAAR signaling restores synaptic plasticity and memory in a mouse model of Down syndrome.Nat. Med. 2015; 21: 318-326Crossref PubMed Scopus (133) Google Scholar]SchizophreniamRNA, CTX (P30)mRNA, CTX (adult)bDecreased level of NKCC1 mRNA, and NKCC1 1–2a, NKCC1b alternative mRNAs together with protein isoform NKCC1b were reported in PBMCs and dorsal lateral prefrontal cortex of patients with schizophrenia, respectively [48,49].mRNA, CTX (E18)mRNA-Prot, HP-CTX (adult)mRNA, HP (E18)[8.Kim H.R. et al.Depolarizing GABAA current in the prefrontal cortex is linked with cognitive impairment in a mouse model relevant for schizophrenia.Sci. Adv. 2021; 7eaba5032Crossref PubMed Scopus (2) Google Scholar,40.Larimore J. et al.Dysbindin deficiency modifies the expression of GABA neuron and ion permeation transcripts in the developing hippocampus.Front. Genet. 2017; 8: 28Crossref PubMed Scopus (13) Google Scholar][45.Dean B. et al.Gene expression profiling in Brodmann's area 46 from subjects with schizophrenia.Aust. N. Z. J. Psychiatry. 2007; 41: 308-320Crossref PubMed Scopus (0) Google Scholar, 46.Hyde T.M. et al.Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia.J. Neurosci. 2011; 31: 11088-11095Crossref PubMed Scopus (202) Google Scholar, 47.Sullivan C.R. et al.Decreased chloride channel expression in the dorsolateral prefrontal cortex in schizophrenia.PLoS ONE. 2015; 10e0123158Crossref PubMed Scopus (6) Google Scholar, 48.Morita Y. et al.Characteristics of the cation cotransporter NKCC1 in human brain: alternate transcripts, expression in development, and potential relationships to brain function and schizophrenia.J. Neurosci. 2014; 34: 4929-4940Crossref PubMed Scopus (39) Google Scholar, 49.Zhang T. et al.Expression of GSK3beta, PICK1, NEFL, C4, NKCC1 and synaptophysin in peripheral blood mononuclear cells of the first-episode schizophrenia patients.Asian J. Psychiatr. 2021; 55102520Crossref PubMed Scopus (2) Google Scholar]FASDProt, HP (P45–P55)cKCC2 upregulation [41,42].[41.Everett J.C. et al.Effects of third trimester-equivalent ethanol exposure on Cl(-) co-transporter expression, network activity, and GABAergic transmission in the CA3 hippocampal region of neonatal rats.Alcohol. 2012; 46: 595-601Crossref PubMed Scopus (0) Google Scholar, 42.Sanderson J.L. et al.Modulation of GABAergic and glutamatergic transmission by ethanol in the developing neocortex: an in vitro test of the excessive inhibition hypothesis of fetal alcohol spectrum disorder.Neuropharmacology. 2009; 56: 541-555Crossref PubMed Scopus (21) Google Scholar, 43.Silvestre de Ferron B. et al.Increase of KCC2 in hippocampal synaptic plasticity disturbances after perinatal ethanol exposure.Addict. Biol. 2017; 22: 1870-1882Crossref PubMed Scopus (1) Google Scholar]Depressive-like behaviorsProt, HP (P14)Prot, HP (P35, P70)dKCC2 upregulation reported at P40 after maternal separation [50].mRNA, amygdala (adult)[50.Hu D. et al.Bumetanide treatment during early development rescues maternal separation-induced susceptibility to stress.Sci. Rep. 2017; 7: 11878Crossref PubMed Scopus (13) Google Scholar, 51.Furukawa M. et al.Neonatal maternal separation delays the GABA excitatory-to-inhibitory functional switch by inhibiting KCC2 expression.Biochem. Biophys. Res. Commun. 2017; 493: 1243-1249Crossref PubMed Scopus (24) Google Scholar, 52.Yang J. et al.Effects of combined brief etomidate anesthesia and postnatal stress on amygdala expression of Cl(-) cotransporters and corticotropin-releasing hormone and alcohol intake in adult rats.Neurosci. Lett. 2018; 685: 83-89Crossref PubMed Scopus (5) Google Scholar, 53.Goubert E. et al.Bumetanide prevents brain trauma-induced depressive-like behavior.Front. Mol. Neurosci. 2019; 12: 12Crossref PubMed Scopus (5) Google Scholar, 54.Gazzo G. et al.Overexpression of chloride importer NKCC1 contributes to the sensory-affective and sociability phenotype of rats following neonatal maternal separation.Brain Behav. Immun. 2021; 92: 193-202Crossref PubMed Scopus (0) Google Scholar]FCD and polymicrogyriaProt, CTX (pediatric and adult)mRNA, CTX (P4)Prot-mRNA, CTX (pediatric)mRNA, CTX (P4)Prot, CTX (pediatric and adult)[56.Shimizu-Okabe C. et al.Changes in the expression of cation-Cl- cotransporters, NKCC1 and KCC2, during cortical malformation induced by neonatal freeze-lesion.Neurosci. Res. 2007; 59: 288-295Crossref PubMed Scopus (0) Google Scholar,57.Wang T. et al.Accumulation of GABAergic neurons, causing a focal ambient GABA gradient, and downregulation of KCC2 are induced during microgyrus formation in a mouse model of polymicrogyria.Cereb. Cortex. 2014; 24: 1088-1101Crossref PubMed Scopus (18) Google Scholar][29.Talos D.M. et al.Altered inhibition in tuberous sclerosis and type IIb cortical dysplasia.Ann. Neurol. 2012; 71: 539-551Crossref PubMed Scopus (100) Google Scholar]; [S1–S5]Dravet syndromemRNA, CTX (1.4–61 yr)[61.Ruffolo G. et al.A novel GABAergic dysfunction in human Dravet syndrome.Epilepsia. 2018; 59: 2106-2117Crossref PubMed Scopus (21) Google Scholar]Neonatal seizuresProt, epileptic hemisphere (P7)eKCC2 upregulation detected after 24 h of PTZ-induced seizures [S8].Prot, CTX-HP (P10)fNKCC1 significantly increased in CTX 24 h after seizures; KCC2 was significantly increased in HP 1 h after seizures and decreased in CTX 24 h after seizures; NKCC1/KCC2 ratio was significantly decreased in CTX 1 h after seizures and increased 12 h (both CTX and HP) and 24 h (CTX) after seizures [S6].[S6–S8]TLEProt-mRNA, HP-CTX (adult)Prot-mRNA, HP (adult)gKCC2 upregulation in pilocarpine-induced model of TLE in mice and in humans [64].Prot, HP(adult)gKCC2 upregulation in pilocarpine-induced model of TLE in mice and in humans [64].Prot-mRNA, HP (adult)Prot-mRNA, HP-PBMCs (adult)[62.Auer T. et al.Functional characterization of novel bumetanide derivatives for epilepsy treatment.Neuropharmacology. 2020; 162107754Crossref PubMed Scopus (8) Google Scholar, 63.Hampel P. et al.Deletion of the Na-K-2Cl cotransporter NKCC1 results in a more severe epileptic phenotype in the intrahippocampal kainate mouse model of temporal lobe epilepsy.Neurobiol. Dis. 2021; 152105297Crossref PubMed Scopus (1) Google Scholar, 64.Karlocai M.R. et al.Enhanced expression of potassium-chloride cotransporter KCC2 in human temporal lobe epilepsy.Brain Struct. Funct. 2016; 221: 3601-3615Crossref PubMed Scopus (0) Google Scholar] [S9–S18][64.Karlocai M.R. et al.Enhanced expression of potassium-chloride cotransporter KCC2 in human temporal lobe epilepsy.Brain Struct. Funct. 2016; 221: 3601-3615Crossref PubMed Scopus (0) Google Scholar,66.Gharaylou Z. et al.A preliminary study evaluating the safety and efficacy of bumetanide, an NKCC1 inhibitor, in patients with drug-resistant epilepsy.CNS Drugs. 2019; 33: 283-291Crossref PubMed Scopus (8) Google Scholar]; [S19–S21]TBIProt-mRNA, HP-CP-CTX (adult)Prot-mRNA, CTX (adult)Prot-mRNA, CTX (adult)[68.Hui H. et al.Inhibition of Na(+)-K(+)-2Cl(-) Cotransporter-1 attenuates traumatic brain injury-induced neuronal apoptosis via regulation of Erk signaling.Neurochem. Int. 2016; 94: 23-31Crossref PubMed Scopus (13) Google Scholar, 69.Zhang M. et al.Astaxanthin alleviates cerebral edema by modulating NKCC1 and AQP4 expression after traumatic brain injury in mice.BMC Neurosci. 2016; 17: 60Crossref PubMed Scopus (28) Google Scholar, 70.Wu H. et al.Melatonin attenuates neuronal apoptosis through up-regulation of K(+) -Cl(-) cotransporter KCC2 expression following traumatic brain injury in rats.J. Pineal Res. 2016; 61: 241-250Crossref PubMed Scopus (43) Google Scholar, 71.Wang F. et al.NKCC1 up-regulation contributes to early post-traumatic seizures and increased post-traumatic seizure susceptibility.Brain Struct. Funct. 2017; 222: 1543-1556Crossref PubMed Scopus (34) Google Scholar,73.Lizhnyak P.N. et al.Traumatic brain injury temporal proteome guides KCC2-targeted therapy.J. Neurotrauma. 2019; 36: 3092-3102Crossref PubMed Scopus (6) Google Scholar]; [S22–S24]SCI, PNI, and neuropathic painProt-mRNA-Ph, DRG, TG, SC (adult)Prot-mRNA, SDH-STNC-SC (adult)Prot, PBMCs (adult)Prot-mRNA, DRG-SC (adult)[76.Yan X. et al.Pretreatment with AQP4 and NKCC1 inhibitors concurrently attenuated spinal cord edema and tissue damage after spinal cord injury in rats.Front. Physiol. 2018; 9: 6Crossref PubMed Scopus (9) Google Scholar, 77.Gao J.L. et al.Suppression of WNK1-SPAK/OSR1 attenuates bone cancer pain by regulating NKCC1 and KCC2.J. Pain