Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family

Mount, David B.; Mercado, Adriana; Song, Luyan; Xu, Jason; George, Alfred L.; Delpire, Eric; Gamba, Gerardo

doi:10.1074/jbc.274.23.16355

Cited by 260 publications

(223 citation statements)

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“…1B). In contrast, we did not detect a coimmunoprecipitation between Neto2 and the other major regulator of neuronal chloride homeostasis, NKCC1 (8), or the other KCC family member abundantly expressed in neurons, KCC3 (24). Reciprocally, anti-KCC2 antibodies coimmunoprecipitated Neto2 from wild-type membrane fraction protein preparations (Fig.…”

Section: Resultsmentioning

confidence: 81%

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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Section: Resultsmentioning

confidence: 81%

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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“…1 the basolateral or secretory isoform of the Na-K2Cl cotransporter (1,2), is a member of the cation-coupled chloride transporter family (3,4), which includes the apical Na-K-2Cl cotransporter of the renal thick ascending limb (5,6), the NaCl cotransporter of the renal distal convoluted tubule (6), and four KCl cotransporters (7)(8)(9). NKCC1 mediates the coupled electroneutral transport of 1 Na ϩ , 1 K ϩ , and 2 Cl Ϫ ions across the plasma membrane (4), driven by the inwardly directed Na ϩ and Cl Ϫ gradients that occur under physiological conditions.…”

Section: Nkcc1mentioning

confidence: 99%

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

Flagella¹,

Clarke²,

Miller³

et al. 1999

Journal of Biological Chemistry

356

349

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In chloride-secretory epithelia, the basolateral Na-K2Cl cotransporter (NKCC1) is thought to play a major role in transepithelial Cl ؊ and fluid transport. Similarly, in marginal cells of the inner ear, NKCC1 has been proposed as a component of the entry pathway for K ؉ that is secreted into the endolymph, thus playing a critical role in hearing. To test these hypotheses, we generated and analyzed an NKCC1-deficient mouse. Homozygous mutant (Nkcc1 ؊/؊ ) mice exhibited growth retardation, a 28% incidence of death around the time of weaning, and mild difficulties in maintaining their balance. Mean arterial blood pressure was significantly reduced in both heterozygous and homozygous mutants, indicating an important function for NKCC1 in the maintenance of blood pressure. cAMP-induced short circuit currents, which are dependent on the CFTR Cl ؊ channel, were reduced in jejunum, cecum, and trachea of Nkcc1 ؊/؊ mice, indicating that NKCC1 contributes to cAMP-induced Cl ؊ secretion. In contrast, secretion of gastric acid in adult Nkcc1 ؊/؊ stomachs and enterotoxin-stimulated fluid secretion in the intestine of suckling Nkcc1 ؊/؊ mice were normal. Finally, homozygous mutants were deaf, and histological analysis of the inner ear revealed a collapse of the membranous labyrinth, consistent with a critical role for NKCC1 in transepithelial K ؉ movements involved in generation of the K ؉ -rich endolymph and the endocochlear potential.

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“…These transporters are involved in cell proliferation (Shen et al, 2001) and in the electro-neutral movement of ions across the plasma membrane, thus controlling ionic and osmotic homeostasis of various cell types. The gene SLC12A6 is located on chromosome 15q13-14 (Hiki et al, 1999;Mount et al, 1999;Race et al, 1999). Several groups have demonstrated that mutations in SLC12A6 are causative for the recessively inherited Andermann syndrome (ACCPN, OMIM %218000), a severe neurological disorder characterized by agenesis of the corpus callosum, peripheral neuropathy (Howard et al, 2002bUyanik et al, 2006) and psychoses (Filteau et al, 1991).…”

Section: Introductionmentioning

confidence: 99%

Functional Analysis of a Potassium-Chloride Co-Transporter 3 (SLC12A6) Promoter Polymorphism Leading to an Additional DNA Methylation Site

Moser

Ekawardhani

Kumsta

et al. 2008

Neuropsychopharmacol

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The human potassium-chloride co-transporter 3 (KCC3, SLC12A6) is involved in cell proliferation and in electro-neutral movement of ions across the cell membrane. The gene (SLC12A6) is located on chromosome 15q14, a region that has previously shown linkage with bipolar disorder, schizophrenia, rolandic epilepsy, idiopathic generalized epilepsy, autism and attention deficit/hyperactivity disorder. Furthermore, recessively inherited mutations of SLC12A6 cause Andermann syndrome, characterized by agenesis of the corpus callosum, which is associated with peripheral neuropathy and psychoses. Recently, we have demonstrated the association of two G/A promoter polymorphisms of SLC12A6 with bipolar disorder in a case-control study, and familial segregation of the rare variants as well as a trend toward association with schizophrenia. To investigate functional consequences of these polymorphisms, lymphocyte DNA was extracted, bisulfite modified, and subsequently sequenced. To investigate SLC12A6 promoter activity, various promoter constructs were generated and analyzed by luciferase reporter gene assays. We provide evidence that the G-allele showed a significant reduction of reporter gene expression. In human lymphocytes, the allele harboring the rare upstream G nucleotide was found to be methylated at the adjacent C position, possibly accountable for tissue-specific reduction in gene expression in vivo. Here we demonstrate functionality of an SNP associated with psychiatric disease and our results may represent a functional link between genetic variation and an epigenetic modification.

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Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family

Cited by 260 publications

References 47 publications

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

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

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

Functional Analysis of a Potassium-Chloride Co-Transporter 3 (SLC12A6) Promoter Polymorphism Leading to an Additional DNA Methylation Site

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Cloning and Characterization of KCC3 and KCC4, New Members of the Cation-Chloride Cotransporter Gene Family

Cited by 260 publications

References 47 publications

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

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

Mice Lacking the Basolateral Na-K-2Cl Cotransporter Have Impaired Epithelial Chloride Secretion and Are Profoundly Deaf

Functional Analysis of a Potassium-Chloride Co-Transporter 3 (SLC12A6) Promoter Polymorphism Leading to an Additional DNA Methylation Site

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

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