Dataset Information

The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2.

ABSTRACT: NKCC and KCC transporters mediate coupled transport of Na⁺+K⁺+Cl^- and K⁺+Cl^- across the plasma membrane, thus regulating cell Cl^- concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cryo-electron microscopy (cryo-EM) structures of the two neuronal cation-chloride cotransporters human NKCC1 (SLC12A2) and mouse KCC2 (SLC12A5), along with computational analysis and functional characterization. These structures highlight essential residues in ion transport and allow us to propose mechanisms by which phosphorylation regulates transport activity.

SUBMITTER: Zhang S

PROVIDER: S-EPMC7889885 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2.

Zhang Sensen S Zhou Jun J Zhang Yuebin Y Liu Tianya T Friedel Perrine P Zhuo Wei W Somasekharan Suma S Roy Kasturi K Zhang Laixing L Liu Yang Y Meng Xianbin X Deng Haiteng H Zeng Wenwen W Li Guohui G Forbush Biff B Yang Maojun M

Communications biology 20210217 1

NKCC and KCC transporters mediate coupled transport of Na++K++Cl- and K++Cl- across the plasma membrane, thus regulating cell Cl- concentration and cell volume and playing critical roles in transepithelial salt and water transport and in neuronal excitability. The function of these transporters has been intensively studied, but a mechanistic understanding has awaited structural studies of the transporters. Here, we present the cry ...[more]

PMID: 33597714

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Project description:Na-K-Cl cotransporter 1 (NKCC1) and K-Cl cotransporter 2 (KCC2) have fundamental roles in neuron differentiation that are integrated with gamma-aminobutyric acid (GABA) and glutamate receptors, GABA synthesized by GAD25/65/67 encoded by GAD1/GAD2 genes, and GABA transporters (GATs). Cells in the eye lens express at least 13 GABA receptor subunits, ?-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl D-aspartate (NMDA) glutamate receptors, GAD1/GAD2, GAT1-4 and vGAT, and NKCC1. NKCC1:KCC2 ratios determine the switch in GABA actions from trophic/growth promoting early in development to their classic inhibitory roles in adult neurons. Lens epithelial cells cover the anterior surface and differentiate to elongated fiber cells in the lens interior with comparable morphology and sub-cellular structures as neurons. NKCC1 is expressed before KCC2 in neuron development and increases cell chloride, which stimulates differentiation and process formation. Subsequently, KCC2 increases and extrudes cell chloride linked with maturation. KCC2 has an additional structural moonlighting role interacting with F-actin scaffolding in dendritic spine morphogenesis. We examined KCC2 versus NKCC1 spatial expression in relation to fiber cell developmental status within the lens.Immunofluorescence and immunoblots were used to detect expression in mouse and rabbit lenses.NKCC1 was restricted to peripheral elongating lens fiber cells in young adult mouse and rabbit lenses. Lens KCC2 expression included the major KCC2b neuronal isoform and was detected in interior fiber cells with decreased NKCC1 expression and localized at the membranes. Lens expression of RE-1 silencing transcription factor (REST) regulated KCC2 is consistent with GAD1 and GAD2, several GABA and glutamate receptor subunits, miR-124, and other REST-regulated genes expressed in lenses.NKCC1 in peripheral elongating fiber cells is superseded by KCC2 expression in interior mature fiber cells that also express >20 additional integral GABA biology genes, AMPA/NMDA glutamate receptors, and an array of accessory proteins that together underlie morphogenesis in neurons. The present findings provide further evidence that this fundamental neuronal regulation is extensively conserved in lens and identify additional parallels in the morphogenetic programs that underlie lens fiber cell and neuronal differentiation and contribute to the development of visual acuity.

Project description:ContextDisturbances in markers of cortical γ-aminobutyric acid neurotransmission are a common finding in schizophrenia. The nature of γ-aminobutyric acid neurotransmission (hyperpolarizing or depolarizing) depends on the local intracellular chloride concentration. In the central nervous system, the intracellular chloride level is determined by the activity of 2 cation-chloride transporters, NKCC1 and KCC2. The activities of these transporters are in turn regulated by a network of serine-threonine kinases that includes OXSR1, STK39, and the WNK kinases WNK1, WNK3, and WNK4.ObjectiveTo compare the levels of NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts in prefrontal cortex area 9 between subjects with schizophrenia and healthy comparison subjects.DesignReal-time quantitative polymerase chain reaction technique was used to measure transcript levels in the prefrontal cortex.SettingHuman brain specimens were obtained from autopsies conducted at the Allegheny County Medical Examiner's Office, Pittsburgh, Pennsylvania.ParticipantsPostmortem brain specimens from 42 subjects with schizophrenia and 42 matched healthy comparison subjects. Brain specimens from 18 macaque monkeys exposed to haloperidol, olanzapine, or sham long-term.Main outcome measuresRelative expression levels for NKCC1, KCC2, OXSR1, STK39, WNK1, WNK3, and WNK4 transcripts compared with the mean expression level of 3 housekeeping transcripts.ResultsOXSR1 and WNK3 transcripts were substantially overexpressed in subjects with schizophrenia relative to comparison subjects. In contrast, NKCC1, KCC2, STK39, WNK1, and WNK4 transcript levels did not differ between subject groups. OXSR1 and WNK3 transcript expression levels were not changed in antipsychotic-exposed monkeys and were not affected by potential confounding factors in the subjects with schizophrenia.ConclusionIn schizophrenia, increased expression levels, and possibly increased kinase activities, of OXSR1 and WNK3 may shift the balance of chloride transport by NKCC1 and KCC2 and alter the nature of γ-aminobutyric acid neurotransmission in the prefrontal cortex.

Dataset Information

The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2.

Publications

The structural basis of function and regulation of neuronal cotransporters NKCC1 and KCC2.

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