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Molecular architecture of potassium chloride co-transporter KCC2.

ABSTRACT: KCC2 is a neuron specific K+-Cl- co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Specific binding of inhibitor compound VU0463271 was demonstrated using surface plasmon resonance (SPR). Mass spectrometry revealed glycosylations and phosphorylations as expected from functional KCC2. We show by electron microscopy (EM) that KCC2 exists as monomers and dimers in solution. Monomers are organized into "head" and "core" domains connected by a flexible "linker". Dimers are asymmetrical and display a bent "S-shape" architecture made of four distinct domains and a flexible dimerization interface. Chemical crosslinking in reducing conditions shows that disulfide bridges are involved in KCC2 dimerization. Moreover, we show that adding a tag to the C-terminus is detrimental to KCC2 function. We postulate that the conserved KCC2 C-ter may be at the interface of dimerization. Taken together, our findings highlight the flexible multi-domain structure of KCC2 with variable anchoring points at the dimerization interface and an important C-ter extremity providing the first in-depth functional architecture of KCC2.

SUBMITTER: Agez M

PROVIDER: S-EPMC5705597 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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Molecular architecture of potassium chloride co-transporter KCC2.

Agez Morgane M Schultz Patrick P Medina Igor I Baker David J DJ Burnham Matthew P MP Cardarelli Ross A RA Conway Leslie C LC Garnier Kelly K Geschwindner Stefan S Gunnarsson Anders A McCall Eileen J EJ Frechard Alexandre A Audebert Stéphane S Deeb Tarek Z TZ Moss Stephen J SJ Brandon Nicholas J NJ Wang Qi Q Dekker Niek N Jawhari Anass A

Scientific reports 20171128 1

KCC2 is a neuron specific K<sup>+</sup>-Cl<sup>-</sup> co-transporter that controls neuronal chloride homeostasis, and is critically involved in many neurological diseases including brain trauma, epilepsies, autism and schizophrenia. Despite significant accumulating data on the biology and electrophysiological properties of KCC2, structure-function relationships remain poorly understood. Here we used calixarene detergent to solubilize and purify wild-type non-aggregated and homogenous KCC2. Spec ...[more]

PMID: 29184062

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Project description:Exceptional mechanical and electrical properties of carbon nanotubes (CNT) have attracted neuroscientists and neural tissue engineers aiming to develop novel devices that interface with nervous tissues. In the central nervous system (CNS), the perinatal chloride shift represents a dynamic change that forms the basis for physiological actions of ?-aminobutyric acid (GABA) as an inhibitory neurotransmitter, a process of fundamental relevance for normal functioning of the CNS. Low intra-neuronal chloride concentrations are maintained by a chloride-extruding transporter, potassium chloride cotransporter 2 (KCC2). KCC2's increasing developmental expression underlies the chloride shift. In neural injury, repressed KCC2 expression plays a co-contributory role by corrupting inhibitory neurotransmission. Mechanisms of Kcc2 up-regulation are thus pertinent because of their medical relevance, yet they remain elusive. Here, it is shown that primary CNS neurons originating from the cerebral cortex, cultured on highly-conductive few-walled-CNT (fwCNT) have a strikingly accelerated chloride shift caused by increased KCC2 expression. KCC2 upregulation is dependent on neuronal voltage-gated calcium channels (VGCC) and, furthermore, on calcium/calmodulin-dependent kinase II, which is linked to VGCC-mediated calcium-influx. It is also demonstrated that accelerated Kcc2 transcription in brain-slices prepared from genetically-engineered reporter mice, in which Kcc2 promoter drives luciferase, when the cerebral cortex of these mice is exposed to fwCNT-coated devices. Based on these findings, whether fwCNT can enhance neural engineering devices for the benefit of neural injury conditions associated with elevated neuronal intracellular chloride concentration-such as pain, epilepsy, traumatic neural injury and ischemia-can now be addressed. Taken together, our novel insights illustrate how fwCNTs can promote low neuronal chloride in individual neurons and thus inhibitory transmission in neural circuits.

Dataset Information

Molecular architecture of potassium chloride co-transporter KCC2.

Publications

Molecular architecture of potassium chloride co-transporter KCC2.

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