Serine 937 phosphorylation enhances KCC2 activity and strengthens synaptic inhibition.

The potassium chloride cotransporter KCC2 is crucial for Cl extrusion from mature neurons and thus key to hyperpolarizing inhibition. Auditory brainstem circuits contain well-understood inhibitory projections and provide a potent model to study the regulation of synaptic inhibition. Two peculiarities of the auditory brainstem are (i) posttranslational activation of KCC2 during development and (ii) extremely negative reversal potentials in specific circuits.

K/Cl cotransporter 2 (KCC2) and Na/ cotransporter 1 (NBCe1) interaction modulates profile of KCC2 phosphorylation.

K/Cl cotransporter 2 (KCC2) is a major Cl extruder in mature neurons and is responsible for the establishment of low intracellular [Cl], necessary for fast hyperpolarizing GABA-receptor mediated synaptic inhibition. Electrogenic sodium bicarbonate cotransporter 1 (NBCe1) is a pH regulatory protein expressed in neurons and glial cells. An interactome study identified NBCe1 as a possible interaction partner of KCC2.

Both chloride-binding sites are required for KCC2-mediated transport.

The K-Cl cotransporter 2 (KCC2) plays an important role in inhibitory neurotransmission, and its impairment is associated with neurological and psychiatric disorders, including epilepsy, schizophrenia, and autism. Although KCCs transport K and Cl in a 1:1 stoichiometry, two Cl coordination sites were indicated via cryo-EM. In a comprehensive analysis, we analyzed the consequences of point mutations of residues coordinating Cl in Cl and Cl.

NKCC1 and KCC2: Structural insights into phospho-regulation.

Inhibitory neurotransmission plays a fundamental role in the central nervous system, with about 30-50% of synaptic connections being inhibitory. The action of both inhibitory neurotransmitter, gamma-aminobutyric-acid (GABA) and glycine, mainly relies on the intracellular Cl concentration in neurons. This is set by the interplay of the cation chloride cotransporters NKCC1 (Na, K, Cl cotransporter), a main Cl uptake transporter, and KCC2 (K, Cl cotransporter), the principle Cl extruder in neurons.

Structural changes in the extracellular loop 2 of the murine KCC2 potassium chloride cotransporter modulate ion transport.

K-Cl cotransporters (KCCs) play important roles in physiological processes such as inhibitory neurotransmission and cell-volume regulation. KCCs exhibit significant variations in K affinities, yet recent atomic structures demonstrated that K- and Cl-binding sites are highly conserved, raising the question of whether additional structural elements may contribute to ion coordination. The termini and the large extracellular domain (ECD) of KCCs exhibit only low sequence identity and were already discussed as modulators of transport activity.

Staurosporine and NEM mainly impair WNK-SPAK/OSR1 mediated phosphorylation of KCC2 and NKCC1.

The pivotal role of KCC2 and NKCC1 in development and maintenance of fast inhibitory neurotransmission and their implication in severe human diseases arouse interest in posttranscriptional regulatory mechanisms such as (de)phosphorylation. Staurosporine (broad kinase inhibitor) and N-ethylmalemide (NEM) that modulate kinase and phosphatase activities enhance KCC2 and decrease NKCC1 activity. Here, we investigated the regulatory mechanism for this reciprocal regulation by mass spectrometry and immunoblot analyses using phospho-specific antibodies.

Phosphoregulation of the intracellular termini of K-Cl cotransporter 2 (KCC2) enables flexible control of its activity.

The pivotal role of K-Cl cotransporter 2 (KCC2) in inhibitory neurotransmission and severe human diseases fosters interest in understanding posttranslational regulatory mechanisms such as (de)phosphorylation. Here, the regulatory role of the five phosphosites Ser, Thr, Ser, Thr, and Thr was investigated by the use of alanine and aspartate mutants. Tl-based flux analyses in HEK-293 cells demonstrated increased transport activity for S932D (mimicking phosphorylation) and T1008A (mimicking dephosphorylation), albeit to a different extent.

Molecular cloning and biochemical characterization of two cation chloride cotransporter subfamily members of Hydra vulgaris.

Cation Chloride Cotransporters (CCCs) comprise secondary active membrane proteins mainly mediating the symport of cations (Na+, K+) coupled with chloride (Cl-). They are divided into K+-Cl- outward transporters (KCCs), the Na+-K+-Cl- (NKCCs) and Na+-Cl- (NCCs) inward transporters, the cation chloride cotransporter interacting protein CIP1, and the polyamine transporter CCC9. KCCs and N(K)CCs are established in the genome since eukaryotes and metazoans, respectively.

Molecular bases of K secretory cells in the inner ear: shared and distinct features between birds and mammals.

In the cochlea, mammals maintain a uniquely high endolymphatic potential (EP), which is not observed in other vertebrate groups. However, a high [K] is always present in the inner ear endolymph. Here, we show that Kir4.

Neto2-null mice have impaired GABAergic inhibition and are susceptible to seizures.

Neto2 is a transmembrane protein that interacts with the neuron-specific K(+)-Cl(-) cotransporter (KCC2) in the central nervous system (CNS). Efficient KCC2 transport is essential for setting the neuronal Cl(-) gradient, which is required for fast GABAergic inhibition. Neto2 is required to maintain the normal abundance of KCC2 in neurons, and increases KCC2 function by binding to the active oligomeric form of this cotransporter.

Molecular and evolutionary insights into the structural organization of cation chloride cotransporters.

Cation chloride cotransporters (CCC) play an essential role for neuronal chloride homeostasis. K(+)-Cl(-) cotransporter (KCC2), is the principal Cl(-)-extruder, whereas Na(+)-K(+)-Cl(-) cotransporter (NKCC1), is the major Cl(-)-uptake mechanism in many neurons. As a consequence, the action of the inhibitory neurotransmitters gamma-aminobutyric acid (GABA) and glycine strongly depend on the activity of these two transporters.

A novel regulatory locus of phosphorylation in the C terminus of the potassium chloride cotransporter KCC2 that interferes with N-ethylmaleimide or staurosporine-mediated activation.

The neuron-specific cation chloride cotransporter KCC2 plays a crucial role in hyperpolarizing synaptic inhibition. Transporter dysfunction is associated with various neurological disorders, raising interest in regulatory mechanisms. Phosphorylation has been identified as a key regulatory process.

Evolution of the cation chloride cotransporter family: ancient origins, gene losses, and subfunctionalization through duplication.

The cation chloride cotransporter (CCCs) family comprises of four subfamilies-K(+)-Cl(-) cotransporters (KCCs), Na(+)-K(+)-2Cl(-) cotransporters (NKCCs), and Na(+)-Cl(-) cotransporters (NCCs)-and possibly two additional members-CCC interacting protein (CIP1) and polyamine transporters (CCC9)-as well. Altogether, CCCs can play essential physiological roles in transepithelial ion reabsorption and secretion, cell volume regulation, and inhibitory neurotransmission and so are present across all domains of life. To gain insight into the evolution of this family, we performed a comprehensive phylogenetic analysis using publically available genomic information.

Egr2::cre mediated conditional ablation of dicer disrupts histogenesis of mammalian central auditory nuclei.

Histogenesis of the auditory system requires extensive molecular orchestration. Recently, Dicer1, an essential gene for generation of microRNAs, and miR-96 were shown to be important for development of the peripheral auditory system. Here, we investigated their role for the formation of the auditory brainstem.

KCC2 transport activity requires the highly conserved L₆₇₅ in the C-terminal β1 strand.

The activity of the neuron-specific K(+), Cl(-) co-transporter 2 (KCC2) is required for hyperpolarizing action of GABA and glycine. KCC2-mediated transport therefore plays a pivotal role in neuronal inhibition. Few analyses have addressed the amino acid requirements for transport-competent conformation.

Opposite temperature effect on transport activity of KCC2/KCC4 and N(K)CCs in HEK-293 cells.

Background: Cation chloride cotransporters play essential roles in many physiological processes such as volume regulation, transepithelial salt transport and setting the intracellular chloride concentration in neurons. They consist mainly of the inward transporters NCC, NKCC1, and NKCC2, and the outward transporters KCC1 to KCC4. To gain insight into regulatory and structure-function relationships, precise determination of their activity is required.

Seasonal variation of the effect of extremely diluted agitated gibberellic acid (10e-30) on wheat stalk growth: a multiresearcher study.

The influence of a homeopathic high dilution of gibberellic acid on wheat growth was studied at different seasons of the year. Seedlings were allowed to develop under standardized conditions for 7 days; plants were harvested and stalk lengths were measured. The data obtained confirm previous findings, that ultrahigh diluted potentized gibberellic acid affects stalk growth.

Effects of elm bark extracts from Ulmus laevis on human chorion carcinoma cell lines.

Purpose: The potential of substances from elm bark extracts to affect cancer has been described in several studies. In this study, the anticancer effects of extracts from Ulmus laevis bark were tested in hormone-dependent gynecological tumours using human chorion carcinoma cell lines.

Methods: The molecular-chemical composition of the bark extract was analysed by pyrolysis-field ionisation mass spectrometry.

Differences in the large extracellular loop between the K(+)-Cl(-) cotransporters KCC2 and KCC4.

K(+)Cl(-) cotransporters (KCCs) play fundamental physiological roles in processes such as inhibitory neurotransmission and cell volume regulation. Mammalian genomes encode four distinct KCC paralogs, which share basic transport characteristics but differ significantly in ion affinity, pharmacology, and relative sensitivity to cell volume. Studies to identify divergence in functional characteristics have thus far focused on the cytoplasmic termini.

Opposite effect of membrane raft perturbation on transport activity of KCC2 and NKCC1.

In the majority of neurons, the intracellular Cl(-) concentration is set by the activity of the Na(+)-K(+)-2Cl(-) cotransporter (NKCC1) and the K(+)-Cl(-) cotransporter (KCC2). Here, we investigated the cotransporters' functional dependence on membrane rafts. In the mature rat brain, NKCC1 was mainly insoluble in Brij 58 and co-distributed with the membrane raft marker flotillin-1 in sucrose density flotation experiments.

CIP1 is an activator of the K+-Cl- cotransporter KCC2.

In most neurons, efficient setting of the intracellular Cl(-)-concentration requires the coordinated regulation of the Cl(-)-inward transporter NKCC1 and the Cl(-)-outward transporter KCC2. Previously, the cation-chloride cotransporter interacting protein 1 (CIP1) was shown to inactivate NKCC1. Here, we investigated its role for KCC2 activity.

Regulation of simultaneous synthesis of floral scent terpenoids by the 1,8-cineole synthase of Nicotiana suaveolens.

The white flowers of N. suaveolens emit a complex bouquet of fragrance volatiles. The dominant compounds are benzenoids (e.