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The neuronal K+Cl- co-transporter 2 (Slc12a5) modulates insulin secretion.

ABSTRACT: Intracellular chloride concentration ([Cl-]i) in pancreatic ?-cells is kept above electrochemical equilibrium due to the predominant functional presence of Cl- loaders such as the Na+K+2Cl- co-transporter 1 (Slc12a2) over Cl-extruders of unidentified nature. Using molecular cloning, RT-PCR, Western blotting, immunolocalization and in vitro functional assays, we establish that the "neuron-specific" K+Cl- co-transporter 2 (KCC2, Slc12a5) is expressed in several endocrine cells of the pancreatic islet, including glucagon secreting ?-cells, but particularly in insulin-secreting ?-cells, where we provide evidence for its role in the insulin secretory response. Three KCC2 splice variants were identified: the formerly described KCC2a and KCC2b along with a novel one lacking exon 25 (KCC2a-S25). This new variant is undetectable in brain or spinal cord, the only and most abundant known sources of KCC2. Inhibition of KCC2 activity in clonal MIN6 ?-cells increases basal and glucose-stimulated insulin secretion and Ca2+ uptake in the presence of glibenclamide, an inhibitor of the ATP-dependent potassium (KATP)-channels, thus suggesting a possible mechanism underlying KCC2-dependent insulin release. We propose that the long-time considered "neuron-specific" KCC2 co-transporter is expressed in pancreatic islet ?-cells where it modulates Ca2+-dependent insulin secretion.

SUBMITTER: Kursan S

PROVIDER: S-EPMC5431760 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

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The neuronal K+Cl- co-transporter 2 (Slc12a5) modulates insulin secretion.

Kursan Shams S McMillen Timothy S TS Beesetty Pavani P Dias-Junior Eduardo E Almutairi Mohammed M MM Sajib Abu A AA Kozak J Ashot JA Aguilar-Bryan Lydia L Di Fulvio Mauricio M

Scientific reports 20170511 1

Intracellular chloride concentration ([Cl-]i) in pancreatic β-cells is kept above electrochemical equilibrium due to the predominant functional presence of Cl- loaders such as the Na+K+2Cl- co-transporter 1 (Slc12a2) over Cl-extruders of unidentified nature. Using molecular cloning, RT-PCR, Western blotting, immunolocalization and in vitro functional assays, we establish that the "neuron-specific" K+Cl-< ...[more]

PMID: 28496181

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Project description: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 GABAA-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. In this study, we investigated the putative effect of KCC2/NBCe1 interaction in baseline and the stimulus-induced phosphorylation pattern and function of KCC2. Primary mouse hippocampal neuronal cultures from wildtype (WT) and Nbce1-deficient mice, as well as HEK-293 cells stably transfected with KCC2WT, were used. The results show that KCC2 and NBCe1 are interaction partners in the mouse brain. In HEKKCC2 cells, pharmacological inhibition of NBCs with S0859 prevented staurosporine- and 4-aminopyridine (4AP)-induced KCC2 activation. In mature cultures of hippocampal neurons, however, S0859 completely inhibited postsynaptic GABAAR and, thus, could not be used as a tool to investigate the role of NBCs in GABA-dependent neuronal networks. In Nbce1-deficient immature hippocampal neurons, baseline phosphorylation of KCC2 at S940 was downregulated, compared to WT, and exposure to staurosporine failed to reduce pKCC2 S940 and T1007. In Nbce1-deficient mature neurons, baseline levels of pKCC2 S940 and T1007 were upregulated compared to WT, whereas after 4AP treatment, pKCC2 S940 was downregulated, and pKCC2 T1007 was further upregulated. Functional experiments showed that the levels of GABAAR reversal potential, baseline intracellular [Cl-], Cl- extrusion, and baseline intracellular pH were similar between WT and Nbce1-deficient neurons. Altogether, our data provide a primary description of the properties of KCC2/NBCe1 protein-protein interaction and implicate modulation of stimulus-mediated phosphorylation of KCC2 by NBCe1/KCC2 interaction-a mechanism with putative pathophysiological relevance.

Project description:BackgroundA significant mechanism of salt-tolerance in rice is the ability to remove Na+ and Cl- in the leaf sheath, which limits the entry of these toxic ions into the leaf blade. The leaf sheath removes Na+ mainly in the basal parts, and Cl- mainly in the apical parts. These ions are unloaded from the xylem vessels in the peripheral part and sequestered into the fundamental parenchyma cells at the central part of the leaf sheath.ResultsThis study aimed to identify associated Na+ and Cl- transporter genes with this salt removal ability in the leaf sheath of rice variety FL 478. From 21 known candidate Na+ and Cl- transporter rice genes, we determined the salt responsiveness of the expression of these genes in the basal and apical parts, where Na+ or Cl- ions were highly accumulated under salinity. We also compared the expression levels of these transporter genes between the peripheral and central parts of leaf sheaths. The expression of 8 Na+ transporter genes and 3 Cl- transporter genes was up-regulated in the basal and apical parts of leaf sheaths under salinity. Within these genes, OsHKT1;5 and OsSLAH1 were expressed highly in the peripheral part, indicating the involvement of these genes in Na+ and Cl- unloading from xylem vessels. OsNHX2, OsNHX3, OsNPF2.4 were expressed highly in the central part, which suggests that these genes may function in sequestration of Na+ and Cl- in fundamental parenchyma cells in the central part of leaf sheaths under salinity. Furthermore, high expression levels of 4 candidate genes under salinity were associated with the genotypic variation of salt removal ability in the leaf sheath.ConclusionsThese results indicate that the salt removal ability in rice leaf sheath may be regulated by expressing various Na+ or Cl- transporter genes tissue-specifically in peripheral and central parts. Moreover, some genes were identified as candidates whose expression levels were associated with the genotypic variation of salt removal ability in the leaf sheath. These findings will enhance the understanding of the molecular mechanism of salt removal ability in rice leaf sheath, which is useful for breeding salt-tolerant rice varieties.

Dataset Information

The neuronal K+Cl- co-transporter 2 (Slc12a5) modulates insulin secretion.

Publications

The neuronal K<sup>+</sup>Cl<sup>-</sup> co-transporter 2 (Slc12a5) modulates insulin secretion.

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