Thierry Coppola

Cloning provides evidence for a family of inward rectifier and G-protein coupled K+ channels in the brain

MbIRK3, mbGIRK2 and mbGIRK3 K+ channels cDNAs have been cloned from adult mouse brain. These eDNAs encode polypeptides of 445, 414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane‐spanning domains M1 and M2 and a pore‐forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMK1 and GIRK1. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRK1 (60%) than to ROMK1 and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K+‐selective currents very similar to IRK1 and GIRK1, respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G‐protein coupled inward rectifyer K+ channels which has been observed through their functional coupling with co‐expressed δ‐opioid receptors. These results provide the first evidence that the GIRK family, as the IRK family, is composed of multiple genes with members specifically expressed in the nervous system.

Spadin, a Sortilin-Derived Peptide, Targeting Rodent TREK-1 Channels: A New Concept in the Antidepressant Drug Design

We found that spadin, a natural peptide derived from sortilin, blocks the mouse TREK-1 channel and might be an efficient and fast-acting antidepressant.

ICER induced by hyperglycemia represses the expression of genes essential for insulin exocytosis

The GTPases Rab3a and Rab27a and their effectors Granuphilin/Slp4 and Noc2 are essential regulators of neuroendocrine secretion. Chronic exposure of pancreatic β‐cells to supraphysiological glucose levels decreased selectively the expression of these proteins. This glucotoxic effect was mimicked by cAMP‐raising agents and blocked by PKA inhibitors. We demonstrate that the transcriptional repressor ICER, which is induced in a PKA‐dependent manner by chronic hyperglycemia and cAMP‐raising agents, is responsible for the decline of the four genes. ICER overexpression diminished the level of Granuphilin, Noc2, Rab3a and Rab27a by binding to cAMP responsive elements located in the promoters of these genes and inhibited exocytosis of β‐cells in response to secretagogues. Moreover, the loss in the expression of the genes of the secretory machinery caused by glucose and cAMP‐raising agents was prevented by an antisense construct that reduces ICER levels. We propose that induction of inappropriate ICER levels lead to defects in the secretory process of pancreatic β‐cells possibly contributing, in conjunction with other known deleterious effects of hyperglycemia, to defective insulin release in type 2 diabetes.

Complexin I regulates glucose-induced secretion in pancreatic β-cells

The neuronal-specific protein complexin I (CPX I) plays an important role in controlling the Ca2+-dependent neurotransmitter release. Since insulin exocytosis and neurotransmitter release rely on similar molecular mechanisms and that pancreatic β-cells and neuronal cells share the expression of many restricted genes, we investigated the potential role of CPX I in insulin-secreting cells. We found that pancreatic islets and several insulin-secreting cell lines express high levels of CPX I. The β-cell expression of CPX I is mediated by the presence of a neuron restrictive silencer element located within the regulatory region of the gene. This element bound the transcriptional repressor REST, which is found in most cell types with the exception of mature neuronal cells and β-cells. Overexpression of CPX I or silencing of the CPX I gene (Cplx1) by RNA interference led to strong impairment in β-cell secretion in response to nutrients such as glucose, leucine and KCl. This effect was detected both in the early and the sustained secretory phases but was much more pronounced in the early phase. We conclude that CPX I plays a critical role in β-cells in the control of the stimulated-exocytosis of insulin.

Molecular cloning of a murine N-type calcium channel α1 subunit: Evidence for isoforms, brain distribution, and chromosomal localization

A cDNA encoding a N‐type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N‐type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N‐type Ca2+ channel gene has been localized on the chromosome 2, band A.A cDNA encoding a N-type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N-type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N-type Ca2+ channel gene has been localized on the chromosome 2, band A.

A role for glutamate transporters in the regulation of insulin secretion.

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.

Research lettersMolecular cloning of a murine N-type calcium channel α1 subunit: Evidence for isoforms, brain distribution, and chromosomal localization

A cDNA encoding a N‐type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N‐type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N‐type Ca2+ channel gene has been localized on the chromosome 2, band A.A cDNA encoding a N-type Ca2+ channel has been cloned from the murine neuroblastoma cell line N1A103. The open reading frame encodes a protein of 2,289 amino acids (257 kDa). Analysis of different clones provided evidence for the existence of distinct isoforms of N-type channels. High levels of mRNA were found in the pyramidal cell layers CA1, CA2 and CA3 of the hippocampus, in the dentate gyrus, in the cortex layers 2 and 4, in the subiculum and the habenula. The N-type Ca2+ channel gene has been localized on the chromosome 2, band A.

Neurotensin protects pancreatic beta cells from apoptosis

The survival of pancreatic beta cells depends on the balance between external cytotoxic and protective molecular systems. The neuropeptide neurotensin (NT) has been shown to regulate certain functions of the endocrine pancreas including insulin and glucagon release. However, the mechanism of action of NT as well as the identification of receptors involved in the pancreatic functions of the peptide remained to be studied. We demonstrate here that NT is an efficient protective agent of pancreatic beta cells against cytotoxic agents. Both beta-TC3 and INS-1E cell lines and the mouse pancreatic islet cells express the three known NT receptors. The incubation of beta cells with NT protects cells from apoptosis induced either by staurosporine or by IL-1beta. In beta-TC3 cells, NT activates both MAP and PI-3 kinases pathways and strongly reduces the staurosporine or the Il-1beta-induced caspase-3 activity by a mechanism involving Akt activation. The NTSR2 agonist levocabastine displays the same protective effect than NT whereas the NTSR1 antagonist is unable to block the effect of NT suggesting the predominant involvement of the NTSR2 in the action of NT on beta cells. These results clearly indicate for the first time that NT is able to protect endocrine beta cells from external cytotoxic agents, a role well correlated with its release in the circulation after a meal.

Antibodies reveal the cytolocalization and subunit structure of the 1,4-dihydropyridine component of the neuronal Ca2+ channel

Rabbit brain synaptosomes bind the 1,4-dihydropyridine derivative (+)[3H]-PN 200-110 with an equilibrium dissociation constant of 0.04 nM and a maximal binding capacity of 400 fmol/mg of protein. Using polyclonal antibodies raised against the different components of the skeletal muscle 1,4-dihydropyridine receptor, we have demonstrated that the brain and muscle receptors share the same subunit composition comprising a large polypeptide chain of Mr 140,000 associated by disulfide bridges with a smaller peptide of Mr 32,000. These antibodies have been used in immunofluorescence staining of brain sections. They reveal a distribution of the Ca2+ channel protein similar to that of 1,4-dihydropyridine binding sites with (+)[3H]PN 200-110 by the autoradiographic technique.

Neurotensin and its receptors in the control of glucose homeostasis

The pharmacological roles of the neuropeptide neurotensin through its three known receptors are various and complex. Neurotensin is involved in several important biological functions including analgesia and hypothermia in the central nervous system and also food intake and glucose homeostasis in the periphery. This review focuses on recent works dealing with molecular mechanisms regulating blood glucose level and insulin secretion upon neurotensin action. Investigations on crucial cellular components involved in the protective effect of the peptide on beta cells are also detailed. The role of xenin, a neurotensin-related peptide, on the regulation of insulin release by glucose-dependent insulinotropic polypeptide is summarized. The last section comments on the future research areas which should be developed to address the function of new effectors of the neurotensinergic system in the endocrine pancreas.