Chloride channels activated by hypotonicity in N2A neuroblastoma cell line

Carpaneto, Armando; Accardi, Alessio; Pisciotta, Marzia; Gambale, Franco

doi:10.1007/s002210050614

Cited by 30 publications

(20 citation statements)

References 14 publications

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“…This value is close to the value obtained by Schmid et al (1998) in cultured pancreatic acinar cells (P glutamate /P Cl 5 0.12) and by Levitan and Garber (1998) in a myeloma cell line (P glutamate /P Cl 5 0.17), but somewhat higher than the value of P glutamate /P Cl 5 0.057 obtained by Carpaneto et al (1999) for the N2A neuroblastoma cell line. Thus, we conclude that the VSOR Cl 2 channel can permit the passage of glutamate anions.…”

Section: Vsor Anion Channels Constituted a Minor Pathway For Glutamatsupporting

confidence: 73%

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress

Liu

Tashmukhamedov

Inoue

et al. 2006

Glia

121

142

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Astrocytes release glutamate upon hyperexcitation in the normal brain, and in response to pathologic insults such as ischemia and trauma. In our experiments, both hypotonic and ischemic stimuli caused the release of glutamate from cultured mouse astrocytes, which occurred with little or no contribution of gap junction hemichannels, vesicle-mediated exocytosis, or reversed operation of the Na-dependent glutamate transporter. Cell swelling and chemical ischemia activated, in cell-attached membrane patches, anionic channels with large unitary conductance (approximately 400 pS) and inactivation kinetics at potentials more positive than +20 mV or more negative than -20 mV. These properties are different from those of volume-sensitive outwardly rectifying (VSOR) Cl- channels, which were also expressed in these cells and exhibited intermediate unitary conductance (approximately 80 pS) and inactivation kinetics at large positive potentials of more than +40 mV. Both maxi-anion channels and VSOR Cl- channels were permeable to glutamate with permeability ratios of glutamate to chloride of 0.21 +/- 0.07 and 0.15 +/- 0.01, respectively. However, the release of glutamate was significantly more sensitive to Gd3+, a blocker of maxi-anion channels, than to phloretin, a blocker of VSOR Cl- channels. We conclude that these two channels jointly represent a major conductive pathway for the release of glutamate from swollen and ischemia-challenged astrocytes, with the contribution of maxi-anion channels being predominant.

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Section: Vsor Anion Channels Constituted a Minor Pathway For Glutamatsupporting

confidence: 73%

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress

Liu

Tashmukhamedov

Inoue

et al. 2006

Glia

121

142

View full text Add to dashboard Cite

show abstract

“…That this is likely to be the case also in vivo is supported by the inhibition of 8-Br-cAMP mediated fluid secretion in perfused inner medullary collecting ducts by chloride channel inhibitors (23), although in this setting inhibition was also seen with bumetanide. A role for chloride channels in osmoregulation has been suggested by their activation by hypertonicity in neuroblastoma cell lines (28), human intestinal cells (29), and HeLa cells (30). Chloride uptake also plays a critical role in the function of the juxtaglomerular apparatus, at least concerning the regulation of the cyclooxygenase 2 expression (31,32).…”

Section: Discussionmentioning

confidence: 99%

Chloride, not sodium, stimulates expression of the γ subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells

Capasso

Rivard

Enomoto

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

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Hypertonicity induced by NaCl, but not by urea or mannitol, up-regulates expression of the ␥ subunit of Na͞K-ATPase in cells of the murine inner medullary collecting duct line (IMCD3) by activation of the Jun kinase 2 (JNK2) pathways. We examined the ionic mediators of the osmosensitive response. An increase in osmolality to 550 milliosmoles per kg of water (mosmol͞kgH 2O) for 48 h by replacement of NaCl with choline chloride did not prevent the up-regulation of the ␥ subunit. Neither Na ؉ ionophores nor inhibitors of cellular Na ؉ uptake altered the up-regulation of the ␥ subunit or JNK activation. Changes in cell cation concentrations driven by incubation in low-K ؉ medium were effective in upregulating the ␣1 subunit of Na͞K-ATPase but did not have any effect on the ␥ subunit. The replacement of NaCl with choline chloride did not down-regulate ␥-subunit expression in cells adapted to hypertonicity. In contrast, the replacement of NaCl with sodium acetate, or pretreatment of cells with the Cl ؊ channel inhibitor 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB) completely blocked ␥-subunit up-regulation, inhibited JNK activation, and caused a significant decrement in cell survival in hypertonic but not isotonic conditions. In adapted cells, replacement of 300 mosmol͞kgH 2O NaCl with sodium acetate resulted in downregulation of the ␥ subunit. In conclusion, we describe a Na ؉ -independent, Cl ؊ -dependent mechanism for hypertonicity-mediated activation of the JNK and the subsequent synthesis of the ␥ subunit of Na͞K-ATPase, which are necessary for cellular survival in these anisotonic conditions.

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“…The osmolarity of both solutions was determined to be 390 mosM. ZnCl 2 was used to block endogenous channel activity specific to Neuro 2A cells (30,31). BoNT reconstitution and channel insertion was achieved by supplementing 5 g/ml BoNT holotoxin or HC to the pipette solution, which was set to an endosomal pH of 5.3.…”

Section: Methodsmentioning

confidence: 99%

Crucial Role of the Disulfide Bridge between Botulinum Neurotoxin Light and Heavy Chains in Protease Translocation across Membranes

Fischer

Montal

2007

Journal of Biological Chemistry

156

167

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Clostridial botulinum neurotoxins (BoNTs) exert their neuroparalytic action by arresting synaptic exocytosis. Intoxication requires the disulfide-linked, di-chain protein to undergo conformational changes in response to pH and redox gradients across the endosomal membrane with consequent formation of a protein-conducting channel by the heavy chain (HC) that translocates the light chain (LC) protease into the cytosol. Here, we investigate the role of the disulfide bridge in the dynamics of protein translocation. We utilize a single channel/single molecule assay to characterize in real time the BoNT channel and chaperone activities in Neuro 2A cells under conditions that emulate those prevalent across endosomes. We show that the disulfide bridge must remain intact throughout LC translocation; premature reduction of the disulfide bridge after channel formation arrests translocation. The disulfide bridge must be on the trans compartment to achieve productive translocation of LC; disulfide disruption on the cis compartment or within the bilayer during translocation aborts it. We demonstrate that a peptide linkage between LC and HC in place of a disulfide bridge is insufficient for productive LC translocation. The disulfide linkage, therefore, dictates the outcome of translocation: productive passage of cargo or abortive channel occlusion by cargo. Based on these and previous findings we suggest a sequence of events for BoNT LC translocation to be HC insertion, coupled LC unfolding, and protein conduction through the HC channel in an N to C terminus orientation and ultimate release of the LC from the HC by reduction of the disulfide bridge concomitant with LC refolding in the cytosol. Clostridium botulinum neurotoxins (BoNTs)2 inhibit synaptic exocytosis in peripheral cholinergic synapses, thereby causing flaccid paralysis (1). BoNTs are synthesized as a single polypeptide chain with a molecular mass of ϳ150 kDa. The BoNT polypeptide is then proteolytically cleaved by bacterial or host proteases into the activated di-chain form: an ϳ50-kDa light chain (LC) and an ϳ100-kDa heavy chain (HC). The LC and HC are cross-linked by a disulfide bond between the two chains. Structurally, BoNTs consist of three modules (1-4): The N-terminal LC is the catalytic domain, and the HC comprises the translocation domain (the N-terminal half) and the receptor-binding domain (the C-terminal half). The LCs of six of the seven isoforms of BoNT, designated A-G, have been crystallized and all share structural similarity to the Zn 2ϩ -metalloprotease thermolysin (2, 4 -11). BoNT LCs are sequencespecific endopeptidases that cleave unique components of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, the synaptic vesicle fusion complex required for membrane fusion (12-14).BoNTs enter cells by receptor-mediated endocytosis (1, 15). It has been widely recognized that, with the exception of BoNT/D, BoNT entry into neuronal cells requires surface receptors involving a specific ganglioside, namely GT1b (16 -1...

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Chloride channels activated by hypotonicity in N2A neuroblastoma cell line

Cited by 30 publications

References 14 publications

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress

Roles of two types of anion channels in glutamate release from mouse astrocytes under ischemic or osmotic stress

Chloride, not sodium, stimulates expression of the γ subunit of Na/K-ATPase and activates JNK in response to hypertonicity in mouse IMCD3 cells

Crucial Role of the Disulfide Bridge between Botulinum Neurotoxin Light and Heavy Chains in Protease Translocation across Membranes

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