New mammalian chloride channel identified by expression cloning

Paulmichl, Markus; Li, Yi; Wickman, Kevin; Ackerman, Michael J.; Peralta, Ernest G.; Clapham, David E.

doi:10.1038/356238a0

Cited by 324 publications

(289 citation statements)

References 27 publications

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“…To identify the protein-protein interactions responsible for this regulation, we synthesized a biotin-tagged peptide corresponding to this sequence and used it as a probe to identify high affinity protein interactions dependent on this motif. From a high content, high density protein array derived from a redundant human brain cDNA expression library (37,200 clones) (43,44,46), we identified a number of novel, potential integrinregulating proteins (Fig. 1A).…”

Section: Kvgffkr-specific Binding Proteins Identified From High Densimentioning

confidence: 99%

“…However, hydrophobicity analysis indicates that ICln lacks the transmembrane helices necessary for channel-pore formation by most vertebrates. When expressed in Xenopus oocytes, it mediates a nucleotide-sensitive outwardly rectifying chloride channel closely resembling the biophysical properties of swelling-dependent chloride channels involved in regulation of cell volume decrease after cell swelling (36,37). It is found in the cytosol in cells-at-rest but is associated with the cell membrane following a hypotonic challenge.…”

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confidence: 99%

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ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

Larkin

Murphy

Reilly

et al. 2004

Journal of Biological Chemistry

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A critical role for the conserved ␣-integrin cytoplasmic motif, KVGFFKR, is recognized in the regulation of activation of the platelet integrin ␣ IIb ␤ 3 . To understand the molecular mechanisms of this regulation, we sought to determine the nature of the protein interactions with this cytoplasmic motif. We used a tagged synthetic peptide, biotin-KVGFFKR, to probe a high density protein expression array (37,200 recombinant human proteins) for high affinity interactions. A number of potential integrin-binding proteins were identified. One such protein, a chloride channel regulatory protein, ICln, was characterized further because its affinity for the integrin peptide was highest as was its expression in platelets. We verified the presence of ICln in human platelets by PCR, Western blots, immunohistochemistry, and its co-association with ␣ IIb ␤ 3 by surface plasmon resonance. The affinity of this interaction was 82.2 ؎ 24.4 nM in a cell free assay. ICln co-immunoprecipitates with ␣ IIb ␤ 3 in platelet lysates demonstrating that this interaction is physiologically relevant. Furthermore, immobilized KVGFFKR peptides, but not control KAAAAAR peptides, specifically extract ICln from platelet lysates. Acyclovir (100 M to 5 mM), a pharmacological inhibitor of the ICln chloride channel, specifically inhibits integrin activation (PAC-1 expression) and platelet aggregation without affecting CD62 P expression confirming a specific role for ICln in integrin activation. In parallel, a cell-permeable peptide corresponding to the potential integrin-recognition domain on ICln (AKFEEE, 10 -100 M) also inhibits platelet function. Thus, we have identified, verified, and characterized a novel functional interaction between the platelet integrin and ICln, in the platelet membrane.

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Section: Kvgffkr-specific Binding Proteins Identified From High Densimentioning

confidence: 99%

mentioning

confidence: 99%

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

Larkin

Murphy

Reilly

et al. 2004

Journal of Biological Chemistry

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“…1 The roles of VSOR in cell volume regulation, proliferation, migration and cell death are well established, but its molecular identity has not fully been clarified until recently. 1,2 In the 1990s, several proteins, including P-glycoprotein, pI cln , and ClC-3, were proposed as molecular identities of VSOR, [3][4][5] but none of them has survived scrutiny. [6][7][8][9][10][11][12] In 2014, 2 research groups independently reported that the leucine-rich repeat containing 8A (LRRC8A), which has 4 transmembrane domains and a leucine-rich repeat (LRR) domain at the C-terminus, is a core factor of VSOR in human cells.…”

Section: Introductionmentioning

confidence: 99%

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

et al. 2016

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The broadly expressed volume-sensitive outwardly rectifying anion channel (VSOR, also called VRAC) plays essential roles in cell survival and death. Recent findings have suggested that LRRC8A is a core component of VSOR in human cells. In the present study, VSOR currents were found to be largely reduced by siRNA against LRRC8A in mouse C127 cells as well. In contrast, LRRC8A knockdown never affected activities of 4 other types of anion channel activated by acid, Ca 2C , patch excision or cAMP. While cisplatin-resistant KCP-4 cells poorly expressed endogenous VSOR activity, molecular expression levels of LRRC8A, LRRC8D and LRRC8E were indistinguishable between VSOR-deficient KCP-4 cells and the parental VSOR-rich KB cells. Furthermore, overexpression of LRRC8A alone or together with LRRC8D or LRRC8E in KCP-4 cells failed to restore VSOR activity. These results show that deficiency of VSOR currents in KCP-4 cells is not due to insufficient expression of the LRRC8A/D/ E gene, suggesting an essential involvement of some other factor(s), and indicate that further study is required to better understand the complexities of the molecular determinants of VSOR, including the precise role of LRRC8 proteins.

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“…Furthermore I Cl(swell) is insensitive to changes in calcium concentrations (33). Despite much effort and several candidates, P-glycoprotein (P-gp) (42), pICln (30) and , none has stood the test of time, and the molecular identity of the channel responsible for I Cl(swell) and its mechanism of activation remain unknown. Extracellular acidification has been reported to both activate and inhibit anion channel activities in different cell types.…”

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Extracellular acidification elicits a chloride current that shares characteristics with I_Cl(swell)

Nobles

Higgins

Sardini

2004

American Journal of Physiology-Cell Physiology

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Nobles, Muriel, Christopher F. Higgins, and Alessandro Sardini. Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell). Am J Physiol Cell Physiol 287: C1426 -C1435, 2004. First published August 11, 2004 doi:10.1152/ ajpcell.00549.2002-A Cl Ϫ current activated by extracellular acidification, ICl(pHac), has been characterized in various mammalian cell types. Many of the properties of ICl(pHac) are similar to those of the cell swelling-activated Cl Ϫ current ICl(swell): ion selectivity (I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ Ͼ F Ϫ ), pharmacology [ICl(pHac) is inhibited by 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (DIDS), 1,9-dideoxyforskolin (DDFSK), diphenylamine-2-carboxylic acid (DPC), and niflumic acid], lack of dependence on intra-or extracellular Ca 2ϩ , and presence in all cell types tested. ICl(pHac) differs from ICl(swell) in three aspects: 1) its rate of activation and inactivation is very much more rapid, currents reaching a maximum in seconds rather than minutes; 2) it exhibits a slow voltage-dependent activation in contrast to the fast voltage-dependent activation and time-and voltage-dependent inactivation observed for ICl(swell); and 3) it shows a more pronounced outward rectification. Despite these differences, study of the transition between the two currents strongly suggests that ICl(swell) and ICl(pHac) are related and that extracellular acidification reflects a novel stimulus for activating I Cl(swell) that, additionally, alters the biophysical properties of the channel.cell swelling-activated chloride current; patch clamp; pH A CHLORIDE CURRENT ACTIVATED by cell swelling (I Cl(swell) ) has been characterized in many different cell types (for review see Refs. 26 and 37), and it is thought to play a role in regulatory cell volume decrease (RVD) in response to hypotonicity. I Cl(swell) exhibits outward rectification, fast voltage-dependent activation, and time-and voltage-dependent inactivation at potentials more positive than ϩ40 mV. The relative anion selectivity for the swelling-activated Cl Ϫ channel is SCN Ϫ Ͼ I Ϫ Ͼ Br Ϫ Ͼ Cl Ϫ Ͼ F Ϫ Ͼ gluconate; this sequence is referred to as "Eisenman sequence I" and indicates a weak interaction between the channel pore and the permeation anion. A number of different substances, including the stilbene derivative 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (DIDS) (9, 35), the anti-inflammatory drug niflumic acid (20), the antiestrogen drug tamoxifen (43), diphenylamine-2-carboxylic acid (DPC) (22), and 1,9-dideoxyforskolin (DDFSK) (9), inhibit I Cl(swell) . Furthermore I Cl(swell) is insensitive to changes in calcium concentrations (33). Despite much effort and several candidates, P-glycoprotein (P-gp) (42), pICln (30) and ClC-3 (12), none has stood the test of time, and the molecular identity of the channel responsible for I Cl(swell) and its mechanism of activation remain unknown. Extracellular acidification has been reported to both activate and inhibit anion channel activities in different cell types. Extracellular...

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New mammalian chloride channel identified by expression cloning

Cited by 324 publications

References 27 publications

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

ICln, a Novel Integrin αIIbβ3-Associated Protein, Functionally Regulates Platelet Activation

Specific and essential but not sufficient roles of LRRC8A in the activity of volume-sensitive outwardly rectifying anion channel (VSOR)

Extracellular acidification elicits a chloride current that shares characteristics with I_Cl(swell)

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