Purification and Reconstitution of Chloride Channels from Kidney and Trachea

Landry, Donald W.; Akabas, Myles H.; Redhead, Christopher; Edelman, Aleksander; Cragoe, Edward J.; Al‐Awqati, Qais

doi:10.1126/science.2472007

Cited by 187 publications

(123 citation statements)

References 28 publications

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“…However, we have previously identified homologues of p64 in neuronal tissue [12]. It is interesting to note that during the drug-affinity purification of p64, a protein of approximately 40 kDa was co-purified [10,23]. Whether RS43 and this 40 kDa protein are related remains to be seen.…”

Section: Discussionmentioning

confidence: 99%

Identification and characterisation of a homologue of p64 in rat tissues

1996

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Previous work has suggested that the gene encoding p64, a component of a bovine kidney intraceHular chloride channel, may be a member of a gene family. We have raised a polyclonai antibody to an E. coli fusion protein which has sequence similarity to p64. Immunoblotting detected a protein in rat brain, kidney, liver and lung. In rat brain, the protein was enriched in cerebellar microsomal membranes. Western blot analyses of denaturing and blue native polyacrylamide gels indicated that the protein is a single non-disulphide-linked polypeptide chain with an apparent Mr of 43 kDa that contributes to a native protein complex with an apparent Mr of 130 kDa.

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Section: Discussionmentioning

confidence: 99%

Identification and characterisation of a homologue of p64 in rat tissues

1996

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“…Cells, DNA, and General Methods-Plasmid pNH⌬P encoding p64 downstream of a T7 promoter has been previously described (1). HeLa cells, recombinant vaccinia encoding T7 RNA polymerase (26), and plasmids encoding mouse Fyn (pBS-Fyn) (27,28), glutathione S-transferase (GST 1 ; pGEX-KG) (29), and GST-Fyn fusion proteins (30) were obtained from Dr. Andrey Shaw, Washington University, St. Louis, MO.…”

Section: Methodsmentioning

confidence: 99%

“…p64 is a chloride channel protein originally discovered by purification of chloride channel activity from bovine kidney microsomes (1). cDNA encoding p64 was subsequently cloned (2).…”

mentioning

confidence: 99%

Regulation of the Bovine Kidney Microsomal Chloride Channel p64 by p59 , a Src Family Tyrosine Kinase

Edwards

Kapadia

2000

Journal of Biological Chemistry

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p64 is a chloride channel of intracellular membranes which is present in regulated secretory vesicles. Mechanisms by which the p64 channel could be regulated are largely unknown. p59fyn is a non-receptor tyrosine kinase of the Src family that has been implicated in a variety of intracellular signaling events. The N-terminal portion of p64 has several potential binding sites for Src family SH2 domains. In this paper, we demonstrate that p64 becomes tyrosine phosphorylated when co-expressed with p59 fyn in HeLa cells. We show that co-expression of p64 with p59 fyn renders p64 a ligand for the SH2 domain of p59 fyn and this SH2 binding is eliminated by treating p64 with alkaline phosphatase. Using sitedirected mutagenesis, we find that tyrosine 33 in the p64 sequence is necessary for SH2 binding. We also characterized p64-p59 fyn interactions using native material from bovine kidney. We found that a small fraction of native kidney p64 can bind Fyn SH2 in vitro. Immunoprecipitation of p64 from solubilized kidney membranes yields a kinase activity with the same mobility by SDSpolyacrylamide gel electrophoresis as authentic bovine p59fyn . Finally, we demonstrate that co-expression of p64 and p59 fyn in HeLa cells results in enhanced p64-associated chloride channel activity.

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“…We therefore cannot yet be sure that the behaviour of CLIC proteins in itro is relevant to their behaviour in i o. p64 itself was first isolated by indanoyloxyacetic acid (IAA) affinity chromatography and identified as a putative anion channel protein because anti-p64 antibodies reduced IAA-sensitive Cl − transport in a kidney microsomal membrane vesicle fraction [36]. However, although kidney microsomes had previously been demonstrated to contain Cl − channels [37], the channels did not correspond to the anion channel activity recorded from p64-containing HeLa cell membranes [38]. Unfortunately, the effect of IAA compounds was not reported in the latter study.…”

Section: Identity Of Intracellular Anion Channelsmentioning

confidence: 99%

Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms

Suginta

Karoulias

Aitken

et al. 2001

Biochemical Journal

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Mammalian chloride intracellular channel (CLIC) (p64-related) proteins are widely expressed, with an unusual dual localization as both soluble and integral membrane proteins. The molecular basis for their cellular localization and ion channel activity remains unclear. To help in addressing these problems, we identified novel rat brain CLIC4 (p64H1) binding partners by affinity chromatography, mass spectrometric analysis and microsequencing. Brain CLIC4 binds dynamin I, α-tubulin, β-actin, creatine kinase and two 14-3-3 isoforms ; the interactions are confirmed in i o by immunoprecipitation. Gel overlay and reverse pull-down assays indicate that the binding of CLIC4 to dynamin I and 14-3-3ζ is direct. In HEK-293 cells, biochemical and immunofluorescence analyses show partial co-localization of

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Purification and Reconstitution of Chloride Channels from Kidney and Trachea

Cited by 187 publications

References 28 publications

Identification and characterisation of a homologue of p64 in rat tissues

Identification and characterisation of a homologue of p64 in rat tissues

Regulation of the Bovine Kidney Microsomal Chloride Channel p64 by p59 , a Src Family Tyrosine Kinase

Chloride intracellular channel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complex containing actin, tubulin and 14-3-3 isoforms

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