A ubiquitous 64-kDa protein is a component of a chloride channel of plasma and intracellular membranes.

Abstract

Chloride channels are present in the plasma and intracellular membranes of most cells. Previously, using the ligand indanyloxyacetic acid (IAA), we purified four major proteins from bovine kidney cortex membrane vesicles. These proteins gave rise to chloride channel activity when reconstituted into phospholipid vesicles. Two ofthese proteins (97 and 27 kDa) were found to be drug-binding proteins by N-terminal sequence analysis. Antibodies raised to the 64-kDa protein stained only this protein on immunoblots, and only this protein was present after purification on an immunoaffinity column. In addition, these same antibodies were able to deplete IAA-94 inhibitable chloride channel activity from solubilized kidney membranes. Of fractions obtained from the gel filtration ofsolubilized kidney membranes, only those containing this 64-kDa protein exhibited measurable chloride channel activity. Immunoblots of a variety of species and cell types, both epithelial and nonepithelial, revealed that this protein is ubiquitous and highly conserved. Immunocytochemistry in CFPAC-1 cells revealed staining for this protein on the apical plasma membrane and in the membranes of intracellular organelles. These results demonstrate that the integral membrane protein p64 is a component of chloride channels present in both epithelial plasma membrane and the membranes of intracellular organelles.

Identification and characterisation of a homologue of p64 in rat tissues

“…We believe that RS43 is distinct from p64 for a number of reasons. Firstly, p64 migrates with an apparent Mr of 64 kDa following SDS-PAGE [10], whereas RS43 migrates with an apparent Mr of 43 kDa. This large difference in Mr makes it unlikely that the two are the same protein.…”

“…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.…”

“…The protein was also unaffected following incubation with endoglycosidase F (our unpublished results), suggesting that it is not glycosylated. In a similar way, p64 protein was purified from microsomal membranes enriched for Golgi markers, and in CFPAC cells it has been immunolocalised to perinuclear vesicles [10]. Finally, there is increasing evidence that C1-channels in endosomes and clathrin-coated vesicles can be activated by protein kinase-A, and phosphorylation of membrane proteins in these locations has caused the appearance of a phosphorylated 65 70 kDa protein [24].…”

“…p64 is a component of a CI-channel and is found in in~racellular membranes [10]. The cDNA encoding the protein 'eas cloned from bovine kidney [11].…”

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

“…The reconstitution of channel activity from pure CLIC1 is highly significant [27], but it is important to stress that the channels have not been shown to correspond to any ion channel normally present in cells. 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].…”