Rat Brain p64H1, Expression of a New Member of the p64 Chloride Channel Protein Family in Endoplasmic Reticulum

Abstract

Many plasma membrane Cl؊ channels have been cloned, including the cystic fibrosis transmembrane conductance regulator and several members of the voltage-gated ClC family. In contrast, very little is known about the molecular identity of intracellular Cl ؊ channels. We used a polymerase chain reaction-based approach to identify candidate genes in mammalian brain and cloned the cDNA corresponding to rat brain p64H1. This encoded a microsomal membrane protein of predicted M r 28,635 homologous to the putative intracellular bovine kidney Cl ؊ channel p64. In situ mRNA hybridization histochemistry showed marked expression in hippocampus and cerebellum, and in vitro expression revealed a large cytoplasmic domain, one membranespanning segment, and a small nonglycosylated N-terminal luminal domain. The predicted protein contained consensus phosphorylation sites for protein kinase C and protein kinase A, and protein kinase C-mediated phosphorylation increased the M r of p64H1 to ϳ43,000, characteristic of the native protein in Western blots. Recombinant p64H1 was immunolocalized to the endoplasmic reticulum of human embryonic kidney 293 and HT-4 cells, and incorporation of human embryonic kidney 293 endoplasmic reticulum vesicles into planar lipid bilayers gave rise to intermediate conductance, outwardly rectifying anion channels. Although p64H1 is the first intracellular Cl ؊ channel component or regulator to be identified in brain, Northern blotting revealed transcripts in many other rat tissues. This suggests that p64H1 may contribute widely to intracellular Cl ؊ transport.

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

“…For example, when recombinant rat brain CLIC4 is transiently expressed in rat hippocampal HT-4 cells, it is directed primarily to the endoplasmic reticulum [17]. In contrast, whereas human CLIC4 partly co-localizes with caveolae in a pancreatic cell line [22], the mouse homologue of CLIC4 (mtCLIC) is markedly, but not exclusively, localized to mitochondria [23].…”

“…For affinity purification of CLIC4-binding proteins, we mixed 100 ml of rat brain cytosol (500 mg of protein) in 0.32 M sucrose (prepared as described previously [17]) with an equal volume of binding buffer [50 mM Tris\HCl (pH 7.4)\50 mM NaCl\1 mM EDTA\1 mM EGTA\5 % (v\v) glycerol] containing protease inhibitors, and precleared it by incubation for 3 h at 4 mC with GST attached to glutathione beads. The precleared cytosol was incubated overnight at 4 mC with CLIC4cd GST fusion protein attached to glutathione beads (35 mg of protein\ml of bead slurry) with continuous gentle mixing.…”

“…A cDNA encoding the cytoplasmic domain (CLIC4cd) corresponding to residues 60-253 of CLIC4 (p64H1 [16,17]) was amplified from rat brain p64H1 cDNA by PCR, using the oligonucleotides CGCGGATCCAAAAGGAAGCCTGCACA-TCTGCAGAACTTGGC and ATACCGCTCGAGCTACTT-GGTAAGTCTCTTGGCGACG. The insert was cloned into pGEX-4T1 (Pharmacia) by using engineered 5h BamH1 sites and 3h XhoI sites (underlined), then sequenced.…”

“…CLIC4, originally named p64H1 because it was the first homologue of p64 to be discovered [16], is a 28.6 kDa protein corresponding to a cDNA cloned from rat brain. CLIC4 mRNA is widely expressed in neuronal and non-neuronal tissues ; cells overexpressing recombinant CLIC4 contain intracellular anion channel activity that is absent from mock-transfected cells [17]. Indirect immunofluorescence, cell fractionation and immunoblotting studies have localized native and recombinant CLIC4 proteins both to the cytosol and to intracellular membranes.…”

“…The cDNA encoding human CLIC3, postulated like CLIC1 to be a nuclear membrane Cl − channel protein, was cloned during a yeast two-hybrid screen [20] for partners interacting with the C-terminal domain of an atypical mitogen-activated protein kinase, ERK-7. CLIC4 cDNA, which was cloned from rat brain by using reverse-transcriptasemediated PCR approaches based on the sequence of p64 [16,17], has both human [21,22] and mouse [23] homologues. The mouse protein was named mtCLIC because it localized in part to mitochondria.…”