A 29 kDa Intracellular Chloride Channel p64H1 Is Associated with Large Dense-Core Vesicles in Rat Hippocampal Neurons

Abstract

A novel class of intracellular chloride channels, the p64 family, has been found on several types of vesicles. These channels, acting in concert with the electrogenic proton pump, regulate the pH of the vesicle interior, which is critical for vesicular function. Here we describe the molecular cloning of p64H1, a p64 homolog, from both human and cow. Northern blot analysis showed that p64H1 is expressed abundantly in brain and retina, whereas the other members of this family (e.g., p64 and NCC27) are expressed only at low levels in these tissues. Immunohistochemical analysis of p64H1 in rat brain, using an affinity-purified antibody, revealed a high level of expression in the limbic system-the hippocampal formation, the amygdala, the hypothalamus, and the septum. Immunoelectron microscopic analysis of p64H1 in hippocampal neurons demonstrated a striking association between p64H1 and large densecore vesicles (LDCVs) and microtubules. In contrast, very low p64H1 labeling was found in perikarya or associated with small synaptic vesicles (SSVs) in axonal profiles. Immunoblot analysis confirmed that p64H1 is colocalized with heavy membrane fractions containing LDCVs rather than the fractions containing SSVs. These results suggest that p64H1-mediated Cl Ϫ permeability may be involved in the maintenance of low internal pH in LDCVs and in the maturation of LDCVs and the biogenesis of functional neuropeptides.

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

“…Indirect immunofluorescence, cell fractionation and immunoblotting studies have localized native and recombinant CLIC4 proteins both to the cytosol and to intracellular membranes. A similar, very unusual, dual localization is characteristic of many CLIC and p64-related proteins [18][19][20][21][22][23][24].…”

“…The brain CLIC4 protein complex also contains α-tubulin. Interestingly, a previous immunohistochemical study [21] revealed a close association of CLIC4 with rat brain microtubules, in addition to partial co-localization with large dense core neurosecretory vesicles. Our results linking CLIC4 and tubulin at the protein level reflect the possibility that dynamin and tubulin might interact in i o, which is consistent with the original characterization of brain dynamin as a microtubule-binding protein [39].…”

“…CLIC4 exists in cells as both a soluble and a membrane-associated protein [16,17,[21][22][23]. Proteolytic digestion of membraneassociated rat brain CLIC4 translated in itro in the presence of microsomes showed the membrane-associated form to be a single-pass integral membrane protein with a small N-terminal luminal domain and a large cytoplasmic domain [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]. Rat brain CLIC4 has also been localized to large dense core vesicles in neurosecretory cells [21]. Such a protean distribution could reflect a shuttling of CLIC4 between the cytoplasm and different endomembrane systems or an involvement of the protein in a widespread cell biological process such as membrane trafficking or vesicle transport.…”

“…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.…”