Molecular Cloning and Expression of a Chloride Ion Channel of Cell Nuclei

Abstract

Ion channels are known to be present on the plasma membrane of virtually all cells and have been found on the membranes of various intracellular organelles. However, until recently they were believed not to occur at the nuclear membrane. In this study we describe the molecular cloning and characterization of a nuclear ion channel protein, designated nuclear chloride channel-27 (NCC27), from the human myelomonocytic cell line, U937. NCC27 is a novel chloride ion channel protein that was found to localize principally to the cell nucleus. Its only known homologue is a bovine chloride ion channel protein (p64) believed to localize to internal organelles. NCC27 therefore represents the first human member of a new class of organellar chloride ion channel proteins.Whereas ion channels have been found on the membranes of various intracellular organelles, it has only been recently that patch clamping studies have suggested their existence at the nuclear membrane. The nuclear pore complexes have been considered the site of communication and exchange between the nucleus and cytoplasm (1, 2). Studies of traffic across the nuclear envelope have in general conformed to the paradigm that ions and small metabolites with diameters of less than 3-4 nm passively diffuse across the nuclear envelope (3). Thus, the concept of nuclear membrane ion channels seems at variance with the generally accepted views of the nuclear envelope, with their function at this location seeming redundant. However, this does not appear to be the case.The first demonstration of ionic conductances in the nuclear membrane were in mouse zygote pronuclei (4). Further evidence then followed with the demonstration of ion-selective channels in avian erythrocytes (5), in mouse oocyte germinal vesicles, in nuclei from two-cell embryos and liver (6), in the nuclei of cardiac myocytes (7), and in rat hepatocyte nuclei (8). Mak and Foskett (9) described the presence of inositol 1,4,5-trisphosphate-dependent receptor channels in isolated nuclei which were activated by inositol 1,4,5-trisphosphate, inhibited by heparin and selective to calcium ions. Similarly, Pasyk and Foskett (10), have shown chloride channel activity in isolated nuclei from CHO 1 cells. More recently, following fractionation and reconstitution of inner and outer nuclear membrane fractions into lipid bilayers, Rousseau et al. (11) have shown the presence of two types of chloride channels. The use of calcium ion imaging techniques has also demonstrated variations in calcium ion concentrations between the nucleus and cytoplasm, again suggesting a selective uptake or retention of these ions by the nucleus (12, 13).With growing electrophysiological data for the existence of nuclear ion channels, it is clear that the cloning and isolation of these proteins will greatly assist in determining their structure and function. This paper describes the molecular cloning and characterization of what we believe to be the first chloride ion channel protein of the nuclear membrane and only the second cloned ion chan...

Glutathione S-Transferase Omega 1-1 Is a Target of Cytokine Release Inhibitory Drugs and May Be Responsible for Their Effect on Interleukin-1औ Posttranslational Processing

“…The first is based on its similarity to CLIC1. Despite our recovery of CLIC1 from the soluble fraction of THP-1 cells, this polypeptide is reported to serve as a chloride channel both in its recombinant state after incorporation into lipid vesicles and in its native state within cells (55,56). GST Omega-1 itself has not been reported to possess ion channel activity, but CLIC1 and GST Omega-1 are related polypeptides; they share sequence homology, possess similar x-ray crystal structures, and are labeled by epoxide-bearing CRIDs.…”

“…Sequencing of multiple peptides led to identification of the 32-kDa polypeptide as CLIC1 (accession no. U93205), a protein belonging to a family of unusual ion channels (55,56). A relationship between GST Omega 1-1 and CLIC1 previously was described based on sequence homology (57).…”

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].…”

“…Human CLIC1 was identified as a putative nuclear membrane Cl − channel [19]. Human CLIC2 is a hypothetical protein encoded by the putative gene XAP121 [25].…”

MIC-1 is a novel TGF-β superfamily cytokine associated with macrophage activation

“…Two of these clones were then selected for more detailed study. One clone turned out to be a novel nuclear membrane chloride ion channel [2]. The second is a TGF-␤ superfamily cytokine that we have called macrophage inhibitory cytokine 1 (MIC-1) [3] and is the subject of this review.…”