Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24 -Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.Chloride ion channels control a variety of cellular processes that are central to normal function and disease states (1). The CLIC 1 family is a recently identified class of Cl Ϫ channel proteins that consists of seven members (p64, parchorin, CLIC1-5) (2, 3). A conserved C-terminal CLIC module of ϳ240 amino acids is present in each member of the family with several members containing additional, unrelated Nterminal domains. Most CLICs are localized to intracellular membranes and have been linked to functions including apoptosis, pH, and cell cycle regulation (4 -6). The CLIC ion channels are unusual in that they possess both soluble and integral membrane forms (2). In this regard they are similar to some bacterial toxins and several classes of intracellular proteins including Bcl-x L and the annexins (7). Our understanding of how such dual natured proteins enter the membrane is limited by the dearth of high resolution structures for key states in this process.We have recently determined the crystal structure of a soluble monomeric form of CLIC1 (8) and found that it is a structural homologue of the GST superfamily of proteins (9). This soluble form of CLIC1 consists of two domains, the N-domain possessing a thioredoxin fold closely resembling glutaredoxin and an all ␣-helical C-domain, which is typical of the GST superfamily. CLIC1 contains an intact glutathione-binding site that was shown to covalently bind glutathione via a conserved CLIC cysteine residue, Cys-24. This led to the suggestion that CLIC1 function may be under redox control, possibly via reactive oxygen or nitrogen species.The structure and stoichiometry of the integral membrane form of the CLIC proteins is still unclear. Electrophysiology of purified, soluble (Escherichia coli-expressed) recombinant CLIC1 in reconstituted artificial bilayers shows that CLIC1 alone is sufficient for chloride ion channel formation (8, 1...

CLIC1 (NCC27) is a member of the highly conserved class of chloride ion channels that exists in both soluble and integral membrane forms. Purified CLIC1 can integrate into synthetic lipid bilayers forming a chloride channel with similar properties to those observed in vivo. The structure of the soluble form of CLIC1 has been determined at 1.4-Å resolution. The protein is monomeric and structurally homologous to the glutathione S-transferase superfamily, and it has a redox-active site resembling glutaredoxin. The structure of the complex of CLIC1 with glutathione shows that glutathione occupies the redox-active site, which is adjacent to an open, elongated slot lined by basic residues. Integration of CLIC1 into the membrane is likely to require a major structural rearrangement, probably of the N-domain (residues 1-90), with the putative transmembrane helix arising from residues in the vicinity of the redox-active site. The structure indicates that CLIC1 is likely to be controlled by redox-dependent processes.Chloride ion channels, located both within the plasma membrane and other internal cell membranes (1, 2), are involved in diverse physiological processes. They are known to participate in the control of secretion and absorption of salt, regulation of membrane potentials, organellar acidification, and cell volume homeostasis (3). Malfunction in these channels can lead to severe disease states (4).Chloride channels fall into several classes based on their sequence relationships. The three best characterized classes are the ligand-gated receptor channels (␥-aminobutyric acid and glycine receptors), the cystic fibrosis transmembrane conductance regulator family, and the ClC chloride ion channels (1, 2). A new class of chloride ion channel, the "chloride intracellular channels" (CLICs), 1 has recently been characterized at a molecular level. To date, there are seven members of the CLIC family: CLIC1 (NCC27) (5), CLIC2 (6), CLIC3 (7), CLIC4 (8), CLIC5 (9), p64 (10), and parchorin (11). All of these proteins exist as soluble globular proteins that can form ion channels in organellar and plasma membranes (5,7,8,(12)(13)(14)(15). Five of the CLIC proteins are each composed of ϳ240 residues, while the longer p64 and parchorin consist of distinct amino-terminal domains followed by the 240-residue CLIC module. This module has recently been shown to share weak sequence homology with the glutathione S-transferase (GST) superfamily (16).The CLIC proteins are expressed in a wide variety of tissues and appear to have diverse physiological functions. p64 is associated with kidney function (17), while CLIC1 and CLIC4 appear to have a broad tissue distribution (5,8,18,19). Several CLICs interact with protein kinases (7,11,20). CLICs are associated with a variety of intracellular membranes including the nuclear membrane (5), the endoplasmic reticular membrane (8), large dense-core vesicles (19), mitochondria (21), trans-Golgi vesicles (22), and secretory vesicles (23). Parchorin forms the chloride channel in water-secreting cells,...

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CLIC1 (NCC27)is an unusual, largely intracellular, ion channel that exists in both soluble and membraneassociated forms. The soluble recombinant protein can be expressed in Escherichia coli, a property that has made possible both detailed electrophysiological studies in lipid bilayers and an examination of the mechanism of membrane integration. Soluble E. coli-derived CLIC1 moves from solution into artificial bilayers and forms chloride-selective ion channels with essentially identical conductance, pharmacology, and opening and closing kinetics to those observed in CLIC1-transfected Chinese hamster ovary cells. The process of membrane integration of CLIC1 is pH-dependent. Following addition of protein to the trans solution, small conductance channels with slow kinetics (SCSK) appear in the bilayer. These SCSK modules then appear to undergo a transition to form a high conductance channel with fast kinetics. This has four times the conductance of the SCSK and fast kinetics that characterize the native channel. This suggests that the CLIC1 ion channel is likely to consist of a tetrameric assembly of subunits and indicates that despite its size and unusual properties, it is able to form a completely functional ion channel in the absence of any other ancillary proteins.

NCC27 belongs to a family of small, highly conserved, organellar ion channel proteins. It is constitutively expressed by native CHO-K1 and dominantly localized to the nucleus and nuclear membrane. When CHO-K1 cells are transfected with NCC27-expressing constructs, synthesized proteins spill over into the cytoplasm and ion channel activity can then be detected on the plasma as well as nuclear membrane. This provided a unique opportunity to directly compare electrophysiological characteristics of the one cloned channel, both on the nuclear and cytoplasmic membranes. At the same time, as NCC27 is unusually small for an ion channel protein, we wished to directly determine whether it is a membrane-resident channel in its own right. In CHO-K1 cells transfected with epitope-tagged NCC27 constructs, we have demonstrated that the NCC27 conductance is chloride dependent and that the electrophysiological characteristics of the channels are essentially identical whether expressed on plasma or nuclear membranes. In addition, we show that a monoclonal antibody directed at an epitope tag added to NCC27 rapidly inhibits the ability of the expressed protein to conduct chloride, but only when the antibody has access to the tag epitope. By selectively tagging either the amino or carboxyl terminus of NCC27 and varying the side of the membrane from which we record channel activity, we have demonstrated conclusively that NCC27 is a transmembrane protein that directly forms part of the ion channel and, further, that the amino terminus projects outward and the carboxyl terminus inward. We conclude that despite its relatively small size, NCC27 must form an integral part of an ion channel complex.

The authors used functional magnetic resonance imaging (fMRI) to determine whether acute intravenous (i.v.) cocaine use would change global cerebral blood flow (CBF) or visual stimulation-induced functional activation. They used flow-sensitive alternating inversion recovery (FAIR) scan sequences to measure CBF and blood oxygen level-dependent (BOLD) sensitive T2* scan sequences during visual stimulation to measure neuronal activation before and after cocaine and saline infusions. Cocaine (0.6 mg/kg i.v. over 30 seconds) increased heart rate and mean blood pressure and decreased end tidal carbon dioxide (CO2). All measures returned to baseline by 2 hours, the interinfusion interval, and were unchanged by saline. Flow-sensitive alternating inversion recovery imaging demonstrated that cortical gray matter CBF was unchanged after saline infusion (-2.4 +/- 6.5%) but decreased (-14.1 +/- 8.5%) after cocaine infusion (n = 8, P < 0.01). No decreases were detected in white matter, nor were changes found comparing BOLD signal intensity in cortical gray matter immediately before cocaine infusion with that measured 10 minutes after infusion. Visual stimulation resulted in comparable BOLD signal increases in visual cortex in all conditions (before and after cocaine and saline infusion). Despite a small (14%) but significant decrease in global cortical gray matter CBF after acute cocaine infusion, specific regional increases in BOLD imaging, mediated by neurons, can be measured reliably.

1 Cisapride is a prokinetic agent which has been associated with QT prolongation, torsades de pointes and cardiac arrest. The cellular mechanism for these observations is high a nity blockade of I Kr (encoded by HERG). 2 In a chronic transfection model using CHO-K1 cells, cisapride inhibited HERG tail currents after a step to +25 mV with similar potency at room and physiological temperatures (IC 50 16.4 nM at 20 ± 228C and 23.6 nM at 378C). 3 Channel inhibition exhibited time-, voltage-and frequency-dependence. In an envelope of tails test, channel blockade increased from 27+8% after a 120 ms depolarizing step to 50+4% after a 1.0 s step. These ®ndings suggested a nity for open and/or inactivated channel states. 4 Inactivation was signi®cantly accelerated by cisapride in a concentration-dependent manner and there was a small (77 mV