Cholesterol promotes interaction of the protein CLIC1 with phospholipid monolayers at the air–water interface

© 2016 by the authors; licensee MDPI, Basel, Switzerland. CLIC1 is a Chloride Intracellular Ion Channel protein that exists either in a soluble state in the cytoplasm or as a membrane bound protein. Members of the CLIC family are largely soluble proteins that possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. The regulatory role of cholesterol in the ion‐channel activity of CLIC1 in tethered lipid bilayers was previously assessed using impedance spectroscopy. Here we extend this investigation by evaluating the influence of cholesterol on the spontaneous membrane insertion of CLIC1 into Langmuir film monolayers prepared using 1‐palmitoyl‐2‐oleoylphosphatidylcholine, 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐ethanolamine and 1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phospho‐L‐serine alone or in combination with cholesterol. The spontaneous membrane insertion of CLIC1 was shown to be dependent on the presence of cholesterol in the membrane. Furthermore, pre‐incubation of CLIC1 with cholesterol prior to its addition to the Langmuir film, showed no membrane insertion even in monolayers containing cholesterol, suggesting the formation of a CLIC1‐cholesterol pre‐complex. Our results therefore suggest that CLIC1 membrane interaction involves CLIC1 binding to cholesterol located in the membrane for its initial docking followed by insertion. Subsequent structural rearrangements of the protein would likely also be required along with oligomerisation to form functional ion channels

Investigating sterol and redox regulation of the ion channel activity of CLIC1 using tethered bilayer membranes

© 2016 by the authors; licensee MDPI, Basel, Switzerland. The Chloride Intracellular Ion Channel (CLIC) family consists of six conserved proteins in humans. These are a group of enigmatic proteins, which adopt both a soluble and membrane bound form. CLIC1 was found to be a metamorphic protein, where under specific environmental triggers it adopts more than one stable reversible soluble structural conformation. CLIC1 was found to spontaneously insert into cell membranes and form chloride ion channels. However, factors that control the structural transition of CLIC1 from being an aqueous soluble protein into a membrane bound protein have yet to be adequately described. Using tethered bilayer lipid membranes and electrical impedance spectroscopy system, herein we demonstrate that CLIC1 ion channel activity is dependent on the type and concentration of sterols in bilayer membranes. These findings suggest that membrane sterols play an essential role in CLIC1’s acrobatic switching from a globular soluble form to an integral membrane form, promoting greater ion channel conductance in membranes. What remains unclear is the precise nature of this regulation involving membrane sterols and ultimately determining CLIC1’s membrane structure and function as an ion channel. Furthermore, our impedance spectroscopy results obtained using CLIC1 mutants, suggest that the residue Cys24 is not essential for CLIC1’s ion channel function. However Cys24 does appear important for optimal ion channel activity. We also observe differences in conductance between CLIC1 reduced and oxidized forms when added to our tethered membranes. Therefore, we conclude that both membrane sterols and redox play a role in the ion channel activity of CLIC1

Versatile multicolor nanodiamond probes for intracellular imaging and targeted labeling

© 2018 The Royal Society of Chemistry. We report on the sizable production of fluorescent nanodiamonds (FNDs) containing a near infrared (NIR) color center-namely the silicon vacancy (SiV) defect, and their first demonstration inside cells for bio-imaging. We further demonstrate a concept of multi-color bio-imaging using FNDs to investigate intercellular processes using two types of FNDs. Due to their specific spectral properties, SiV FNDs can be distinguished from common nitrogen-vacancy (NV) FNDs and show a distinct initial spreading throughout the cell interior. The reported results are the first demonstration of multi-color labeling with FNDs that are especially interesting for in vivo bio-imaging due to their stable fluorescence

Evidence of the Key Role of H<inf>3</inf>O⁺ in Phospholipid Membrane Morphology

© 2016 American Chemical Society. This study explains the importance of the phosphate moiety and H3O+ in controlling the ionic flux through phospholipid membranes. We show that despite an increase in the H3O+ concentration when the pH is decreased, the level of ionic conduction through phospholipid bilayers is reduced. By modifying the lipid structure, we show the dominant determinant of membrane conduction is the hydrogen bonding between the phosphate oxygens on adjacent phospholipids. The modulation of conduction with pH is proposed to arise from the varying H3O+ concentrations altering the molecular area per lipid and modifying the geometry of conductive defects already present in the membrane. Given the geometrical constraints that control the lipid phase structure of membranes, these area changes predict that organisms evolving in environments with different pHs will select for different phospholipid chain lengths, as is found for organisms near highly acidic volcanic vents (short chains) or in highly alkaline salt lakes (long chains). The stabilizing effect of the hydration shells around phosphate groups also accounts for the prevalence of phospholipids across biology. Measurement of ion permeation through lipid bilayers was made tractable using sparsely tethered bilayer lipid membranes with swept frequency electrical impedance spectroscopy and ramped dc amperometry. Additional evidence of the effect of a change in pH on lipid packing density is obtained from neutron reflectometry data of tethered membranes containing perdeuterated lipids

Members of the chloride intracellular ion channel protein family demonstrate glutaredoxin-like enzymatic activity

© 2015 Al Khamici et al. The Chloride Intracellular Ion Channel (CLIC) family consists of six evolutionarily conserved proteins in humans. Members of this family are unusual, existing as both monomeric soluble proteins and as integral membrane proteins where they function as chloride selective ion channels, however no function has previously been assigned to their soluble form. Structural studies have shown that in the soluble form, CLIC proteins adopt a glutathione S-transferase (GST) fold, however, they have an active site with a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. We demonstrate that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity. CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. Mutagenesis experiments identify cysteine 24 as the catalytic cysteine residue in CLIC1, which is consistent with its structure. CLIC1 was shown to reduce sodium selenite and dehydroascorbate in a glutathione-dependent manner. Previous electrophysiological studies have shown that the drugs IAA-94 and A9C specifically block CLIC channel activity. These same compounds inhibit CLIC1 oxidoreductase activity. This work for the first time assigns a functional activity to the soluble form of the CLIC proteins. Our results demonstrate that the soluble form of the CLIC proteins has an enzymatic activity that is distinct from the channel activity of their integral membrane form. This CLIC enzymatic activity may be important for protecting the intracellular environment against oxidation. It is also likely that this enzymatic activity regulates the CLIC ion channel function

A Measurement of Rb using a Double Tagging Method

The fraction of Z to bbbar events in hadronic Z decays has been measured by the OPAL experiment using the data collected at LEP between 1992 and 1995. The Z to bbbar decays were tagged using displaced secondary vertices, and high momentum electrons and muons. Systematic uncertainties were reduced by measuring the b-tagging efficiency using a double tagging technique. Efficiency correlations between opposite hemispheres of an event are small, and are well understood through comparisons between real and simulated data samples. A value of Rb = 0.2178 +- 0.0011 +- 0.0013 was obtained, where the first error is statistical and the second systematic. The uncertainty on Rc, the fraction of Z to ccbar events in hadronic Z decays, is not included in the errors. The dependence on Rc is Delta(Rb)/Rb = -0.056*Delta(Rc)/Rc where Delta(Rc) is the deviation of Rc from the value 0.172 predicted by the Standard Model. The result for Rb agrees with the value of 0.2155 +- 0.0003 predicted by the Standard Model.Comment: 42 pages, LaTeX, 14 eps figures included, submitted to European Physical Journal

Measurement of the B+ and B-0 lifetimes and search for CP(T) violation using reconstructed secondary vertices

The lifetimes of the B+ and B-0 mesons, and their ratio, have been measured in the OPAL experiment using 2.4 million hadronic Z(0) decays recorded at LEP. Z(0) --> b (b) over bar decays were tagged using displaced secondary vertices and high momentum electrons and muons. The lifetimes were then measured using well-reconstructed charged and neutral secondary vertices selected in this tagged data sample. The results aretau(B+) = 1.643 +/- 0.037 +/- 0.025 pstau(Bo) = 1.523 +/- 0.057 +/- 0.053 pstau(B+)/tau(Bo) = 1.079 +/- 0.064 +/- 0.041,where in each case the first error is statistical and the second systematic.A larger data sample of 3.1 million hadronic Z(o) decays has been used to search for CP and CPT violating effects by comparison of inclusive b and (b) over bar hadron decays, No evidence fur such effects is seen. The CP violation parameter Re(epsilon(B)) is measured to be Re(epsilon(B)) = 0.001 +/- 0.014 +/- 0.003and the fractional difference between b and (b) over bar hadron lifetimes is measured to(Delta tau/tau)(b) = tau(b hadron) - tau((b) over bar hadron)/tau(average) = -0.001 +/- 0.012 +/- 0.008