The Journal of Membrane Biology

Yan, Yangyang; Yang, Youshan; Bian, Shumin; Sigworth, Fred

doi: 10.1007/s00232-012-9425-7pmid: 22729647

The slack (slo2.2) gene codes for a potassium-channel α-subunit of the 6TM voltage-gated channel family. Expression of slack results in Na+-activated potassium channel activity in various cell types. We describe the purification and reconstitution of Slack protein and show that the Slack α-subunit alone is sufficient for potassium channel activity activated by sodium ions as assayed in planar bilayer membranes and in membrane vesicles.

Wang, Chao; Wu, He-ming; Jing, Xiao-rong; Meng, Qiang; Liu, Bei; Zhang, Hua; Gao, Guo-dong

doi: 10.1007/s00232-012-9436-4pmid: 22772441

The chronic mild stress (CMS) protocol is widely used to evoke depression-like behaviors in the laboratory. Some animals exposed to CMS are resistant to the development of anhedonia, whereas the remaining are responsive, CMS-resilient and CMS-sensitive, respectively. The aim of this study was to examine the effects of chronic stress on oxidative parameters in the rat brain. The consumption of sweet food, protein and lipid oxidation levels and superoxide dismutase and catalase activities in the rat hippocampus, cortex and cerebellum were assessed. We found a significant increase in protein peroxidation (hippocampus and cortex), a significant increase in catalase activity (cortex, hippocampus and cerebellum) and a decrease in superoxide dismutase activity (cortex, hippocampus and cerebellum) in the CMS-sensitive group compared to the CMS-resilient group and normal controls as well as an increase in lipid peroxidation (cerebellum) in the CMS-sensitive and CMS-resilient groups compared to normal controls. However, there was no significant difference in protein peroxidation (cerebellum) and lipid peroxidation (cortex and hippocampus) among the three groups. In conclusion, our results indicate that the segregation into CMS-sensitive and -resilient groups based on sucrose intake is paralleled by significant differences in oxidative parameters. CMS induces oxidative damage and alterations in the activity of antioxidants which may lead to increased oxidative damage, irrespective of the anhedonia-like status of the stressed animals.

Kuvichkin, Vasily

doi: 10.1007/s00232-012-9437-3pmid: 22644390

Recent discovery of the role of nuclear pores in transcription, predicted by our early DNA-membrane complex (DMC) model, makes membrane-bound DNA (MBD) isolation from the cell nucleus and analysis of the MBD actual. The method of MBD isolation proposed by us retains DMC integrity during isolation. We used HeLa cells for DMC extraction. Changing the ionic composition of the isolation medium and replacing DNase I, used commonly for chromatin destruction, with a set of restriction enzymes allowed us to isolate the MBD. Treatment of a nuclear membrane with proteinase K and ultrasound has been used to increase the yield of MBD. Electron microscopic analysis of the purified fraction of isolated DMC supports our previous model of nuclear envelope lipid–chromatin interaction in the nuclear pore assembly.

Park, Jin-Won

doi: 10.1007/s00232-012-9438-2pmid: 22622287

Spherical phospholipid bilayers, or vesicles, were prepared layer by layer using a double-emulsion technique, which allows the outer layer of the vesicles to be formed with two phospholipids that have different head groups: phosphatidylcholine (PC) and phosphatidylethanolamine. At the outer layer of the vesicles, the phospholipase D (PLD) catalyzed for the conversion of PC to phosphatidic acid. The reaction caused by PLD induced the curvature change of the vesicles, which eventually led to the rupture of the vesicles. Before the investigation, the ratio of dioleoylphosphatidylethanolamine to oleoylhydroxyphosphatidylethanolamine was found as a condition such that the vesicles made with the mixed lipids were as stable as those made with pure dioleoylphosphatidylcholine. Response time from the PLD injection to vesicle rupture was monitored by the composition of the outer layer by the fluorescence intensity change of pH-sensitive dye encapsulated in the vesicles. The response time began to be slowed at approximately 30 % PC. The response times for the compositions were associated with the surface density of PC at the outer layer. These results also seem to be determined by the size of PLD, specifically the PLD active site.

Uydu, Hüseyin; Yıldırmış, Sermet; Örem, Cihan; Calapoglu, Mustafa; Alver, Ahmet; Kural, Birgül; Örem, Asım

doi: 10.1007/s00232-012-9441-7pmid: 22706680

The statins, most commonly used in the treatment of hyperlipidemia, have certain beneficial effects including improved endothelial function, plaque stability and decreased oxidative stress and inflammation, beyond their lipid-lowering effect in plasma. We evaluated the pleiotropic impact of atorvastatin on erythrocyte structural/mechanical properties and lipid peroxidation in dyslipidemics. The study group included 44 patients with dyslipidemia and was divided into subgroups according to triglyceride and cholesterol levels as hypercholesterolemic (n = 29) and mixed-type hyperlipidemic (n = 15). Subjects were given 10 mg atorvastatin per day for 12 weeks. Changes in serum lipid composition, lipid contents, Na+/K+-ATPase activity and osmotic fragility in erythrocytes and oxidative stress parameters of erythrocytes and plasma were studied. Atorvastatin therapy improved the serum lipid profile of both subgroups. This alteration was accompanied by a decreased level of cholesterol in erythrocyte membranes. Moreover, enhanced activity of Na+/K+-ATPase in erythrocytes reflected the improvements in membrane lipids of both subgroups. However, a significant change was observed in osmotic fragility values of the mixed-typed dyslipidemic group. This treatment lowered the lipid peroxidation in plasma and erythrocytes and increased plasma total antioxidant capacity in all groups. The present study shows that the use of atorvastatin reversed the structural and functional features of erythrocyte membranes in dyslipidemic subjects. Also, hypolipidemic therapy had a beneficial impact on a balance between oxidant and antioxidant systems.

Kangjian, Wang; Nianhua, Dan; Shiwei, Xiao; Yichun, Ye; Weihua, Dan

doi: 10.1007/s00232-012-9442-6pmid: 22722761

Collagen (Col)–chitosan (Chi) membrane was modified by a hot dehydrogenation cross-linking method. Carbodiimide was added for further crossing modification. Chondroitin sulfate (CS) was added so that Col–Chi sulfate composite membranes were prepared. The structure of the composite membranes was characterized by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy, and its mechanical properties, degradation, and cytotoxicity were characterized. The composite membrane was applied to a full-thickness skin injury in animal experiments performed in rabbits. Strong interactions and good compatibility among Col, Chi, and CS in the composite membrane were present. The good mechanical properties, biocompatibility, digestion resistance, and wound healing promotion of the composite membrane make it a potential wound dressing or skin scaffold for tissue engineering.

Val, Coral; White, Stephen; Bondar, Ana-Nicoleta

doi: 10.1007/s00232-012-9452-4pmid: 22836667

We combined systematic bioinformatics analyses and molecular dynamics simulations to assess the conservation patterns of Ser and Thr motifs in membrane proteins, and the effect of such motifs on the structure and dynamics of α-helical transmembrane (TM) segments. We find that Ser/Thr motifs are often present in β-barrel TM proteins. At least one Ser/Thr motif is present in almost half of the sequences of α-helical proteins analyzed here. The extensive bioinformatics analyses and inspection of protein structures led to the identification of molecular transporters with noticeable numbers of Ser/Thr motifs within the TM region. Given the energetic penalty for burying multiple Ser/Thr groups in the membrane hydrophobic core, the observation of transporters with multiple membrane-embedded Ser/Thr is intriguing and raises the question of how the presence of multiple Ser/Thr affects protein local structure and dynamics. Molecular dynamics simulations of four different Ser-containing model TM peptides indicate that backbone hydrogen bonding of membrane-buried Ser/Thr hydroxyl groups can significantly change the local structure and dynamics of the helix. Ser groups located close to the membrane interface can hydrogen bond to solvent water instead of protein backbone, leading to an enhanced local solvation of the peptide.

Kawaguchi, Riki; Zhong, Ming; Kassai, Miki; Ter-Stepanian, Mariam; Sun, Hui

doi: 10.1007/s00232-012-9463-1pmid: 22815070

Vitamin A has diverse biological functions and is essential for human survival. STRA6 is the high-affinity membrane receptor for plasma retinol binding protein (RBP), the principle and specific carrier of vitamin A (retinol) in the blood. It was previously shown that STRA6 couples to lecithin retinol acyltransferase (LRAT) and cellular retinol binding protein I (CRBP-I), but poorly to CRBP-II, for retinol uptake from holo-RBP. STRA6 catalyzes both retinol release from holo-RBP, which is responsible for its retinol uptake activity, and the loading of free retinol into apo-RBP, which can cause retinol efflux. Although STRA6-catalyzed retinol efflux into apo-RBP can theoretically deplete cells of retinoid, it is unclear to what extent this efflux happens and in what context. We show here that STRA6 can couple strongly to both CRBP-I and CRBP-II for retinol efflux to apo-RBP. Strikingly, pure apo-RBP can cause almost complete depletion of retinol taken up by CRBP-I in a STRA6-dependent manner. However, if STRA6 encounters both holo-RBP and apo-RBP (as in blood), holo-RBP blocks STRA6-mediated retinol efflux by competing with apo-RBP’s binding to STRA6 and by counteracting retinol efflux with influx. We also found that STRA6 catalyzes efficient retinol exchange between intracellular CRBP-I and extracellular RBP, even in the presence of holo-RBP. STRA6’s retinol exchange activity may serve to refresh the intracellular retinoid pool. This exchange is also a previously unknown function of CRBP-I and distinguishes CRBP-I from LRAT.

Lin, Bor-Ruei; Hsieh, Ying-Hsin; Jiang, Chun; Tai, Phang

doi: 10.1007/s00232-012-9477-8pmid: 22854753

We have developed a sensitive method to detect the opening of SecA-dependent, protein-conducting channels in Xenopus oocytes. In this study, we determined the ionic current activities of the SecA-dependent channel from membrane vesicles depleted of SecYEG. We found that these SecYEG-depleted membranes produced SecA-dependent ionic currents in the oocytes, as did membranes containing SecYEG. However, reconstituted membranes depleted of SecYEG required higher concentrations of SecA to elicit ionic currents like those in membranes containing SecYEG. In contrast to membranes containing SecYEG, the proofreading capacity of signal peptides was lost for those membranes lacking SecYEG. These findings are consistent with loss of signal peptide specificity in channel activity from membranes of SecY suppressor or SecY plug domain mutants. The signal peptide specificity of the reconstituted membranes, like SecA-liposomes, can be restored by the addition of SecYEG proteoliposomes. On the other hand, the channel activity efficiency of reconstituted membranes was fully restored, while SecA-liposomes could only be partially enhanced by the addition of SecYEG, indicating that, in addition to SecYEG, other membrane proteins contribute to the efficiency of channel activity. The SecA-dependent channels in membranes that lacked SecYEG also lost ion selectivity to monovalent cations but retained selective permeability to large anions. Thus, the electrophysiological evidence presented here indicates that SecYEG is not obligatory for the channel activity of Escherichia coli membranes, as previously shown for protein translocation, and that SecYEG is important for maintenance of the efficiency and specificity of SecA-dependent channels.

Busath, David; Woodbury, Dixon; Frost, Adam

doi: 10.1007/s00232-012-9439-1pmid: 22653449