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1.  Atox1 Contains Positive Residues That Mediate Membrane Association and Aid Subsequent Copper Loading 
The Journal of membrane biology  2013;246(12):10.1007/s00232-013-9592-1.
Copper chaperones bind intracellular copper and ensure proper trafficking to downstream targets via protein-protein interactions. In contrast to the mechanisms of copper binding and transfer to downstream targets, the mechanisms of initial copper loading of the chaperones are largely unknown. Here we demonstrate that antioxidant protein 1 (Atox1 in human cells), the principal cellular copper chaperone responsible for delivery of copper to the secretory pathway, possesses the ability to interact with negatively charged lipid headgroups via distinct surface lysine residues. Moreover, loss of these residues lowers the efficiency of copper loading of Atox1 in vivo, suggesting that the membrane may play a scaffolding role in copper distribution to Atox1. These findings complement the recent discovery that the membrane also facilitates copper loading of the copper chaperone for superoxide dismutase 1 and provide further support for the emerging paradigm that the membrane bilayer plays a central role in cellular copper acquisition and distribution.
doi:10.1007/s00232-013-9592-1
PMCID: PMC3827972  PMID: 24036897
Atox1; Chaperone; Copper Trafficking; Copper Homeostasis; Membrane Scaffold
2.  The Effect of Fatal Carbon Monoxide Poisoning on the Equilibria Between Cell Membranes and the Electrolyte Solution 
The effect of fatal carbon monoxide poisoning on equilibria between cell membranes and surrounding ions was described using a theoretical four-equilibria model. The model was developed to obtain parameters characterizing the interactions between solution ions and erythrocyte or thrombocyte membrane surface. The parameters are the total surface concentrations of both acidic and basic groups CA, CB and their association constants with solution ions KAH, KBOH. These parameters were used to calculate the theoretical values of surface charge density. The model was validated by comparison of these values to experimental data, which were determined from the electrophoretic mobility measurements of the blood cells. The experimental and theoretical surface charge density values agree at pH 2–8, and at higher pH, the deviation was observed.
doi:10.1007/s00232-014-9753-x
PMCID: PMC4300427  PMID: 25416423
Acid–base equilibria; Surface charge density; Microelectrophoresis; Erythrocytes; Thrombocytes; Fatal carbon monoxide poisoning
3.  The Connexin46 Mutant, Cx46T19M, Causes Loss of Gap Junction Function and Alters Hemi-channel Gating 
An N-terminal mutant of connexin46 (T19M) alters a highly conserved threonine and has been linked to autosomal dominant cataracts. To study the cellular and functional consequences of substitution of this amino acid, T19M was expressed in Xenopus oocytes and in HeLa cells. Unlike wild-type Cx46, T19M did not induce intercellular conductances in Xenopus oocytes. In transfected HeLa cells, T19M was largely localized within the cytoplasm, with drastically reduced formation of gap junction plaques. Expression of rat T19M was cytotoxic, as evidenced by an almost complete loss of viable cells expressing the mutant protein by 48–72 h following transfection. When incubated in medium containing physiological concentrations of divalent cations, T19M-expressing cells showed increased uptake of DAPI as compared with cells expressing wild-type Cx46, suggesting aberrant connexin hemi-channel activity. Time-lapse and dye uptake studies suggested that T19M hemi-channels had reduced sensitivity to Ca2+. Whole cell patch clamp studies of single transfected HeLa cells demonstrated that rat T19M formed functional hemi-channels with altered voltage-dependent gating. These data suggest that T19M causes cataracts by loss of gap junctional channel function and abnormally increased hemi-channel activity. Furthermore, they implicate this conserved threonine in both gap junction plaque formation and channel/hemi-channel gating in Cx46.
Electronic supplementary material
The online version of this article (doi:10.1007/s00232-014-9752-y) contains supplementary material, which is available to authorized users.
doi:10.1007/s00232-014-9752-y
PMCID: PMC4300453  PMID: 25404239
Cataract; Lens; Gap junction; Hemi-channel; Cx46
4.  Cell-specific targeting strategies for electroporation-mediated gene delivery in cells and animals 
The Journal of membrane biology  2013;246(10):737-744.
The use of electroporation to facilitate gene transfer is an extremely powerful and useful method for both in vitro and in vivo applications. One of its great strengths is that it induces functional destabilization and permeabilization of cell membranes throughout a tissue leading to widespread gene transfer to multiple cells and cell types within the electric field. While this is a strength, it can also be a limitation in terms of cell-specific gene delivery. The ability to restrict gene delivery and expression to particular cell types is of paramount importance for many types of gene therapy, since ectopic expression of a transgene could lead to deleterious host inflammatory responses or dysregulation of normal cellular functions. At present, there are relatively few ways to obtain cell-specific targeting of nonviral vectors, molecular probes, small molecules, and imaging agents. We have developed a novel means of restricting gene delivery to desired cell types based on the ability to control the transport of plasmids into the nuclei of desired cell types. In this article, we discuss the mechanisms of this approach and several applications in living animals to demonstrate the benefits of the combination of electroporation and selective nuclear import of plasmids for cell-specific gene delivery.
doi:10.1007/s00232-013-9534-y
PMCID: PMC3726570  PMID: 23525583
transfection; nuclear import; nucleus; gene delivery; trafficking; transcription; electroporation
5.  Developmental Expression of the Outer Hair Cell Motor Prestin in the Mouse 
The Journal of membrane biology  2007;215(1):49-56.
The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (Cv), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, Cv was about 70% of the mature level. Cv saturated at P18 when hearing shows full maturation. On the other hand, Clin, which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor’s operating voltage midpoint , Vpkcm, tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, Vpkcm reached a steady-state level near −67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate Vpkcm, may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.
doi:10.1007/s00232-007-9004-5
PMCID: PMC4154540  PMID: 17415610
Development; Mouse outer hair cell; Motor protein; Hearing; Cochlear amplification
6.  Evaluation of voltage-sensitive fluorescence dyes for monitoring neuronal activity in the embryonic central nervous system 
The Journal of membrane biology  2013;246(9):679-688.
Using an optical imaging technique with voltage-sensitive dyes (VSDs), we have been investigating the functional organization and architecture of the central nervous system (CNS) during embryogenesis. In the embryonic nervous system, a merocyanine-rhodanine dye, NK2761, has proved to be the most useful absorption dye for detecting neuronal activity because of its high signal-to-noise ratio (S/N), low toxicity, and small dye bleaching. In the present study, we evaluated the suitability of voltage-sensitive fluorescence dyes for optical recording in the embryonic CNS. We screened eight styryl (hemicyanine) dyes in isolated brainstem-spinal cord preparations from 7-day old chick embryos. Measurements of voltage-related optical signals were made using a multiple-site optical recording system. The signal size, S/N, photobleaching, effects of perfusion, and recovery of neural responses after staining were compared. We also evaluated optical responses with various magnifications. Although the S/N was lower than with the absorption dye, clear optical responses were detected with several fluorescence dyes, including di-2-ANEPEQ, di-4-ANEPPS, di-3-ANEPPDHQ, di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA, and di-2-ANEPPTEA. Di-2-ANEPEQ showed the largest S/N, whereas its photobleaching was faster and the recovery of neural responses after staining was slower. Di-4-ANEPPS and di-3-ANEPPDHQ also exhibited a large S/N, but required a relatively long time for recovery of neural activity. Di-4-AN(F)EPPTEA, di-2-AN(F)EPPTEA, and di-2-ANEPPTEA showed smaller S/Ns than di-2-ANEPEQ, di-4-ANEPPS, and di-3-ANEPPDHQ, but the recovery of neural responses after staining was faster. This study demonstrates the potential utility of these styryl dyes in optical monitoring of voltage changes in the embryonic CNS.
doi:10.1007/s00232-013-9584-1
PMCID: PMC4096138  PMID: 23975337
optical recording; voltage-sensitive dye; embryo; nervous system; fluorescence; screening
7.  Reptation-Induced Coalescence of Tunnels and Cavities in Escherichia Coli XylE Transporter Conformers Accounts for Facilitated Diffusion 
The Journal of Membrane Biology  2014;247(11):1161-1179.
Structural changes and xylose docking to eight conformers of Escherichia Coli XylE, a xylose transporter similar to mammalian passive glucose transporters GLUTs, have been examined. Xylose docks to inward and outward facing conformers at a high affinity central site (Ki 4–20 µM), previously identified by crystallography and additionally consistently docks to lower affinity sites in the external and internal vestibules (Ki 12–50 µM). All these sites lie within intramolecular tunnels and cavities. Several local regions in the central transmembrane zone have large positional divergences of both skeleton carbon Cα positions and side chains. One such in TM 10 is the destabilizing sequence G388-P389-V390-C391 with an average RMSD (4.5 ± 0.4 Å). Interchange between conformer poses results in coalescence of tunnels with adjacent cavities, thereby producing a transitory channel spanning the entire transporter. A fully open channel exists in one inward-facing apo-conformer, (PDB 4ja4c) as demonstrated by several different tunnel-finding algorithms. The conformer interchanges produce a gated network within a branched central channel that permits staged ligand diffusion across the transporter during the open gate periods. Simulation of this model demonstrates that small-scale conformational changes required for sequentially opening gate with frequencies in the ns-μs time domain accommodate diffusive ligand flow between adjacent sites with association–dissociation rates in the μs-ms domain without imposing delays. This current model helps to unify the apparently opposing concepts of alternate access and multisite models of ligand transport.
Electronic supplementary material
The online version of this article (doi:10.1007/s00232-014-9711-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s00232-014-9711-7
PMCID: PMC4207944  PMID: 25163893
Xylose; XylE structure; GLUT1; Facilitated diffusion; Docking
8.  Maturational Changes in Rabbit Renal Brush Border Membrane Vesicle Osmotic Water Permeability 
The Journal of membrane biology  1998;164(2):177-185.
We have recently shown that the osmotic water permeability (Pf) of proximal tubules from neonatal rabbits is higher than that of adults (AJP 271:F871-F876, 1996). The developmental change in Pf could be due to differences in one or more of the components in the path for transepithelial water transport. The present study examined developmental changes in water transport characteristics of the proximal tubule apical membrane by determining Pf and aquaporin 1 (AQP1) expression in neonatal (10–14 days old) and adult rabbit renal brush border membrane vesicles (BBMV). AQP1 abundance in the adult BBMV was higher than the neonatal BBMV. At 25°C the Pf of neonatal BBMV was found to be significantly lower than the adult BBMV at osmotic gradients from 50 to 250 mOsm/kg water. The activation energy for osmotic water movement was higher in the neonatal BBMV than the adult BBMV (9.19 ± 0.37 vs. 5.09 ± 0.57 kcal • deg−1 • mol−1, P < 0.005). Osmotic water movement in neonatal BBMV was inhibited 17.9 ± 1.3% by 1 mm HgCl2 compared to 34.3 ± 3.8% in the adult BBMV (P < 0.005). These data are consistent with a significantly greater fraction of water traversing the apical membrane lipid bilayer in proximal tubules of neonates than adults. The lower Pf of the neonatal BBMV indicates that the apical membrane is not responsible for the higher transepithelial Pf in the neonatal proximal tubule.
PMCID: PMC4134809  PMID: 9662561
9.  The Mechanism of Enterohepatic Circulation in the Formation of Gallstone Disease 
The Journal of Membrane Biology  2014;247(11):1067-1082.
Bile acids entering into enterohepatic circulating are primary acids synthesized from cholesterol in hepatocyte. They are secreted actively across canalicular membrane and carried in bile to gallbladder, where they are concentrated during digestion. About 95 % BAs are actively taken up from the lumen of terminal ileum efficiently, leaving only approximately 5 % (or approximately 0.5 g/d) in colon, and a fraction of bile acids are passively reabsorbed after a series of modifications in the human large intestine including deconjugation and oxidation of hydroxy groups. Bile salts hydrolysis and hydroxy group dehydrogenation reactions are performed by a broad spectrum of intestinal anaerobic bacteria. Next, hepatocyte reabsorbs bile acids from sinusoidal blood, which are carried to liver through portal vein via a series of transporters. Bile acids (BAs) transporters are critical for maintenance of the enterohepatic BAs circulation, where BAs exert their multiple physiological functions including stimulation of bile flow, intestinal absorption of lipophilic nutrients, solubilization, and excretion of cholesterol. Tight regulation of BA transporters via nuclear receptors (NRs) is necessary to maintain proper BA homeostasis. In conclusion, disturbances of enterohepatic circulation may account for pathogenesis of gallstones diseases, including BAs transporters and their regulatory NRs and the metabolism of intestinal bacterias, etc.
doi:10.1007/s00232-014-9715-3
PMCID: PMC4207937  PMID: 25107305
Bile acids metabolism; Enterohepatic circulation; Transporters; Nuclear receptor; Gallstone formation
10.  Diffusional Water Permeability (PDW) of Adult and Neonatal Rabbit Renal Brush Border Membrane Vesicles 
The Journal of membrane biology  2002;187(3):167-174.
We have shown that there is a maturational increase in osmotic water permeability (Pf) of rabbit renal brush border membrane vesicles (BBMV). The purpose of the present study was to further investigate the changes in proximal tubule water transport that occur during postnatal development. Diffusional water permeability (PDW) has not been measured directly in adult or neonatal BBMV. We validated the method described by Ye and Verkman (Simultaneous optical measurement of osmotic and diffusional water permeability in cells and liposomes. Biochemistry 28:824-829, 1989) to measure PDW in red cell ghosts and liposomes, to examine the maturational changes in PDW in BBMV. This method utilizes the sensitivity of 8-amino-naphtalene-1,3,6-trisulfonic acid (ANTS) fluorescence to the D2O-H2O content of the solvent. ANTS-loaded neonatal (11 days old) and adult BBMV were rapidly mixed with two volumes of isoosmotic D2O solution using a stopped-flow apparatus at 5°–37°C. PDW was lower in neonatal than adult BBMV at 5° (3.77 ± 0.34 vs. 5.35 ± 0.43 μm/sec, respectively, p < 0.05) and 20°C (7.03 ± 0.40 vs. 9.04 ± 0.25 μm/ sec, respectively, p < 0.001), but was not different at 30° and 37° C. The activation energy (Ea) was higher in neonatal than in adult BBMV (9.29 ± 0.56 kcal/ mol vs. 6.46 ± 0.56 kcal/mol, p < 0.001). In adult BBMV, PDW was inhibited by 0.5 mm HgCl2 by 46.6 ± 3.6%, while it was not affected in neonatal BBMV (p < 0.001). The results indicate that PDW can be measured in rabbit renal BBMV. There are significant changes in water transport across the apical membrane during postnatal development, consistent with a maturational increase in channel-mediated water transport.
doi:10.1007/s00232-001-0161-7
PMCID: PMC4124817  PMID: 12163975
Stop-flow kinetics; Development; Fluorescence; Membrane transport
11.  Differential and Isomer-Specific Modulation of Vitamin A Transport and the Catalytic Activities of the RBP Receptor by Retinoids 
The Journal of membrane biology  2013;246(8):647-660.
Retinoids are vitamin A derivatives with diverse biological functions. Both natural and artificial retinoids have been used as therapeutic reagents to treat human diseases, but not all retinoid actions are understood mechanistically. Plasma retinol binding protein (RBP) is the principal and specific carrier of vitamin A in the blood. STRA6 is the membrane receptor for RBP that mediates cellular vitamin A uptake. The effects of retinoids or related compounds on the receptor’s vitamin A uptake activity and its catalytic activities are not well understood. In this study, we dissected the membrane receptor-mediated vitamin A uptake mechanism using various retinoids. We show that a subset of retinoids strongly stimulates STRA6-mediated vitamin A release from holo-RBP. STRA6 also catalyzes the exchange of retinol in RBP with certain retinoids. The effect of retinoids on STRA6 is highly isomer-specific. This study provides unique insights into the RBP receptor’s mechanism and reveals that the vitamin A transport machinery can be a target of retinoid-based drugs.
doi:10.1007/s00232-013-9578-z
PMCID: PMC3777752  PMID: 23811822
Membrane receptor; Membrane transport; Vitamin A transport; Retinoid
12.  Basic Features of a Cell Electroporation Model: Illustrative Behavior for Two Very Different Pulses 
The Journal of Membrane Biology  2014;247(12):1209-1228.
Science increasingly involves complex modeling. Here we describe a model for cell electroporation in which membrane properties are dynamically modified by poration. Spatial scales range from cell membrane thickness (5 nm) to a typical mammalian cell radius (10 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}m), and can be used with idealized and experimental pulse waveforms. The model consists of traditional passive components and additional active components representing nonequilibrium processes. Model responses include measurable quantities: transmembrane voltage, membrane electrical conductance, and solute transport rates and amounts for the representative “long” and “short” pulses. The long pulse—1.5 kV/cm, 100 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}s—evolves two pore subpopulations with a valley at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sim}$$\end{document}5 nm, which separates the subpopulations that have peaks at \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sim}$$\end{document}1.5 and \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${\sim}$$\end{document}12 nm radius. Such pulses are widely used in biological research, biotechnology, and medicine, including cancer therapy by drug delivery and nonthermal physical tumor ablation by causing necrosis. The short pulse—40 kV/cm, 10 ns—creates 80-fold more pores, all small (\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$<$$\end{document}3 nm; \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\sim$$\end{document}1 nm peak). These nanosecond pulses ablate tumors by apoptosis. We demonstrate the model’s responses by illustrative electrical and poration behavior, and transport of calcein and propidium. We then identify extensions for expanding modeling capability. Structure-function results from MD can allow extrapolations that bring response specificity to cell membranes based on their lipid composition. After a pulse, changes in pore energy landscape can be included over seconds to minutes, by mechanisms such as cell swelling and pulse-induced chemical reactions that slowly alter pore behavior.
Electronic supplementary material
The online version of this article (doi:10.1007/s00232-014-9699-z) contains supplementary material, which is available to authorized users.
doi:10.1007/s00232-014-9699-z
PMCID: PMC4224743  PMID: 25048527
Cell electroporation; Computational model; Electrical behavior; Poration behavior; Solute transport
13.  Maturational Changes in Rabbit Renal Basolateral Membrane Vesicle Osmotic Water Permeability 
The Journal of membrane biology  2000;174(1):53-58.
We have recently demonstrated that while the osmotic water permeability (Pf) of neonatal proximal tubules is higher than that of adult tubules, the Pf of brush-border membrane vesicles from neonatal rabbits is lower than that of adults. The present study examined developmental changes in the water transport characteristics of proximal tubule basolateral membranes by determining aquaporin 1 (AQP1) protein abundance and the Pf in neonatal (10–14 days old) and adult rabbit renal basolateral membrane vesicles (BLMV). At 25°C the Pf of neonatal BLMV was significantly lower than the adult BLMV at osmotic gradients ranging from 40 to 160 mOsm/kg water. The activation energies for osmotic water movement were identical in the neonatal and adult BLMV (8.65 ± 0.47 vs. 8.86 ± 1.35 kcal · deg−1 · mol−1). Reflection coefficients for sodium chloride and sodium bicarbonate were identical in both the neonatal and adult BLMV and were not different from one. Mercury chloride (0.5 mm) reduced osmotic water movement by 31.3 ± 5.5% in the adult BLMV, but by only 4.0 ± 4.0% in neonatal vesicles (P < 0.01). Adult BLMV AQP1 abundance was higher than that in the neonate. These data demonstrate that neonatal BLMV have a lower Pf and AQP1 protein abundance than adults and that a significantly greater fraction of water traverses the basolateral membrane lipid bilayer and not water channels in neonates compared to adults. The lower Pf of the neonatal BLMV indicates that the basolateral membrane is not responsible for the higher transepithelial Pf in the neonatal proximal tubule.
PMCID: PMC4089855  PMID: 10741432
14.  Cx43 Associates with Nav1.5 in the Cardiomyocyte Perinexus 
The Journal of membrane biology  2012;245(7):411-422.
Gap junctions (GJs) are aggregates of channels that provide for direct cytoplasmic connection between cells. Importantly, this connection is thought responsible for cell-to-cell transfer of the cardiac action potential. The GJ channels of ventricular myocytes are composed of connexin43 (Cx43). Interaction of Cx43 with zonula occludens-1 (ZO-1) is localized not only at the GJ plaque, but also to the region surrounding the GJ, the perinexus. Cx43 in the perinexus is not detectable by immunofluorescence, yet localization of Cx43/ZO-1 interaction to this region indicated the presence of Cx43. Therefore, we hypothesized that Cx43 occurs in the perinexus at a lower concentration per unit membrane than in the GJ itself, making it difficult to visualize. To overcome this, the Duolink protein–protein interaction assay was used to detect Cx43. Duolink labeling of cardiomyocytes localized Cx43 to the perinexus. Quantification demonstrated that signal in the perinexus was lower than in the GJ but significantly higher than in nonjunctional regions. Additionally, Duolink of Triton X-100-extracted cultures suggested that perinexal Cx43 is nonjunctional. Importantly, the voltage gated sodium channel Nav1.5, which is responsible for initiation of the action potential, was found to interact with perinexal Cx43 but not with ZO-1. This work provides a detailed characterization of the structure of the perinexus at the GJ edge and indicates that one of its potential functions in the heart may be in facilitating conduction of action potential.
doi:10.1007/s00232-012-9465-z
PMCID: PMC4085045  PMID: 22811280
Connexin43; Duolink; Gap junction; Hemichannel; Nav1.5; Perinexus; Sodium channel
16.  Overexpression of the polycystin-1 (PC-1) C-tail enhances sensitivity of M-1 cells to ouabain 
The Journal of membrane biology  2013;246(7):581-590.
Cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD) are abnormally sensitive to ouabain, responding to physiological ouabain concentrations with enhanced proliferation and increased forskolin-induced transepithelial fluid secretion. This requires activation of the epidermal growth factor receptor (EGFR), Src kinase, and the extracellular regulated kinases MEK and ERK. Here, we have determined if the ADPKD phenotype obtained in mouse cortical collecting duct cells by stable overexpression of the C-terminal domain of polycystin-1 (PC-1 C-tail) also elicits the ADPKD-like response to ouabain in the cells. M-1 C20 cells expressing the PC-1 C-tail, and M-1 C17 cells, lacking expression of this construct, were treated with physiological concentrations of ouabain, and cell proliferation, activation of the EGFR-Src-MEK-ERK pathway, forskolin-induced transepithelial Cl− secretion, and the sensitivity of the Na,K-ATPase to ouabain were explored. M-1 C20 cells responded to ouabain with increased cell proliferation and ERK phosphorylation. Ouabain also augmented forskolin-induced and cystic fibrosis transmembrane conductance regulator (CFTR)-mediated apical secretion of Cl− in M-1 C20 cells. These effects required activation of EGFR, Src and MEK. In contrast, ouabain had no significant effects on M-1 C17 cells. Interestingly, approximately 20 % of the Na,K-ATPase from M-1 C20 cells presented an abnormally increased sensitivity to ouabain. Overexpression of PC-1 C-tail in M-1 C20 cells is associated with a ouabain sensitive phenotype and an increased ability of the cells to proliferate and secrete anions upon ouabain stimulation. This phenotype mimics the ouabain sensitivity of ADPKD cells and may help promote their cystogenic potential.
doi:10.1007/s00232-013-9573-4
PMCID: PMC4051140  PMID: 23784065
Na,K-ATPase; ouabain signaling; cystic epithelia; polycystic kidney disease; polycystin-1
17.  Biophysical Mechanism of the Protective Effect of Blue Honeysuckle (Lonicera caerulea L. var. kamtschatica Sevast.) Polyphenols Extracts Against Lipid Peroxidation of Erythrocyte and Lipid Membranes 
The Journal of Membrane Biology  2014;247(7):611-625.
The aim of the present research was to determine the effect of blue honeysuckle fruit and leaf extracts components on the physical properties of erythrocyte and lipid membranes and assess their antioxidant properties. The HPLC analysis showed that the extracts are rich in polyphenol anthocyanins in fruits and flavonoids in leaves. The results indicate that both extracts have antioxidant activity and protect the red blood cell membrane against oxidation induced by UVC irradiation and AAPH. The extracts do not induce hemolysis and slightly increase osmotic resistance of erythrocytes. The research showed that extracts components are incorporated mainly in the external part of the erythrocyte membrane, inducing the formation of echinocytes. The values of generalized polarization and fluorescence anisotropy indicate that the extracts polyphenols alter the packing arrangement of the hydrophilic part of the erythrocyte and lipid membranes, without changing the fluidity of the hydrophobic part. The DSC results also show that the extract components do not change the main phase transition temperature of DPPC membrane. Studies of electric parameters of membranes modified by the extracts showed that they slightly stabilize lipid membranes and do not reduce their specific resistance or capacity. Examination of IR spectra indicates small changes in the degree of hydration in the hydrophilic region of liposomes under the action of the extracts. The location of polyphenolic compounds in the hydrophilic part of the membrane seems to constitute a protective shield of the cell against other substances, the reactive forms of oxygen in particular.
doi:10.1007/s00232-014-9677-5
PMCID: PMC4052015  PMID: 24862869
Blue honeysuckle polyphenol extracts; HPLC analysis; Antioxidant activity; Erythrocyte membrane anisotropy; Model lipid membranes; Lipid phase transition
18.  Physicochemical Aspects of Reaction of Ozone with Galactolipid and Galactolipid–Tocopherol Layers 
The Journal of Membrane Biology  2014;247(7):639-649.
The impact of reaction of galactolipids with ozone on the physicochemical properties of their monolayers was examined. In Megli and Russo (Biochim Biophys Acta, 1778:143–152, 2008), Cwiklik and Jungwirth (Chem Phys Lett, 486:99–103, 2010), Jurkiewicz et al. (Biochim Biophys Acta, 1818:2388–2402, 2012), Khabiri et al. (Chem Phys Lett, 519:93–99, 2012), and Conte et al. (Biochim Biophys Acta, 1828:510–517, 2013), the properties of layers formed from model mixtures composed of chosen lipids and selected oxidation products were studied, whereas in this work, question was raised as to how the oxidation reactions taking place in situ affect the physical properties of the galactolipid layers. So, set experiment should take into account the effect of all reaction products. The mechanical characteristics of monolayers of monogalactosyldiacyl-glycerol (MGDG) and digalactosyldiacylglycerol (DGDG) were determined by Langmuir trough technique, and the electrical properties of liposomes formed from these lipids by measuring their electrophoretic mobility. Considerable loss of galactolipid molecules forming monolayers was found at ozone concentrations (in aqueous medium) higher than 0.1 ppm with a stronger effect measured for MGDG. That goes along with the greater amounts of MDA found in the extracts of oxidized MGDG films compared with DGDG. Based on this, it was concluded that an additional galactose group present in DGDG molecules acts protectively under oxidative conditions. The surface tension of the solutions (of small volume) contacting the oxidized galactolipids films was significantly reduced, indicating the presence of soluble in polar media, surface active reaction products. The presence of α-tocopherol in mixtures with tested galactolipids at a molar ratio of lipid to tocopherol equal to 1.7:1 caused some inhibition of lipid oxidation, reducing the decrease of amount of lipid particles forming the monolayer. Here, also protective effect of α-tocopherol was greater for the MGDG compared to DGDG.
doi:10.1007/s00232-014-9681-9
PMCID: PMC4052016  PMID: 24862871
Galactolipids; Monolayers; Zeta potential; Oxidation; Protective action of α-tocopherol
19.  Affinity of Alkylphosphocholines to Biological Membrane of Prostate Cancer: Studies in Natural and Model Systems 
The Journal of Membrane Biology  2014;247(7):581-589.
The effectiveness of two alkylphosphocholines (APCs), hexadecylphosphocholine (miltefosine) and erucylphosphocholine to combat prostate cancer has been studied in vitro with artificial cancerous membrane, modelled with the Langmuir monolayer technique, and on cell line (Du-145). Studies performed with the Langmuir method indicate that both the investigated drugs have the affinity to the monolayer mimicking prostate cancer membrane (composed of cholesterol:POPC = 0.428) and the drug-membrane interactions are stronger for erucylphosphocholine as compared to hexadecylphosphocholine. Moreover, both studied drugs were found to fluidize the model membrane, which may lead to apoptosis. Indeed, biological studies confirmed that in Du-145 cell line both investigated alkylphosphocholines cause cell death primarily by apoptosis while necrotic cells constitute only a small percentage of APC-treated cells.
doi:10.1007/s00232-014-9674-8
PMCID: PMC4052013  PMID: 24848301
Alkylphosphocholines; Model membrane; Langmuir monolayers; Prostate cancer
20.  APols-Aided Protein Precipitation: A Rapid Method for Concentrating Proteins for Proteomic Analysis 
The Journal of Membrane Biology  2014;247(9-10):941-947.
Abstract
Amphipols (APols) are a newly designed and milder class of detergent. They have been used primarily in protein structure analysis for membrane protein trapping and stabilization. We have recently demonstrated that APols can be used as an alternative detergent for proteome extraction and digestion, to achieve a “One-stop” single-tube workflow for proteomics. In this workflow, APols are removed by precipitation after protein digestion without depleting the digested peptides. Here, we took further advantage of this precipitation characteristic of APols to concentrate proteins from diluted samples. In contrast with tryptic peptides, a decrease in pH leads to the unbiased co-precipitation of APols with proteins, including globular hydrophilic proteins. We demonstrated that this precipitation is a combined effect of acid precipitation and the APols’ protein interactions. Also, we have been able to demonstrate that APols-aided protein precipitation works well on diluted samples, such as secretome sample, and provides a rapid method for protein concentration.
Electronic supplementary material
The online version of this article (doi:10.1007/s00232-014-9668-6) contains supplementary material, which is available to authorized users.
doi:10.1007/s00232-014-9668-6
PMCID: PMC4196042  PMID: 24838764
Amphipols; Proteomics; Protein precipitation; Concentrating protein; Mass spectrometry
21.  Mechanistic Basis for Type 2 Long QT Syndrome Caused by KCNH2 Mutations that Disrupt Conserved Arginine Residue in the Voltage Sensor 
The Journal of membrane biology  2013;246(5):355-364.
KCNH2 encodes the Kv11.1 channel, which conducts the rapidly activating delayed rectifier K+ current (IKr) in the heart. KCNH2 mutations cause type 2 long QT syndrome (LQT2), which increases the risk for life-threatening ventricular arrhythmias. LQT2 mutations are predicted to prolong the cardiac action potential (AP) by reducing IKr during repolarization. Kv11.1 contains several conserved basic amino acids in the fourth transmembrane segment (S4) of the voltage sensor that are important for normal channel trafficking and gating. This study sought to determine the mechanism(s) by which LQT2 mutations at conserved arginine residues in S4 (R531Q, R531W or R534L) alter Kv11.1 function. Western blot analyses of HEK293 cells transiently expressing R531Q, R531W or R534L suggested that only R534L inhibited Kv11.1 trafficking. Voltage-clamping experiments showed that R531Q or R531W dramatically altered Kv11.1 current (IKv11.1) activation, inactivation, recovery from inactivation and deactivation. Coexpression of wild type (to mimic the patients’ genotypes) mostly corrected the changes in IKv11.1 activation and inactivation, but deactivation kinetics were still faster. Computational simulations using a human ventricular AP model showed that accelerating deactivation rates was sufficient to prolong the AP, but these effects were minimal compared to simply reducing IKr. These are the first data to demonstrate that coexpressing wild type can correct activation and inactivation dysfunction caused by mutations at a critical voltage-sensing residue in Kv11.1. We conclude that some Kv11.1 mutations might accelerate deactivation to cause LQT2 but that the ventricular AP duration is much more sensitive to mutations that decrease IKr. This likely explains why most LQT2 mutations are nonsense or trafficking-deficient.
doi:10.1007/s00232-013-9539-6
PMCID: PMC3706098  PMID: 23546015
Long QT syndrome; Potassium ion channel; Arrhythmia; Channel gating; Trafficking
22.  Bioinformatic Characterization of the Trimeric Intracellular Cation-Specific Channel Protein Family 
The Journal of membrane biology  2011;241(2):77-101.
Trimeric intracellular cation-specific (TRIC) channels are integral to muscle excitation–contraction coupling. TRIC channels provide counter-ionic flux when calcium is rapidly transported from intracellular stores to the cell cytoplasm. Until recently, knowledge of the presence of these proteins was limited to animals. We analyzed the TRIC family and identified a profusion of prokaryotic family members with topologies and motifs similar to those of their eukaryotic counterparts. Prokaryotic members far outnumber eukaryotic members, and although none has been functionally characterized, the evidence suggests that they function as secondary carriers. The presence of fused N- or C-terminal domains of known biochemical functions as well as genomic context analyses provide clues about the functions of these prokaryotic homologs. They are proposed to function in metabolite (e.g., amino acid/ nucleotide) efflux. Phylogenetic analysis revealed that TRIC channel homologs diverged relatively early during evolutionary history and that horizontal gene transfer was frequent in prokaryotes but not in eukaryotes. Topological analyses of TRIC channels revealed that these proteins possess seven putative transmembrane segments (TMSs), which arose by intragenic duplication of a three-TMS polypeptide-encoding genetic element followed by addition of a seventh TMS at the C terminus to give the precursor of all current TRIC family homologs. We propose that this family arose in prokaryotes.
doi:10.1007/s00232-011-9364-8
PMCID: PMC3980945  PMID: 21519847
TRIC channel; Sarcoplasmic reticulum; Prokaryotic homolog; Potassium metabolite transport; Evolutionary origin; Topology
23.  What do aquaporin knockout studies tell us about fluid transport in epithelia? 
The Journal of membrane biology  2013;246(4):297-305.
The investigation of near-isosmotic water transport in epithelia goes back over 100 years; however debates over mechanism and pathway still remain. Aquaporin (AQP) knockouts have been used by various research groups to test the hypothesis of an osmotic mechanism, as well as to explore the paracellular vs transcellular pathway debate. Non-proportional reductions in the water permeability of a water-transporting epithelial cell (e.g. a reduction of around 80–90%) compared to the reduction in overall water transport rate in the knockout animal (e.g. a reduction of 50–60%) are commonly found. This non-proportionality has led to controversy over whether AQP knockout studies support or contradict the osmotic mechanism. Arguments raised for and against an interpretation supporting the osmotic mechanism typically have partially-specified, implicit or incorrect assumptions. We present a simple mathematical model of the osmotic mechanism with clear assumptions and, for models based on this mechanism, establish a baseline prediction of AQP knockout studies. We allow for deviations from isotonic/isosmotic conditions and utilize dimensional analysis to reduce the number of parameters that must be considered independently. This enables a single prediction curve to be used for multiple epithelial systems. We find that a simple, transcellular-only osmotic mechanism sufficiently predicts the results of knockout studies and find criticisms of this mechanism to be overstated. We note, however, that AQP knockout studies do not give sufficient information to definitively rule out an additional paracellular pathway.
doi:10.1007/s00232-013-9530-2
PMCID: PMC3622118  PMID: 23430220
aquaporin knockouts; epithelial transport; osmosis
24.  Automated Procedure for Contact-Map-Based Protein Structure Reconstruction 
The Journal of Membrane Biology  2014;247(5):409-420.
Knowledge of the three-dimensional structures of ion channels allows for modeling their conductivity characteristics using biophysical models and can lead to discovering their cellular functionality. Recent studies show that quality of structure predictions can be significantly improved using protein contact site information. Therefore, a number of procedures for protein structure prediction based on their contact-map have been proposed. Their comparison is difficult due to different methodologies used for validation. In this work, a Contact Map-to-Structure pipeline (C2S_pipeline) for contact-based protein structure reconstruction is designed and validated. The C2S_pipeline can be used to reconstruct monomeric and multimeric proteins. The median RMSD of structures obtained during validation on a representative set of protein structures, equaled 5.27 Å, and the best structure was reconstructed with RMSD of 1.59 Å. The validation is followed by a detailed case study on the KcsA ion channel. Models of KcsA are reconstructed based on different portions of contact site information. Structural feature analysis of acquired KcsA models is supported by a thorough analysis of electrostatic potential distributions inside the channels. The study shows that electrostatic parameters are correlated with structural quality of models. Therefore, they can be used to discriminate between high and low quality structures. We show that 30 % of contact information is needed to obtain accurate structures of KcsA, if contacts are selected randomly. This number increases to 70 % in case of erroneous maps in which the remaining contacts or non-contacts are changed to the opposite. Furthermore, the study reveals that local reconstruction accuracy is correlated with the number of contacts in which amino acid are involved. This results in higher reconstruction accuracy in the structure core than peripheral regions.
doi:10.1007/s00232-014-9648-x
PMCID: PMC3983884  PMID: 24682239
Ion channel; Protein structure; Contact-maps; Protein structure reconstruction; Protein electrostatics
25.  Post-translational modifications in connexins and pannexins 
The Journal of membrane biology  2012;245(0):319-332.
Post-translational modifications are a common cellular process that is used by cells to ensure a particular protein function. This can happen in a variety of ways, for example from the addition of phosphates or sugar residues to a particular amino acid ensuring proper protein life cycle and function. In this review, we assess evidence for ubiquitination, glycosylation, phosphorylation, S-nitrosylation as well as other modifications on connexins and pannexin proteins. Based on the literature, we find that post-translational modifications are an important component of connexin and pannexin regulation.
doi:10.1007/s00232-012-9453-3
PMCID: PMC3954810  PMID: 22739962
post-translational modifications; connexins; pannexin

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