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At physiological pH, the head groups of PtdSer and PtdIns have an overall negative charge
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but I'm sitting here tossing the basketball around and walking over to find a magnet buried under some dirt and what do I think? you have it... thinking of PS ! lol
more than a few articles I come across mentioning that PS is "negatively" charged...
Inversion of membrane surface charge by trivalent cations probed with a cation-selective channel
Nov 2012
Philip A. Gurnev and Sergey M. Bezrukov
Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892
*Corresponding Author: Philip A. Gurnev; Program in Physical Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health,
9000 Rockville Pk., Bldg. 9, Rm. 1E-106; Bethesda, MD 20892, phone: +1-301-451-2275; fax: +1-301-496-2172; Email: vog.hin.liam@pvenrug
Abstract
We demonstrate that the cation-selective channel formed by gramicidin A can be used as a reliable sensor for studying the multivalent ion accumulation at the surfaces of charged lipid membranes and the “charge inversion” phenomenon. In asymmetrically charged membranes with the individual leaflets formed from pure negative and positive lipids bathed by 0.1 M CsCl solutions the channel exhibits current rectification which is comparable to that of a typical n/p semiconductor diode. We show that even at these highly asymmetrical conditions the channel conductance can be satisfactorily described by the electrodiffusion equation in the constant field approximation but, due to predictable limitations, only when the applied voltages do not exceed 50 mV. Analysis of the changes in the voltage-dependent channel conductance upon addition of trivalent cations allows us to gauge their interactions with the membrane surface. The inversion of the sign of the effective surface charge takes place at the concentrations which correlate with the cation size. Specifically, these concentrations are close to 0.05 mM for lanthanum, 0.25 mM for hexaamminecobalt, and 4 mM for spermidine.
Keywords: molecular diode, nano-sensing, current rectification, bilayer lipid membrane, gramicidin A
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1. INTRODUCTION
Long-range electrostatic forces are crucially involved in many interactions within and between biomolecules. Charged groups play the well-recognized roles in functioning of proteins, nucleic acids, phospholipids and their supra-molecular assemblies. Phospholipid molecules, building blocks of cellular membranes, mostly contain zwitterionic or negatively charged headgroups exposed on the membrane/water interface. Membrane surface potential is crucially involved in regulation of membrane transport, cell-cell recognition, and membrane-bound enzymes1. In the presence of multivalent cations the surface charge screening may be accompanied by its overcompensation, or the so-called “charge inversion” at the membrane surface. This phenomenon has been observed experimentally 2–12, and discussed in a number of theoretical studies, e.g. 13–18. Charge inversion by polyvalent ions is thought to be highly relevant for the number of biological processes, including action of drugs 19, gene delivery 20, DNA condensation7, and viral packing 21, 22. However, despite the extensive experimental and theoretical work, problems regarding electrostatics at the charged membrane interfaces, counter-ion screening, and charge inversion phenomenon remain a subject of intense discussions, e.g. 11, 23.
The motivation for this study is two-fold. First, in addition to the biological processes mentioned above, charge inversion could also be involved in regulation of channel function during membrane fusion, which is shown to require the presence of highly charged polypeptide chains 24. Second, most of the approaches for studying charge inversion published so far deal with different modifications of electrokinetic measurements, which involve the notion of a sliding plane. The present study is different in this respect as there is no need to postulate the position of the sliding plane, though we have to use other adjustable parameters as described below. We study the accumulation of multivalent ions at the charged surfaces by using the conductance of an ion channel as a sensor of the potential at the surface of the membrane hosting the channel. With the planar lipid bilayer membranes as established models of cell membranes, the technique of lipid monolayer opposition 25 allows obtaining artificial bilayers with asymmetrical distribution of lipids between the two monolayers. The charge state of the bilayer surface may be probed by measuring ion currents in the presence of lipophilic ions 26, ion transporters 27, 28, or ion pores induced by short peptides 29–34 and channel-forming proteins 35–39.
The conductive pore of a benchmark channel formed by pentadecapeptide gramicidin A is composed of two ß-helical 15 amino acid monomers and is exclusively permeable for monovalent cations 40. As shown in a number of publications, the ion conductance of the gramicidin pore reports well on the charged state of surface bilayer groups. Indeed, charges on the membrane surface attract counter-ions and reduce the concentration of co-ions near the entrance of the pore, thus increasing or decreasing the number of cations, depending on the sign of the surface charge. The effects of the surface charge are particularly strong in electrolytes of low ionic strength.
In the present work we measure the ion conductance of the gramicidin A channel to assess changes in the charge state of phospholipid bilayers as a function of concentration of trivalent ions in the bulk. With 0.1 M cesium as the current-mediating ion we first study the channel conductance in asymmetric bilayers where one monolayer is formed from negatively charged phosphatidylserine (PS) and the other from either neutral phosphatidylcholine (PC) or positively charged trimethylammonium propane (TAP). We demonstrate that at small voltages the gramicidin A channel in asymmetrically charged membranes displays current rectification which is close to that of a typical solid-state diode. We also show that even at these highly asymmetrical conditions, our measurements are reasonably well described by a simple theoretical model of biased diffusion with only two adjustable parameters.
We then investigate the effects of three cations: lanthanum chloride, hexaamminecobalt chloride, and spermidine chloride on negatively charged bilayers. At asymmetrical, one side addition of these trivalent cations the channel conductance becomes asymmetric in applied voltage. Most pronounced reduction of the channel conductance is observed at the voltages that are positive from the side of trivalent cation addition. To estimate the bilayer surface charge in the presence of trivalent cations, we compare the corresponding conductance-voltage relationships with those obtained in control experiments with asymmetrical bilayers formed from lipid monolayers of different charge. All these cations are able to overcompensate the effective surface charge of the PS monolayer at the concentrations that correlate with their size. The most potent is lanthanum, which neutralizes the surface charge felt by the channel at 0.05 mM concentration, followed by hexaamminecobalt with about five-fold higher concentration and then by spermidine with about hundred-fold higher concentration of charge inversion.
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2. EXPERIMENTAL METHODS
Bilayer lipid membranes were prepared from diphytanoylphosphoserine (PS), diphytanoylphosphocholine (PC), and dioleoyl trimethylammonium propane (TAP), Avanti Polar Lipids, Alabaster, AL, using monolayer-opposition technique by Montal and Mueller 25. The Teflon chamber, with two (cis and trans) compartments of 1.7 ml, was divided by a 15-µm-thick Teflon partition with a 60–70-µm diameter aperture. PS, PC and TAP monolayers were made from 2 mg/ml aliquots of lipids in pentane. After bilayer formation gramicidin A (a generous gift from O. S. Andersen, Weill Cornell University Medical College) was added from 1–10 nM ethanol stock solutions to both aqueous compartments at the amount sufficient to produce a single channel activity. Buffer solutions contained 100 mM CsCl, 5 mM HEPES at pH 7. Lanthanum chloride (Sigma), hexaamminecobalt chloride (Fluka), and spermidine chloride (Fluka) were admixed from the aliquots to the cis compartment of the membranes, made from PS monolayers. The choice of CsCl over KCl or NaCl was dictated by the fact that the gramicidin channel conductance in CsCl is about 1.8 higher than in KCl and 2.9 higher than in NaCl of the same molarity41.
The membrane potential was maintained using Ag/AgCl electrodes with 2 M KCl and 15 % (w/v) agarose bridges. The membrane chamber and headstage were isolated from external noise sources with a double metal screen (Amuneal Manufacturing Corp., Philadelphia, PA). Conductance measurements were performed using an Axopatch 200B amplifier (Molecular Devices, Foster City, CA) in the voltage clamp mode. Data were filtered by a low-pass 8-pole Butterworth filter (Model 9002, Frequency Devices, Inc., Haverhill, MA) at 5 kHz, directly saved into the computer memory with a sampling frequency of 10 kHz, and analyzed using pClamp 10 software. All measurements were made at room temperature, T = (23 ± 1) °C. Gramicidin A amplitudes at a given transmembrane voltage were collected from individual single-channel events and calculated by Gaussian fitting of a histogram of ~50 single events.
Liposomes were prepared by sonication according to the Morrissey Laboratory protocol (Morrissey, J. H. 2001. Morrissey laboratory protocol for preparing phospholipid vesicles (SUV) by sonication. tf7.org/suv.pdf). Liposome buffer was the same as for bilayer measurements. Liposomes diameter (75–120 nm) was checked with light scattering. Measurement of the liposome ?-potential and sizing of the liposomes were performed with Zeta-Plus ?-potential analyzer (Brookhaven Instruments Corporation, Holtsville, NY). Diluted (1:10) PS or TAP liposome solutions in 1.5 ml cuvettes were injected with appropriate amounts of trivalent cations and transferred into the analyzer. PC liposomes were used as a standard of neutrality of the liposome surface in ?-potential measurements. The measurements were performed at room temperature, T = (23 ± 1) °C.
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3. RESULTS AND DISCUSSION
The results of a control experiment with gramicidin A channels incorporated into asymmetric planar lipid bilayers made of the negatively charged lipid (PS, the cis monolayer) and the positively charged lipid (TAP, the trans monolayer) are shown in Figure 1. Raw data in panels A and B and the corresponding current-voltage curves in panel C demonstrate that in asymmetric membranes the channel exhibits a highly non-linear behavior with a significant asymmetry resembling that of a solid-state diode rectifier. The current at +150 mV applied from the side of the negative monolayer is more than an order of magnitude higher than the current at the opposite polarity. Non-linear current-voltage dependencies were first reported for gramicidin A channel in membranes with only one lipid leaflet charged in a classical study by Frohlich 29. Here, in Fig. 1B, C we present experiments with the membranes where the leaflets are oppositely charged.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4307797/
sitting here tossing the basketball around and walking over to find a magnet buried under some dirt and what do I think? you have it... thinking of PS !
New tools reveal how to make cells speak Notch
New biochemical techniques have revealed that conversing in a language called Notch requires “speaking” cells to physically tug on “listening” cells. Image: Ugreen/iStock
By STEPHANIE DUTCHENJune 4, 2015
Scientists have solved a decades-old mystery about how our cells communicate with one another to build and maintain our bodies.
They found that in order to converse in a language called Notch, the “speaking” cell must physically tug on the “listening” cell.
The results of their research, which required combining specialties and inventing new experimental techniques, should help scientists better understand how cells convey information through Notch signaling.
The findings are reported June 4 in Developmental Cell.
Get more HMS news here.
Notch is important for processes such as determining cell fate during embryonic development. In addition, mutations of the gene encoding Notch are found in some diseases, including more than half of all cases of T-cell acute lymphoblastic leukemia (T-ALL).
The study could therefore inform researchers’ understanding of what is going wrong in such diseases, said co-corresponding author Joseph Loparo, assistant professor of biological chemistry and molecular pharmacology at Harvard Medical School.
The tools the team created should help scientists investigate the role physical force plays in regulating a variety of biological systems.
“Knowing that this ancient system for cell-cell communication has built into it a dependence on mechanical force has relevance to the fundamental question of how Notch signaling guides normal developmental decisions in multicellular organisms, and should provide insight into mutations that likely bypass the force requirement in disease,” said Stephen Blacklow, the Gustavus Adolphus Pfeiffer Professor and chair of the Department of Biological Chemistry and Molecular Pharmacology at HMS, who led the study with Loparo.
“I am excited that our work provides new insights into Notch signaling, and we hope that the tools we developed will be useful to our colleagues in the Notch signaling field,” said Wendy Gordon, who conducted the work as a postdoctoral researcher in the Blacklow lab. Gordon is now an assistant professor at the University of Minnesota.
“Furthermore, altered mechanical forces in the microenvironment of cells in disease states like cancer is an emerging concept that necessitates new tools like the ones we developed to understand how other proteins on the surface of cells use mechanical force to communicate signals to the inside of cells,” she said.
Open sesame
Before this study, researchers understood the basic order of events needed to convey a Notch message. But pieces were missing.
They knew that a molecule called Delta on the surface of the “speaking” cell binds to a molecule called Notch on the surface of the “listening” cell. Then an enzyme swoops in, and, like a pair of scissors, snips off a piece of Notch, releasing the signal into the cell.
Notch’s “cut here” site is initially hidden. It wasn’t clear how it becomes available to the enzyme scissors. Experiments showed that the mere binding of Delta and Notch molecules wasn’t enough to expose it.
Fifteen years ago, scientists proposed that a mechanical force might be needed. In the current study, Blacklow and Loparo’s teams became the first to directly test the idea—and show that it’s probably correct.
“You need to pull on Notch,” said Blacklow.
Magnetic pull
To make their discoveries, the researchers first set up simplified Notch systems on a glass slide. They attached tiny magnetic beads to individual Notch molecules tethered to the slide and bathed them in the Notch-cutting enzymes.
At first, nothing happened. Then they used what they called magnetic tweezers to pull on the beads. When they achieved enough force, the Notch molecules were cut and the beads floated away.
“It was a really clever idea,” said Blacklow of Gordon’s experimental design.
When pulled, fluorescently tagged magnetic beads are cut free of Notch and float away. Experiment shown at 100 times normal speed. Video: Wendy Gordon
The tests revealed that the force needed to reveal the cutting site was within the realm of what might occur in a real cell.
The researchers pursued their findings in a second set of experiments conducted in real cells using complete Notch and Delta molecules. They confirmed that magnetically tugging at Delta within a certain range of forces revealed Notch’s cutting site and released the signal without tearing apart either molecule.
Joining forces
Finally, the researchers built synthetic alternatives to the natural Delta-Notch connections to test whether anything about the natural bond other than the pull force might help expose the cutting site.
“Is it just a tether?” asked Loparo. “Or does something else in the connection lower the barrier to the door opening?”
Gordon designed one synthetic system. Norbert Perrimon, the James Stillman Professor of Developmental Biology at HMS, and members of his lab designed another.
Both systems showed that the pull force alone was sufficient to reveal the cutting site and release the signal. The experiments also revealed which parts of the Delta-Notch system are essential.
As they slot one more piece into the puzzle of Notch signaling, the research teams look forward to exploring new questions their work has raised.
“We came to this problem with two complementary areas of expertise,” said Loparo. “Neither of our labs could’ve done this on their own. It’s a fun and exciting way to do science.”
This work was supported by the National Institutes of Health (grants R01 CA092433 and P01 CA119070) and the Howard Hughes Medical Institute.
https://hms.harvard.edu/news/pull-open
Inhibitors of Notch signaling can be used not only as direct anti-cancer agents but also as a sensitizer to current therapy.
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