• • Asymmetrical membrane. Inside: negatively charged PLs; outside: neutral PLs; both sides: cholesterol (~30%). • Both the PL composition and distribution, and the presence of proteins of influence on the membrane properties. Cell membrane: bilayer of various phospholipids (PLs) with proteins ((ion) channels => transport of molecules across the membrane) [1]. DOPC/PE membranes PC/PS membranes PI/PS membranes Investigation of the influence of the composition of the membrane on its electrical properties ð Better understanding of the properties of the cellular membranes ð Towards more controlled and reproducible cellular experiments. BLMs = models for cell membranes BLM preparation and charaterization Used (phospho)lipids (found in natural cell membranes) Painting technique + Easy - Membrane reproducibility? - Smaller membrane area Montal-Mueller (MM) technique + + Asymmetrical membranes + Larger membrane area - Tedious Membrane reproducibility DOPC PS PI PE PC CH Iris van Uitert*, Yanina Cesa**, Hans de Boer*, Johan Bomer*, Martin Bennink**, Séverine Le Gac*, and Albert van den Berg* * BIOS the Lab-on-a-Chip group, ** Biophysical Engineering, MESA+ Institute for Nanotechnnology, THE NETHERLANDS E-mail: [email protected], Url: http://www.bios.ewi.utwente.nl DOPC/PS membranes DOPC/PI membranes 0 50 100 150 200 250 300 350 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 X=DOPC/PI Voltage (mV) 0 20 40 60 80 100 120 140 160 180 Capacitance (pF) Breakthrough Voltage Capacitance Cellular membrane Bilayer lipid membrane • • Characterisation of membrane stability: measurement of the breakthrough voltage for many different membrane compositions, using binary membranes and membranes with more complex compositions mimicking the leaflets of biological cell membranes (MDCK cells). • Membrane stability related to PL packing density and consequently to the number of insaturations in the hydrocarbon tails, the bulkiness of the headgroup and their charge. • Insertion of a protein channel (áHL) in BLMs and first studies on the membrane stability. Preparation of BLMs with mixtures of (phospho)lipids using the painting and MM techniques. This work is funded by the POF group at the University of Twente. [1] Alberts, [2] Montal, M.; Mueller, P. Proceedings of the National Academy of Sciences of the United States of America 1972, 69, pp. 3561-3566. [3] Krylov, A. V.; Pohl, P.; Zeidel, M. L.; Hill, W. G. Journal of General Physiology 2001, 118, pp. 333-339. B. et al. (1989). Molecular biology of the cell. 2 ed.,New York: Garland Publishing Inc. • Preparation of asymmetrical membranes using different (phospho)lipid compositions. • Further electrical characterisation of BLMs that contain protein channels (á-HL, gramicidin). • Study of the influence of the amount of inserted proteins on the stability of the membrane. Influence of cholesterol in a membrane 0 50 100 150 200 250 300 350 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 X=PC/PS Voltage (mV) 0 20 40 60 80 100 120 140 160 180 Capacitance (pF) Breakthrough Voltage Capacitance 0 50 100 150 200 250 300 350 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 X=PI/PS Voltage (mV) 0 20 40 60 80 100 120 140 160 180 Capacitance (pF) Breakthrough Voltage Capacitance Influence of the presence of proteins Pure lipid membranes 0 50 100 150 200 250 300 350 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 X=DOPC/PS Voltage (mV) 0 20 40 60 80 100 120 140 160 180 Capacitance (pF) Breakthrough Voltage Capacitance L- -Phosphatidylethanolamine (Heart, Bovine) a L- -Phosphatidylinositol (Liver, Bovine-Sodium Salt) a L- -Phosphatidylcholine (Heart, Bovine) a L- -Phosphatidylserine (Brain, Porcine-Sodium Salt) á 1,2-Diphytanoyl-sn-Glycero-3-Phosphocholine 0 50 100 150 200 250 300 350 0 2 4 6 8 10 12 % CH Voltage (mV) Breakthrough voltage Properties of BLMs a mixture of 2 different PLs painted prepared from Parameters influencing membrane stability • Negative charge ð electrostatic repulsion • Unsaturations in the hydrocarbon tails ð “kinking” of the tail • Bulky headgroup • PE and DOPC have the same headgroup. • PE has one lipid tail with more unsaturations. Linear of the and of the of the membrane upon addition of PE (increased number of unsaturations) in the BLM due to a in the . decrease breakthrough voltage stability decrease PL packing density Decrease breakthrough voltage stability decrease PL packing density of the and of the of the membrane upon addition of PI (addition of unsaturations, bulky headgroups and negative charges) in the BLM that causes a in the . Linear of the and of the of the membrane upon addition of PI (more unsaturations, bulky headgroups and charges) in the BLM that causes a in the . decrease breakthrough voltage stability decrease PL packing density • • Lower packing density for 100% PS membranes. Mixed membranes (50:50) than monoPL membrane: stabilizing charge interactions between the headgroups of DOPC and PS + (ammonium NH moiety of DOPC with carboxylic 3 moiety of PS) causing an in the . more stable increase PL packing density Properties of BLMs more complex lipid mixtures Towards mimicking the membrane of cells prepared from ð Increase breakthrough voltage stability of the and of the of the membrane upon addition of cholesterol. BLMs mimicking the inner/outer leaflets • • Outer leaflet than the inner leaflet (good agreement with results reported in the literature). Mixed membranes than monoPL membranes. more stable more stable a-hemolysin (áHL) = channel protein. + • Rapid efflux of K and small molecules, influx of + + Na , Ca and molecules with MW < 1 kDa. 2 • Pore-diameter: 1.5 nm. • áHL used as a model of proteins found in cell membranes. • Jumps in the current = insertion of pore proteins (1 jump = 1 protein inserted). TOWARDS A BETTER UNDERSTANDING OF BIOLOGICAL MEMBRANE PROPERTIES: INFLUENCE OF THEIR COMPOSITION ON THEIR STABILITY BLMs created on a flat surface with a μhole Cholesterol DOPC: no unsaturation, neutral ð PI: 4 unsaturations, charged, bulky head ð highest ð most stable membrane V bt lowest ð least stable membrane V bt 0 50 100 150 200 250 300 350 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 X=DOPC/PE Voltage (mV) 0 20 40 60 80 100 120 140 160 180 Capacitance (pF) Breakthrough Voltage Capacitance DOPC • no unsaturation • neutral PE • 4 unsaturations • neutral DOPC • no unsaturation • neutral PI • 4 unsaturations • negative charge • bulky head PI • 4 unsaturations • negative charge • bulky head PS • 1 unsaturation • negative charge DOPC • no unsaturation • neutral PS • 1 unsaturation • negative charge PS • 1 unsaturation • negative charge PC • 2 unsaturations • neutral PL Unsaturations Charge V bt (mV) DOPC 0 x 204.00 PC 2 x 172.00 PS 1 168.33 PE 4 x 160.00 PI 4 91.67 GSL: Total Cerebrosides (Brain, Porcine). SM: Sphingomyelin (Egg, Chicken) DOPC membranes; MM technique, insertion of áHL. Stabilizing interactions between cholesterol and the hydrocarbon tails of the PLs [1]. 350 μm aperture PMMA substrate PMMA substrate PMMA substrate holder Device for BLM preparation Device fitting on a microscope stage PMMA Membrane Introduction Experimental Results Conclusion Outlook Acknowledgements References Hydrocarbon tail Headgroup Cis-double bond resulting in • • Similar packing density for monoPL membrane: commpensation between the number of unsaturations and the charge. Mixed membranes (50:50) than monoPL membrane (see above). more stable • Coupling electrical and optical measurements using a home made set-up. Decrease PL packing density reduced stability in the resulting in a of the membrane. Goal of this work PLs (% in mix) PE (48.3%) PS (25.2%) PI (3.6%) CH (22.6%) Mix V bt (mV) 160.00 168.33 91.67 - 270.00 PLs (% in mix) GSL (26.8%) SM (23.8% ) PC (12.0% ) CH (37.3% ) Mix V bt (mV) - - 172.00 - 340.00 Inner leaflet Outer leaflet Painting of inner/outer mixtures of MDCK cells [3] Painting of a CH- DOPC mixture Stability of the membranes determined by measuring its breakthrough voltage (V ) bt