Coupling optical and electrical measurements in artificial membranes: Lateral diffusion of lipids and channel forming peptides in planar bilayers
Biological Procedures Online ?
Coupling Optical and Electrical Measurements in Artificial Membranes: Lateral Diffusion of Lipids and Channel Forming Peptides in Planar Bilayers
Duclohier H. 0
Helluin O. 0
Lea E. 1
Mackie A.R. 2
S. Ladha 2
0 UMR 6522 CNRS-Universite? de Rouen, Bd M. de Broglie , 76821 Mont-Saint-Aignan , France
1 School of Biological Sciences, University of East Anglia , Norwich NR4 7TJ , England. Present Address: The Johnson Research Foundation for Molecular Biophysics and Structural Biology, Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine , Philadelphia, PA 19104-6059 , USA
2 Institute of Food Research, BBSRC , Colney Research Park, Norwich NR4 7UA, England
Planar lipid bilayers (PLB) were prepared by the Montal-Mueller technique in a FRAP system designed to simultaneously measure conductivity across, and lateral diffusion of, the bilayer. In the first stage of the project the FRAP system was used to characterise the lateral dynamics of bilayer lipids with regards to phospholipid composition (headgroup, chain unsaturation etc.), presence of cholesterol and the effect of divalent cations on negatively-charged bilayers. In the second stage of the project, lateral diffusion of two fluorescently-labelled voltage-dependent pore-forming peptides (alamethicin and S4s from Shaker K+ channel) was determined at rest and in the conducting state. This study demonstrates the feasibility of such experiments with PLBs, amenable to physical constraints, and thus offers new opportunities for systematic studies of structure-function relationships in membrane-associating molecules.
The need to couple functional measurements (nearly exclusively electrical measurements for channels)
with other physical methods informing about membrane dynamics or structure (with respect to both
lipid matrix and protein or peptide effectors) is a long-standing issue in Biophysics. It requires
ingenious experimental set-ups that often compromise between the high sensitivity and rapid kinetics
allowed by electrophysiological recordings (at the single-channel level, 1-10 molecules can be
functionally monitored in a membrane area of, say, 1 mm2 on a millisecond timescale) and the usually
low signal to noise ratio associated with optical or spectroscopic methods. Refer to the review by
MacDonald and Wraight (1). Nevertheless, beginning with the classical papers on natural excitable
membranes of the sixties, eg. (2), successful and meaningful studies have been reported ever since,
perhaps with fewer such attempts reported recently despite technological improvements. Some of these
technologies are still in development, for example, near-field optical microscopy and fluorescence
correlation spectroscopy, among others.
In spite of the fact that planar lipid bilayers are still the best-suited artificial membrane system for the
study of reconstituted ion channels and receptors, data dealing with their physical characterisation,
especially as regards dynamics are scant. The dynamics of molecules in membranes (order parameter,
degree of freedom of lipid aliphatic chains, lateral and rotational diffusions), often referred to as
membrane fluidity, have been associated with modulation of the activity of many important functions of
proteins in biological membranes (for a review, see 3). Following cold or heat adaptation and diet
changes, the membrane lipid composition can readjust via metabolic pathways so as to provide an
optimal lipid environment (the ?homeoviscous theory?, see e.g. 4-5). Alterations in the microviscosity
(and presumably the lateral mobility) can also be strongly correlated to some pathologies (
fluidity changes were also shown, through monitoring of the rate of excimer formation, to occur in
pyrene labeled nerve fibers during action potentials (
). Lateral diffusion within natural or artificial
membranes is one of the most well-characterised parameters related to the dynamic state of the
membrane (see review 8). This diffusion has fundamental implications in functional coupling between
membrane components through collisional mechanisms as, for example, in the photosynthetic electron
), in visual transduction (
) as well as in receptor-mediated endocytosis (
) and in
intercellular adhesion (
). Recently, new techniques such as ?single particle tracking? demonstrate
that lateral diffusion is not homogeneous throughout the plasma membrane of most cells (
Specific examples include cell junctions of vascular endothelium (
) and neurons (
Over the last fifteen years, fluorescence recovery after photobleaching (FRAP) has become the most
direct and elegant way of measuring the rate of lipid and protein lateral diffusion, mostly within
biological membranes such as red blood cells (
). Significant theoretical and methodological
improvements such as the ?fringe or periodic pattern bleaching? (
), later improved (
) and more
recently ?scanning microphotolysis? (
) complement the basic technique.
Although most spectroscopic investigations dealing with membrane models have been carried out on
populations of lipid vesicles, planar lipid bilayers have become popular model systems for
characterising the function of purified voltage-gated channels, receptors and their peptide models (see
e.g. 22-24). Pioneering work on direct measurement of the lateral diffusion coefficient (D) of
fluorescent probes in lipid bilayers was carried out with bilayers supported on a circular platinum loop
) and on electron microscopy grids (
). However, little effort has since been devoted to
characterising D in more ?realistic? model membranes used in reconstitution studies. The present work
gives the first determinations of D on ?virtually solvent- free? Montal-Mueller bilayers (referred
hereafter as PLB for planar lipid bilayers) formed in a conventional manner (
), and which are
amenable to simultaneous fluorescence recovery after photobleaching (FRAP) as well as conductance
MATERIALS AND METHODS
The lipids were purchased from Avanti Polar Lipids (Birmingham, AL) and used without further
purification. Cholesterol (CHOL) was purchased from Sigma Chemical Company (St Louis, MI). The
N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE) was purchased from Molecular Probes (Eugene, OR). Fluorescently labelled
alamethicin (Alm-Gly-FITC) was prepared as previously described (
). Fluorescently labelled S4
Shaker K+ (N-(7-nitrobenzoyl-2-oxa-1,3-diazol-4-yl) -S4 Shaker (NBD-S4 Shaker) was a gift from Dr
Y. Shai (
Virtually solvent-free planar lipid bilayer formation
A 25m m thick PTFE septum (Goodfellow, Cambridge, UK) with a hole of 200-300 m m diameter (made
by an electric arc discharge) in the centre was clamped into a specially designed chamber allowing
simultaneous electrical (conductance and capacitance) and FRAP measurements (Figure 1).
The body of the cell was machined from two blocks of PTFE. The cell was assembled by carefully
positioning the PTFE septum between the cell halves with the hole located centrally. Optical windows
were fitted in the recesses of the outer faces of the cell. These in turn were held in position by a
temperature controlled brass housing which was clamped against each face of the cell. Prior to
membrane formation, the hole in the septum was coated with 1 ?l of 1% (v/v) hexadecane in hexane on
each side. The hexane was allowed to evaporate. To form Montal and Mueller bilayers, buffer was
added to each side of the chamber such that the level was above the hole in the septum. Lipid
containing 1 mole% NBD-PE was spread from a hexane-ethanol (9:1 v/v) solution on the buffer surface
in the chambers and allowed to stand for a few minutes to allow evaporation of the solvent. The buffer
level on the trans-side was lowered below the hole in the PTFE septum and then raised back to its
original level. PLB formation was monitored visually and by capacitance measurements.
Capacitance and conductance measurements.
Conductance and capacitance measurements were performed from the same experimental set-up. A DC
voltage or a mixed DC-AC voltage from a signal generator (20 mHz Pulse/Function generator, model
628, Dynatech, Nevada) is applied in the cis side of the bilayer via Ag/AgCl electrode. A second
Ag/AgCl trans electrode is connected to an I-V converter (RAP Montgomery, model HAMK2, London,
UK) with a 1 GW feedback resistor. The output signal is then filtered by a dual variable filter (Kemo,
model VBF4, Beckenham, UK) and sent to an oscilloscope, a X-Y recorder or a microcomputer. For the
conductance measurements, a DC or a low frequency (0.01 Hz) triangular voltage is applied to ensure
steady-state instantaneous current responses. For capacitance measurements, a triangular wave of high
frequency (amplitude 10 mV, frequency 100Hz) was applied to the bilayer. If the conductance of the
bilayer remains low, the capacitance of the system could be directly obtained from the rms value of the
squared output ( I = C dV/dt) , capacitance being equal to V/K, with K =10 mV pF-1 The system was
calibrated with capacitors of known values and the contribution of the line (cables, connections ...
outside the bilayer) was substracted.
Fluorescence recovery after photobleaching (FRAP)
Measurement of the lateral diffusion coefficient of the fluorophore in planar lipid bilayers was achieved
using the FRAP method. A schematic diagram of the apparatus (Figure 2) was developed from our
conventional spot photobleaching FRAP set-ups that are based on upright (
) and inverted (
Argon ion laser
FFigiguurree 22. .SchSecmhaetimcdaiatigcradmiaogfrthaemproinfcitphael cpormipnocniepnatsl ocfotmhepFolunoernestcsenocfe tPhheotFobllueoacrheisncgence
Photobleaching (FRAP) apparatus
Essentially the apparatus was constructed using components from a Nikon Optiphot microscope
mounted on its side (i.e. with a horizontal optical axis). The nosepiece and trinocular eyepiece of the
microscope were mounted on a special Nikon bracket which was bolted to an optical rail (Newport,
Microcontrole) on the laser table (Photon Control, Cambridge). The PLB cell was mounted on a
separate bracket attached to a micrometer-controlled translation stage to allow focusing, and the stage
was again bolted to the optical rail.
The intensity of the beam of a 10W argon ion laser (Innova 100-10) was attenuated by reflection off
glass flats and by passing through a neutral density filter as shown in Figure 2. When the fast electronic
shutter (Uniblitz) (c) is closed, only the monitoring laser beam (a) illuminates the sample. When the
shutter is open the intense, bleaching beam (b) which is transmitted through two of the glass flats
passes through to the sample. The beam provided by the modulator (Coherent Innova, model 304A)
passed through a beam monitor (beam splitter and photodiode), the signal from which is used to
electronically compensate for minor fluctuations in laser beam intensity. The laser beam then passed
through a pinhole aperture (A1) located at the image plane, at the entrance port of the epi-illumination
attachment of the fluorescence microscope. A dichroic mirror (DM) and long pass filter (LPF) were
used to ensure only emitted light reached the photon counting photomultiplier tube (PMT; Thorn-EMI
9816B) positioned at the camera port of the trinocular eyepiece. The PMT was protected during the
bleaching pulse by an electronic gating circuit and a mechanical shutter (MS). Prior to entering the
detector, the emitted light beam passed through a second aperture (A2) again positioned at the image
plane. The laser beam profile and spot radius at the point of focus were determined using a beam
scanner (Photon Inc, BeamScan model 2180, California, USA). The laser beam was of Gaussian cross
sectional intensity with half-width at 1/e2 height of the laser beam at its point of focus equal to 3.3 m m
(spot radius). System timing and control, data acquisition and data analysis are performed using a VME
microcomputer system (Motorola 68020). The acquisition software was developed in house. The
microscope and cell were enclosed with an aluminium box which acted as a Faraday cage and
minimised noise in the conductance measurements.
All FRAP experiments were performed at a controlled room temperature of 23oC. The laser spot (spot
radius 3.3 m m) was directed at the centre of the PLB and was sufficiently small for the surrounding
bilayer to act as a ?near infinite reservoir?. In the experiments described below, generally ten FRAP
curves were collected for each set of conditions and averaged before analysis. FRAP data were analysed
by non-linear least squares fitting to an expression defining the time dependence of fluorescence
recovery observed with a circular beam of Gaussian cross sectional intensity and had the form (
F(0) + F(? ) t
? b t D ? ,
1 + ? t b t D ?
where F(t) is the observed fluorescence as a function of time, F(0) is the intensity of the fluorescence
immediately after the bleach pulse, F(? ) is the fluorescence at infinite time after the bleach pulse, b is
the depth of bleach parameter and tD is the characteristic diffusion time. The lateral diffusion
coefficient, D, is given by D = w2/4tD, where w (spot radius) is the half-width at 1/e2 height of the laser
beam at its point of focus on the membrane. The percentage mobile fraction (%R; %recovery) is given
% R =
F(? ) - F(0)
F(t < 0) - F(0)
where F(t<0) is the prebleach fluorescence (
RESULTS AND DISCUSSION
At the initial stages of discussing the concept of combining the FRAP and bilayer set-up, two
configurations were discussed. Firstly, having the FRAP system on the vertical axis and the bilayer
setup on the horizontal axis. The second configuration involved developing a novel FRAP system on the
horizontal axis and forming the bilayer in the conventional manner in the vertical axis. The first
configuration was attractive because at the Institute of Food Research (IFR) we already had well
established FRAP systems using the conventional inverted (
) and upright (
Therefore, we had to design a novel bilayer system which would allow formation of PLBs on the
horizontal axis and use it like a ?specimen slide? to interrogate the bilayer with the FRAP system.
However, attempts at Rouen University to develop a system which would allow the formation of the
PLBs on the horizontal axis showed that this approach was problematical. Therefore, we decided to
proceed with the second configuration and mounted the microscope horizontally on an optical rail and
then connected the necessary electronics to carry out FRAP.
Initial tests of the horizontal FRAP layout, indicated that problems with the alignment of the optics had
to be solved. Briefly, in the FRAP set-up the bleach laser beam, the monitoring laser beam and the
detection of fluorescence by the PMT have to be coincident at the point focus on the planar bilayer. At
the IFR we had considerable expertise in aligning the optics of the inverted (
) and upright (
microscopes to successfully carry out FRAP experiments. However, with the microscope essential
dismantled and mounted horizontally all the usual reference points, used to align the optics, were no
longer present. To overcome this difficulty a special viewer was designed to allow the fluorescent spot
to be monitored at the exit pinhole and to ensure that all the optics were in register to give optimal
Introduction of the bilayer set-up involved the designing and construction of a bilayer chamber which
was compatible with the FRAP set-up. The design shown in Figure 1 has proved to be excellent. It has
to noted that the distance from the centre of the chamber where the hole in the septum is located to the
optical window is 1cm and therefore requires the use of long working distance objectives in order to
observe the formation of the PLBs. To minimise the electrical interference from the laser equipment,
the current-voltage converter used to measure capacitance and conductance of the PLBs was installed
as close as possible to the chamber between the incident and collecting parts of the microscope with the
whole being enclosed in a Faraday box. Special software was also developed to link the bilayer and
FRAP detection and analysis systems in order to synchronise the data collection.
In conjunction with establishing the equipment we also transferred the techniques necessary to make
PLBs from Rouen and Essex Universities to IFR. Three techniques commonly used to form bilayers
were tested for the value to this project:
1) The liposome method (
) proved the most reliable way to form a bilayer. Although conductivity
measurements could be performed with this membrane, an excess of liposomes in the light path made
FRAP measurements impossible.
2) The "painting" method (
) formed bilayers which allowed both conductivity and FRAP
measurements. The main disadvantage of the bilayers formed by this technique was that they contained
large amounts of spreading solvent and the physical properties of such bilayers were not constant.
3) The Montal-Mueller method (
) of forming bilayers was ideal for both FRAP and conductivity
The bilayers were usually prepared with the fluorophore incorporated into the lipid phase. However,
some fluorescently labelled peptides were added directly into the bulk phase and incorporation into the
bilayer was monitored by conductance and fluorescence measurements. The fluorophores were chosen
such that there was little or no fluorescence from the free fluorophore in bulk aqueous phase, but, when
incorporated into the lipid phase, there was a significant enhancement in fluorescence. This assisted in
minimising the interference from the fluorescence of the fluorophore in the bulk aqueous phase and
ensured that only fluorescence from the fluorophore incorporated into the bilayer was being monitored.
However, if the choice of fluorophore was restricted such that there were appreciable amounts of
fluorescence in the bulk aqueous phase, then we used a rapid perfusion system to exchange the aqueous
phase in the chamber and then proceeded with the experiment. This procedure would eliminate
interference from the fluorescence of the fluorophore from the bulk aqueous phase and allow the
measurement of fluorescence of the fluorophore associated with the bilayer.
The new experimental set-up was tested for standard lipid diffusion regimes in nude bilayers (without
?membrane effectors?) with different lipids, cholesterol, and added calcium (
). We then proceeded to
illustrate how important information about the pore forming mechanism of alamethicin could be
derived from the approach described in this article. Alamethicin, a natural peptaibol (rich in the
noncoded alpha-aminoisobutyric acid), being still the prototype (the most extensively-studied) of highly
voltage-dependent pore-formers is useful in modelling aspects of physiologically-important ion
channels and receptors (i.e. the action potential Na+, K+ and Ca2+ channels). As a result, the
?barrelstave? model (popular for describing the pore forming mechanisms of alamethicin and by later
extension of a number of natural or designed amphipathic peptides) for dynamic intramembrane
conducting aggregates applies strictly to channel- and pore-formers whose open state (or active
conformation) is driven by voltage. In this model, the central conducting part of the pore is made up by
the juxtaposition of the hydrophilic sectors of neighbouring helices forming a transmembrane bundle.
The uptake and release of alamethicin monomers by this bundle, via lateral diffusion in the bilayer, is
the simplest explanation accounting for the non-integral conductance increments. There is general
agreement between conductance values and a geometric model for a pore of varying size, depending
upon the number of monomers or helical rods. In this scheme, it is obvious that the lateral diffusion of
monomers within the lipid bilayer is an important process in regulating the pore structure. To this end,
we prepared fluorescently labelled pore forming peptides and determined their lateral diffusion
properties at rest and in the conducting state.
Results show that the voltage-induced ?building up? of peptide conducting aggregates (whether
) or S4s, the gating element of Shaker potassium channels) leads to a slight but
significant reduction in the lateral diffusion of the peptide. The voltage-dependent process can be
divided into i) a voltage-driven partition of the peptide from the aqueous bulk and/or insertion into the
bilayer, and ii) an intrinsic voltage-sensitivity (gating proper) of the embedded aggregates. The two do
not match exactly and the apparent high voltage-dependence observed in macroscopic conductance
experiments (see below) mainly reflects the first process.
G = I / V = ? Nae (V) . g . Po(V)
where G and I are macroscopic conductance and current, V the applied voltage, ? Nae (V) the number of
channels expressing or opened at a given voltage, g the average single-channel conductance?or the
conductance of the most probable substate?and Po(V) the probability of opening at the specified
voltage. Thus the reduced lateral diffusion coefficients observed with these two different peptides (quite
unrelated in their sequence and amphipathy) most probably result from the further
membraneembedment of the pore-formers under the influence of voltage (on the helical dipoles), the first step
leading to ?peptide channels?.
This study illustrates the feasibility of coupled optical and electrical measurements on reconstituted
systems, i.e. ion channels or peptide models interacting with planar lipid bilayers. This is an exquisite
experimental/biophysical configuration which allows precise control of the chemical species involved
(lipids, peptides etc) and of electrolytes on both sides of the bilayer, and it is unique in maintaining
gradients (electrical and chemical). These parameters are much to harder to control in the conventional
lipid vesicles (or proteolipid vesicles). As recently suggested (1), combining spectroscopic and
electrical recording techniques offers real opportunities in the investigation of structure-function
relationships of membrane interacting peptides.
The programme was initiated in 1991 thanks to a modest Franco-British grant (Alliance scheme, no. 91
073) which allowed bilateral reciprocal visits for discussion (couple of days) and working sessions
(couple of weeks). A number of oral and poster communications were soon presented and although the
granted period ended in 1993, the main articles were published in 1996 and 1997 and the programme
has the potential to expand. Altogether this research involved (albeit partly) around 10 scientists. This
has been a real interdisciplinary project truly relevant to Membrane Biophysics, involving physical
chemists, organic or peptide chemists, biochemists, and electrophysiologists with interests both in
natural channels and their peptide models reconstituted into planar lipid bilayers (PLBs). We also like
to thank David Clark for his helpful discussions during the initial stages of this project. Dr S. Ladha and
A. Mackie were supported by CSG funding from the BBSRC.
Macdonald A.G. and P.C. Wraight 1995 . Combined spectroscopic and electrical recording techniques in membrane research: prospects for single channel studies . Prog. Biophys. Molec. Biol.
Cohen , L.B. , Landowne , D. and B.M. Salzberg . 1990 . Optical measurements on squid axons . In ? Squid as experimental animals? (edited by D .L. Gilbert , W.J. Adelman , Jr. and J.M. Arnold).
Plenum Press, New York. pp 161 - 170 .
Shinitzky , M. 1984a . Membrane fluidity and cellular functions . In Physiology of Membrane Fluidity , Vol. I. M. Shinitzky , editor. CRC Press, Inc., Boca Raton , Florida.
Cossins , A. R. 1983 . The adaptation of membrane structure and function to changes in temperature . In Cellular Adaptation to Environmental Changes. A.R. Cossins and P. Sheterline, editors. Cambridge University Press, Cambridge, London, New York. 3- 32 .
Cuculescu , M. , D. Hyde and K. Bowler 1995 . Temperature acclimation of marine crabs: changes in plasma membrane fluidity and lipid composition . J. Therm. Biol . 20 , 207 - 222 .
6. Shinitzky , M. 1984b . Membrane fluidity in malignancy: adversative and recuperative . Biochim. Biophys. Acta 738 , 251 - 261 .
7. Georgescauld , D. and H. Duclohier . 1978 . Transient fluorescence signals from pyrene labeled pike nerves during action potentials. Possible implications for membrane fluidity changes . Biochem. Biophys. Res. Commun . 83 , 1186 - 1191 .
8. Tocanne , J. F. , L. Dupou-C?zanne and A. Lopez . 1994 . Lateral diffusion of lipids in model and natural membranes . Prog. Lipid Res . 33 , 203 - 237 .
9. Blackwell , M., C. Gubas , S. Gygax , D. Roman and B. Wagner . 1994 . The planoquinone diffusion coefficient in chloroplasts and its mechanistic implications . Biochim. Biophys. Acta 1183 , 553 - 543 .
10. Lamb , T. D. 1994 . Stochastic simulation of activation in the G-protein cascade of phototransduction . Biophys. J . 67 , 1439 - 1454 .
11. Schlessinger , J. 1993 . Lateral and rotational diffusion of EGF-receptor complex relationship to receptor mediated endocytosis . Biopolymers 22 , 47 - 353
12. Leckband , D. E. , J. N. Israelachvili , F. J. Schmitt and W. Knoll . 1992 . Long-range attraction and molecular-rearrangements in receptor-ligand interactions . Science 255 , 1419 - 1421 .
13. Anderson , C. M. , G. N. Georgiou , I. E. G. Morrison , G. V. W. Stevenson and R. J. Cherry . 1992 . Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge coupled device camera - low density lipoprotein and influenza virus receptor mobility at 4oC . J.Cell Sci . 101 , 415 - 425 .
14. Cherry , R. J., G. N. Georgiou and I. E. G. Morrison . 1994 . New insights into the structure of cell membranes from single particle tracking experiments . Biochem. Soc. Trans . 22 , 781 - 784 .
15. Jacobson , K. , Sheets , E.R. and Simpson , R. 1995 . Revisiting the fluid mosaic model . Science 268 , 1441 - 1442 .
16. Tournier , J. F. , A. Lopez , N. Gas and J. F. Tocanne . 1989 . The lateral motion in the apical plasma membrane of endothelial cells is reversibly affected by the presence of cell junctions . Exp. Cell Res . 181 , 375 - 384 .
17. Joe , E.H. and Angelides , K.J. 1993 . Clustering and mobility of voltage-dependent sodium channels during myelination . J. Neurosci . 13 , 2993 - 3005 .
18. Bloom , J. A. and W. W. Webb . 1983 . Lipid diffusibility in the intact erythrocyte membrane . Biophys. J . 42 , 295 - 305 .
19. Smith , B. A. and H. M. McConnell . 1978 . Determination of molecular motion in membranes using periodic pattern photobleaching . Proc. Natl. Acad. Sci . 75 , 2759 - 63 .
20. Davoust , J. , P. F. Devaux and L. Leger . 1982 . Fringe pattern photobleaching, a new method for the measurement of transport coefficients of biological macromolecules . EMBO J . 1 , 1233 - 1238 .
21. Wedekind , P. , U. Kubitschek, and R. Peters . 1994 . Scanning microphotolysis: a new photobleaching technique based on fast intensity modulation of a scanned laser beam and confocal imaging . J. Microsc . 176 , 23 - 33 .
22. Krueger , B. K. , J. F. Worley and R. J. French . 1983 . Single sodium channel from rat brain incorporated into planar lipid bilayers . Nature 303 , 172 - 175
23. Hanke , W. and W. R. Schlue . 1993 . Planar lipid bilayers. Methods and applications . Academic Press. London, San Diego.
24. Chang , H. M. , R. Reitsjetter , R. P. Mason and R. Gruener . 1995 . Attenuation of channel kinetics and conductance by cholesterol: an interpretation using structural stress as a unifying concept . J. Membrane Biol . 143 , 51 - 63 .
25. Koppel , D.E. , Axelrod , D. , Schlessinger , J. , Elson , E.L. and W.W. Webb . 1976 . Dynamics of fluorescence marker concentration as a probe of mobility . Biophys. J . 16 , 1315 - 1329 .
26. Fahey , P. F. and W. W. Webb . 1978 . Lateral diffusion in phospholipid bilayer membranes and multilamellar liquid crystals . Biochemistry . 17 , 3046 - 3053 .
27. Montal , M. and P. Mueller . 1972 . Formation of bimolecular membranes from monolayers and study of their properties . Proc. Natl. Acad. Sci. USA 69 , 3561 - 3566 .
28. Helluin , O. , Dugast , J-Y , Molle, G. , Mackie , A. R. , Ladha , S. and Duclohier , H. ( 1997 ) Lateral diffusion and conductance properties of a fluorescein-labelled alamethicin in planar lipid bilayers . Biochimica et Biophysica Acta , 1330 , 284 - 292
29. Peled , Z.H. , Arkin , I.T. , Engelman , D.M. and Y. Shai . 1996 . Coassembly of synthetic segments of Shaker K+ channel within phospholipid membranes . Biochemistry 35 , 6828 - 6838 .
30. Ladha , S. , A. R. Mackie and D. C. Clark . 1994 . Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady state anisotropy . J.Membrane Biol ., 142 , 223 - 229 .
31. Clark , D. C. , R. Dann , A. R. Mackie , J. Mingins , A. C. Pinder , P. W. Purdy , E. J. Russell , L. J. Smith and D. R. Wilson . 1990 . Surface diffusion in SDS stabilized thin liquid films . J. Colloid Interf. Sci. , 138 , 195 - 206 .
32. Yguerabide , J. , J. A. Schmidt and E. E. Yguerabide . 1982 . Lateral mobility in membranes as detected by fluorescence recovery after photobleaching . Biophys. J . 39 , 69 - 75 .
33. Wolf , D. E. 1989 . Designing, building and using a fluorescence recovery after photobleaching instrument . Methods Cell Biol . 30 , 271 - 306 .
34. Schindler , H. 1980 . Formation of planar bilayers from artificial or native membrane vesicles . FEBS letters 122 , 77 - 79 .
35. Mueller , P. , Rudin , D. O. , Tien , H. T. and Westcott , W. C. 1962 . Reconstitution of cell membrane structure in vitro and its transformation into an excitable system . Nature 194 , 979 - 980 .
36. Ladha , S. , Mackie , A. , Harvey , L. , Clark , D. , Lea , E. , Brullemans , M. and Duclohier , H. ( 1996 ) Lateral diffusion in planar lipid bilayers. A FRAP investigation of its modulation by lipid composition, cholesterol or alamethicin content and divalent cations . Biohys. J . 71 , 1364 - 1373 .