Interleaflet Diffusion Coupling When Polymer Adsorbs onto One Sole Leaflet of a Supported Phospholipid Bilayer

ReceiVed October 9, 2006 ReVised Manuscript ReceiVed January 11, 2007 Introduction. Lipid mobility in fluid-phase lipid bilayers attracts much interest not just because this problem is fundamental but also because it is so closely related to biological processes such as membrane ion channel formation, ligandreceptor binding, and membrane adhesion and fusion.1-3 Here we consider lipid lateral diffusion in bilayers that are supported on a solid substrate. Literature on this general question is extensive, including various aspects such as free Brownian motion in singleand multicomponent bilayers,4,5 anomalous subdiffusion in heterogeneous bilayers,6 obstructed diffusion in phase-separated bilayers,7 and hop diffusion in compartmentalized cell membranes.8 However, the fact that any lipid bilayer is comprised of an inner leaflet and an outer leaflet raises the question of whether lipid moves synchronously in the two leafletssa problem of interleaflet coupling in phospholipid bilayers. Here we focus on discriminating between diffusion on the two sides of the bilayer. Previous studies of interleaflet coupling in phospholipid bilayers did not investigate the situation where the adsorbate was located onto one sole side. Important prior studies investigated lipid raft formation in the two leaflets due to temperature decrease or incorporated cholesterol molecules.9,10 Also, in the absence of adsorbate, lipid diffusion in the outer and inner leaflets of a planar-supported lipid bilayer was studied with the conclusion that strong coupling exists between diffusion in the inner and outer leaflets.11 We address the basic question of what happens when adsorbate is present. As adsorbate, we allow polymers to adsorb at low surface coverage to the outer leaflet, and using iodide quenching of diffusion in the outer leaflet, we discriminate between lipid diffusion in the outer and inner leaflets. Experimental Section. For study, phospholipid DLPC, 1,2dilauroyl-sn-glycero-3-phosphocholine, was purchased from Avanti Polar Lipids (Alabaster, AL), into which we doped at 10 ppm (0.001%) molar concentration DMPE, 1,2-dimyristoylsn-glycero-3-phosphoethanolamine, with polar head group labeled by rhodamine B. Supported bilayers of the mixture were formed on hydrophilic quartz using known protocols based on the fusion of small unilamellar vesicles.12 Fully quaternized poly(4-vinylpyridine), QPVP, was prepared by us from parent poly(vinylpyridine) (Polymer Source Inc., Quebec, Canada) by reaction with an excess of ethyl bromide.13 The surface coverage of QPVP on DLPC bilayers was quantified using Fourier transform infrared spectroscopy in the mode of attenuated total reflection (FTIR-ATR) using known methods.13 All measurements were made in PBS buffer (10 mM, pH 6.0). The lateral diffusion of dye-labeled lipids was measured by fluorescence correlation spectroscopy (FCS) in the two-photon excitation mode using a home-built apparatus.14 As illustrated in Figure 1, near-infrared femtosecond pulses from a Ti:sapphire laser (800 nm, 82 MHz, pulse width ∼100 fs) were directed into a water immersion objective lens (Zeiss Axiovert 135 TV, 63x, NA ) 1.2) via a dichroic mirror and focused onto the sample, giving an excitation spot whose diffraction-limited diameter was ∼0.35 μm, thus affording measurements that were spatially resolved depending on where the laser beam was focused. Fluorescence from the sample was collected by the same objective and split into two channels after passing through the dichroic mirror and the emission filter. Each channel was connected to a single photon counting module (Hamamatsu). The photon counting output was recorded by an integrated FCS data acquisition board (ISS, Champaign, IL) and was analyzed to give an autocorrelation function curve, G(τ), which one can fit to standard equations to give the translational diffusion coefficient (D).15,16 For the planar-supported phospholipid bilayer system studied here, it is appropriate to fit data of this kind to a 2-dimensional Gaussian model with two-photon excitation using the following equation17