Dynamic force spectroscopy on supported lipid bilayers: effect of temperature and sample preparation.

Biological membranes are constantly exposed to forces. The stress-strain relation in membranes determines the behavior of many integral membrane proteins or other membrane related-proteins that show a mechanosensitive behavior. Here, we studied by force spectroscopy the behavior of supported lipid bilayers (SLBs) subjected to forces perpendicular to their plane. We measured the lipid bilayer mechanical properties and the force required for the punch-through event characteristic of atomic force spectroscopy on SLBs as a function of the interleaflet coupling. We found that for an uncoupled bilayer, the overall tip penetration occurs sequentially through the two leaflets, giving rise to two penetration events. In the case of a bilayer with coupled leaflets, penetration of the atomic force microscope tip always occurred in a single step. Considering the dependence of the jump-through force value on the tip speed, we also studied the process in the context of dynamic force spectroscopy (DFS). We performed DFS experiments by changing the temperature and cantilever spring constant, and analyzed the results in the context of the developed theories for DFS. We found that experiments performed at different temperatures and with different cantilever spring constants enabled a more effective comparison of experimental data with theory in comparison with previously published data.

Changes in single K(+) channel behavior induced by a lipid phase transition.

We show that the activity of an ion channel is correlated with the phase state of the lipid bilayer hosting the channel. By measuring unitary conductance, dwell times, and open probability of the K(+) channel KcsA as a function of temperature in lipid bilayers composed of POPE and POPG in different relative proportions, we obtain that all those properties show a trend inversion when the bilayer is in the transition region between the liquid-disordered and the solid-ordered phase. These data suggest that the physical properties of the lipid bilayer influence ion channel activity likely via a fine-tuning of its conformations. In a more general interpretative framework, we suggest that other parameters such as pH, ionic strength, and the action of amphiphilic drugs can affect the physical behavior of the lipid bilayer in a fashion similar to temperature changes resulting in functional changes of transmembrane proteins.

Supported lipid bilayers on mica and silicon oxide: comparison of the main phase transition behavior.

The usual biophysical approach to the study of biological membranes is that of turning to model systems. From these models, general physical principles ruling the lateral membrane structure can be obtained. A promising model system is the supported lipid bilayer (SLB) which could foresee the simultaneous investigation of the structure and physical properties of lipid bilayers reconstituted with membrane proteins. A complete exploitation of the model system to retrieve biologically relevant information requires an in-depth knowledge of the possible effect that experimental parameters could have on the behavior of the SLB. Here we used atomic force microscopy (AFM) to study the effect of different types of substrates on the behavior of SLBs as far as their main phase transition is concerned. We found that different substrates (mica and silicon oxide) can affect in dissimilar ways the interleaflet coupling of the bilayer, which might represent a sort of lipid signaling allowing communication between receptors on the extracellular leaflet and cytoplasmic components. By decreasing the interaction between the SLB and the substrate the interleaflet coupling is preserved independently of the bilayer preparation strategy. Moreover, we investigated by time-lapse AFM an isothermal phase transition induced by a pH change on a SLB. We established that the presence of a pH gradient across the bilayer can weaken the strength of the interleaflet coupling which is present in symmetrical pH conditions.

Phase-transition-induced protein redistribution in lipid bilayers.

We report an atomic force microscopy study on the lateral spatial redistribution of an integral membrane protein reconstituted in supported lipid bilayers (SLBs) subjected to a thermally induced phase transition. KcsA proteins were reconstituted in proteoliposomes of POPE/POPG (3:1, mol/mol), and SLBs, including the proteins, were then obtained by the vesicle fusion technique on mica. By decreasing the temperature, the lipid bilayer passed from a liquid disordered (l(d)) phase in which the proteins are homogeneously distributed to a coexistence of solid ordered (s(o)) and l(d) domains with the proteins preferentially distributed in the l(d) domains. The inhomogeneous distribution eventually led to protein clustering. The obtained results are discussed in light of the role that the lipid/protein interaction can have in determining the function of integral membrane proteins.

How anesthetics, neurotransmitters, and antibiotics influence the relaxation processes in lipid membranes.

We used pressure perturbation calorimetry to investigate the relaxation time scale after a jump into the melting transition regime of artificial lipid membranes. This time is equivalent to the characteristic rate of domain growth. The studies were performed on single-component large unilamellar and multilamellar vesicle systems with and without the addition of small molecules such as general anesthetics, neurotransmitters, and antibiotics. These drugs interact with membranes and affect melting points and profiles. In all systems, we found that heat capacity and relaxation times are linearly related to each other in a simple manner, and we outline the theoretical origin of this finding. Thus, the influence of a drug on the time scale of domain formation processes can be understood on the basis of their influence on the heat capacity profile. This allows estimations of the characteristic relaxation time scales in biological membranes.

Diffusion in two-component lipid membranes--a fluorescence correlation spectroscopy and monte carlo simulation study.

Using fluorescence correlation spectroscopy, calorimetry, and Monte Carlo simulations, we studied diffusion processes in two-component membranes close to the chain melting transition. The aim is to describe complex diffusion behavior in lipid systems in which gel and fluid domains coexist. Diffusion processes in gel membranes are significantly slower than in fluid membranes. Diffusion processes in mixed phase regions are therefore expected to be complex. Due to statistical fluctuations the gel-fluid domain patterns are not uniform in space and time. No models for such diffusion processes are available. In this article, which is both experimental and theoretical, we investigated the diffusion in DMPC-DSPC lipid mixtures as a function of temperature and composition. We then modeled the fluorescence correlation spectroscopy experiment using Monte Carlo simulations to analyze the diffusion process. It is shown that the simulations yield a very good description of the experimental diffusion processes, and that predicted autocorrelation profiles are superimposable with the experimental curves. We believe that this study adds to the discussion on the physical nature of rafts found in biomembranes.