Whole-GUV patch-clamping.

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

Mechano-capacitive properties of polarized membranes.

Biological membranes are capacitors that can be charged by applying a field across the membrane. The charges on the capacitor exert a force on the membrane that leads to electrostriction, i.e. a thinning of the membrane. Since the force is quadratic in voltage, negative and positive voltage have an identical influence on the physics of symmetric membranes. However, this is not the case for a membrane with an asymmetry leading to a permanent electric polarization. Positive and negative voltages of identical magnitude lead to different properties. Such an asymmetry can originate from a lipid composition that is different on the two monolayers of the membrane, or from membrane curvature. The latter effect is called 'flexoelectricity'. As a consequence of permanent polarization, the membrane capacitor is discharged at a voltage different from zero. This leads to interesting electrical phenomena such as outward or inward rectification of membrane permeability. Here, we introduce a generalized theoretical framework, that treats capacitance, polarization, flexoelectricity, piezoelectricity and thermoelectricity in the same language. We show applications to electrostriction, membrane permeability and piezoelectricity and thermoelectricity close to melting transitions, where such effects are especially pronounced.

Lipid ion channels and the role of proteins.

In the absence of proteins, synthetic lipid membranes can display quantized conduction events for ions that are virtually indistinguishable from those of protein channels. The phenomenological similarities between typical conductances are striking: they are of equal order and show similar lifetime distributions and current histograms. They can include conduction bursts, flickering, and multistep conductance. Lipid channels can be gated by voltage and blocked by drugs. They respond to changes in lateral membrane tension and temperature. Thus, they behave like voltage-gated, temperature-gated, and mechano-sensitive protein channels, or like receptors. The similarity between lipid and protein channels poses an important problem for the interpretation of protein channel data. For example, the Hodgkin-Huxley theory for nerve pulse conduction requires a selective mechanism for the conduction of sodium and potassium ions. To this end, the lipid membrane must act both as a capacitor and as an insulator. Nonselective ion conductance by mechanisms other than the gated protein channels challenges the proposed mechanism for pulse propagation. Nevertheless, textbooks rarely describe the properties of the lipid membrane surrounding the proteins in their discussions of membrane models. These similarities lead to important questions: Do these similarities in lipid and protein channels result from a common mechanism, or are these similarities fortuitous? What distinguishes protein channels from lipid channels, if anything? In this Account, we document experimental and theoretical findings that show the similarity between lipid and protein channels. We discuss important cases where protein channel function strongly correlates with the properties of the lipid. Based on statistical thermodynamics simulations, we discuss how such correlations could come about. We suggest that proteins can act as catalysts for lipid channel formation and that this hypothesis can explain some of the unexplained correlations between protein and lipid membrane function.

Fluctuations of systems in finite heat reservoirs with applications to phase transitions in lipid membranes.

In an adiabatically shielded system, the total enthalpy is conserved. Enthalpy fluctuations of an arbitrarily chosen subsystem must be buffered by the remainder of the total system which serves as a heat reservoir. The magnitude of these fluctuations depends on the size of the reservoir. This leads to various interesting consequences for the physical behavior of the subsystem. As an example, we treat a lipid membrane with a phase transition that is embedded in an aqueous reservoir. We find that large fluctuations are attenuated when the reservoir has finite size. This has consequences for the compressibility of the membrane since volume and area fluctuations are also attenuated. We compare the equilibrium fluctuations of subsystems in finite reservoirs with those in periodically driven systems. In such systems, the subsystem has only finite time available to exchange heat with the surrounding medium. A larger frequency therefore reduces the volume of the accessible heat reservoir. Consequently, the fluctuations of the subsystem display a frequency dependence. While this work is of particular interest for a subsystem displaying a transition such as a lipid membrane, some of the results are of a generic nature and may contribute to a better understanding of relaxation processes in general.

Non-invasive detection of animal nerve impulses with an atomic magnetometer operating near quantum limited sensitivity.

Magnetic fields generated by human and animal organs, such as the heart, brain and nervous system carry information useful for biological and medical purposes. These magnetic fields are most commonly detected using cryogenically-cooled superconducting magnetometers. Here we present the first detection of action potentials from an animal nerve using an optical atomic magnetometer. Using an optimal design we are able to achieve the sensitivity dominated by the quantum shot noise of light and quantum projection noise of atomic spins. Such sensitivity allows us to measure the nerve impulse with a miniature room-temperature sensor which is a critical advantage for biomedical applications. Positioning the sensor at a distance of a few millimeters from the nerve, corresponding to the distance between the skin and nerves in biological studies, we detect the magnetic field generated by an action potential of a frog sciatic nerve. From the magnetic field measurements we determine the activity of the nerve and the temporal shape of the nerve impulse. This work opens new ways towards implementing optical magnetometers as practical devices for medical diagnostics.

The Effect of the Nonlinearity of the Response of Lipid Membranes to Voltage Perturbations on the Interpretation of Their Electrical Properties. A New Theoretical Description.

Our understanding of the electrical properties of cell membranes is derived from experiments where the membrane is exposed to a perturbation (in the form of a time-dependent voltage or current change) and information is extracted from the measured output. The interpretation of such electrical recordings consists in finding an electronic equivalent that would show the same or similar response as the biological system. In general, however, there is no unique circuit configuration, which can explain a single electrical recording and the choice of an electric model for a biological system is based on complementary information (most commonly structural information) of the system investigated. Most of the electrophysiological data on cell membranes address the functional role of protein channels while assuming that the lipid matrix is an insulator with constant capacitance. However, close to their melting transition the lipid bilayers are no inert insulators. Their conductivity and their capacitance are nonlinear functions of both voltage, area and volume density. This has to be considered when interpreting electrical data. Here we show how electric data commonly interpreted as gating currents of proteins and inductance can be explained by the nonlinear dynamics of the lipid matrix itself.