Trapped Pore Waters in the Open Proton Channel HV 1.

The voltage-gated proton channel, HV 1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10-12 cm3 s-1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the HV 1 wild-type channel and the D174A mutant open at 0 mV. The conductance of 1400 H+ s-1 per wild-type channel agrees with the calculated diffusion limit for a ≈2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while interrupting the water wire across the membrane.

Modeling of SGLT1 in Reconstituted Systems Reveals Apparent Ion-Dependencies of Glucose Uptake and Strengthens the Notion of Water-Permeable Apo States.

The reconstitution of secondary active transporters into liposomes shed light on their molecular transport mechanism. The latter are either symporters, antiporters or exchangers, which use the energy contained in the electrochemical gradient of ions to fuel concentrative uptake of their cognate substrate. In liposomal preparations, these gradients can be set by the experimenter. However, due to passive diffusion of the ions and solutes through the membrane, the gradients are not stable and little is known on the time course by which they dissipate and how the presence of a transporter affects this process. Gradient dissipation can also generate a transmembrane potential (VM). Because it is the effective ion gradient, which together with VM fuels concentrative uptake, knowledge on how these parameters change within the time frame of the conducted experiment is key to understanding experimental outcomes. Here, we addressed this problem by resorting to a modelling approach. To this end, we mathematically modeled the liposome in the assumed presence and absence of the sodium glucose transporter 1 (SGLT1). We show that 1) the model can prevent us from reaching erroneous conclusions on the driving forces of substrate uptake and we 2) demonstrate utility of the model in the assignment of the states of SGLT1, which harbor a water channel.

Photoswitching of model ion channels in lipid bilayers.

Membrane proteins can be regulated by alterations in material properties intrinsic to the hosting lipid bilayer. Here, we investigated whether the reversible photoisomerization of bilayer-embedded diacylglycerols (OptoDArG) with two azobenzene-containing acyl chains may trigger such regulatory events. We observed an augmented open probability of the mechanosensitive model channel gramicidin A (gA) upon photoisomerizing OptoDArG's acyl chains from trans to cis: integral planar bilayer conductance brought forth by hundreds of simultaneously conducting gA dimers increased by typically >50% - in good agreement with the observed increase in single-channel lifetime. Further, (i) increments in the electrical capacitance of planar lipid bilayers and protrusion length of aspirated giant unilamellar vesicles into suction pipettes, as well as (ii) changes of small-angle X-ray scattering of multilamellar vesicles indicated that spontaneous curvature, hydrophobic thickness, and bending elasticity decreased upon switching from trans- to cis-OptoDArG. Our bilayer elasticity model for gA supports the causal relationship between changes in gA activity and bilayer material properties upon photoisomerization. Thus, we conclude that photolipids are deployable for converting bilayers of potentially diverse origins into light-gated actuators for mechanosensitive proteins.

Scattering versus fluorescence self-quenching: more than a question of faith for the quantification of water flux in large unilamellar vesicles?

The endeavors to understand the determinants of water permeation through membrane channels, the effect of the lipid or polymer membrane on channel function, the development of specific water flow inhibitors, the design of artificial water channels and aquaporins for the use in industrial water filtration applications all rely on accurate ways to quantify water permeabilities (P f). A commonly used method is to reconstitute membrane channels into large unilamellar vesicles (LUVs) and to subject these vesicles to an osmotic gradient in a stopped-flow device. Fast recordings of either scattered light intensity or fluorescence self-quenching signals are taken as a readout for vesicle volume change, which in turn can be recalculated to accurate P f values. By means of computational and experimental data, we discuss the pros and cons of using scattering versus self-quenching experiments or subjecting vesicles to hypo- or hyperosmotic conditions. In addition, we explicate for the first time the influence of the LUVs size distribution, channel distribution between vesicles and remaining detergent after protein reconstitution on P f values. We point out that results such as the single channel water permeability (p f) depend on the membrane matrix or on the direction of the applied osmotic gradient may be direct results of the measurement and analysis procedure.

Micropipette Aspiration-Based Assessment of Single Channel Water Permeability.

Measurements of the unitary hydraulic conductivity of membrane channels, pf , may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for pf measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving pf values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. pf amounted to 2.4 ± 0.1 × 10-13 cm³ s-1 for aquaporin-1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on pf on a single sample.

Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP.

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

Intrinsic Membrane Permeability to Small Molecules.

Spontaneous solute and solvent permeation through membranes is of vital importance to human life, be it gas exchange in red blood cells, metabolite excretion, drug/toxin uptake, or water homeostasis. Knowledge of the underlying molecular mechanisms is the sine qua non of every functional assignment to membrane transporters. The basis of our current solubility diffusion model was laid by Meyer and Overton. It correlates the solubility of a substance in an organic phase with its membrane permeability. Since then, a wide range of studies challenging this rule have appeared. Commonly, the discrepancies have their origin in ill-used measurement approaches, as we demonstrate on the example of membrane CO2 transport. On the basis of the insight that scanning electrochemical microscopy offered into solute concentration distributions in immediate membrane vicinity of planar membranes, we analyzed the interplay between chemical reactions and diffusion for solvent transport, weak acid permeation, and enzymatic reactions adjacent to membranes. We conclude that buffer reactions must also be considered in spectroscopic investigations of weak acid transport in vesicular suspensions. The evaluation of energetic contributions to membrane translocation of charged species demonstrates the compatibility of the resulting membrane current with the solubility diffusion model. A local partition coefficient that depends on membrane penetration depth governs spontaneous membrane translocation of both charged and uncharged molecules. It is determined not only by the solubility in an organic phase but also by other factors like cholesterol concentration and intrinsic electric membrane potentials.

The Effect of Buffers on Weak Acid Uptake by Vesicles.

The assessment of weak acid membrane permeability (Pm) frequently involves large unilamellar vesicles. It relies on measurements of the intravesicular pH drop, ΔpHin, in response to a sudden augmentation of external acid concentration. However, ΔpHin may be primarily governed by non-instantaneous protonation and deprotonation reactions of (i) the acid itself, (ii) the buffer molecules, and (iii) the fluorescent pH reporter dye. Moreover, buffer concentration and acid gradient also serve as determinants of ΔpHin, as we show here. The uniexponential time constant (τ) of ΔpHin(t) is an invalid measure of Pm as Arrhenius plots of Pm and τ reveal different activation energies for acid influx. We calculate Pm by fitting a mathematical model to experimental stopped-flow traces. The model takes into account not only the time course of total internal buffer capacity but also (i) water self-dissociation, (ii) volume changes due to acid induced osmotic water flow, and (iii) the spontaneous membrane proton leak. It allows extracting a Pm of 30.8 ± 3.5 µm/s for formic acid for 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicles.

Passive Permeability of Planar Lipid Bilayers to Organic Anions.

The membrane permeability P of organic ions was reported to be governed by the structure of the permeating molecule. Thus far, it is unclear whether the ion structure alters membrane partition or translocation proper across the membrane. Here, we obtained P values for 24 anionic compounds (18 concrete values, 6 upper limits) measuring the current that they carry through folded planar lipid bilayers. The P values range over more than 10 log units. Our measured permeability values correlate well (r = 0.95; logRMSE 0.74) with the hexadecane/water partition coefficients of the respective chemicals predicted by the COSMO-RS theory. Other attempts to predict P from the partition coefficient of the neutral molecule and from the solvation energy (Born energy) that opposes transfer into the membrane once the molecule is charged were unsuccessful. The uncertainties in assigning an effective radius to nonspherical molecules were much too large. The observation underlines that the actual structure of the molecules needs to be considered to predict partition and thus P by the solubility-diffusion model.

Membrane Permeabilities of Ascorbic Acid and Ascorbate.

Vitamin C (VC)-a collective term for the different oxidation and protonation forms of ascorbic acid (AscH)-is an essential micronutrient that serves as (i) a potent antioxidant and (ii) a cofactor of a manifold of enzymatic processes. Its role in health is related to redox balance maintenance, which is altered in diseases such as obesity, cancer, neurodegenerative diseases, hypertension, and autoimmune diseases. Despite its importance, VC uptake has been poorly investigated. Available literature values for the passive membrane permeability P of lipid bilayers for AscH scatter by about 10 orders of magnitude. Here, we show by voltage clamp that P - of AscH's anionic form (ascorbate Asc - ) is negligible. To cross the membrane, Asc - picks up a proton in the membrane vicinity and releases it on the other side of the membrane. This leads to a near-membrane pH drop that was visualized by scanning pH microelectrodes. The AscH concentration dependent pH profiles indicated P   =   1.1   ±   0.1   ×   10 - 8   cm / s . Thus, AscH's P is comparable to that of sorbitol and much lower than that of other weak acids like acetic acid or salicylic acid. The observation suggests that the capacity of the passive transcellular transport pathway across the lipid matrix does not suffice to ensure the required VC intake from the gastrointestinal tract.