Solid-supported Membranes Research Articles

Stimulation of Ca2+ -ATPase Transport Activity by a Small-Molecule Drug.

The sarco(endo)plasmic reticulum Ca2+−ATPase (SERCA) hydrolyzes ATP to transport Ca2+ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small‐molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP‐dependent Ca2+ translocation. The concentration dependence of the CDN1163 effect provided an EC50=6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca2+ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca2+ release into the SR lumen.

Engineering and functional characterization of a proton-driven \u03b2-lactam antibiotic translocation module for bionanotechnological applications

Novel approaches in synthetic biology focus on the bottom-up modular assembly of natural, modified natural or artificial components into molecular systems with functionalities not found in nature. A possible application for such techniques is the bioremediation of natural water sources contaminated with small organic molecules (e.g., drugs and pesticides). A simple molecular system to actively accumulate and degrade pollutants could be a bionanoreactor composed of a liposome or polymersome scaffold combined with energizing- (e.g., light-driven proton pump), transporting- (e.g., proton-driven transporter) and degrading modules (e.g., enzyme). This work focuses on the engineering of a transport module specific for β-lactam antibiotics. We previously solved the crystal structure of a bacterial peptide transporter, which allowed us to improve the affinity for certain β-lactam antibiotics using structure-based mutagenesis combined with a bacterial uptake assay. We were able to identify specific mutations, which enhanced the affinity of the transporter for antibiotics containing certain structural features. Screening of potential compounds allowed for the identification of a β-lactam antibiotic ligand with relatively high affinity. Transport of antibiotics was evaluated using a solid-supported membrane electrophysiology assay. In summary, we have engineered a proton-driven β-lactam antibiotic translocation module, contributing to the growing toolset for bionanotechnological applications.

Expanding the Potential of the Solvent-Assisted Method to Create Bio-Interfaces from Amphiphilic Block Copolymers.

Artificial membranes, as materials with biomimetic properties, can be applied in various fields, such as drug screening or bio-sensing. The solvent-assisted method (SA) represents a straightforward method to prepare lipid solid-supported membranes. It overcomes the main limitations of established membrane preparation methods, such as Langmuir-Blodgett (LB) or vesicle fusion. However, it has not yet been applied to create artificial membranes based on amphiphilic block copolymers, despite their enhanced mechanical stability compared to lipid-based membranes and bio-compatible properties. Here, we applied the SA method on different amphiphilic di- and triblock poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PDMS-b-PMOXA) copolymers and optimized the conditions to prepare artificial membranes on a solid support. The real-time membrane formation, the morphology, and the mechanical properties have been evaluated by a combination of atomic force microscopy and quartz crystal microbalance. Then, selected biomolecules including complementary DNA strands and an artificial deallylase metalloenzyme (ADAse) were incorporated into these membranes relying on the biotin-streptavidin technology. DNA strands served to establish the capability of these synthetic membranes to interact with biomolecules by preserving their correct conformation. The catalytic activity of the ADAse following its membrane anchoring induced the functionality of the biomimetic platform. Polymer membranes on solid support as prepared by the SA method open new opportunities for the creation of artificial membranes with tailored biomimetic properties and functionality.

Selection of Transporter-Targeted Inhibitory Nanobodies by Solid-Supported-Membrane (SSM)-Based Electrophysiology.

Single domain antibodies (nanobodies) have been extensively used in mechanistic and structural studies of proteins and they pose an enormous potential as tools for developing clinical therapies, many of which depend on the inhibition of membrane proteins such as transporters. However, most of the methods used to determine the inhibition of transport activity are difficult to perform in high-throughput routines and depend on labeled substrates availability thereby complicating the screening of large nanobody libraries. Solid-supported membrane (SSM) electrophysiology is a high-throughput method, used for characterizing electrogenic transporters and measuring their transport kinetics and inhibition. Here we show the implementation of SSM-based electrophysiology to select inhibitory and non-inhibitory nanobodies targeting an electrogenic secondary transporter and to calculate nanobodies inhibitory constants. This technique may be especially useful for selecting inhibitory nanobodies targeting transporters for which labeled substrates are not available.

The structural basis of promiscuity in small multidrug resistance transporters

By providing broad resistance to environmental biocides, transporters from the small multidrug resistance (SMR) family drive the spread of multidrug resistance cassettes among bacterial populations. A fundamental understanding of substrate selectivity by SMR transporters is needed to identify the types of selective pressures that contribute to this process. Using solid-supported membrane electrophysiology, we find that promiscuous transport of hydrophobic substituted cations is a general feature of SMR transporters. To understand the molecular basis for promiscuity, we solved X-ray crystal structures of a SMR transporter Gdx-Clo in complex with substrates to a maximum resolution of 2.3 Å. These structures confirm the family’s extremely rare dual topology architecture and reveal a cleft between two helices that provides accommodation in the membrane for the hydrophobic substituents of transported drug-like cations.

Correlation between adhesion strength and phase behaviour in solid-supported lipid membranes

Fundamental understanding of vesicle adhesion in the size range ≤ 200 nm is of major importance when addressing biologically relevant processes involving the presence of small vesicles like exosomes or endosomes. Using quartz crystal microbalance with dissipation monitoring, we investigate the correlation between vesicle deformation and eventual membrane rupture on surfaces with different adhesion levels, as well as their respective thermotropic phase transitions. In particular, phase transitions of solid-supported membranes on Au resemble the cooperative behaviour of lipid membrane transitions in bulk. In contrast, solid-supported membranes on SiO2 exhibit broadened ‘double-peak’ transitions, rendering a ‘decoupling’ effect during melting due to stronger interactions with SiO2. This paper provides a comprehensive view of the correlation between size, geometry and phase transitions observed in the layer of adsorbed lipid vesicles/membranes. It paves the way to explore structural changes on more complex biointerfaces by acoustic-based sensors.

Electrophysiology Measurements of Metal Transport by MntH2 from Enterococcus faecalis.

Transition metals are essential trace elements and their high-affinity uptake is required for many organisms. Metal transporters are often characterised using metal-sensitive fluorescent dyes, limiting the metals and experimental conditions that can be studied. Here, we have tested whether metal transport by Enterococcus faecalis MntH2 can be measured with an electrophysiology method that is based on the solid-supported membrane technology. E. faecalis MntH2 belongs to the Natural Resistance-Associated Macrophage Protein (Nramp) family of proton-coupled transporters, which transport divalent transition metals and do not transport the earth metals. Electrophysiology confirms transport of Mn(II), Co(II), Zn(II) and Cd(II) by MntH2. However, no uptake responses for Cu(II), Fe(II) and Ni(II) were observed, while the presence of these metals abolishes the uptake signals for Mn(II). Fluorescence assays confirm that Ni(II) is transported. The data are discussed with respect to properties and structures of Nramp-type family members and the ability of electrophysiology to measure charge transport and not directly substrate transport.

Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases.

P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

Rethinking the Bounds of Ion-Coupled Transport

Ion-coupled transporters are involved in numerous physiological processes, including nutrient acquisition, ion homeostasis, and toxin excretion, but the mechanistic details of how the energy stored in ion gradients is translated to uphill substrate transport are still poorly understood. This puzzle is especially interesting in the case of multidrug transporters, which must be able to couple ion flux to the transport of chemically diverse substrates. Using nuclear magnetic resonance (NMR) spectroscopy, our lab discovered that the proton-coupled multidrug transporter EmrE from E. coli must be capable of coupling drug and proton with multiple different stoichiometries. Here, we use mass action kinetic simulations and solid supported membrane (SSM) electrophysiology to rethink the bounds of ion-coupled transport. These results suggest the intriguing possibility that it may be possible to reverse the direction of driven transport, hijacking promiscuous transporters for targeted drug delivery. Our work has broad implications for the mechanism, evolution, and design of ion-coupled transporters.

Functional Characterization of SLC Transporters Using Solid Supported Membranes.

Here, we present a protocol for the functional characterization of the H+-coupled human peptide transporter PepT1 and sufficient notes to transfer the protocol to the Na+-coupled sugar transporter SGLT1, the organic cation transporter OCT2, the Na+/Ca2+ exchanger NCX, and the neuronal glutamate transporter EAAT3.The assay was developed for the commercially available SURFE2R N1 instrument (Nanion Technologies GmbH) which applies solid supported membrane (SSM)-based electrophysiology. This technique is widely used for the functional characterization of membrane transporters with more than 100 different transporters characterized so far. The technique is cost-effective, easy to use, and capable of high-throughput measurements.SSM-based electrophysiology utilizes SSM-coated gold sensors to physically adsorb membrane vesicles containing the protein of interest. A fast solution exchange provides the substrate and activates transport. For the measurement of PepT1 activity, we applied a peptide concentration jump to activate H+/peptide symport. Proton influx charges the sensor. A capacitive current is measured reflecting the transport activity of PepT1 . Multiple measurements on the same sensor allow for comparison of transport activity under different conditions. Here, we determine EC50 for PepT1-mediated glycylglycine transport and perform an inhibition experiment using the specific peptide inhibitor Lys[Z(NO2)]-Val.

Using Force Spectroscopy to Probe Coiled-Coil Assembly and Membrane Fusion.

Force spectroscopy allows the manipulation of single molecules and the characterization of their properties and interactionsthereby rendering it a powerful tool for biological sciences. Force spectroscopy at the level of individual molecules requires force resolution in the piconewton regime as achieved by optical tweezers (OT), magnetic tweezers (MT), and atomic force microscopy (AFM) with AFM providing the largest force range from tenth of piconewton to several micronewton. In membrane probe spectroscopy the commonly used sharp cantilever tip is replaced by a lipid-coated glass sphere. This technique expands the scope of force spectroscopy to processes at and between lipid bilayers, like the formation of coiled coils between SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins as well as subsequent membrane fusion. To this end, two solid-supported membranes equipped with SNARE proteins or fusion peptides are separately deposited on a flat glassy surface and on a micrometer glass sphere attached to the end of a tipless AFM cantilever. These two membranes are rapidly brought into contact until a defined force is reached. The AFM deflection readout is used to monitor the distance between the two bilayers, which allows to observe and identify fusion processes of the two lipid membranes, while the forces needed to separate the two surfaces give insights into the formation of SNARE complexes. By changing the contact pressure one can access fusion kinetics and to some extent reconstruct the energy landscape of membrane fusion. In this chapter we describe the preparation of membrane-coated colloidal probes attached to AFM cantilevers, experimental procedures, and necessary data analysis to perform membrane probe spectroscopy in the presence of fusogenic peptides or proteins.

A Comparative Study of Phosphatidylcholine versus Phosphatidylserine-Based Solid Supported Membranes for the Preparation of Liposome-Rich Interfaces.

Solid supported membranes (SSMs) are usually formed by an hybrid octadecanethiol/phosphatidylcholine (PC) bilayer supported by a gold electrode. Recently, it was shown that phosphatidylserine (PS) in place of PC can promote a more effective accumulation of lipid vesicles on the SSM surface when Ca2+ and Mg2+ ions are present in the external environment. Here we performed a detailed comparative study of the vesicle adsorption process onto PC- and PS-SSMs by employing surface plasmon resonance (SPR), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). SPR analysis has demonstrated a higher affinity of the PS-SSM surface for the phospholipid vesicles. Both SPR and EIS measurements suggest that adsorption of lipid vesicles on the PC-SSM tends to a saturating value, whereas a continuous and progressive vesicle adsorption occurs on the PS-SSM surface following subsequent liposome additions. AFM analysis pointed out a systematic flattening of the adsorbed vesicles on the PS-SSM surface. We interpreted our results as due to the strong coordinating action of the high amount of divalent cations accumulated at the negatively charged PS-SSM surface, whereas a lower amount of cations is present on the dipolar PC-SSM surface, which can therefore adsorb only a limited number of vesicles.

Quartz Crystal Microbalance With Dissipation Monitoring: A Versatile Tool to Monitor Phase Transitions in Biomimetic Membranes

Solid-supported lipid membranes are popular models that connect biological and artificial materials used in bio-technological applications. Controlling the lipid organization and the related functions of these model systems entails understanding and characterizing their phase behavior. Quartz crystal microbalance with dissipation (QCM-D) is an acoustic-based surface-sensitive technique which is widely used in bio-interfacial science of solid-supported lipid membranes. Its sensitivity to mass and energy dissipation changes at the solid-lipid layer-liquid interface allows the detection of phase transformations of solid-supported membrane geometries. In this perspective, we highlight this valuable feature and its related methodology, review current advances and briefly discuss future perspectives. Furthermore, a specific example on the ability of QCM D to detect lipid organization changes of cholesterol containing solid-supported lipid vesicle layers (SVLs) upon the addition of aspirin is also provided

EAAT3 Investigated using SSM-Based HTS Electrophysiology on the SURFE2R 96SE

The excitatory amino acid transporter 3 (EAAT3) is involved in the neuronal re-uptake of glutamate and plays a central role in the regulation of excitatory neurotransmission and synaptic plasticity. EAAT3 also transports cysteine, necessary for the synthesis of glutathione and GABA. EAAT3 is expressed not only throughout the brain, but in many organs such as intestines, liver and heart. Here it seems to provide the main pathway of aspartate. Several connections of EAAT3 to severe neuronal disorders like epilepsy and schizophrenia have been described, as well as to metabolic disturbances concerning in the maintenance of aspartate and cysteine levels. This makes EAAT 3 not only an important target for functional research, but also a potential drug target. Here, we present data on EAAC1, a mouse homologue of EAAT3, using a novel high throughput instrument for SSM (solid supported membrane)-based electrophysiology, the SURFE2R 96SE. SSM-based electrophysiology is a label-free electrical measuring method with very high sensitivity which enables the resolution of low turnover transport and even binding-events. Using the purified membrane of EAAC1 expressing CHO cells, we were able to determine substrate affinities and their interaction and to compare the effect of six known inhibitors directly with each other. We evaluated the assay stability and success rate. Furthermore, we were able to resolve substrate binding and to confirm the described anion conductance of the transporter. Using the SURFE2R 96SE with CHO cells we were able to generate an efficient, robust and very flexible assay, which is an ideal tool for the biophysical and pharmacological characterization of EAAC1 and even suitable for drug screening approaches.

Conformational memory in the association of the transmembrane protein phospholamban with the sarcoplasmic reticulum calcium pump SERCA

The sarcoplasmic reticulum Ca2+-ATPase SERCA promotes muscle relaxation by pumping calcium ions from the cytoplasm into the sarcoplasmic reticulum. SERCA activity is regulated by a variety of small transmembrane peptides, most notably by phospholamban in cardiac muscle and sarcolipin in skeletal muscle. However, how phospholamban and sarcolipin regulate SERCA is not fully understood. In the present study, we evaluated the effects of phospholamban and sarcolipin on calcium translocation and ATP hydrolysis by SERCA under conditions that mimic environments in sarcoplasmic reticulum membranes. For pre-steady-state current measurements, proteoliposomes containing SERCA and phospholamban or sarcolipin were adsorbed to a solid-supported membrane and activated by substrate concentration jumps. We observed that phospholamban altered ATP-dependent calcium translocation by SERCA within the first transport cycle, whereas sarcolipin did not. Using pre-steady-state charge (calcium) translocation and steady-state ATPase activity under substrate conditions (various calcium and/or ATP concentrations) promoting particular conformational states of SERCA, we found that the effect of phospholamban on SERCA depends on substrate preincubation conditions. Our results also indicated that phospholamban can establish an inhibitory interaction with multiple SERCA conformational states with distinct effects on SERCA's kinetic properties. Moreover, we noted multiple modes of interaction between SERCA and phospholamban and observed that once a particular mode of association is engaged it persists throughout the SERCA transport cycle and multiple turnover events. These observations are consistent with conformational memory in the interaction between SERCA and phospholamban, thus providing insights into the physiological role of phospholamban and its regulatory effect on SERCA transport activity.

The influence of solid scaffolds on flat and curved lipid membranes

Solid-supported membranes have become a common tool to study lipid membrane properties in a controlled environment. One particular example is the study of membrane curvature and its effect on lipid sorting. Here we simulate solid-supported membranes using the coarse grain molecular dynamics Martini force field. We characterize basic properties of the solid surfaces and lipid membranes deposited on them. Subsequently we construct large, solid ridges and use them to induce curvature in DOPC membranes. We study membrane properties, such as lateral diffusion and tail order parameters, relative to the curved membrane. Finally, we study the effect of the induced curvature on lateral lipid sorting in a ternary lipid membrane. Thus, we obtain comprehensive and microscopic insight into the impact of curvature on a lipid membrane in terms of structure and dynamics.

Lipid bilayer membrane technologies: A review on single-molecule studies of DNA sequencing by using membrane nanopores

Nanopores based on α-hemolysin and MspA represent attractive sensing platforms due to easy production and operation with relatively low background noise. Such characteristics make them highly favorable for sequencing nucleic acids. Artificial lipid bilayer membranes, also referred to as black lipid membranes, in conjunction with membrane nanopores, can be applied to both the detection and highly efficient sequencing of DNA on a single-molecule level. However, the inherently weak physical properties of the membrane have impeded progress in these areas. Current issues impeding the ultimate recognition of the artificial lipid bilayer as a viable platform for detection and sequencing of DNA include membrane stability, lifespan, and automation. This review (with 105 references) highlights attempts to improve the attributes of the artificial lipid bilayer membrane starting with an overview on the present state and limitations. The first main section covers lipid bilayer membranes (BLM) in general. The following section reviews the various kinds of lipid bilayer membrane platforms with subsections on polymer membranes, solid-supported membranes, hydrogel-encapsulated membranes, shippable and storable membrane platforms, and droplet interface bilayers. A further section covers engineered biological nanopore sensor applications using BLMs with subsections offering a comparative view of different DNA sequencing methods, a detailed look at DNA Sequencing by synthesis using alpha-hemolysin nanopores, sequencing by synthesis using the MspA nanopore and quadromer map, and on limitations of sequencing based on synthesis technology. We present an outlook at the end that discusses current research trends on single-molecule sequencing to highlight the significance of this technology and its potential in the medical and environmental fields.

From Gene to Function: Cell-Free Electrophysiological and Optical Analysis of Ion Pumps in Nanodiscs

Nanodiscs that hold a lipid bilayer surrounded by a boundary of scaffold proteins have emerged as a powerful tool for membrane protein solubilization and analysis. By combining nanodiscs and cell-free expression technologies, even completely detergent-free membrane protein characterization protocols can be designed. Nanodiscs are compatible with various techniques, and due to their bilayer environment and increased stability, they are often superior to detergent micelles or liposomes for membrane protein solubilization. However, transport assays in nanodiscs have not been conducted so far, due to limitations of the two-dimensional nature of nanodisc membranes that offers no compartmentalization. Here, we study Krokinobacter eikastus rhodopsin-2 (KR2), a microbial light-driven sodium or proton pump, with noncovalent mass-spectrometric, electrophysiological, and flash photolysis measurements after its cotranslational insertion into nanodiscs. We demonstrate the feasibility of adsorbing nanodiscs containing KR2 to an artificial bilayer. This allows us to record light-induced capacitive currents that reflect KR2’s ion transport activity. The solid-supported membrane assay with nanodisc samples provides reliable control over the ionic condition and information of the relative ion activity of this promiscuous pump. Our strategy is complemented with flash photolysis data, where the lifetimes of different photointermediates were determined at different ionic conditions. The advantage of using identical samples to three complementary approaches allows for a comprehensive comparability. The cell-free synthesis in combination with nanodiscs provides a defined hydrophobic lipid environment minimizing the detergent dependence often seen in assays with membrane proteins. KR2 is a promising tool for optogenetics, thus directed engineering to modify ion selectivity can be highly beneficial. Our approach, using the fast generation of functional ion pumps incorporated into nanodiscs and their subsequent analysis by several biophysical techniques, can serve as a versatile screening and engineering platform. This may open new avenues for the study of ion pumps and similar electrogenic targets.

Mechanisms of charge transfer in human copper ATPases ATP7A and ATP7B.

ATP7A and ATP7B are Cu+ -transporting ATPases of subclass IB and play a fundamental role in intracellular copper homeostasis. ATP7A/B transfer Cu+ ions across the membrane from delivery to acceptor proteins without establishing a free Cu+ gradient. Transfer of copper across the membrane is coupled to ATP hydrolysis. Current measurements on solid supported membranes (SSM) were performed to investigate the mechanism of copper-related charge transfer across ATP7A and ATP7B. SSM measurements demonstrated that electrogenic copper displacement occurs within ATP7A/B following addition of ATP and formation of the phosphorylated intermediate. Comparison of the time constants for cation displacement in ATP7A/B and sarcoplasmic reticulum Ca2+ -ATPase is consistent with the slower phosphoenzyme formation in copper ATPases. Moreover, ATP-dependent copper transfer in ATP7A/B is not affected by varying the pH, suggesting that net proton counter-transport may not occur in copper ATPases. Platinum anticancer drugs activate ATP7A/B and are subjected to ATP-dependent vectorial displacement with a mechanism analogous to that of copper. © 2016 IUBMB Life, 69(4):218-225, 2017.

SSM-Based Electrophysiology for Transporter Research.

Functional characterization of transport proteins using conventional electrophysiology can be challenging, especially for low turnover transporters or transporters from bacteria and intracellular compartments. Solid-supported membrane (SSM)-based electrophysiology is a sensitive and cell-free assay technique for the characterization of electrogenic membrane proteins. Purified proteins reconstituted into proteoliposomes or membrane vesicles from cell culture or native tissues are adsorbed to the sensor holding an SSM. A substrate or a ligand is applied via rapid solution exchange. The electrogenic transporter activity charges the sensor, which is recorded as a transient current. The high stability of the SSM allows cumulative measurements on the same sensor using different experimental conditions. This allows the determination of kinetic properties including EC50, IC50, Km, KD, and rate constants of electrogenic reactions. About 100 different transporters have been measured so far using this technique, among them symporters, exchangers, uniporters, ATP-, redox-, and light-driven ion pumps, as well as receptors and ion channels. Different instruments apply this technique: the laboratory setups use a closed flow-through arrangement, while the commercially available SURFE2R N1 resembles a pipetting robot. For drug screening purposes high-throughput systems, such as the SURFE2R 96SE enable the simultaneous measurement of up to 96 sensors.