Published: May 11th, 2013
Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.
The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.
Here we demonstrate a new electrophysiological approach based on a solid supported membrane (SSM) for characterization of electrogenic membrane proteins.
The solid support consists of a thin gold layer on a glass slide, the sensor chip. The hydrophilic gold surface is used to bind the thiol group of an alcanethiol reagent. Afterwards, selfassembly of a phosphatidylcholine monolyer completes the formation of the SSM.
To measure electrogenic reactions of membrane proteins, proteoliposomes or membrane fragments are adsorbed to the SSM (Figure 1). The protein containing membrane and the SSM then form a capacitively coupled membrane system. Therefore, charge translocation at the protein containing membrane can be detected by capacitive coupling via the SSM. This method yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
The sensor chip is mounted into a cuvette system (Figure 2). The cuvette has a cylindrical cuvette volume of 17 μl (net volume with o-ring mounted). A spring contact pin creates the contact to the amplifier. An outlet connector is screwed to the top of the main part and carries the reference electrode, a chlorinated silver wire.
The cuvette is mounted in a Faraday cage. It is connected to a fluid pathway , which is used to induce the transport activity of the membrane protein in response to a fast solution exchange (Figure 3). In the single solution exchange protocol two solutions, one non-activating and one activating solution, are required. The flow is controlled by pressurized air using a valve control software on a computer or manual switches on an interface box.
1. Setting up an Apparatus for SSM-based Electrophysiology
Details are given in two of our technical publications, which also contain schematic drawings and photographs of our cuvettes and set up 1, 2. Different solution exchange configurations and flow protocols are also discussed in our method paper 2.
In the following we add a few recent improvements and technical details which are of direct relevance for the video presentation.
Valve control and data acquisition
The interface box contains a commercial USB digital output / analog input interface (NI USB 6009 National Instruments) and the valve drivers. It controls the solution flow and is responsible for data acquisition. Valves are normally driven with 12 V. However, for fast switching the valves can be driven with a voltage of up to 18 V. In the video we use a 12 V power supply.
The valve driver circuit board can operate four valves. It was manufactured by the workshop of the Max Planck Institute of Biophysics. Valves can be controlled by computer or manually via switches at the front panel. The latter is convenient for flushing- and cleaning-procedures. During measurement, the interface box is computer-controlled using a valve control and data acquisition software (SURFE2R software, IonGate Biosciences).
In this section the different protocols for the preparation of a SSM-based electrophysiology experiment are mentioned.
2.1 Making the Lipid solution for formation of the SSM
2.2 Chlorination of the reference electrode
The reference electrodes have to be chlorinated in regular intervals due to abrasion.
2.3 Preparation of the polyacrylamide gel bridge
2.4 Preparation of the measuring solutions
Due to the strong interaction of the solutes with the SSM, electrical artifacts are generated when solutions of different composition are exchanged. Therefore, the preparation of the solutions is a critical step. Take the following precautions during the solution preparation process to minimize solution exchange artifacts.
3. A SSM-based Electrophysiology Experiment
Here we present a typical protocol for a SSM-based electrophysiological experiment.
3.1 Preparing the SSM Setup
While the SSM Setup is not in use the tubes are filled with a 30% ethanol/water solution to avoid bacterial growth.
3.2 Mounting the cuvette
3.3 Measuring membrane parameters
To check the quality of the SSM, capacitance and conductance are measured using the function generator.
3.4 Checking for solution exchange artifacts
After the membrane parameters have been checked, the measuring buffers should be tested for solution exchange artifacts.
3.5 Adding the protein sample
Usually proteoliposomes or membrane fragments are stored frozen at -80 °C. For one measurement an aliquot of approximately 30 μl is needed.
3.6 General Procedure of an SSM-based experiment
3.7 Control Measurements
A signal rundown during a series of measurements could occur due to protein degradation or a loss of adsorption. Each SSM experiment should include a rundown control. This is done by repeated measurements with identical solutions. The minimal rundown control means one measurement at the beginning and one at the end of the experiment, using the same solution.
We recommend to do the rundown control under conditions where you expect the highest peak amplitude in the experiment. This makes it easier to quantify the rundown of the signal.
The signal rundown can be quantified by a comparison of the peak amplitudes of the two rundown controls. The resulting percentage value can be used to correct the measured signals by assuming a linear time dependence of the rundown.
3.8 Artifact controls
If possible try to validate your results by inhibiting the protein of interest after the experiment. Remaining signals are probably solution exchange artifacts. The signals then have to be corrected for the measured artifacts.
3.9 After the experiment
Up to now, SSM-based electrophysiology was used to characterize more than 20 transporters, mostly of prokaryotic origin, e.g. MelB 3, NhaA 4 and PutP 5 (summarized in 6). But also eukaryotic transporters from intracellular membranes, e.g. ClC7 7 as well as ion channels, e.g. the nicotinic acetylcholine receptor 8 have been investigated.
Here we present as an example measurements of the sugar/H+ cotransporter LacY from Escherichia coli using proteoliposomes with at least 85% right side out oriented LacY at a LPR of 5 9, 10. We focus on the main steps leading to a minimal kinetic model of this transport protein.
1. Electrophysiological characterization of wildtype LacY
The first step is a general characterization of the electrogenic reaction by measuring different pH values (Figure 4), substrate concentrations (Figure 5) and substrates. To validate the technique, KM and pK values given in literature were reproduced.
The pH dependence of LacY shows two different electrogenic reactions. The capacitively coupled current increases between pH 5 and pH 8.5, but also changes its shape. Under alkaline conditions steady state transport is observed, while for acidic pH values only a rapid electrogenic reaction remains. To distinguish the steady state signals from the fast electrogenic reactions we used circuit analysis to reconstruct transporter currents (Figure 6).
At pH 7 the monophasic signal becomes biphasic. This also indicates that there are two different electrogenic reactions. Estimated from the transferred charge (integral of the signals), the two reactions have about 6% and 94% of the total electrogenicity of the transport cycle.
2. Assignment of the electrogenic reactions
To assign the two electrogenic reactions to particular steps in the reaction cycle of LacY, the E325A LacY variant was measured (Figure 7). This variant only shows lactose exchange but is deficient for all active transport modes, because of the inhibition of proton release. The signal of E325A LacY represents a fast transient and is constant for all measured pH values. The shape and amplitude of the signal is similar to the signal of wildtype LacY under acidic conditions. In addition we observe a small negative phase after the declining peak current, which is characteristic for rapid transient currents measured in capacitively coupled systems. Because lactose binding and release is not electrogenic, the fast transient must correlate with an electrogenic conformational transition following lactose binding.
It is known that the proton release step is rate limiting for LacY turnover in the lactose concentration gradient driven transport mode. In this case we expect an increase of the transport rate with alkaline pH, because proton release is favored. This is the case for the steady state transport correlated signal of wildtype LacY. Moreover it could be shown by pH gradient measurement that only the inner pH influences the steady state transport of LacY (unpublished). Therefore the large electrogenic reaction could be assigned to the proton release step.
3. Measurements for kinetic analysis
After identification of the electrogenic reactions on and off transport rates were determined using a SSM Setup with a high time resolution of about 4.5 msec. This is only possible under conditions, when one reaction dominates the signal. In this case the rate constants can be derived from the transient currents under consideration of the time resolution of the signals using an iterative least-squares deconvolution algorithm (Figure 8). The determined rate constants as well as the proposed minimal kinetic model (Figure 9) finally were used for the simulation of the transporter kinetics. The simulated curves reproduce the SSM data which additionally proves the kinetic model.
Figure 1. Adsorption geometry of proteoliposomes on the SSM. The SSM is formed on a sensor chip, a structured gold coated glass slide. To measure electrogenic reactions of membrane proteins, proteoliposomes or membrane fragments are adsorbed to the SSM. The two membranes form a capacitively coupled system. This is why only transient currents are detected.
Figure 2. The SSM cuvette carries the sensor chip and the reference electrode. The sensor chip is sandwiched between the cuvette base and the cuvette head. The reference electrode is isolated from the flow pathway by a polyacrylamide gel salt bridge.
Figure 3. Fluid Pathway and Electrical Circuit of the SSM Setup. In the single solution exchange protocol only one non-activating (NA) and one activating (A) solution are required. The flow is controlled by two 2-way valves (V1) and one terminal valve (V2). The terminal valve switches between non-activating and activating solutions, directing one of the solutions to the cuvette and the other solution to a waste container (W). The sensor chip (SSM) is connected to the amplifier (I/U), while the reference electrode (AgCl) is connected to the function generator (F) in the external electrical circuit. A computer (PC) and an interface box are used for valve operation and data acquisition.
Figure 4. Transient currents measured with wild-type LacY proteoliposomes after 100 mM lactose concentration jumps at different pH values. The non-activating solution contained 100 mM glucose while the activating solution contained 100 mM lactose. All solutions were prepared in 100 mM potassium phosphate buffer at the indicated pH with 1 mM DTT. For details of measurement conditions in this and the following figures please refer to 9 and 10.
Figure 5. Averaged normalized Peak currents measured with wild-type LacY proteoliposomes after lactose concentration jumps at different concentrations and pH values. The KM values are obtained from the hyperbolic fits.
Figure 6. Reconstruction of transporter currents. The transient currents (A) used to reconstruct the transporter currents (B) were measured with wild-type LacY proteoliposomes after 100 mM lactose concentration jumps at pH 8.5 (blue trace) and pH 5.2 (red trace). At pH 8.5 continuous turnover is observed while at pH 5.2 a fast electrogenic reaction occurs. This is clearly observed in the reconstructed current (B).
Figure 7. Transient current measured with E325A LacY proteoliposomes after a 100 mM lactose concentration jump at pH 5.2. This LacY variant is inhibited in the proton release step of the transport cycle and therefore transport-deficient. The small negative component observed is characteristic for rapid transient currents measured in capacitively coupled systems and is caused by the discharge of the membrane capacitance after a fast electrogenic reaction. Its time constant π0 is determined by the capacitances and conductances of the system (Figure 1).
Figure 8. Transient currents measured with wild-type LacY proteoliposomes after different lactose concentration jumps at pH 5.2. Under these conditions no steady state transport is observed, but an electrogenic conformational transition upon lactose binding. The dashed line shows the transfer function representing the time resolution of the system. The inset shows the determination of the on and off rate constants from the observed rate constant kobs with a hyperbolic fit to the data. The observed rate constant was determined from the transient currents with an iterative least-squares deconvolution algorithm 10.
Figure 9. A kinetic model for the reaction cycle of LacY. Two electrogenic steps could be identified and characterized by SSM measurements. A strongly electrogenic reaction representing ~94% of the total charge displacement in the reaction cycle is only observed at neutral and basic pH values. It could be assigned to the proton release step (blue arrow). A weakly electrogenic step is observed at acidic pH when continuous turnover of wild-type LacY is inhibited. We assigned this reaction to an electrogenic conformational transition upon lactose binding, which represents 6% of the total charge displacement in the reaction cycle.
1. Advantages of SSM-based electrophysiology compared to conventional methods
SSM-based electrophysiology has proven itself as a valuable tool of the electrophysiological toolbox. It is especially useful in cases where convention electrophysiology, namely patch clamp and voltage clamp methods, cannot be applied: Apart from a few rare exceptions bacterial transporters can not be investigated using voltage clamp or patch clamp methods because of the small size of bacteria and because they are difficult to express in mammalian cells or oocytes. But also physiologically relevant mammalian transporters can be investigated. In this case SSM-based electrophysiology is attractive for transporters from intracellular membranes and for screening applications in drug discovery because of its robustness and its potential for automation.
SSM-basedUsing conventional electrophysiology, time resolved characterization of transporters is challenging. Since the turnover of transporters is low a 'giant patch' or a 'whole cell' configuration is required, which have an inherently low time resolution in a solution exchange experiment. The complication can be overcome using photolytic substrate release. However, only a limited number of substrates are suited for this approach. Here the rapid solution exchange at the SSM offers the unique opportunity to perform electrophysiological studies with a high time resolution using arbitrary substrates.
2. Limitations and critical steps
In contrast to patch clamp and voltage clamp techniques, SSM-based electrophysiology cannot be used to apply a potential. Transporter characterization is therefore restricted to transport modes which do not rely on a membrane potential.
In general, SSM-based electrophysiology has no limitations concerning the type of the (electrogenic) transporter. But voltage clamp or patch clamp methods can have advantages, if intracellular components like binding proteins are required for protein functionality.
Limitations can arise, if solution exchange creates large artifact currents. This happens when the substrate interacts strongly with the SSM like in the case of lipophilic compounds. Artifact controls can be used to correct the measured signals. Moreover high salt background in all measuring buffers can be used to reduce the artifacts. But in cases, where the size of the artifact is comparable to the protein signal, it is almost impossible to isolate the protein related signal from the artifact. Fortunately, high artifacts are unusual in an optimized solution exchange.
There are a few steps which are critical for the successful realization of a SSM-based electrophysiology experiment. The preparation of the protein sample is the most important part. If proteoliposomes are used, be sure the reconstitution process yields a clean, reproducible sample of a sufficient LPR and the transporter is oriented in the correct way. The LPR can be checked by freeze fracture electron microscopy and orientation by an ELISA experiment if antibodies are available.
Only use a SSM which shows optimal parameters for incubating the protein sample. The injection of the protein is another critical step. Sonication is essential and air bubbles should be avoided during the injection. After sample incubation the measurements itself are critical, because air bubbles will remove the adsorbed protein sample from the sensor chip. Therefore always remove air bubbles after changing solutions. Nevertheless a signal rundown can occur. To correct a possible signal rundown, it is essential to accomplish rundown controls during the experiment.
3. Specialized Systems
The SSM-Setup can be modified according to its application. Moreover there are completely different, highly specialized setups available.
There is the possibility to measure protein signals under asymmetrical conditions, e.g. under a pH gradient. To establish asymmetrical buffer compositions inside and outside the proteoliposomes a third solution, the resting solution, has to be introduced and this requires a double exchange configuration. Here an additional three-way valve switching between non-activating and resting solutions is required.
To increase the time resolution of the system we developed an alternative flow pathway lacking the terminal valve, but using a different type of cuvette. Here the junction of activating and non-activating solution is located inside the cuvette, 3 mm in front of the SSM. This setup is well suited for kinetic analysis of fast transport processes. It could be shown that a time resolution as low as 2 msec is possible.
Commercial fully automated systems are available aiming at a significantly higher throughput for drug screening. A movable unit collects solutions and injects them on the sensor surface in 96-well plates in a standard microtiter plate format.
No conflicts of interest declared.
We thank J. Garcia-Celma, I Smirnova and R. Kaback for contributions to the LacY measurements and E. Bamberg for support and helpful discussions.
|RK 52 H
|Hielscher Ultrasonics GmbH
|NResearch, West Caldwell, USA
|NResearch, West Caldwell, USA
|GDH 14 AN
|Max Planck Institute of Biophysics
|Max Planck Institute of Biophysics
|100 ml solution containers
|Seal Science Inc.
|Max Planck Institute of Biophysics
|Max Planck Institute of Biophysics
|SAINT-GOBAIN Performance Plastics
|Fraunhofer Institut für Schicht und Oberflächentechnik
|Max Planck Institute of Biophysics
|USB analog-to-digital converter
|Avanti Polar Lipids, Inc.
|VWR AnalaR NORMAPUR
|Natural E. coli lipids polar extract
|Avanti Polar Lipids, Inc.
|for LacY reconstitution
|Potassium phosphate dibasic
|Potassium phosphate monobasic
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