Введение в твердом носителе мембранный электрофизиологии

So supported membrane based electrophysiology abbreviated to SSM based electrophysiology. It's a new electrophysiological approach. It offers for a number of applications a significant improvement compared to the conventional methods.

The principle tool of conventional electrophysiology is the electrolyte filled microparticle electrode, whereas as its M based electrophysiology detects charge translocation via a solid supported membrane electrode. It is extremely useful in cases where conventional electrophysiology cannot be used, such as in the case of bacterial transporters, which apart from a few rare exceptions, cannot be investigated by voltage clamp or patch cam methods. In such cases, SSM based electrophysiology has proved very successful.

It can also be employed to investigate physiologically relevant eukaryotic transporters in intracellular membranes. There are many ways of carrying out SSM based electrophysiology experiments. In basic research, it can be applied to determine kinetic parameters and clarify transport mechanism, but its robustness and potential for automation make it ideal for screening applications in track discovery.

The SSM electrode carries a compound membrane system consisting of the underlying SSM and the adopt proteasomes or membrane fragments containing your transport protein of interest. The electrical properties of the compound membrane can be described by an equivalent circuit in which the membranes form a capacitively coupled system. Note that the electrical behavior of the system is essentially the same whether membrane fragments or protea liposomes are used.

The charge displacement in the transport protein is transmitted to the measuring circuit via the capacitance of the bladder membrane. A detection principle called capacitive coupling. In a typical experiment, rapid solution exchange at VSSM provides the substrate for the transport protein V ensuing electrogenic transport activity of the protein generates a current, which is ly coupled to the measuring system via the SSM capacitance.

At the same time, the electrogenic protein activity gradually charges the liposomal membrane leading to an inhibition of the transport protein and the declining current in the recording circuit, the measured current is a result of simultaneous transport reactions of millions of protea liposomes absorbed to the Enzo. Each Containing about 100 transporters. The setup consists of a Faraday cage with a solution pathway, external circuits for valve control, signal amplification, plus data acquisition and analysis.

The EC cage includes containers, valves, and tubes required for solution flow control, as well as a cubit with a XOR chip. The XOR chip carries the SSM electrode and the absorbed prot liposomes. The vete is connected to the amplifier and function generator of the external electrical circuit.

SSM measurements, wreak fire, two solutions, one activating and one non activating apart from the substrate, which is present only in the activating solution. Both solutions have the same composition in the single exchange configuration. Two, two-way valves control the flow of the non reactivating and the activating solutions.

The terminal valve, a mechanically coupled three times three-way valve, is used to switch between the non activating and the activating solutions, directing one of the solutions to waste container and the other solution to the qve. When the terminal valve is switched, the flow of the non activating solution is rerouted to the waste and the activating solution flows to the qve. This configuration allows a simultaneous continuous flow of both solutions.

At any time during the flow protocol, the flow pathway can be optimized according to need. However, one has to keep in mind that the distance between the SSM and the junction of the non activating and activating solutions should be kept as short as possible. Since it determines the timer solution of the solution exchange.

The current amplifier outside the power deck cage is connected to a sensor chip. Typically, this is set to an amplification of 10 to the power of eight to 10, to the power of nine volt per amper and rest time of one to 10 milliseconds. A function generator is connected to the reference electrode and a computer with valve control and data acquisition software and an interface box complete the system.

The interface box contains the valve drivers data acquisition and the digital output hardware solution flow is maintained by pressurized air of oh 0.22. One bar pressure is monitored using a digital millimeter. It can be maintained by filling a steel container installed between the air supply and the solution containers in the EC Cage via a needle valve.

The CVIC consists of four parts. The zen chip is sandwiched between the vet base and the vet head. A cylindrical outlet connector is screwed to the vet head and carries the reference electrode assembly.

The reference electrode consists of a chlorinated silver wire with a miniature SMC connector and is isolated from the flow pathway by a poly acry gel salt bridge. The not in use, the gel bridge assembly is stored in a 100 millimolar potassium phosphate pH seven solution containing 100 millimolar potassium chloride. First, the reference electrode has to be mounted into the reference electrode assembly containing the geo bridge.

It is important to avoid air bubbles while installing the reference electrode. Now this part of the vete is attached to the outlet connector, which has been prefilled with buffer solution. All connections have to be sealed with O rings.

This SM is formed on a zor chip, a structured goat coated glass light. This a sematic road is one millimeter and diameter and is connected to the contact pad via a oh 0.2 millimeter white contact strip. A spring contact pin creates a contact to the amplifier.

The zen chip is stored in the dark in a 10 LAR Okta Deccan thiol ethanol solution. To maintain its arcane thi layer to remove the Okta deccan viral molecules, which have not been absorbed, the electrode is rinsed with pure ethanol and the electrode dried in nitrogen gas. Now the phosphocholine mono layer can be assembled.

The lipid solution contains 15 milligram per milliliter DFI choline and 250 microgram per milliliter. Okta two A mine in decay and is stored at minus 20 degree. The electrode is now placed accurately on the base of SA cubit.

One microliter of the solution is added to the SSM electrode without touching the gold layer. Immediately after adding the lipid solution, the vete can be assembled. The vete volume sealed by an O ring is cylindrical with an internal volume of 17 microliter when mounted.

The SSM should be Centered under the inlet bore. Before connecting the vet, the fluid system has to be prefilled with 100 millimolar potassium phosphate buffer solution. It is important to ensure that there are no air bubbles in the tubing and the valves.

Now the vet can be mounted into the thyroid day cage, connect the amplifier, the inlet and outlet of the solution flow pathway and the function generator. Finally, the cubit can be rinsed with a 100 millimeter potassium phosphate buffer. This leads to spontaneous SSM formation to check the quality of the SSM.

Capacitance and conductance are measured using the function generator to measure the conductance 100 millivolt DC voltage is used. The current decay shows the charging of the membrane capacitor after the capacitor is fully charged. The measured current yields the membrane conductance using M'S law.

Here we observe a conductance of oh 0.17 nano Siemens. The capacitance can be measured using triangular AC voltage of 50 millivolt peak to peak amplitude and a frequency of oh 0.5 hertz. The amplitude of the resulting square wave current represents the judging current of the capacitor.

The capacitance is equal to the transferred charge divided by the imposed voltage. This membrane shows a capacitance of 2.3 nano, a high conductance and a low capacitance indicates that the SSM is not correctly formed. In these cases, the membrane should be discarded and another SSM prepared Using a new electrode chip.

Usually prot liposomes are stored frozen at Minus 80. TRE th the sample on ice sonication is essential because an aggregated sample absorbs pulley to the SSM to obtain a homogeneous prot liposome suspension. Three ten second sonication cycles are alternated with ten second cooling intervals on ice.

Rinse their SSM with non activating solution. Then unscrew the outlet connector together with the reference electrode sample. Using a pipette, inject 30 microliters of the protein sample through the outlet of the vete into the ette volume.

First mount the pipette tip to the outlet bore. Then open the manual above in the non activating pathway and inject the protea liposomes. Before demoting the pipette tip.

Close the manual above to prevent further solution flow. Allow the protea liposomes to absorb to the SSM for an incubation time of one To two hours. Use the flow protocol Window of the valve control and data acquisition software to define your flow protocol.

If you want to measure under symmetrical conditions, like for determination of substrate specificity, KM value, or pH dependency, choose the single solution exchange protocol. Set the flow duration for each solution to oh 0.5 to one seconds. The current is recorded throughout the entire flow protocol.

The amplification is typically set to 10 to the power of nine, and for the sample rate, we choose 2000 data points per second. Finally, save your new protocol under the title NA a and a. In the second tap of the flow protocol window, you can define the open or closed state of specific valves to control flow of non activating or activating solution Through qubit.

Here we demonstrate the measurement Of protea liposomes, the sugar proton import like Y from E coli. The activating solution contains the transported substrate lactose, while the non activating solution contains the same amount of the non transported glucose as a compensatory compound. In addition to the sugars, the solutions contain 100 millimolar potassium phosphate at pH 7.6 and one millimolar DTT.

After changing solutions, use the manual valve to make sure that air bubbles are removed. Before starting a flow protocol, adjust the pressure just before starting the measurement. We routinely use the pressure of O 0.6 bar, which at our specific valve and tube configuration yields a flow rate of approximately one milliliter per second.

Then select a safe flow protocol here, the NAA protocol, and press the start button To start each measurement. During the first few measurements loosely Absorbed, protea liposomes are removed and therefore the peak current may decrease. Repeat the measurement until the peak current remains constant.

During the first phase of the non activating solution flow, the current is zero. The exchange from non activating to activating solution, which contains lactose induces the electrogenic transport activity of the membrane. Protein positive charge accumulates inside the prote liposomes and the amplifier detects a positive transient current due to positive coupling via the SSM.

This positive positively coupled current is called the on signal V exchange from the activating solution. Back to the non activating solution induces proton sugar code transport out of the liposomes. In this case, a negative LY coupled current is displayed the so-called off signal for protein kinetic analysis.

Only the on signal is used since the off signal is poorly defined due to the charge state of the membrane valve switching creates mechanical and electrical artifacts. Fortunately, these are restricted to a time range before the protein induced signals are observed. Note that the 50 milliseconds delay between valve switching artifacts and transporter signals is the time the solution needs to propagate from the terminal valve to the surface of the SSM Rapid Solution Exchange, Such as that performed on the SSM represents a typical relaxation experiment.

In such experiments, the current relaxes through a time dependent pre steady state phase into a constant steady state. Current kinetic details of the reaction cycle may be derived from the shape and magnitude of the pre steady state current. While the steady state current provides information about the continuous membrane protein turnover.

In the ly coupled system where protea liposomes are absorbed to their SSM, the pre steady state currents are virtually unaltered. While a constant steady state current is never obtained, instead due to charging of the liposomes, the measured current gradually decreases. The transporter current can be calculated from the measured ly coupled current By means of circuit analysis.

Here we demonstrate Representative results using the sugar proton co-transporter, like Y in the PA range between 8.5 and 7.6. The transporter current is dominated by an electrogenic reaction late in reaction cycle. Under these conditions, the pre steady state face consists of a simple current rise to its steady state value.

This then decays due to capacitive coupling. We are interested in the steady state transport current. In the absence of a membrane potential, this is represented to a good approximation by the peak current.

The Ali Mein kinetics can therefore be analyzed by blotting the peak currents for different substrate concentrations, but consider that peak current amplitudes can only be compared rigorously if they are obtained from a single sensor in one experiment. One reason is the variability of the absorption efficiency. When the pH is further reduced, the monophasic signal becomes biphasic.

And finally, a rapid transient remains for transport deficient lag Y mutant. The rapid transient is followed by a small, slower negative component. The negative component is characteristic for rapid transient currents measured in ly coupled systems and is caused by membrane capacitance discharge.

Here, the peak current does not represent steady state transport, but rather an electrogenic confirmation of transition early in the ion cycle, which becomes apparent because a la y mutant shows no steady state transport, and for wildtype lag, y steady state transport is inhibited by a low pH. The experimental evidence provides a mechanistic interpretation of charge translocation in the reaction cycle of lag y, which is shown in the kinetic model. The fast transient observed for the transport deficient mutant, or in the case of wildtype lag Y at acidic conditions is due to a rapid weekly electrogenic reaction following lactose binding at higher pH values, the signal is dominated by the main electrogenic reaction late in the reaction cycle, which was proposed to represent pseudo plasmic proton release in the capacitively coupled system.

This leads to transient currents representing steady state transport of Lag y due to the strong interaction Of the Utes. With the SSM, electrical artifacts are generated when solutions of different composition are exchanged. Make sure non activating and activating solutions are as similar as possible, especially with regard to their pH and ionic strengths.

Control should be carried out because substrates can also produce artifacts. Always test your specific buffer system and flow protocol for artifacts using the bare SSM before the protein sample is added. After adding the protein sample control measurements can be done with solutions that contain non substrates, which are structurally similar to the substrate.

Moreover, there's a possibility to test substrate for artifacts using liposomes or membrane fragments without the protein of interest. You can also add inhibitors to your solutions or pipette them. After the experiment directly to the outlet of the vete to measure transporter signal inhibition, the protein specific signals will then disappear, leaving only solution exchange artifacts.

The protein signal then can be easily corrected. Make sure the loss of absorbed prot, liposomes or protein degradation does not affect your measurement. By performing repeated control measurements under identical conditions, a signal rundown of up to 20%is acceptable and can be corrected for by assuming a linear time Dependence of the rundown.

This setup is the result of 20 years of technical development in our lab. Also, specialized systems with higher timer solution for kinetic analysis and lower solution consumption for expensive substrates are available. For drug screening, we use fully automatic setups with higher throughput.

To date, more than 20 transporters have been characterized using this method. These include transport APAs, respiratory chain complexes, cool transporters, exchangers, and even iron channels. So when you're unable to solve your problem by standard electrophysiology, consider using SS Mase electrophysiology instead.