Rekonstitution der ein Transmembranprotein, das spannungsgesteuerten Ionenkanal, KvAP, in Riesen unilamellare Vesikel für Mikroskopie und Patch-Clamp-Studien

The overall goal of the following experiment is to incorporate voltage gated ion channels into giant uni lamellar vesicles and characterize their activity with patch clamp measurements. This is achieved by first partially dehydrating, a solution of small vesicles containing the ion channel KVAP to create a lipid protein film. Next, the lipid film is rehydrated, which causes the formation and growth of cell sized giant ALR vesicles or gvs containing the channel.

Then the gvs are transferred to our recording chamber and a piece of GUV membrane is excised with a patch pipet in order to measure ion currents. The results show that the ion channels in the GUV membrane activate in response to changes in membrane voltage based on the analysis of the current recordings. Giant unilateral physicals can help answer key questions in the field of biophysics related to membrane protein interactions.

For instance, the interplay of transmembrane proteins with the physical properties of the lipid membrane. Though this method can provide insight into the function of the voltage gated iron channel KVAP, it can also be applied to other iron channels, transporters, and pumps, and transmembrane proteins in general. Visual demonstration of this method is critical as the steps involving the manipulation of giant vesicles are.

How to pick up just reading articles After preparing solutions and small unal vesicles containing KVAP. According to the text protocol, prepare the electro formation chamber by first inserting the wires through the holes and rotating and wiping the wires to make sure they are clean, sunation and drying under a stream of nitrogen or air. Prepare 30 microliters of three milligram per milliliter SUV suspension in SUV buffer as described in the text protocol, mix the solution vigorously to deposit the SUV solution.

Use a two microliter pipette or five microliter glass syringe to place small droplets of the SUV solution on the wires. Make sure the drops are small enough and spaced far enough apart that they do not touch or fuse. Let the deposited SUVs dry for about 30 minutes in open air.

When the droplets have settled, rotate the wire so the lipid deposits will be easier to observe with the microscope. Next, use a syringe to apply vacuum grease to the bottom of the chamber around the three wells and gently press a 40 millimeter by 22 millimeter cover slip against it to seal the chamber bottom so that it adheres without a gap. Then use ceiling paste to seal the sides of the chamber and apply vacuum grease on the top of the chamber outlining the three wells.

Slowly add growth buffer until each well is filled to the top. Avoid any rapid movement of the solution in the wells as this can. Strip the lipid film off the electrodes.

Close the chamber by gently pressing the top cover slide onto the grease. Taking care not to dislodge the bottom cover slip. Use two alligator clips to connect the signal generator to the wires.

Set the frequency according to the salt concentration of the buffer being used and with a multimeter measure and adjust the voltage across the wires. Also according to your buffer, use aluminum foil to cover the chamber to protect the flora fours from light. Leave the G UUVs to grow for two to three hours in the low salt buffer and 12 hours or overnight for the high salt buffer.

After the incubation, disconnect the chamber from the generator and carefully place it on an inverted microscope. To evaluate GUV growth plasma, clean a cover slide for one minute so that the ARO solution will spread nicely on it. Within 15 minutes of cleaning the cover slide, apply 200 microliters of warm aro solution prepared according to the text protocol so that it wets the entire surface.

Tilt the slide vertically and touch the lower edge on a tissue to remove excess liquid, leaving a thin, smooth layer of aros on the slide. Place the slide on a hot plate or oven at 60 degrees Celsius and leave it to dry for at least 30 minutes. Next, place an AROS coated cover slip in a standard 3.5 centimeter petri dish.

Then after preparing the SUV solution, gently apply about 15 microliters in about 30 very small drops onto the aros surface. Take care not to distort the aros layer too much. Place the slide under a gentle stream of nitrogen for about 10 to 15 minutes and follow the evaporation of the buffer by eye.

As the droplets dry as soon as the SUVs have dried, add about one milliliter of growth buffer to cover the slide surface. Allow the swelling to proceed for about 30 minutes, and then use an inverted microscope with phase contrast or DIC To examine the growth of the GU UUVs in the growth chamber to patch clamp gu UUVs ate the chamber by adding a five milligram per milliliter beta Caine solution, which prevents the Gus from adhering, spreading and rupturing. Incubate for five minutes and rinse, insert the ground electrode and use observation buffer to fill the chamber.

Next, transfer the G UUVs to the observation chamber for electro formed GU UUVs. Open the growth chamber by gently removing the top cover slip. Place the pipet tip directly above each wire and to detach the GU UUVs slowly aspirate about 10 microliters while moving the pipette tip along the wire for gel assisted swelling GU UUVs.

First tap the side of the Petri dish a few times to detach the GU UUVs from the cover slip surface. Position the pipette tip just above the cover slip and aspirate 10 microliters while pulling the tip back over the surface directly transfer the harvested GVS to the observation chamber. Wait a few minutes for the Gus to settle to the bottom to patch clamp the GVS.

Use observation buffer to fill a fresh patch pipette and mount it on the patch clamp head stage. Search through the chamber to locate a defect-free GUV and check that it contains fluorescent protein. Apply a constant positive pressure of more than 100 pascals to keep the patch pipette interior clean and insert the patch pipette into the chamber.

Bring the patch pipette into the field of view and apply test pulses to measure or compensate the pipette voltage offset and resistance. Then examine the pipette under fluorescent illumination to confirm that the tip is clean. Bring the patch pipette towards the GUV and if necessary simultaneously reduce the positive pressure so that the outward flow from the patch pipette does not make the GUV run away.

When the patch pipette is close to the GUV, apply negative pressure to pull the GUV against the patch pipette. Monitor the resistance as the tongue of the GUV membrane enters the patch pipette and the giga seal forms. When the inside out membrane patch has been excised from the GUV and the giga seal is stable, switch off the test pulses and apply a voltage protocol as outlined in the text.

This figure shows DIC and epi fluorescence images of a defect-free GUV. The uniform protein fluorescence in the GUV membrane confirms that KVAP is incorporated in the GUV rather than remaining in the lipid film and has not formed aggregates Gus produced using the three methods are seen here. The fluorescent lipid and protein signals have been scaled to the same average density so that g UUVs with a low or high protein density have a magenta or green shade in the overlay images while GVS with an average protein density are white.

Isolated defect-free G UUVs were identified and the GUV size distribution is shown here. Typically, electro formation produces more defect-free G UUVs than gel assisted swelling, but the GUS produced by electro formation are smaller. Shown here is the protein density distribution inferred from GUV fluorescence.

Electro formation with high salt buffer produces GU UUVs with varying protein densities. The density varies much less for electro formation with low salt buffer and the protein density of GU UUVs produced by gel Assisted swelling is remarkably uniform depending on the protein concentration in the excised patch. The current traces show either single channels or an ensemble of channels.

KVAP shows voltage dependent opening. While attempting this procedure, it is important to remember the giant vesicles of fragile objects not resistant to sheer and osmotic stress. Following this procedure, as a methods like pulling lipid nano tubes can be performed in order to answer additional questions like curvature sensing of proteins.

After watching this video, you should have a good understanding of how to produce GVS containing functionally reconstituted proteins.