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The overall goal of the following experiment is to examine the mechano sensitivity of the trip V four ion channel. This can be achieved by transforming yeast cells with the trip V four gene and examining the hypothetically induced calcium entry through transgenic a foreign using a luminometer. This can also be examined with the electrophysiological approach.

Trip V four CRNA is first generated and injected into xenopus cytes after incubation. Cytes are examined by two electrode voltage clamp upon the hypotonic challenge. To examine the mechano sensitivity at the single channel level, suction is applied to the membrane patches excised from the cytes.

I am Professor Jin Kung. Previously, the activities of TRIP B four ion channels were largely shown by URA to fluorescence myth developed here, provide powerful and quantitative alternatives to examine the mechanical sensitivity of trip B four. The first method we adopted the use of lum lum is through transgenic aqua to examine the mechanical sensitivity of Trip V four in budding yeast.

In the second method with electrophysiological approach, the properties of Trip V four are examined apt expression of tribute V four CRNA in XUS Cytes. The basic method of cyte electrophysiology, such as the removal of the vilin membrane and gig own seal formation have already been demonstrated in a work of brown and coworker in this very journal in 2008 here will show additional steps needed to study the mechanical sensitivity of Triv four. This is my coworker, Dr.Jin Fcon.

She'll demonstrate the use of T lum geometry as well as the fabrication of the injection pipettes, filling pipettes with CRNA solution and injecting the solution into the old sites. In addition, she'll demonstrate patch clam recording in on cell and in excised inside out mode. She'll also showed the activation of the mechano sensitive channel native to the O site membrane, as well as the results from the activation of heterologous.

Lay expressed TV four Begin this procedure by culturing two milliliters of yeast cells overnight to post logarithmic phase in DCD Lu Yura dropout medium, supplemented with one molar sorbitol in a 30 degree Celsius shaker. The next day inoculate 200 microliters of the culture into 1, 800 microliters of fresh medium, supplemented with two micromolar Lucifer. Risine then grow in the dark at room temperature for another 24 hours without shaking right before dilution.

Measure the osmolarity of the one molar sorbitol enriched culture and the sodium chloride using a vapor pressure OSM ter. Next aliquot 20 microliters of fresh culture into a 12 millimeter luminometer tube for a single tube luminometer. For the hypotonic shock add 200 microliters of solution containing 25 millimolar sodium EGTA at pH 7.2 or sodium MES at pH 7.2 and 500 to 100 millimolar sodium chloride to 20 microliters of the culture at 1, 400 Milli Osmo continuously monitor the luminescence before, during, and at least 120 seconds after the osmotic down shock interrupted only by the brief dilution operation register the luminometer output on the computer as relative luminescence units.

In this procedure, pull a micro pipette with a pipette puller. The micro pipette should have a long shank under a dissecting microscope. Carefully pinch the shank of the pulled micro pipette at the appropriate point to create an opening.

The micro pipette opening should be about 30 micrometers in diameter to facilitate the filling and ejection of CRNA solution. Here are the micro injector and pipette holder. This is the control with the uptake and ejection functions clearly marked.

After back filling the pipette with mineral oil mounted onto the holder, then lower it into a drop of CRNA solution. Subsequently fill the pipette by micro injector automatically under the dissecting microscope, select an oocyte. Then lower the filled micro pipette to penetrate the oocyte.

After the micro pipette has been inserted in it, start the CRNA injection. After about 30 cytes have been injected, transfer them to the ND 96 medium containing ruthenium red and Gentamycin. Next, transfer them individually to a 96 well plate containing the ND 96 medium at the end.

Place them in the incubator for several days to allow the expression of trip V four channels. Two electrophysiological methods are used to examine the mechanism sensitivity of trip V four. The first method is the two electrode voltage clamp, and this is the setup in this step.

Pull the recording pipettes for voltage measurement and current injection from the 100 microliter bolic glasses with a pipette pull. The pipette electrodes should have a tip aperture of about one micron in diameter. Next, backfill the electrodes with two molar potassium chloride, which will result in 0.1 to 0.2 mega resistance.

Mount them on the HS two a head stages. These two are the micro manipulators that hold the voltage control electrode and the current injection electrode, which together with a VG two x 100 virtual grounded bath clamp are connected to a gene clamp 500 amplifier interface through a digit 1440 digitizer. And the data is acquired using P clamp 10 software.

Sitting at the stage of the inverted microscope is the recording chamber to perform a recording, a xenopus cyte is placed in the chamber and the two electrodes are inserted in it through micro manipulation. The solution bathing the cyte in the recording chamber is in a continuous flow through gravity. Feed the inlet into the chamber is connected through tubing into a manifold fabricated with a bank of syringe shells.

Each shell is filled with a different solution. The content of the bath is changed by closing or opening of different clamps on the tubing. Changing the bath from a high to low osmolarity delivers one form of mechanical stimulation.

Now, fill the pulled glass pipettes with the internal solution. The setup for patch clamp is similar to that for voltage clamp with the electrode mounted on the head stage and then secured on the micro manipulator. Attach the patch clamp pipette to a five milliliter syringe and a manometer.

The patch clamp electrode is manipulated onto the surface of the oocyte. After the removal of the Vitale membrane, apply continuous suction to adhere the patch to the pipette tip, which would increase the electric resistance to the test current pulse. When the resistance is large enough indicating the successful formation of a giga seal, the electrode with the patch is then withdrawn from the oocyte to establish the excised inside out recording mode.

Then apply suction pulses into the excised patch. There is a 40 pico Siemens mechano sensitive channel native to the xenopus oocyte. This figure shows the activation of these channels when a suction pulse is applied in the membrane, patches excised from oocytes successfully expressing trip V four.

The activation of the 90 pico Siemens unitary conductance of trip V four can be observed in a similar manner. Shown here is the rat trip V four expressed in yeast responding to hypotonic shock. A 750 milliosmole hypotonic shock triggers a large luminescence increase in trip V four T performance, but not in T performance of an empty plasmid or plasmid bearing a trip V four with a mutation in its ion filter.

And here is the dose response relation between the hypotonic shock and the peak response. The whole oocyte macroscopic current responses to hypotonic stimuli were examined with a two electrode voltage clamp. The peak currents from an oocyte expressing very high levels of wild type trip V four upon 100 millisecond voltage steps in response to the removal of 100 millimolar sorbitol from the 250 milli Osmo bath solution and addition of three micromolar ruthenium red.

The repeated peak current increases upon hypotonic stimuli and it decreases upon the return to isotonic solution or the addition of the channel blocker. This figure shows the direct activation of wild type trip V four by membrane stretch seen under patch clamp, a sample of raw traces of average quality from a patch excised from a trip V four expressing oocyte showing activation by 60 millimeters of mercury suction applied to an excised inside out patch held at positive 50 volts. The uppermost trace is displayed at a faster time base to show the unitary current transition between closed and an open level.

This video tape should give you more concrete understanding on how yeast lumin, geometry, and O site electrophysiological methods can be applied to the study of mechanical sensitivity of triv four channels.