電気生理学測定の電気中立運送者の活動を研究への応用

The method of giant patches combined with ion-selective microelectrodes is a versatile method used to detect and quantify ion fluxes. Specifically, it is used to measure the proton fluxes generated by the activity of the electro-neutral ion transporter, the sodium-hydrogen exchanger or NHE, which is expressed on the cell membrane. The sodium-hydrogen exchanger generates a gradient of protons in close proximity to the plasma membrane, indicated here as an orange ring.

This gradient of protons across the cell membrane is measured by whole cell patch clamp recording combined with a technique that uses a proton selective microelectrode. The proton selective microelectrode is moved close to the cell surface, where it records the flux of protons moving out from the cells, shown here in position A.And away from the cell surface, where it records the free proton concentration present in the solution indicated by position B.And then, back to position A, close to the cell membrane, in a repetitive, oscillatory movement. The voltage difference from A to B is recorded and is dependent on the representative of the NHE activity.

Transporter ions such as protons across the cell membrane affect process essential to life. Therefore, changes in pH are tightly regulated by an array of molecules, including the sodium-proton exchanger, a plasma membrane proton exclusion system. The oscillating pH microelectrode combined with whole cell patch clamping is a novel approach to study the proton flux mediated by the sodium-proton exchanger activity.

Application of this methods overcome some of the major limitation of typically used techniques to measure sodium-proton exchanger activity, such as moderate sensitivity, and time resolution and another good control of the intracellular environment. I will give a demonstration of the methods together with my collaborator, Dr Victor Babich. Dr Matthew Henry will be the narrator.

We are going to demonstrate an application of this method by measuring the activity of the sodium-proton exchanger isoform three that is a major regulator of absorption of salt in water across the intestine and renal epithilium. The proton selective microelectrodes for the measurement of the proton concentration are fabricated from standard borosilicated glass tubing. Borosilicate glass electrodes having a 1.2 millimeter external diameter and without filament are pulled with a vertical puller, using a conventional double pull protocol.

A pipette tip of two to three micrometers in diameter is obtained. The pipettes are placed in a jar holder with the pipette tip facing up. Next, a mixture is prepared to siliconize the pipettes.

It is poured into a jar and the lid is closed tightly. Pipettes are incubated with the siliconization mixture for 24 to 48 hours. Use caution, be sure to prepare the mixture and siliconize the pipettes at room temperature in a well ventilated chemical fumehood.

Back fill the pipette with the appropriate ion selective resin and carefully place it vertically in a jar holder with the pipette tip facing down. To prepare for patch clamping, begin by pulling patch clamping pipettes from thin-walled borosilicate glass with an external diameter of two millimeters and internal diameter of 1.4 millimeters in a vertical puller using conventional double pull protocol. Use a microforge mounted on a stage of an inverted microscope to make a wide pipette tip.

Position the pipette close to the thick platinum wire of the microforge. Turn on the heat using a foot switch that controls the current on and off. Heat the low melting point glass that covers the middle of the thick platinum wire of the microforge and then move the pipette tip close to the melted soft glass.

The heating moves the platinum wire slightly towards the center. The platinum wire returns to the initial position and the pipette tip breaks. Repeat the procedure until the desired tip diameter is obtained and finally, apply heat to smooth the tip.

Pipette tips are 10 micrometers in diameter after polishing with the microforge. Using the thick platinum wire is a benefit in that there is little expansion after turning on the electricity and less possibility of overheating the wire and pipette with excessive current. After the cells are dissociated using trypsin and trypsinization is blocked, cells are maintained on a shaker with rocking to prevent formation of clumps.

This is done for up to two hours following trypsinization. The electrophysiology set up consists of a temperature controller to regulate the temperature of the solution in the perfusion system. All experiments are performed at about 37 degrees Celsius.

And an optical filter switcher to control the filter wheel for fluorescence imaging. A perfusion system valve controller to regulate the opening and closing of the perfusion system valves. And the temperature controller to regulate the temperature of the recording chamber.

An oscilloscope to observe waveforms and a patch clamp amplifier. A computer running this Capmeter software. And an electrometer to record ion selective microelectrode signal.

And a differential amplifier to amplify the signal of the electrometer. The electrophysiology setup is equipped also with an inverted microscope with stage controller, two micro-manipulators with controllers positioned on the left and right sites of the microscope, the perfusion system with inline solution heater. After the ion selective microelectrode pipette is mounted to the micromanipulator, back fill the patch clamping pipette with the intracellular solution.

Make sure that the solution reaches the tip of the pipette. It might require several gentle taps to eliminate bubbles in the tip of the pipette. Then mount the patch clamping pipette to the head stage of the electrophysiology setup.

Gently transfer healthy appearing cells to the recording chamber filled with a defined volume of bath solution. It is important to keep the head stage at a 45 degree angle to the horizontal axis. This ensures an entry angle for the pipette that is suitable for the formation of a highly resistant seal.

While the patch clamp pipette is lowered in the bath solution, apply a small amount of positive pressure to the pipette to reduce the chance of blocking the tip. Select a cell and approach it with a small amount of positive pressure. Stop moving the patch clamp pipette once the tip is close to the cell.

Lower the ion selective microelectrode in the bath solution until it is the same focal plane of the patch clamp pipette tip. When the pipette is centrally positioned over the cell, initiate the seal by applying a small amount of negative pressure. A mega-ohm seal is obtained within a few seconds.

Make sure that the holding potential is maintained by the amplifier at zero millivolts. Finally, rupture the cell membrane with a series of short pulses of suction and start the recording. Using the giant patch recording method, it is possible to perfuse solutions rapidly in the glass pipette via an intrapipette perfusion system to modify the intracellular content of ions, lipids, and proteins.

Position the ion selective microelectrode pipette close to the cell held by the patch clamp pipette and start the oscillation of the ion selective microelectrode pipette by moving it to and from the cell. Application of this technique allows one to study the molecular regulation of the sodium-hydrogen exchanger. Taking advantage of the giant patches method, the intracellular composition of ATP and lipids was modified by an intrapipette perfusion system.

Intracellular perfusion of apyrase, which hydrolyzes intracellular ATP and decreases phosphoinositide content caused a 50%decrease in NHE isoform three activity. The apyrase effect on NHE3 activity was not reversed by intracellular perfusion of the phosphoinositide, PIP2. However, it was reversed by the intracellular perfusion of the phophoinositide, PIP3.

Measurement of NHE activity takes advantage of combining the proton selective microelectrode and patch clamping methods. This allows for the study of the molecular basis behind the differential regulation of NHE by phosphoinositides and downstream signal cascades. After watching this video, you should have a good comprehension on how to measure the activity of the sodium-proton exchanger using an oscillating pH microelectrode combined with all cell patch clamping.

And an example of possible application of the methods.