Calcium release activated calcium channels or crack channels, which are located in the plasma membrane play an important role in T-cell proliferation and gene expression stimulation of T-cell receptors with an antigen results in formation of the second messenger, anatol tri phosphate and activation of calcium release from the store via anatol RIF phosphate receptors. Reduction in calcium concentration within the store leads to activation of crack channels and consequent increase in the cytosol calcium concentration. Abnormalities in crack channel function in T cells are linked to a number of human diseases, including severe combined immunodeficiency disorders and autoimmune diseases.
Here a method is presented that allows for reliable and reproducible recording of endogenous calcium and sodium crack channel currents from human T cells. First, FSA garin, a blocker of Sarco endoplasmic reticulum. Calcium ATPase is applied in calcium free extracellular or solution containing three millimolar magnesium to deplete the store.
This procedure opens crack channels but does not induce crack current because of the poor permeability of crack channels to magnesium. A whole cell voltage clamp is then established on a T-cell membrane. The cell is perfused with a pipette solution containing a high concentration of the calcium chelator pta.
Calcium containing or divalent cation free extracellular solutions are then applied sequentially to activate measurable calcium and sodium currents via crack channels. The resulting data demonstrate the level of functional expression of crack channels in resting human T cells. Investigation of membrane currents via crack channels may provide valuable information on crack channel regulation and functions and establish the role of crack channels in normal immune responses and immunological diseases.
Generally, individuals new to this method will struggle due to the small size of human T lymphocytes and low amplitude of endogenous crack currents in these cells. The CD three positive human T lymphocytes for this experiment were obtained from a peripheral blood sample from a healthy volunteer and isolated using tep human T-cell enrichment cocktail and TEP density medium. According to the instructions in the accompanying written protocol, cells were maintained in T-cell culture medium at 37 degrees Celsius in a carbon dioxide incubator.
Prepare the following solutions bat solution number one containing three millimolar magnesium and no calcium bat solution Number two, containing 20 millimolar calcium bat solution number three containing 10 millimolar calcium chelator, H-E-D-T-A and no divalent ions and pipette solution containing 12 millimolar calcium chelator bta. Store the solutions at four degrees Celsius for up to one week. On the day of the experiment, pull pipettes with tip diameter of approximately two microns using glass capillaries and a pipette puller.
Use silica tubing with filament with the specification shown here and listed in the accompanying text. After pulling the pipette tips are coated with PEC R 61 0 1 semiconductor protective coating and fire polished. Store them in a dust protected place such as a pipette holder.
Open the software controlling the patch clamp amplifier here. An E PC 10 patch clamp amplifier is controlled by pulse software. Configure and save the setup on cell and whole cell macros used for giga seal formation and establishing wholesale configuration.
Next in the acquisition software, configure and save the stimulation protocol here to record crack currents. The acquisition protocol in the pulse generation window is set as a voltage ramp from minus 120 to plus 100 millivolts of 50 milliseconds. In duration.
Create two stimulation protocols with ramp voltage stimulation frequencies of 0.5 hertz and five hertz, and save them in the pulse generation window under the names ramp one and ramp two respectively. To prepare cells for recording plate 500 microliters of the cell suspension containing 0.2 to 0.5 million cells on a recording chamber consisting of a polyol lysine coated cover slip with an attached silicon O-ring. Incubate for 20 minutes at 37 degrees Celsius.
Following the incubation, secure the recording chamber in a chamber holder. Here, a custom made chamber holder is used in which the base part supports the cover slip on the bottom of the chamber and the top part presses against the top of the cover slip. Place the chamber holder on the stage of the inverted microscope using a 40 x oil immersion objective and bright field illumination.
Find a field of cells and focus. Place the silver silver chloride reference electrode into the bath. Then align a multi barrel gravity driven perfusion system by placing the tip of the inflow tube at 45 degrees to the cover slip close to the field of view, the suction tube is inserted into the top part of the chamber holder.
Rotate the top part of the chamber holder until the suction tube is opposite the inflow tube to perform correct channel. Current recordings begin by opening the valve on the perfusion system to perfuse the recording chamber with calcium free. Three millimolar magnesium containing bat solution number one, supplemented with 0.5 to one micromolar AP argon for eight to 10 minutes to block the circa pump.
This step depletes the calcium store. Next, use an einor micro loader with a P 20 air displacement pipette. To fill the pipette with the pipette solution, insert the pipette into the holder on the amplifier head stage here.
A custom made pressure suction device is connected to pressurized air and the vacuum lines. To create positive or negative pressure in a patch pipette, apply a small positive pressure about three inches of water inside the pipette by connecting the pipette to the pressure suction device outlet via the connective tube. Click the setup control key in the EPC 10 patch clamp window on the computer screen.
To execute the setup macro. Find a round cell of an average size with a smooth outer surface that is firmly attached to the cover slip. Then lower the patch electrode into the bath under visual control via the microscope in the oscilloscope window.
The current flowing through the pipette can be viewed after immersing the pipette into the bath solution. A step like change in the current amplitude is evoked by the five millivolt voltage test pulse applied to the pipette by the setup macro. The pulse software automatically calculates pipette resistance and displays it in the membrane resistance window.
On the computer screen, the pipette resistance is usually five to six mega ohm. Click on the auto control key in the amplifier window on the computer's screen to correct the offset potential generated between the pipette and the reference electrode. The baseline current will shift to a zero current level.
Use the micro manipulator to bring the pipette tip in to touch the cell membrane. Once contact is made, there will be a decrease in the amplitude of the current evoked by the test pulse. Apply negative pressure 20 to 40 inches of water to the patch pipette.
Monitor the giga seal formation in the oscilloscope window. The amplitude of the current evoked by the test pulse should gradually decrease as the value of the pipette resistance increases to more than five giga ohm. Usually giga seal contact forms quickly, but it may also take one to two minutes to achieve a stable giga seal.
Once the giga seal is achieved, disconnect the pipette holder from a pressure suction device by removing the connective tube from the outlet. Click the onsell control key on the computer screen to execute the onsell macro, which was set prior to the experiment. According to the EPC 10 manual, compensate for the pipette capacitance by clicking on the auto control key in the cfat window.
In the oscilloscope window, the current transient will disappear and the value of the pipette capacitance will appear in the cfat window to break the cell membrane Inside the pipette, connect the pipette to a 20 cubic centimeter syringe via the connective tube and pull back the plunger to apply additional negative pressure to the pipette. When the cell membrane inside the pipette ruptures two current transients charging the cell membrane to a new voltage level will appear. Click on the whole cell control key on the computer screen to execute the preset whole cell macro.
Then to set the holding potential type plus 30 millivolts into the V membrane window, positive holding potential helps prevent calcium dependent crack channel in activation. Next, click on the auto control key in the C slow window. To compensate the cell capacitance in the oscilloscope window, the current transient will disappear.
The values for cell capacitance and series resistance will appear in the C slow and R series windows respectively. Series resistance is typically within the range of seven to 20 mega. The C slow values which correspond to values of cell capacitance are typically in the range of 1.5 to 2.5 pico fer.
In resting human T cells, click on the ramp one control key in the oscilloscope window to apply a series of voltage ramps with frequency of 0.5 hertz. This will execute the stimulation protocol generated and saved prior to the experiment in the oscilloscope window. The currents evoked by the voltage ramps are shown.Record.
These current traces in calcium free solution number one containing three millimolar magnesium. In this solution, the crack current is very small due to the poor permeability of crack channels for magnesium Saban. Use these traces in future analysis as leak currents.
The absolute amplitude of the leak at minus 100 millivolts should be equal or less than five pka amps. If the cell is leaky. Stop the experiment and start over with a new patch pipette and a new cell.
Next to record calcium current via crack channels switch the perfusion valves to replace bath solution number one with bath solution number two containing 20 millimolar calcium. This allows for calcium ions to flow via crack channels and produce an inward current in the oscilloscope window. An inwardly rectifying calcium current via crack channels develops current amplitude at negative voltages, continues to increase for about one minute due to calcium dependent potentiation of the crack current.
Click on the ramp two control key in the oscilloscope window to execute a preset stimulation protocol, which changes the frequency of stimulation with voltage ramps to five hertz increase in frequency of stimulation helps to record maximal sodium current via crack channels, which is transient in nature. Next, switch the perfusion valves again to replace bath solution number two with sodium containing divalent cation free solution number three in the oscilloscope window. A transient larger amplitude inwardly rectifying sodium current via crack channels develops.
Once the experiment is over, close the file, it saves the recorded current traces for further analysis to analyze the data, retrieve the saved current records into analysis software. Here pulse software is used for analysis using pairs of cursors in the oscilloscope window of the pulse program. Measure the current amplitudes at the beginning and the end of voltage ramps here.
The current amplitudes are measured at minus 100 millivolts and at plus 100 millivolts. Export the values of the current amplitudes into a graphic program such as origin scientific graphing and analysis software. For further analysis and graphical presentation, crack currents were measured in CD three positive resting T cells from a healthy human subject using the method described here, this figure shows a time course of crack currents recorded in whole cell voltage clamp configuration at minus 100 millivolts indicated by the filled circles and plus 100 millivolts indicated by the open circles.
Prior to the giga seal formation, the cell was pre incubated for approximately 10 minutes in calcium free. Three millimolar magnesium containing bat solution number one containing 0.5 micromolar orthogon after break-in bat. Solutions were sequentially applied as follows, calcium free three millimolar magnesium containing bat solution number one, followed by 20 millimolar calcium containing bat solution number two, followed by divalent cation free bath solution number three, followed by 20 millimolar calcium containing bath solution Number two, the cell was stimulated with a series of voltage ramps from minus 120 millivolts to plus 100 millivolts as shown here.
Voltage ramps were applied every 200 milliseconds or at a frequency of five hertz in divalent cation free bat solution number three and every two seconds or at a frequency of 0.5 hertz in all other solutions. Note the slow development of calcium current via crack channels after application of 20 millimolar calcium containing bat solution number two, and fast transient development of sodium current via crack channels in divalent cation free bat solution number three. This figure shows representative current traces recorded during voltage ramps in calcium free.
Three millimolar magnesium containing bat solution number one 20 millimolar calcium containing bat solution number two, and divalent cation free bat solution. Number three, the calcium and sodium current traces during the individual voltage ramps can be obtained by subtracting currents recorded in calcium free three millimolar magnesium containing bat solution number one from currents recorded in 20 millimolar calcium containing bath solution number two, and divalent cation free bath solution Number three, the resulting current traces displaying current voltage relationships typical for calcium and sodium currents via crack. Channels are seen here.
Once mastered this technique and all the states of the procedure are performed properly, one can record crack channel currents from five to 10 cells a day. After watching this video, you should have a good understanding of how to record correctional current in rest in human T cells. Following this procedure, you should be also able to record correct currents in other cells of immune systems such as activated human T cells, monocytes, macrophages, and also cell lines.Electrophysiological.
Recording of crack channel currents in the cells of immune system may enable researchers to establish their role in calcium homeostasis in immune cells.
