The overall goal of this procedure is to measure electrical current through a single ion channel. This is accomplished by first heterologous expressing the ion channel of interest in cells that are easy to work with and are conducive to the experimental goals. The second step is to use a fire polished pipette to obtain a stable seal on the cell membrane.
Next, the experimenter should determine the number of channels present in the patch, and if it is single channel record current, the final step is to process and analyze the data. Ultimately, recording current from single ion channels is used to investigate the biophysical properties by which these membrane proteins act, which forms the basis for how neuronal and other excitable cells communicate. In contrast to methods that allow you to measure currents through multiple active ion channels, the advantage of this approach is that measuring currents through an individual ion channel allows you to visualize and separate a wide range of response patterns inherent to a particular channel.
This method can be used to answer key questions in the neuroscience field, such as physiological and therapeutic modulators influence communication between neurons. Generally, individuals who are new to this technique will struggle because it's difficult to obtain low noise recordings that contain only a single ion channel. When first attempting this method, it's really useful to see somebody do it because to obtain a really stable seal, which is the most important thing in this method, you need to approach the cell gently at the particular angle, and most importantly, you have to be persistent To prepare for transfection.
HEK 2 93 cells are plated in 35 millimeter dishes one day in advance to bring them to 50 to 60%confluence in a sterile tube. Prepare the transfection mixture. Add one microgram of each CDNA of interest, 315 microliters of double distilled water and 350 microliters of 42 millimolar heaps.
Finally, add 35 microliters of calcium chloride dropwise to form the precipitate. Vortex the tube for five seconds and add 175 microliters of transfection suspension to each of the four dishes. Plated the day before.
Incubate the cells at 37 degrees Celsius for two hours. Following the incubation, remove the media and wash with PBS. Next, add two milliliters of growth medium, supplemented with two millimolar magnesium chloride to prevent an MDA receptor mediated excitotoxicity.
First pull two symmetrical recording pipettes from boy silicate glass using a vertical puller. Next, fire polishing is necessary to obtain the appropriate diameter and resistance. Next set up for recording by opening the QUB data acquisition software and selecting the acquisition window.
Under layout. Open a new QUB data file by clicking new data and enter the experimental parameters to be used. Adjust the amplitude scaling to 0.1 volts per pico amp by right clicking the data file, selecting properties, and clicking the data tab.
After replacing the media with two milliliters of PBS containing calcium and magnesium, mount the dish of cells onto the microscope stage. Focus on the cellular field using phase contrast microscopy to evaluate that cells are healthy and well attached to the dish in monolayer fashion. Switch to fluorescence detection to verify that the transfection was successful.
Next, set the parameters for single channel recording. Set the mode to voltage clamp and applied voltage to plus 100 millivolts with the voltage holding command. In the off position, select the seal test button based on fluorescence and overall appearance.
Choose a cell to patch verify under phase contrast that the cell is well attached to the dish has a large portion of the cell surface exposed for patching and otherwise looks healthy. Fill a freshly polished pipette with a solution suitable for NMDA receptors and cell attached experiments and flick gently to dislodge any air bubbles. Secure the recording pipette onto the amplifier head stage and make sure that the silver wire is immersed in the pipette solution to prevent bath solution from entering the pipette.
During the approach, use a small plastic syringe connected to the pipette holder to gently apply positive pressure. Next, use the micro manipulator to direct the pipette into the bath. Closing the electrical circuit, position it directly over the cell selected for patching.
Take note of the oscilloscope upon entry into the bath. The pipette resistance should be within the optimal range while monitoring the pipe's position visually in the microscope and its resistant electrically on the oscilloscope. Continue the approach in small increments until the pipette impinges gently on the cell.
The test signal will decrease slightly to indicate increased resistance. To form a seal, pick up the syringe and gently apply slight negative pressure through the lateral tubing. By pulling the syringe plunger seal formation is indicated by a complete flattening of the test signal waveform on the oscilloscope.
On the amplifier. Switch the external command toggle from seal test to off. Switch the voltage holding command from off to positive and increase the gain from 10 x to 100 x.
Observe the oscilloscope for channel activity, which if present will be displayed as square downward deflections from the previously flat baseline. If channel activity is displayed on the oscilloscope, start to acquire data into the previously opened digital file in QUB by pressing the play button, followed by the record button. To stop acquiring data, press the stop button in QUB and save the QDF file a signal that does not flatten completely, or a baseline that becomes noisy and unstable may indicate a weak seal.
In this case, remove and discard the pipette and repeat the proceeding steps with a fresh pipette until an adequate seal is obtained. To begin the data analysis process, open the recorded data file in QUB and inspect the current trace in the pre interface. Under layout.
Remove the digital filter to display the recorded trace in full bandwidth by unchecking the box labeled fc. Visually scan the trace to spot irregularities and artifacts Brief. Current spikes can be corrected by highlighting an adjacent clean region of the same conductance class.
Right clicking and selecting set erase buffer first, zoom in on the spike until individual sample points are visible. Select the region to be replaced by highlighting only this region. Right click and select erase to correct the baseline for the entire record.
Define the zero current baseline by selecting an early portion of the record where the baseline is stable. Highlight right click and select set baseline. When the base guideline appears, confirm by visual inspection that it accurately represents the baseline level.
Identify points in the record where the raw data baseline deviates visibly from the set baseline. Correct this by selecting a small section of baseline within the deviating region. Right click and select the add a baseline node command.
When the record contains occasional periods with excess noise or artifacts, which cannot be easily corrected, these portions should be deleted, but first need to be broken into consecutive segments. Highlight immediately before the region to be discarded. Right click and select break segment.
Following this, highlight the section to be discarded right click, select, delete, and hit enter to idealize records. Switch to the MOD interface under layout. In the data display panel, select a small portion of the record containing both closed and open events.
Move the cursor into the high resolution panel below and within the selected trace, highlight a clean segment representative of the baseline. Move the cursor to the model panel below, right click the black square, which will represent the closed state and select grab. Repeat the procedure for channel openings by highlighting a portion of the traits that represents the open conductance.
Right click the red square in the model panel and select grab. Perform the idealization for the selected portion of the record by selecting the SEL button under data source. Then right click on the idealized tab underneath the modeling section on the pop-out panel.
Verify that the desired parameters for analysis are correct and then click run. The result of the idealization along with amplitude histogram for the selected portion is overlaid with the data to allow for visual inspection of the idealization. If necessary, adjust idealization parameters for the best result and repeat, perform the idealization for the entire file by selecting the file button under data source.
Then right click on the idealized tab and click run. The results for the entire file is displayed. Overlaid with the data, carefully inspect the match between the idealized trace and the recorded data.
Occasionally events are falsely identified that can be excluded from the idealized trace. Select the false event right click and select join IDL. Shown here are calcium ion only currents through a single NMDA receptor in a HEK 2 93 cell.
Using the cell attached patch clamp technique, data idealization produces amplitude histograms, which fit nicely into two distinct conductance classes in this cell. Attached recording from HEK 2 93 cells, one Meno sensitive channel is activated by minus 20 millimeters. Mercury pressure through the patch pipette during constant application of voltage.
The red dotted line indicates the zero current level. These recordings from native channels show continuous NMDA receptor activity recorded from the soma of a neuron that was dissociated from the prefrontal cortex of a rat embryo and maintained in culture for five or 27 days. Once mastered, this procedure can be done in 30 minutes to one hour depending on the duration of the recording.
Following this procedure, other methods like kinetic analysis and state modeling can be performed to determine a channel's gating mechanism and to estimate reaction rates. After Watching this video, you should have a pretty good understanding of how to record and to begin analyzing currents through one ion channel protein.
