One-channel Cell-attached Patch-clamp Recording

Summary

Described here is a procedure for obtaining long stretches of current recording from one ion channel with the cell-attached patch-clamp technique. This method allows for observing, in real time, the pattern of open-close channel conformations that underlie the biological signal. These data inform about channel properties in undisturbed biological membranes.

Abstract

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate depends on properties intrinsic to each channel protein as well as the mechanism by which it is generated and controlled and represents an important area of current research. Information about the operational dynamics of ion channel proteins can be obtained by observing long stretches of current produced by a single molecule. Described here is a protocol for obtaining one-channel cell-attached patch-clamp current recordings for a ligand gated ion channel, the NMDA receptor, expressed heterologously in HEK293 cells or natively in cortical neurons. Also provided are instructions on how to adapt the method to other ion channels of interest by presenting the example of the mechano-sensitive channel PIEZO1. This method can provide data regarding the channel’s conductance properties and the temporal sequence of open-closed conformations that make up the channel’s activation mechanism, thus helping to understand their functions in health and disease.

Introduction

Fast communication across biological membranes relies almost exclusively on oligomeric pore forming membrane proteins, commonly referred to as channels. These proteins differ widely in activation signals, gating mechanisms, and conductance properties. Channel proteins whose pores are selective to ions are classified as ion channels; their activation produces ionic currents across the membrane, and their responses can be recorded with high resolution in real time using electrophysiologic techniques. The activation signals span a broad array of chemical and physical inputs including concentration gradients, mechanical and electrical forces, and temperature; thus, further classifying ion channels into ligand gated, mechanosensitive, voltage gated, or heat sensitive types. In this article, protocols are described to record one-channel activity from a ligand gated channel, the NMDA receptor, and a mechanosensitive channel, PIEZO1, using the patch-clamp technique. 

Patch-clamp electrophysiology is the first and most widely used experimental method sufficiently sensitive to permit the observation of single molecules^1,2. In addition to this exquisite sensitivity, it has vastly expanded the biological preparations amenable to electrophysiologic recording and also has allowed the observation of ion channels in intact membranes. First, because both voltage clamping and current recording are accomplished with the same electrode, it can be used to record signals across small cells or membranes patches. The technique revealed that ion channels are not restricted to excitable membranes of frog muscles, eel electroplaques, or squid giant axons^3,4, but rather that they represent ubiquitous fixtures of transmembrane signaling mechanisms and are intrinsic to all cellular membrane types of uni- or multicellular organisms, and also to intracellular membranes. Importantly, the capability to record transmembrane currents by simply attaching a glass pipette to an intact cell provided the unprecedented opportunity to record activity from ion channels in their native undisrupted membranes. Thus, the cell attached patch-clamp technique, which is described in this protocol, permits monitoring the activity of ion channels continuously for tens of min or longer in their native environment.

Under normal thermal fluctuations, all proteins, including ion channel proteins, undergo structural changes over a broad time scale, with the fastest and most frequent rearrangements represented most likely by side-chain movements and much slower, less frequent changes represented by the repositioning of entire domains or subunits, or in some cases by post translational modifications or protein-protein interactions^5,6. Observing long periods of activity generated by one molecule can help to understand the functional dynamics of ion channels in intact physiological membranes and provides valuable information about the operational mechanism of the molecule observed. 

In contrast to the growing understanding of the diversity of ion channels across cell types and developmental stages, knowledge about the molecular composition of ion channels in native membranes is still limited. All ion channels are multimeric proteins and the majority of native ion channels assemble from several types of subunits producing proteins of wide molecular diversity, which is often accompanied with diverse conductance and gating properties. For this reason, ion channels of defined molecular composition are studied upon expression in heterologous systems. In particular, HEK293 cells, which are a clonal line of immortalized human embryonic kidney cells⁷, gained widespread acceptance as the preferred system for heterologous expression of recombinant ion channels. Among the many advantages that elevated  HEK293 cells as the choice system for ion channel electrophysiology are the ease and affordability of culturing and maintaining long-lived stable cultures, their ability to carry out post-translational folding, processing and trafficking of mammalian proteins, and in many cases, their low level or even absence of endogenous expression for the channel of interest^7,8. Expressing recombinant ion channels and studying their functional properties in HEK293 cells continues to be a valuable approach to obtain information about structure-function properties of ion channels as well as the specific properties of ion channel isoforms and their roles in native tissue. The protocols described in this article can be applied equally well to recombinant ion channels expressed in HEK293 cells and to native ion channels.

In summary, the patch-clamp technique, through its unprecedented capacity to resolve signals from one molecule remains, to date, the most direct method for observing the behavior of single molecules. In its cell-attached mode, patch-clamp recording allows long observation periods which, when done for one molecule, can provide exceptional insight into the operation of ion channels. Below is presented a protocol for obtaining high resolution current recordings from cell attached patches containing one ion channel protein.

Protocol

1. Cell Culture and Protein Expression

1. Maintain HEK293 cells (ATCC number CRL-1573) between passages 22 and 40, which includes passages performed by ATCC, in monolayer culture in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin mixture at 5% CO₂ and 37 °C. Between experiments, passage cells into T25 flasks at 5-20 fold dilutions in a final volume of 10 ml. Note: Using cells during these passages guarantees favorable cell health which will allow for optimal seal formation and patch stability.
2. For transfection, plate HEK293 cells in 35 mm dishes at a density of ~10⁵ cells/dish, which corresponds to ~0.5-0.6 ml of cell suspension per dish and grow cells in 2 ml medium for 18-24 hr.
3. In a sterile 1.5 ml centrifuge tube, prepare the transfection mixture for four 35 mm dishes by adding (in this order): (1) 1 μg of each cDNA (GluN1, GluN2A, and GFP); (2) 315 μl of double distilled water (ddH₂O); (3) 350 μl of 42 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), and (4) 35 μl of 2.5 M CaCl₂ drop wise, which is used to form the precipitate.
4. Vortex tube for 5 sec and add 175 μl of transfection suspension in each of the four 35 mm dishes plated the day before, which now contain cells at 50-60% confluence, and incubate the cells at 37 °C for 2 hr.
5. Aspirate off the transfection medium, wash with PBS and replace with 2 ml of growth medium supplemented with 2 mM MgCl₂ to prevent NMDA receptor mediated excitotoxicity⁹. Note: The cells can be used for electrophysiological recordings 24-48 hr post transfection.

2. Electrode Preparation

1. Generate 2 symmetrical recording pipettes by pulling on borosilicate glass tubes with a vertical puller. Based on the size and geometry of the resulting tip, further shape pipettes using a polisher. Note: The tip size can be evaluated visually and electrically. Visually, the outer diameter should be in the 1.4-5.6 μm range. Electrically, an optimum size tip, when filled with extracellular solution, should produce resistances in the 12-24 MΩ range (see Figure 2B).
2. Use a pipette solution, which for cell attached experiments represents the extracellular milieu that will produce maximal channel activity and high current amplitudes. Note: For NMDA receptors, this corresponds to a solution containing saturating concentrations of agonists (glutamate and glycine), 1 mM EDTA, which chelates divalent cationic inhibitors and blockers, and has physiologic concentrations of sodium (150 mM) as the sole permeant ion (in mM: 1 glutamate, 0.1 glycine, 150 NaCl, 2.5 KCl, 1 EDTA, and 10 HEPBS, buffered at pH 8.0 with 1 N NaOH).

3. Cell-attached Patch-clamp Recording

1. Open QuB data acquisition software (www.qub.buffalo.edu), and select the acquisition window under ‘layout’. Open a new QuB data file (QDF) by clicking ‘New Data’ under the drop down menu entitled ‘File’ and enter the following initial parameters: sampling rate, 40 kHz; A/D scaling, 3,000; output channel, 1; and A/D data size, 2. Adjust the amplitude scaling to 0.1 V/pA by right-clicking the data file, selecting properties, and clicking the ‘data’ tab. Note: These parameters can be refined for each setup using a model-cell in the cell-attached mode. Additional instruction on data acquisition with QuB software and QDF properties are online at www.qub.buffalo.edu.
2. Select a 35 mm dish containing ion channel expressing cells and replace growth medium with 2 ml PBS containing calcium and magnesium; mount the dish onto the microscope stage and focus on the cellular field using phase contrast microscopy to evaluate that cells are healthy and well attached to the dish in monolayer fashion (Figure 1A). Switch to fluorescence detection to verify that the transfection was successful (Figure 1B). Note: The range of fluorescence intensities can be used as a crude measure of protein expression.
3. For single channel recording, set the amplifier output gain to x10, patch configuration to β = 1, analog filter to 10 kHz, mode to voltage-clamp and applied voltage to +100 mV. With the voltage holding command in the OFF position, select the ‘seal test’ button.
4. Based on fluorescence and cell health, 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. Note: Maintain phase contrast illumination to avoid bleaching the cell during the approach and patch-clamping.
5. Fill a freshly polished pipette with just enough solution so it makes contact with the electrode and shake gently to dislodge air bubbles that may be trapped in the tip. Secure the recording pipette onto the amplifier headstage by tightening the sealing screw of the pipette holder and making sure that the silver wire is immersed in the pipette solution.
6. Using a small (5 cc) plastic syringe, which is connected to the pipette holder by tubing, gently apply positive pressure to prevent impurities from entering and clogging the tip during the approach (Figure 2A).
7. Using the micromanipulator, direct the pipette into the bath and position it directly over the cell selected for patching (Figure 1C), closing the electrical circuit. Take note of the oscilloscope, which should indicate a rectangular waveform corresponding to the amplifier’s seal test signal (Figure 2B). Note: Upon entry into the bath, the pipette resistance within the optimal range is 12-24 MΩ. For a 5 mV test signal, the measured current range corresponds to ~20-40 pA.
8. While monitoring pipette position visually through the microscope and pipette resistance electrically on the oscilloscope, continue the approach in small increments until the pipette impinges gently on the cell, and the test signal decreases slightly to indicate increased resistance (Figure 2B).
9. To form a seal, pick up the syringe and apply slight negative pressure through the lateral tubing by pulling the syringe plunger, which pulls the cellular membrane into the tip of the recording pipette and initiates the formation of a GΩ resistance seal¹⁰. Take note of the test signal waveform on the oscilloscope, in which seal formation is indicated by its complete flattening, with only capacitive transients visible (Figure 2B). Note: If signal does not flatten completely or the baseline becomes noisy, this indicates a weak seal with substantial current leak around the seal. This will prevent resolving one channel currents. If this is the case, withdraw the pipette from the cell and away from the bath, remove it from the head stage and discard it. Repeat the process with a freshly polished pipette until obtaining a seal in the adequate range (≥ 1 GΩ).
10. On the amplifier, switch the external command toggle from ‘seal test’ to ‘off’; switch the voltage holding command to positive (which was set previously to ‘off’); and increase the gain to x100. Observe the oscilloscope for channel activity, which, if present, will be displayed as square upward deflections from the previously flat baseline (Figure 2C).
11. If channel activity is displayed on the oscilloscope, 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 to an easily retrievable location on the computer hard drive by selecting ‘Save Data As…’ in the drop down menu entitled ‘File’.

4. Data Preprocessing and Idealization

Note: Important information can be extracted from single channel recordings by statistical analyses which assigns each data point to an appropriate conductance class (in the simplest case, closed or open). This process is referred to as data idealization and a brief description of data idealization with the segmental k means (SKM) method¹¹ in QuB is described below.

1. Open the data file in QuB, and view the current traces in the ‘Pre’ interface under ‘layout’. Display the recorded file unfiltered (remove digital filter) by unchecking the box labeled ‘Fc.’
2. Visually scan the record to spot irregularities and artifacts (Figure 4A). Correct brief current spikes that occur within the trace (Figure 4A) by selecting an adjacent clean region of the same conductance class, highlighting this region, right-clicking and selecting ‘set erase buffer.’ Zoom in on the spike until individual sample points are visible, select the region to be replaced and erase by highlighting only this region and then right-click ‘erase’.
3. Define the zero current baseline for the entire record by selecting an early portion of the record where the baseline is stable, highlight, right-click and select ‘set baseline’. Verify that the guideline which appears accurately represents the baseline level (purple in Figure 4B)
4. 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.
5. Identify regions of the recording containing excess noise or artifacts that cannot be easily corrected (Figure 4C). Highlight the region to be discarded, right-click, and select ‘delete.’ Note: In this case kinetic information will be lost: the processed record will be shorter and the points occurring after the splice point will appear to be continuous with the pre-noise region.
6. To idealize records using the SKM algorithm¹¹, highlight a portion of the trace containing both open and closed events and enter the ‘Mod’ interface under ‘Layout.’ In the “model” panel, highlight a clean portion of the trace that is representative of the baseline throughout the file, right-click the black square (closed state) and select “grab”. Do the same for channel openings by highlighting the open conductance, right click the red square in the “model” panel and select “grab”.
7. Perform the idealization for the entire record by selecting the ‘File’ button under ‘Data Source.’ Right-click on the ‘Idealize’ tab underneath the ‘Modeling’ section to verify that the desired parameters for analysis are correct, and then click ‘Run.’ The result of the idealization, along with amplitude histogram for the entire file, is overlaid with the data to allow for visual inspection of the idealization (Figure 5). Note: Inadequate idealization may lead to the generation of ‘false events,’ in which QuB detects channel openings and closures that do not actually occur. Within the recording resolution, which is set by the amplifier’s analog filter and the sampling rate, the resolution with which SKM detects open and closed events can be controlled by setting a dead time for the analyses. Optimal dead times must be selected by trial and error and will depend on sampling rate, however, a good rule of thumb is to select a dead time that is 2-3 samples¹².
8. To verify that the idealization accurately represents the raw data, manually scan the idealized trace. Upon identification of errors in the idealization, highlight the trace over the false event, right click and select ‘Join Idl.’ Note: For additional options and a detailed explanation of various commands and functions in QuB, consult the QuB Manual online (www.qub.buffalo.edu).

Results

Recombinant NMDA Receptors

NMDA receptors bind and respond to the concomitant action of two co-agonists: glutamate and glycine. They assemble as heterotetramers of two glycine binding GluN1 subunits and two glutamate binding GluN2 subunits. GluN2 subunits are encoded by four genes (A-D) and of these the most widely transcribed forms in brain are GluN2A in adult and GluN2B in juvenile animals. Because of the diversity of NMDA receptor subtypes in native preparations, expressing...

Discussion

In the ion channel field, an important area of research is dedicated to understanding the sequence of events that leads to channel opening or the channel’s gating mechanism. For most channels, this process is complex and involves several kinetic steps that cannot be deduced from a macroscopic multi-channel signal. In contrast, experiments can be designed where observing the sequence of open/closed events in single channel record can produce more detailed information about gating mechanisms. In the methods described...

Materials

Name	Company	Catalog Number	Comments
Chemicals	Sigma	Various
Borosillicate Glass	Sutter	BF-150-86-10
Bright field inverted microscope	Olympus	1x51	Nikon also has similar microscopes
Fluroescent box	X-cite	Series 120
Liquid Light Guide	X-cite	OEX-LG15
Micromanipulator	Sutter Instruments	MP-225
Oscilloscope	Tektronix	TDS1001
Amplifier	Molecular Devices	Axon Axopatch 200B
Table	TMC	63561
NIDAQ card	National Instruments	776844-01
Puller	Narishige	PC-10
Polisher	Narishige	Microforge MF-830
Faraday Cage	TMC	8133306
High Speed Pressure Clamp	ALA Scientific Instruments	ALA HSPC
Pressue/Vaccuum Pump	ALA Scientific Instruments	ALA PV-PUMP	For HSPC-1