Automated Patch Clamp Analysis of nAChα7 and Na(V)1.7 Channels.

Automated Patch Clamp Analysis of nAChα7 and NaV1.7 Channels

UNIT 11.13

Alison Obergrussberger,1 Claudia Haarmann,1 Ilka Rinke,1 Nadine Becker,1 David Guinot,1 Andrea Brueggemann,1 Sonja Stoelzle-Feix,1 Michael George,1 and Niels Fertig1 1

Nanion Technologies, Munich, Germany

ABSTRACT Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (NaV 1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of NaV 1.7, and activation and allosteric modulation of nAChα7R. Curr. Protoc. Pharmacol. 65:11.13.1-11.13.48. C 2014 by John Wiley & Sons, Inc. Keywords: automated patch clamp r NaV 1.7 r nicotinic acetylcholine receptors

INTRODUCTION The glass pipet patch clamp technique was first described by Neher and Sakmann (1976). However, the utility of this approach for drug discovery is limited because the assay is low throughput and technically demanding. Automation of the patch clamp technique has increased throughput and ease of use (Dunlop et al., 2008; Farre et al., 2009; Stoelzle et al., 2011a). Thanks in part to these technical advances, automated patch clamp (APC) devices are now commonly employed in academic and industrial laboratories (Milligan et al., 2009; Balansa et al., 2010). Continued improvement and development of APC devices has made them for useful for conducting more complex experiments and data analysis. More recent advances in this technology include APC devices that allow for ultra-fast perfusion and temperature control, which make expansion in the range of experimental protocols possible. Provided in this unit is information on three devices with varying degrees of throughput. 1. The Port-a-Patch device records from one cell at a time using a planar borosilicate glass chip. The device is easy to use and increases throughput compared with the conventional glass patch clamp. Accessories are available, including the External Perfusion System for recording ligand-gated ion channels and the Fast Perfusion System described below (Basic Protocol 1 and Alternate Protocol 1). 2. The Patchliner device records from eight cells simultaneously. In this case, the glass chips contain micro-fluidic channels for fast perfusion. The Patchliner is particularly popular because it allows for a great deal of flexibility in designing experimental protocols. The system includes a precisely controlled and fast solution exchange, with a temperature-control feature that allows for measurements in a more physiologically Electrophysiological Techniques Current Protocols in Pharmacology 11.13.1-11.13.48, June 2014 Published online June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471141755.ph1113s65 C 2014 John Wiley & Sons, Inc. Copyright

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relevant environment. Experiments involving both of these features are described in this unit (Basic Protocol 2 and Alternate Protocols 2 and 3). 3. The SyncroPatch 96 records from 96 cells in parallel. This makes it possible to conduct more complex experiments. A sophisticated pipetting system ensures rapid solution exchange and low compound exposure times. These properties make the SyncroPatch 96 an ideal high throughput system for both ligand- and voltage-gated ion channels (Basic Protocol 3 and Alternate Protocol 4). In the present unit, these systems are described as approaches for studying NaV 1.7 and nAChα7R. A voltage-gated sodium channel, NaV 1.7 is expressed primarily in the peripheral nervous system. It is thought to play a role in nociception and believed to be involved in various pain states, including inflammatory pain (Nassar et al., 2004; Dib-Hajj et al., 2013) and hypersensitivity to heat following burn injury (Shields et al., 2012). As NaV 1.7 undergoes fast desensitization, complex voltage protocols are needed to test compound potency on the inactivated state of this channel. Protocols are provided in this unit for recording hNaV 1.7 expressed in CHO or HEK cells with particular attention focused on recording activation and inactivation kinetics and measuring differences in potency on the inactivated versus the activated state of the channel recorded on the Patchliner. The nAChα7R is a particularly challenging drug target for screening because of its rapid desensitization kinetics. This necessitates minimizing exposure times in order to reliably and reproducibly measure nAChα7R currents in response to the application of acetylcholine (ACh) or nicotine. In addition, it is known that temperature influences the allosteric modulation of nAChα7R (Sitzia et al., 2011). Given the importance of this channel in cognitive function, and its potential role in mediating some symptoms of Alzheimer’s disease and schizophrenia (Pohanka, 2012), it is a popular target for drug discovery. Not only is it necessary to have the capacity to reliably record this channel at room temperature, but it may also be critical to test drug candidates at physiological temperature. Protocols are provided in this unit for examining the activation of nAChα7R by ACh or nicotine with different patch clamp systems, as well as the use of Patchliner for studying the allosteric modulation of nAChα7R by PNU120596 at different temperatures. BASIC PROTOCOL 1

ACTIVATION AND INACTIVATION KINETICS OF NaV 1.7 USING THE PORT-A-PATCH Because the potency of many compounds at NaV 1.7 is altered in a state-dependent manner, it is important to establish the voltage-dependence of channel activation and inactivation before performing pharmacological studies on these voltage-gated sodium channels (Bean et al., 1983; Kaczorowski et al., 2011). Detailed in this protocol is the use of the planar patch clamp device, the Port-a-Patch (Fig. 11.13.1A), to record the voltage dependence of activation and inactivation of NaV 1.7. Additionally, an experiment is described for constructing a concentration-response curve for tetrodotoxin (TTX).

Materials

Automated Patch Clamp Analysis of Ion Channels

Nanion standard internal solution for Na+ channels (NSIntSNa; see recipe) CHO cells expressing hNaV 1.7 (Anaxon AG) or HEK cells expressing hNaV 1.7 (EMD Millipore) Nanion standard external solution for Na+ channels (NSExtSNa; see recipe) Seal enhancing solution (SES; see recipe) 3 nM, 10 nM, 30 nM, 100 nM, 300 nM and 1 μM TTX (Tocris) prepared in NSExtSNa. Prepare the 1 nM TTX stock solution in NSExtSNa. Store aliquots


Current Protocols in Pharmacology

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Figure 11.13.1 (A) The planar patch clamp set-up, the Port-a-Patch, is composed of the Port-aPatch unit, Suction Control, amplifier, and a desktop computer. (B) The Fast Perfusion System for the Port-a-Patch. The Port-a-Patch is shown with the Fast Perfusion Chamber attached. There are four tubes that enter the manifold of the Fast Perfusion Chamber, and these contain either control external recording solution or a solution of ligand/compound. The tube at the right of the manifold is attached to the waste removal pump. There is little dead volume in the manifold and minimal distance between the cell and where the solution enters the bath, thus achieving an exchange time of less than 20 msec and an exposure time of 100 msec. (C) The Patchliner. Automated patch clamp device where up to eight cells can be recorded simultaneously. Amplifiers and computer not shown. (D) The SyncroPatch 96. Automated patch clamp device where up to 96 cells can be recorded in parallel.

at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment for preparing the TTX concentrations by serial dilution. Amplifier (e.g., EPC10 USB, HEKA Elektronik) Computer running the following software: PatchControl software (Nanion Technologies) PatchMaster software (HEKA Elektronik) Igor software (Wavemetrics) Excel (Microsoft) NPC -1 chips, resistance 2 to 3.5 MΩ (Nanion Technologies) Port-a-Patch recording station (Nanion Technologies) R

Electrophysiological Techniques

11.13.3 Current Protocols in Pharmacology

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Port-a-Patch suction control (Nanion Technologies) External perfusion system for the Port-a-Patch, including a laminar flow chamber (Nanion Technologies; optional) R

Prepare setup and load NPC -1 chip with solutions 1. Activate the computer and amplifier. Allow the amplifier to warm up for 10 min before beginning the experiment.

2. Double click the PatchControl icon on the computer desktop to start PatchControl and PatchMaster. With the latter, the user is prompted to provide a file name for the data to be stored. Provide an appropriate name for that day’s experiments to create a new file (e.g., YYMMDD_NaV17), and ensure the data can be retrieved. 3. Load the parameters file Nanion fragile.ppf if not already loaded. Nanion provides three main parameter files that are supplied with the instrument: Nanion fragile.ppf, Nanion intermediate.ppf, and Nanion strong.ppf. Depending on the strength of the membrane, the most appropriate parameter file should be selected as the starting point. It is possible to alter parameters such as strength of suction and time spent in each step to optimize the parameter file for a particular cell line (see below). To load a different parameter file, go to ‘Experiment->Load parameter file’ in PatchControl. The parameter file contains a series of pre-programmed steps for automating cell capture as well as sealing and rupture of the cell membrane. Thresholds are predefined for seal resistance, suction, holding potential, and time for each step. If the procedure is not modified by the user, the software will go through each step in turn until a seal is formed and whole-cell configuration is attained. The thresholds may be altered and the suction controlled manually by pressing ‘pause’ during the course of the experiment. Once the thresholds are changed, the experiment continues with the new thresholds. Should these new values provide better results with this cell line, the parameter file may be saved under a new name for use whenever this cell line is employed.

4. Using a pipet, add 5 μl NSIntSNa to the inside of the chip (Fig. 11.13.2A). The small droplet will cling to the glass due to surface tension. Grasp the sides of the chip without touching the glass surface, as fingers may leave behind grease and dust that could compromise cell capture and sealing.

5. Turn the chip over and screw it onto the chip-mounting chamber of the Port-a-Patch. Be sure that the internal solution makes contact with the internal electrode. Internal and external electrodes for the Port-a-Patch are composed of silver. A layer of silver chloride is required on the silver electrodes. To achieve this, the electrodes are carefully sanded with fine sandpaper to remove any residual silver chloride and are placed in bleach for approximately 10 min. The electrodes should be re-chloridized once to twice per week depending on the frequency of use. As freshly chloridized electrodes are black, white electrodes need to be re-chloridized.

6. Put on the Faraday top. If using the External Perfusion System for the Port-a-Patch, the Laminar Flow Chamber is used instead of the standard Faraday top (Fig. 11.13.2B). The tubes should be pressed tightly into the manifold of the Laminar Flow Chamber and primed with solution before initiating the experiment. This ensures that no air bubbles are present. Make certain that the External Perfusion panel is switched to manual mode, and then open each of the valves in turn while the external waste removal pump is switched on. This step can be performed prior to initiating an experiment using an old chip. Automated Patch Clamp Analysis of Ion Channels

7. Add 5 μl of NSExtSNa to the top of the chip (approximately in the middle) and position the external electrode so that it is inside this droplet (see Fig. 11.13.2C).



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Figure 11.13.2 (A) The internal solution is pipetted onto the inside of the chip. The user grasps the outside edge (made of plastic) of the chip and pipets 5 μl of internal solution carefully onto the inside of the glass chip. (B) The Laminar Flow Chamber mounted on the Port-a-Patch. (C) Once the chip is screwed onto the Port-a-Patch chip mounting station, the standard Faraday top is added and 5 μl external solution is carefully pipetted onto the middle of the chip. Note the position of the external electrode, which must be in the external droplet.

Alternatively, if the External Perfusion System is employed, open the valve containing NSExtSNa and turn on the waste removal pump until solution fills the chamber. Close the valve and turn the pump off. A square pulse should be observed on the screen of the oscilloscope window of the dataacquisition software, indicating electrical contact between the internal and external electrodes. If this pulse is not observed, move the external electrode to ensure it is positioned in the solution.

8. Press the ‘play’ button in the PatchControl software. At this point the ‘wait for contact’ step will be run in the pre-programmed protocol. The PatchControl software will instruct the PatchMaster software to display the chip resistance in the top box of the PatchControl software. If this value is greater than 0 and less than 10 MΩ (or other threshold set by the user), then the Offset potential (VpOffset) will be automatically adjusted, and the system is ready for cells to be added. The threshold chosen depends on chip resistance. A threshold of greater than 0 and less than 10 MΩ is sufficient for the standard resistance of chips in the ranges 1 to 2 MΩ; 2 to 3.5 MΩ, and 3 to 5 MΩ. If customized chips are used that are likely to have a resistance greater than 10 MΩ (e.g., chips with a resistance range of 8 to 12 MΩ), the threshold



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in the step ‘wait for contact’ must be increased, e.g., up to 18 MΩ. The experiment will terminate if the criteria are not met. However, the user has the option to change the threshold and restart the experiment if the threshold is set too low for a high-resistance chip. If the resistance is very high, e.g., greater than 50 MΩ or even in the GΩ range, it is likely the electrodes are not in solution, the patch clamp aperture is blocked, or there is a problem with the solutions. First check that the electrodes are in the solution. If this is not the problem, repeat the experiment with a fresh chip and/or solutions.

Perform cell capture and sealing procedure 9. Pipet the cell suspension up and down a few times to ensure that cells are not clustered at the bottom of the microcentrifuge tube (for cell harvesting protocol see Support Protocol 3). Aspirate 5 μl of the cell suspension using a pipet. 10. Press the ‘Add cells’ button in the PatchControl software. Suction is applied to the chip after the software adjusts the VpOffset again in PatchMaster. The ‘wait for cell’ step is now activated in PatchControl. 11. Add the 5 μl of aspirated cell suspension into the middle of the external solution droplet after the Suction Control has applied negative pressure. Aim the pipet tip towards the center of the chip (i.e., the location of the patch clamp aperture). Some force may be required to direct cells to the patch clamp aperture.

12. By the use of suction, a cell will be attracted automatically to the patch clamp aperture, thereby increasing chip resistance. This typically occurs within a few seconds of adding the cells. The software automatically recognizes this increase in resistance and will proceed to ‘wait for seal’, the next step of the sealing procedure. During the ‘wait for cell’ step, parameters are defined for the chip threshold resistance to be considered cell capture. An increase of resistance up to approximately 10 MΩ (depending on initial chip resistance) is usually sufficient to indicate cell capture. Strength of suction and suction increase are also defined in the software. Should a cell not be captured immediately, the suction will be increased by a defined amount until a cell is attracted to the hole and the resistance increased. Should no increase in resistance be detected in a predetermined period of time (default 120 sec), the software will assume that no cell is captured and the experiment will be terminated. The default values for the suction applied in the ‘wait for cell’ step in Nanion fragile.ppf are −40 mbar to −100 mbar, increasing by 10 mbar every 10 sec. If a cell is routinely not captured using these values, the values may be increased by pressing Edit, e.g., −50 mbar to −150 mbar increasing by 20 or 50 mbar every 10 sec. Once the values are optimized for the cell line being studied, the parameter file can be saved and loaded every time that cell line is used.

13. Once a cell is captured, the holding potential of the cell automatically ramps down to −40 mV (voltage increased by 2 mV every 0.3 sec) in the step ‘wait for seal’. At this stage, 20 μl of SES is added to the chip and then 20 μl of solution is removed. The solution should be added gently to one side of the droplet (external side), adding to the existing droplet but away from the center of the chip to avoid disruption of the seal. The pipet should be then placed at the other side of the droplet and 20 μl of solution removed and discarded. This creates a ‘washing’ motion of the cell, helping to improve the seal. Alternatively, when using the External Perfusion System, the valve containing the SES is opened and the external waste removal pump switched on. The valve is closed after 5 sec and the pump switched off. The presence of calcium in the SES and fluoride in the internal solution helps to improve the seal by an unknown mechanism.


The default seal threshold to be reached is 100 MΩ, with the default values for suction being −40 mbar to −50 mbar, increasing by 5 mbar every 5 sec. The timeout for this step is 20 sec. Once the cell is captured, the amount of suction should be reduced (as shown here, −40 mbar) to ensure that the cell does not go spontaneously into a wholecell configuration prior to forming a good seal. The addition of SES, coupled with the



reduction in holding potential, ensures that the 100-Ω threshold is attained within the 20-sec timeout period. If the threshold of 100 MΩ is not reached within the timeout period, the software automatically moves on to the next step. With this protocol, only in the steps ‘wait for contact’ and ‘wait for cell’ will the experiment be terminated if the timeout is reached.

14. Once a resistance threshold is attained (as defined in the software), PatchControl will automatically proceed to the Improve Seal 1 step. In this step, holding potential is ramped down to −80 mV at a rate of 2 mV every 0.3 sec and suction is increased until defined thresholds for resistance or maximum suction are reached. The software then moves automatically to the Improve Seal 2 step. The threshold of resistance to be reached in Improve Seal 1 is 100 MΩ, with the default parameters for suction ranging from −40 mbar to −100 mbar, increasing by 10 mbar every 10 sec. The timeout is 60 sec. The important component of the step Improve Seal 1 is ramping down the holding potential to −80 mV. This usually has a significant influence on the improvement of the seal. If the timeout is reached before the seal threshold, the software moves to the next step. It is possible that the seal will improve over time and can improve to a GΩ seal even after the whole-cell configuration is attained. Even if the seal is relatively low at this stage, it is worth continuing with the experiment until the last step in case the seal improves at a later stage. Alternatively, the software can be paused and the suction controlled manually. The user can switch the suction to atmosphere or apply increased suction to assess whether the seal can be improved manually.

15. The rate of seal improvement is calculated during the Improve Seal 2 step. The suction will remain constant if the seal improves at or above a certain (user-defined) rate. The suction will automatically be altered should the seal not improve, or improves at a lower rate than anticipated. The suction will be switched to atmospheric pressure and the rate of improvement measured again. If the rate of seal improvement does not reach or exceed the defined rate, pressure will be re-applied at an increased value. This will continue until the seal threshold is attained or the timeout is reached. The software then automatically moves onto the ‘wait for whole cell’ step. In the step Improve Seal 2, the default seal threshold is 600 MΩ, the timeout is 150 sec, the rate of seal improvement 50 MΩ/min, the maximum pressure is −200 mbar, the minimum duration is 10 sec (i.e., minimum time spent in this step), and maximum iterations are 50 (i.e., number of times the program will switch between atmosphere and pressure, increasing the pressure each time). The majority of these parameters are appropriate for most cell types. However, if the cells are particularly fragile, the maximum pressure should be reduced to −100 or −150 mbar, whereas for cells with a particularly tough membrane, the maximum pressure should be increased to −250 or −300 mbar. As indicated above (step 14), if the timeout or maximum number of iterations is reached before the seal threshold is attained, the software will move automatically to the next step. The experiment should proceed even with a low seal, as it is possible the seal will improve upon rupture of the membrane. It is also possible to press ‘pause’ and control the Suction Control manually, switching between atmosphere and pressure. Once a new maximum pressure value is established by observing the seal and controlling the suction manually, this value can be substituted in the system and the parameter file saved.

16. During the ‘wait for whole cell’ step, atmospheric pressure is applied to the cell while a set amount of pressure is reached in the reservoir of the Port-a-Patch Suction Control unit. This is then applied to the cell for a few seconds (usually 4 sec), and atmospheric pressure applied again. Now the pressure in the reservoir is increased and then applied to the cell. At this point the ‘zap’ function of the amplifier may be used so that zap is applied in combination with the suction pulses. These cycles of suction application are continued until the whole-cell parameters are attained. The amount the suction should increase with each cycle is defined by the user, with default being an increase of 25 mbar per cycle. If the cells are particularly weak and



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whole-cell configuration is attained very quickly, this step increase can be decreased to, for example, 15 mbar. If the cell membranes are particularly tough, the amount may be increased. The number of times the zap function is used can also be defined in this step. Default is once after four pulses, or the function can be removed completely by setting ‘0 times after 4 pulses’. While the software automatically goes through each step based on the established parameters, the user can pause the protocol at any time and control the suction manually. This is particularly useful when employing a new cell type, or if cells are found to be fragile on a particular day. Once new cell behaviors have been observed, the protocol parameters can be altered to perform better for that particular cell type. The protocol can be saved and loaded each time that cell type is studied. Parameters used for defining whole cells are by default slow capacitance (Cslow) greater than 5 pF, and series resistance (Rseries) less than 40 MΩ, to ensure that the protocol enters the last step (‘maintain whole cell’) as soon as the cell achieves whole-cell configuration. Note that the Rseries is usually less than 12 MΩ once the recording begins. If the whole-cell parameters are already attained in steps ‘wait for seal’, Improve Seal 1, or Improve Seal 2, the software will go quickly through the remaining steps to the last (‘maintain whole cell’).

17. Once whole-cell configuration is reached, the software automatically pauses in the step ‘maintain whole cell’, leaving a small amount of suction applied (or atmospheric pressure; user defined), and a negative holding potential remains applied to the cell. For NaV 1.7, a holding potential of -100 mV is used. At this stage, if the seal resistance is still low (e.g., less than 100 MΩ), i.e., the software has gone through the steps and the seal threshold has not been reached, and the leak current is very large, the user must decide whether to terminate or continue the experiment. Typically, the type of experiment (recordings of ligand-gated ion channels can usually tolerate a lower seal than those of voltage-gated ion channels) and the amount of leak influence this decision. If it is decided to continue with the experiment, the data can be discarded at a later time.

18. The SES should now be washed away by adding 20 μl NSExtSNa to the chip and removing 20 μl solution as described previously (step 13). This is repeated three to five times to ensure full exchange of the solution. Alternatively, if the External Perfusion System is used, the valve containing NSExtSNa is opened and the waste removal pump switched on. The solution can be constantly perfused over the cell during the course of the experiment when the External Perfusion System is employed. In this case, the user should carefully monitor the NSExtSNa level in the syringe to ensure that it does not run dry. Alternatively, the valve can be closed and the waste removal pump switched off until the beginning of the pharmacology experiment (see step 22).

19. The user now automatically compensates for the capacitance transients by pressing the ‘Auto’ button of the HEKA EPC10USB amplifier next to CSlow and RSeries values. This should be done two to three times to ensure that the compensation is adequate. RSeries compensation can then be set to 80% compensation, 100 μsec (a description of CSlow, RSeries, and RSeries compensation terms can be found in The Axon Guide; Sherman-Gold, 2012: http://mdc.custhelp.com/euf/assets/content/ Axon%20Guide%203rd%20edition.pdf). The system is now ready for acquisition of electrophysiological data.


Acquire data 20. With PatchMaster, the voltage protocol to elicit a current-voltage (IV) plot is predefined by the user in the ‘pulse generator file’ (pgf). Press the Na-IV-P4leak button to start the voltage steps. The corresponding online analysis (onl) is also pre-loaded to automatically plot the maximum peak amplitude versus the test potential each time the Na-IV-P4leak protocol is executed.



Figure 11.13.3 Screenshot of the PatchMaster pgf file for the NaV 1.7 current-voltage plot. Number of sweeps (in this case 12), time interval (in this case 2 sec between each sweep), and sample interval (i.e., sampling rate; in this case 50 μsec which is equivalent to a sampling rate of 20 kHz) are set in the top left section under ‘Timing’. The parameters for the voltage steps are set in the section ‘Segments’. Here, the voltage in mV, duration in msec, and step increase in mV are set. A visual representation of the voltage step protocol is shown at the bottom. In this case, the first segment is the holding potential for 11 msec followed by a second segment which steps to −60 mV for 20 msec, and this voltage is increased by 10 mV with each subsequent sweep; finally, the third segment sets the voltage back to the holding potential for 10 msec. On the right, the online analysis file is chosen, which will be automatically performed when the voltage protocol is run. The segment of the voltage protocol to be used for the analysis is also set; in this case, this is segment 2. The parameters for the leak subtraction protocol are shown under ‘Leak Pulses’.

The pulse generator file is accessed in PatchMaster using the F8 shortcut key. In the case of the Na-IV-P4leak protocol, the voltage is stepped from the −120 mV holding potential to a test potential of −60 mV for 20 msec and then back to the holding potential. With each subsequent step, the test potential is increased by 10 mV to a maximum of 50 mV (total number of steps 12), with a sweep interval of 2 sec. By also employing a P/4 leak-subtraction protocol, the leak current is calculated and subtracted to maintain a constant baseline and to subtract any capacitances that remain. For this, a small voltage step is used before the test pulse (one-quarter of the test step size in a hyperpolarized direction) from the holding potential. It is important that the parameters chosen for the leak subtraction step not activate channels. This step is repeated four times and the sum of the 4 × ¼ amplitude traces is subtracted from the recorded data in PatchMaster. The displayed data are the leak-subtracted data. Displayed on Figure 11.13.3 is a screenshot of the PatchMaster voltage protocol.



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Figure 11.13.4 Screenshot of the PatchMaster onl files for online analysis of the current-voltage plot for NaV 1.7. At the bottom under ‘Analysis Functions’ the parameters to be analyzed are chosen. In this case, extremum is chosen (i.e., peak amplitude whether that is positive or negative), and amplitude, which is the voltage. The cursor bounds are set in percentage, in this case 1.2% to 31.2% of segment 2 of the relevant voltage protocol (shown in Fig. 11.13.3). This means that the peak is sought in segment 2 in the region which is 1.2% to 31.2% from the start of the step. A graph can be displayed automatically during recording and the parameters to be used for graphical representation are chosen under ‘Graph Entries’. In this case voltage (Ampl_1) is plotted on the X axis and peak amplitude (Extr_1) is plotted on the Y axis.

The online analysis file is accessed in PatchMaster using the F7 shortcut key. In this case, the peak amplitude (extremum, i.e., maximum peak amplitude whether positive or negative) within the region 1.2% to 31.2% after step to the test potential is measured and plotted against the test potential (Fig. 11.13.4).

21. The Na inactivation voltage protocol is also now run in PatchMaster. Here, the pre-defined protocol Na Inact is run. This voltage protocol uses a 5-sec pre-pulse to different test potentials followed by a 10-msec step to 0 mV, with a sweep interval of 20 sec (Fig. 11.13.5 ). The online protocol Na_Inact is linked via the pulse generator file and is automatically run during the data acquisition process. The peak amplitude in the 10-msec step to 0 mV is plotted online against the test potential in the 5-sec pre-pulse.


22. The pre-defined voltage protocol ‘NaPharmP4leak’ is now initiated in PatchMaster. This protocol steps from the holding potential of −100 mV to 0 mV for 20 msec, and is repeated every 3 sec (using a P/4 leak subtraction). Once the current amplitude is stable as visually observed and determined by the user, the external solution is changed to a solution containing the pharmacological agent of interest (in this case, TTX). To do this, 20 μl of the new solution is added to the chip and removed as described in step 13, and this is repeated three to five times to completely exchange the solution. Alternatively, when using the External Perfusion System for the Port-a-Patch, the external solution may be exchanged continuously. It is important to first open the valve containing NSExtSNa (with the waste removal pump also switched on) to record data when the control solution is perfusing. Then, open the valve containing the lowest concentration of TTX (3 nM), with the next highest concentration added once the current has stabilized. Repeat the process with increasing concentrations of TTX. Following the addition of the highest concentration of TTX (1 μM), exchange the solution back to NSExtSNa to reverse the effect of TTX.



Figure 11.13.5 Screenshot of the PatchMaster pgf file for the NaV 1.7 inactivation plot. Number of sweeps (in this case 16), time interval (in this case 20 sec between each sweep), and sample interval (i.e., sampling rate; in this case 50 μsec which is equivalent to a sampling rate of 20 kHz) are set in the top left section under ‘Timing’. The parameters for the voltage steps are set in the section ‘Segments’. Here, the voltage in mV, duration in msec, and step increase in mV are set. A visual representation of the voltage step protocol is shown at the bottom. In this case, the first segment is the holding potential for 11 msec followed by a second segment which steps to −120 mV for 5 sec, and this voltage is increased by 10 mV with each subsequent sweep; a third segment steps to 0 mV for 10 msec, and finally the fourth segment sets the voltage back to the holding potential for 10 msec. On the right, the online analysis file is chosen, which will be automatically performed when the voltage protocol is run. The segment of the voltage protocol to be used for the analysis is also set; in this case this is segment 2 for the X axis and segment 3 for the Y axis. The parameters for the leak subtraction protocol are shown under ‘Leak Pulses’.

Analyze data 23. Once data are acquired and visualized online, they are analyzed further offline. Each time the raw data are displayed in PatchMaster, the online analysis results are written in the Notebook of PatchMaster. These values may then be exported to Microsoft Excel. Other export functions (e.g., export of traces and stimulus in ASCII format, as Igor wave, or in Binary format) are also available with PatchMaster. 24. Using Excel, average the data from several cells and calculate the standard error of mean (S.E.M). Export these values to Igor. 25. In Igor, fit the averaged data for the IV and inactivation IV using the Boltzmann equation using an Igor macro.



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26. In Igor, fit the average data for the TTX concentration response curve according to the Hill equation using Patcher’s Power Tools. ‘Patcher’s Power Tools’ is a software package adding a series of external operations, macros, and experiments to Igor. The software package was developed by Dr. Francisco Mendez and Frank Würriehausen in the Department of Membrane Biophysics at MPI Biophysical Chemistry in Göttingen, Germany. It is available for download free of charge at http://www3.mpibpc.mpg.de/groups/neher/index.php?page=software. ALTERNATE PROTOCOL 1

ACTIVATION OF nAChα7R ON THE PORT-A-PATCH USING THE FAST PERFUSION CHAMBER The nAChα7R is a challenging target for drug discovery because of its fast desensitization kinetics. Thus, during exposure of the receptor to a ligand, the channels open and then quickly enter a desensitized state in which they cannot open again. Because recovery from this desensitized state takes time, repetitive activation of the channel for pharmacology experiments is difficult. This makes it necessary to minimize the time of exposure of the ion channel to the ligand. Described below is a Port-a-Patch Fast Perfusion Chamber protocol for recording nAChα7R expressed in HEK cells.

Additional Materials (see also Basic Protocol 1) HEK cells expressing nAChα7R (Induced; Galantos Pharma) Nanion standard external solution for nAChα7R (NSExtSnAChα7R; see recipe) Nicotine (Sigma) 70 μM prepared in NSExtSnAChα7R (see recipe). The stock solution for nicotine is 10 mM in DMSO. Store aliquots at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the required nicotine concentration in NSExtSnAChα7R. R

Prepare setup and load NPC -1 chip with solutions 1. Follow steps 1 to 5 of Basic Protocol 1 to activate the system (if not already on) and R to load the NPC -1 chip with internal solution. 2. Mount the Fast Perfusion Chamber (Fig. 11.13.1B), and switch the perfusion system to manual mode. Run NSExtNa through the chamber and turn on the pump to remove the waste solution. Before performing the experiment, make certain that the tubes of the Fast Perfusion System are tightly pressed into the manifold. Prime with the solutions to ensure that no air bubbles are present in the tubing, as bubbles can disrupt the cell on the patch clamp aperture, causing premature termination of the experiment. Make sure that the perfusion pump is attached via a digital cable to the back of the HEKA EPC10USB amplifier for later use in the auto mode. NSExtSNa is used for cell capture as the solution for nAChα7R interferes with cell capture because of its high Ca2+ content.

3. Terminate perfusion and check the oscilloscope screen for the square pulse indicating electrical contact between the internal and external electrodes. 4. Follow step 8 of Basic Protocol 1.

Perform cell capture and sealing procedure 5. Pipet the nAChα7R-expressing HEK cell suspension up and down several times to ensure that cells are not clustered at the bottom of the microcentrifuge tube. Aspirate into a pipet 20 μl of cell suspension.


6. Press the ‘Add cells’ button in the PatchControl software. Initially, the software adjusts the VpOffset again in PatchMaster, and then suction is applied to the chip. The ‘wait for cell’ step is now activated in PatchControl. Add the 20 μl of aspirated cell suspension into the middle of the external solution droplet after the Suction Control has applied negative pressure.



A

B

Figure 11.13.6 Screenshot of the PatchMaster pgf file for perfusion of nicotine to activate the nAChα7R. (A) Channel 1 defines the voltage step (in this case constant holding potential of –80 mV). Importantly, the timing is defined here. (B) Channel 2 defines the commands for the perfusion system. In this case, the perfusion switches from line 1 (digital word 11) to line 3 (digital word 13) for 100 msec and then back to line 1.


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7. Follow step 12 of Basic Protocol 1. 8. In the ‘wait for seal’ step of the PatchControl software, open the valve of the perfusion system containing the NSExtSnAChα7R and activate the waste removal pump. It is only necessary to flush the solution through for approximately 5 sec. This ensures complete exchange of the solution.

9. Continue with steps 14 to 17 of Basic Protocol 1. 10. Once whole-cell parameters are attained, the software automatically pauses in the ‘maintain whole cell’ step, leaving a small amount of suction applied (or atmospheric pressure; user defined), and a negative holding potential remains applied to the cell. In this case, −80 mV was used. Suction remains applied during the recording to maintain good electrical access to the cell, i.e., to prevent resealing of the membrane. A negative holding potential is employed to maintain the health of the cell during the recording and to ensure a large current amplitude.

11. The user now automatically compensates for the capacitance transients by pressing the ‘Auto’ button of the HEKA EPC10USB amplifier next to CSlow and RSeries values. This should be repeated two to three times to ensure that the compensation is adequate. The system is now ready to acquire electrophysiological data. No wash is necessary in this case, as the cell is already in the recording solution.

Acquire data 12. The required concentration of nicotine in the NSExtSnAChα7R (in this case 70 μM) should be present in line 3 of the perfusion system. 13. Switch the perfusion pump to auto mode, making certain that the switch at the side of the pump is set to digital. In PatchMaster, press the nACh button to begin the experiment. The voltage is maintained constant, and a second channel in the pgf controls the perfusion panel. Nicotine is perfused over the cell when valve 3 is opened for 100 msec and rinsed away when valve 1 is opened (NSExtSnAChα7R without nicotine). A second channel in the pgf file is used to control the perfusion system. The timings are set using the first channel where voltage is also determined. In the second channel the ‘digital word’ is used to control the perfusion panel. Thus, the valve numbers can be entered for regulation, as well as for the waste removal pump. The voltage remains constant for the entire experiment (Fig. 11.13.6). An onl file can also be pre-defined to plot peak amplitude versus sweep count or time.

Analyze data 14. For data analysis follow steps 23 to 24 of Basic Protocol 1. SUPPORT PROTOCOL 1


CELL CULTURE FOR USE ON THE PORT-A-PATCH, PATCHLINER, AND SyncroPatch 96 The patch clamp technique, conventional or automated, involves puncturing a small hole in the cell membrane and measuring ionic conductance across a live cell membrane. For this reason, a healthy cell population is important for all electrophysiological experiments. This is particularly true for automated patch clamp experiments. When using an automated patch clamp device, the cells, which usually adhere to the bottom of a culture dish, must first be lifted into suspension, requiring that cells be as healthy as possible



before initiating the experiments. Described in this protocol are the main steps for culturing of CHO or HEK cells expressing hNaV 1.7 and HEK or GH4C1 cells expressing nAChα7R for use on the Port-a-Patch, Patchliner, and SyncroPatch 96. This protocol can be used for other cell types as well, including ND7-23 and other cell lines, although some modifications, e.g., choice of enzyme and length of incubation in enzyme, may be required for certain cells/ion channels to obtain the most robust and reproducible results. In particular, when harvested, the cells should be unclustered, since a cell suspension containing cell clusters will hinder the capture and sealing rate. To ensure the cells do not cluster, they should be well dispersed during cell passaging. The cells should also be passaged every 2 to 3 days, so that the membrane does not become too tough, which makes whole-cell access difficult. Confluency should not be allowed to reach 100%, as this affects ion-channel expression.

Materials Ion-channel overexpressing cell lines, e.g.: CHO cells expressing hNaV 1.7 (Anaxon AG) HEK cells expressing hNaV 1.7 (EMD Millipore) GH4C1 cells expressing nAChα7R (cells kindly provided by Thomas Seeger, Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany) HEK cells expressing nAChα7R (inducible cell line; Galantos Pharma) Cell culture medium (see recipe) PBS-EDTA: PBS (PAA Laboratories, GmbH) containing 2 mM EDTA 1× trypsin-EDTA (PAA Laboratories, GmbH) 100-mm culture dishes (PAA Laboratories, GmbH) 75-cm2 (T-75) culture flasks (PAA Laboratories, GmbH) Light microscope 15-ml conical tubes (Falcon-style tubes; PAA Laboratories, GmbH) Tabletop centrifuge Passage cells 1. Split cells every 2 to 3 days into new 100-mm culture dishes or 75-cm2 flasks. Depending on what kind of ion channel the user wants to investigate, one or more of the cell lines listed in the materials list above can be used.

2. Examine the cells under a microscope to determine split ratio (optimal confluency 50% to 80%). Do not allow the cells to reach 100% confluency as this can decrease ion channel expression. Cells are split on Mondays, Wednesdays, and Fridays. The split ratio is determined as follows: For HEK cells: Visualize the cells under the microscope to determine what the split ratio should be (best 70% to 80% confluency): Split ratio (from a 70% to 80% confluent dish) on Mondays and Wednesdays:

1:2: 4 ml cell suspension into new flask/dish 1:3: 3 ml cell suspension into new flask/dish 1:10: 1.2 ml cell suspension into new flask/dish Split ratio (from a 70% 80% confluent dish) on Fridays:



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1:3: 3 ml cell suspension into new flask/dish 1:5: 2 ml cell suspension into new flask/dish 1:10: 1 ml cell suspension into new flask/dish For CHO cells: Visualize the cells under the microscope to determine what the split ratio should be (best not exceeding 60% confluency). Split ratio (from a 60% confluent dish) on Mondays and Wednesdays:

1:3: 3 ml cell suspension into new flask/dish 1:5: 2 ml cell suspension into new flask/dish 1:10: 1 ml cell suspension into new flask/dish 1:20: 0.5 ml cell suspension into new flask/dish Split ratio (from a 60% confluent dish) on Fridays:

1:6: 1.5 ml cell suspension into new flask/dish 1:10: 1 ml cell suspension into new flask/dish 1:20: 0.5 ml cell suspension into new flask/dish 3. Prepare the new flasks/dishes by adding fresh medium. The 75-cm2 flask requires 15 to 20 ml medium and the 100 × 20 mm dish requires 10 ml medium. Prepare flasks and media under sterile technique in a cell culture laminar flow hood to minimize contamination risks. Sterilize glass pipets or use sterile plastic pipets. Spray gloves/hands and other exposed surfaces with 70% ethanol.

4. Aspirate the medium from the dish/flask containing cells and discard waste medium. 5. Add 3 ml PBS-EDTA to rinse away remaining medium and gently rotate the plate. Aspirate away the PBS-EDTA. 6. Repeat step 5. 7. Add 2 ml of 1× trypsin-EDTA, and gently swirl the plate around. Aspirate off approximately 90% of the trypsin solution, leaving a thin film on the surface of the plate. 8. Place plate in incubator at 37°C (95% O2 /5% CO2 ) and pre-warm for approximately 3 min. 9. Remove the plate from the incubator, tap gently and, using the microscope, make certain that all cells are detached. If not, replace the plate in the incubator for an additional 30 sec to 1 min. 10. Once cells are detached, add 5 to 10 ml of medium to the cells, gently pipetting the cells up and down five to ten times. 11. Transfer the cells to a 15-ml conical tube and centrifuge 2 to 3 min at 100 × g, room temperature. The cells will pellet at the bottom of the tube.

12. Discard the supernatant. 13. Add 10 ml of medium to the cell pellet and gently pipet up and down three to five times. Automated Patch Clamp Analysis of Ion Channels


14. Dispense an appropriate volume of cells (0.5 to 4 ml) to the new flasks/dishes. Gently swirl and pipet up and down twice. 15. Place flask/dish into the incubator (37°C; 95% O2 /5% CO2 ). Current Protocols in Pharmacology

16. Harvest cells for electrophysiological experiments 1 to 4 days after plating.

INDUCTION OF HEK nAChα7R CELL LINE The HEK cell line containing nAChα7R must be induced to express the ion channel.

SUPPORT PROTOCOL 2

Materials HEK cells expressing nAChα7R (inducible cell line; Galantos Pharma) Culture medium (see recipe) Inducer Supplemented Media (Galantos Pharma) 1. After 1 to 2 days culturing, remove one culture dish containing HEK cells expressing nAChα7R from the incubator. 2. Exchange the medium for Inducer Supplemented Media (Galantos Pharma). 3. Return to the 37°C incubator (95% O2 /5% CO2 ) for 24 hr. 4. At the end of the 24 hr, remove the cells from the incubator and exchange the medium for normal DMEM/Ham’s F12 medium. 5. Place the cells in the 30°C incubator (95% O2 /5% CO2 ) for at least 24, and up to 48 hr before using them for patch clamp experiments. Placing the culture dish/flask at 30°C for at least 24 hr increases expression of the receptor by an unknown mechanism. As cells do not grow much at this temperature, a 50% to 80% confluent dish should be used for induction.

CELL HARVESTING FOR USE ON THE PORT-A-PATCH, PATCHLINER, AND SyncroPatch 96

SUPPORT PROTOCOL 3

Cells must be harvested into a cell suspension for use with an automated patch clamp device. For best results, the cells should be widely dispersed and unclustered to ensure fast capture at the patch clamp aperture of the glass chip. Cells should be handled carefully to ensure that they are as healthy as possible for use in long-lasting patch clamp experiments. A cell suspension with many cell clusters or cell debris is likely to have a reduced capture and sealing rate. When employing a new cell line, it may be necessary to optimize factors such as choice of lifting agent, e.g., trypsin, Accutase, or PBS-EDTA, length of time in lifting agent, centrifugation, and cold incubation/recovery time after harvesting. Cells should be gently, not vigorously, pipetted up and down, with care taken not to introduce air bubbles when pipetting.

Materials Ion-channel overexpressing cell lines, e.g.: CHO cells expressing hNaV 1.7 (Anaxon AG) HEK cells expressing hNaV 1.7 (EMD Millipore) GH4C1 cells expressing nAChα7R (cells kindly provided by Thomas Seeger, Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany) HEK cells expressing nAChα7R (induced; Galantos Pharma) DMEM/Ham’s F12 medium including glutamine (PAA Laboratories, GmbH) PBS-EDTA: PBS (PAA Laboratories, GmbH) containing 2 mM EDTA 1× trypsin-EDTA (PAA Laboratories, GmbH) Nanion standard external solution for Na+ channels (NSExtSNa; see recipe) Light microscope 15-ml Falcon tube Small centrifuge Current Protocols in Pharmacology



1. Remove a dish/flask from the incubator and check the degree of confluency under the microscope. Depending on what kind of ion channel the user wants to investigate, one or more of the cell lines listed in the materials list above can be used. For best results, harvested cells should be at 50% to 80% confluency.

2. Aspirate and discard the medium from dish/flask. 3. Add 3 ml PBS-EDTA to the dish/flask to rinse away remaining medium. Gently rotate the plate. Aspirate and discard the PBS-EDTA. 4. Repeat step 3. 5. Add 2 ml of 1× trypsin (to the CHO or HEK NaV 1.7 cells) or 2 ml of PBS-EDTA (to the GH4C1 or the induced HEK nAChα7R cells), and gently swirl the solution around the plate. Aspirate off and discard 90% of the trypsin or PBS-EDTA. Trypsin is not used for the GH4C1 or HEK nAChα7R cell lines, as recommended by the cell providers, since trypsin may digest away parts of the ion channel, rendering it non-functional. Because of the fragility of the cells, PBS-EDTA is used as the lifting agent for these cells.

6. Place CHO or HEK NaV 1.7 cells in the incubator at 37°C for approximately 3 min. Leave the GH4C1 or induced HEK nAChαR cells at room temperature for approximately 3 min. 7. At the end of the 3-min time period, gently tap the plate and, using the microscope, ensure that all cells are detached. If some are still adherent, place the cells back into the incubator for 30 sec to 1 min. 8. Add medium/NSExtSNa (50/50 v/v mix) to the CHO or HEK NaV 1.7 cells to obtain a final cell density of approximately 50,000 to 1,000,000 cells per ml. Gently pipet the cells up and down three to five times. These cells are now ready for use. Cell density requirements are higher for the Port-a-Patch as compared to the Patchliner and SyncroPatch 96. Cell density for the Port-a-Patch should be closer to 1,000,000 cells per ml and for the Patchliner and SyncroPatch 96, 50,000 to 500,000 cells per ml.

9. Add 5 to 10 ml medium to the GH4C1 or the induced HEK nAChα7R cells and gently pipet the cells up and down five to ten times. 10. Transfer the GH4C1 or the induced HEK nAChα7R cells to a 15-ml Falcon tube and centrifuge 2 to 3 min at 100 × g, room temperature, to create a pellet. 11. Discard the supernatant. 12. Add NSExtSNa to the cell pellet to yield a final cell density of approximately 50,000 to 1,000,000 cells per ml. Gently pipet the cells up and down three to five times. The GH4C1 or induced HEK nAChα7R cells are now ready for use. BASIC PROTOCOL 2


ELECTROPHYSIOLOGICAL MEASUREMENTS OF NaV 1.7 AT Vhalf INACTIVATION ON THE PATCHLINER The Patchliner is an automated planar patch clamp device for recording up to eight cells simultaneously (Fig. 11.13.1C). Measurements of the potency of compounds on hNaV 1.7 at Vhalf , the potential at which half of the channels are inactivated, may be critical, as many compounds display a higher affinity for the inactivated as compared to the activated



state of the channel (Hille, 1977; Bean et al., 1983; Castle et al., 2009; Kaczorowski et al., 2011). Using pre-programmed complex analysis protocols, the Patchliner calculates the Vhalf value for each cell using a different holding potential for each cell when measuring hNaV 1.7 at the Vhalf value. Although values for Vhalf will be similar across all cells, there are slight variations from cell to cell. Variations of just ±5 mV in this region can have a significant influence on channel inactivation and current amplitude, where some of the cells are already in the inactivated state while others still show the maximum current. For this reason, it is best to calculate Vhalf individually for each cell rather than use an average value. Detailed in this protocol are the steps involved in conducting an experiment on the Patchliner to measure the activation and inactivation properties of hNaV 1.7, the potency of TTX using Vhalf as the holding potential, and the potency of tetracaine at Vhalf and −120 mV as the holding potential to compare IC50 values.

Materials NSIntSNa (see recipe) NSExtSNa (see recipe) SES (see recipe) TTX (Tocris) 1 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM prepared in NSExtSNa (see recipe), with the highest concentration being the stock solution. Store aliquots at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the various TTX concentrations by serial dilution. Tetracaine (Sigma) 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, 1 mM made up in NSExtSNa. The 10 mM tetracaine stock solution is prepared in NSExtSNa (see recipe). Store aliquots at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the needed tetracaine concentrations by serial dilution CHO cells expressing hNaV 1.7 (Anaxon AG) or HEK cells expressing hNaV 1.7 (EMD Millipore) Patchliner recording station (Nanion Technologies) Two EPC10 Quadro amplifiers (HEKA Elektronik) Computer running the following software: PatchControlHT software (Nanion Technologies) PatchMaster software (HEKA Elektronik) Igor software (Wavemetrics) R

NPC -16 chips, resistance 2-3.5 MΩ (Nanion Technologies) General experimental set-up 1. Turn on computer, amplifiers, and Patchliner. Allow the amplifiers to warm up for 10 min prior to use. Make certain there is ample distilled water in the wash bottle and that the waste bottle is empty. Also make certain the external waste removal system is attached and that the small bottle for the waste is empty.

2. Double click the PatchControlHT icon on the computer desktop to open PatchControlHT and PatchMaster. 3. Load the pre-programmed tree NaV17 Vhalf_1303.xml in PatchControlHT. Select the Edit mode to browse the experiment steps and to make any modifications. The tree is the experimental procedure used by PatchControlHT to run the experiment. It contains all the commands required to fill the chip with solutions, capture and seal the cells, and complete the experiment. A full list of commands is available in the Edit mode of PatchControlHT. These can be added to the current tree using a ‘drag-and-drop’ principle. The steps of chip fill and cell capture/sealing change little between experiments. The folder containing commands specific for the current experiment are found in the red



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compound storage area

cell hotel

chip wagon NPC-16 Chip

measure head

Figure 11.13.7 The Patchliner platform seen from above. The positions of the measure-head, cell hotel, chip wagon, NPC -16 chips, and compound plates are indicated. R

‘experiment’ folder. Here, the PatchMaster pgf or protocol is defined, as is the compound addition routine. The compound positions are defined in the Joblist. The compound names and concentrations are defined in the Compoundlist.

4. Prepare compounds and solutions and place them in the appropriate rack. Shown in Figure 11.13.7 is an annotated picture of the Patchliner showing different positions. Compounds must be placed in the rack defined in the Joblist. The robot runs through the Joblist using the solutions in the order they are defined.

5. Place three chips on the chip wagon (resistance 2 to 3.5 MΩ). 6. Harvest cells and place the cell suspension in the cell hotel. Place a blue plastic pipet tip onto the appropriate nozzle in the cell hotel. The cells are aspirated every 30 sec to maintain viability, prevent settling at the bottom of the cell hotel, and prevent clustering. The parameters of the cell hotel can be set individually. Volume, speed, and frequency are defined based on the particular cell type. Typically a total volume of 1 ml of cell suspension is added to the cell hotel. The default values for the settings for the cell hotel are: volume: 500 μl; speed: 100 μl/sec; frequency: once every 30 sec. These default values are appropriate for most cell types. If cells have a tendency to cluster, frequency and speed may be increased. If the volume of the cell suspension in the cell hotel becomes low (100 MΩ indicating a blocked hole, these will be turned off and not serviced further. Atmospheric pressure is applied for the remainder of the experiment, and further solution additions do not occur at these channels. This happens very rarely.

14. The pipet collects cells from the cell hotel for the first channel. Suction is applied to the first channel as the pipet adds the cells. Once a cell is captured, suction is reduced. The pipet repeats this procedure for all other channels, with cells collected individually for each channel of the chip. 15. Holding potential is reduced to −40 mV. 16. SES is added to all eight channels automatically by the pipet. 17. Holding potential is reduced to −80 mV. 18. Suction is applied to the cells until the whole-cell configuration is attained. 19. Atmospheric pressure is applied to each cell, with CSlow and RSeries compensated automatically. 20. The external solution is exchanged for NSExtSNa. A quality control function may be built into the tree to automatically turn off channels with a low seal resistance. These cells will not be serviced further by the pipet. Alternatively, the user can manually switch the channels on or off.

Perform activation and inactivation plot experiments 21. The command ‘start NaIV’ is run in PatchControlHT (as part of the tree). PatchMaster runs the pgf NaIV (same parameters as those used for the Port-a-Patch; see step



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20 in Basic Protocol 1), the voltage steps required to create a current-voltage plot. The onl file associated with the pgf automatically generates the IV in PatchMaster. The pgf in PatchMaster is defined for the first channel (same voltage protocol as for the Port-a-Patch shown in step 20 of Basic Protocol 1). In PatchControlHT, the function ‘Modify pgf – record all channels’ copies the voltage protocol for all other channels. The same is true for the online file. The first channel is defined in PatchMaster, and then the function ‘Adjust online file’ in PatchControlHT copies the online analysis to all other channels. During the recording, the function ‘Get all online results’ exports all online results (peak amplitude versus voltage in this case) into Excel. For the results to be correctly exported, certain parameters in the function ‘Get all online results’ must be properly set. Among these are the number of online results and time between results.

22. When the NaIV is complete, PatchControlHT automatically starts the PatchMaster protocol NaIV-inactivation. The NaIV-Inact voltage protocol is the same as that used for the Port-a-Patch in Basic Protocol 1, step 21. Online results are exported as detailed in step 21 of this protocol, above.

23. PatchControlHT uses a specialized analysis function to calculate the Vhalf for each cell individually. These Vhalf values are then used as the holding potentials for each cell.

Study pharmacology of hNaV 1.7 using TTX 24. Once the IV plots have been completed, PatchControlHT automatically initiates the pharmacology protocol. The voltage protocol ‘RecNaPharm’ is activated. In this case, the holding potential is the Vhalf value calculated from the previous step. The voltage is then stepped to 0 mV for 20 msec (this step is used for analysis), then to −120 mV for 2 sec (see Critical Parameters), and finally back to Vhalf . The sweep interval is 10 sec. The online analysis ‘NaPharm’ runs simultaneously during the recording, the peak amplitude is plotted against time and the peak amplitude is automatically exported to an Excel spreadsheet in the correct format to load into Igor for analysis. The pipet collects the solution from the position indicated in the Joblist. The volume and speed of addition are also defined in the Joblist. The compound name and concentration, which were defined previously in the Compoundlist, are also exported into the Excel spreadsheet along with the peak amplitude and sweep number. A label may be automatically placed in the PatchMaster file in the relevant sweep. The function ‘Label Sweep: Compound;concentration’ is used in the tree.

25. The pipet of the Patchliner adds the concentrations of TTX defined in the Joblist. The solutions are added in increasing concentrations, with the pipet washed between each addition. 26. After all the concentrations of TTX have been added, an optional wash step with the addition of NSExtSNa can be integrated into the tree. In this case, the wash step is performed now. If a wash step has not been integrated but the user wishes for it to be performed, the PatchControlHT software can be stopped and the function ‘Add External Solution’ dragged from the list of commands into the tree. Highlight this function and press ‘play’ to initiate the wash step.


27. The experiment is now complete if the robot was programmed to perform only a single experiment. The chip wagon will move out of the recording chamber, and the pipet will be washed at the wash station.



The robot can be programmed to continually run the same experiment with a different compound each time. In this case, the robot will always move to the next position of the chip, then the next chip, until all chips are used. With each round it uses a different compound. The robot will stop automatically if the compounds are all screened before the chips are used up. Alternatively, the ‘multiples’ option can be selected, wherein a minimum number of cells per compound is set in the Joblist. If the minimum number of cells per compound is not recorded in the experiment, the next position of the chip will be filled and the experiment started again. This will repeat until the minimum number of cells has been exposed to each compound. The robot will then stop and wait for further instructions from the user.

Compare pharmacology of tetracaine at Vhalf versus −120 mV 28. Once the IV plots are complete, PatchControlHT will automatically start the pharmacology protocol. The voltage protocol ‘RecNaPharm’ or ‘NaPharm’ is initiated, as defined in the tree. Depending on which voltage protocol is started, the holding potential is either −120 mV or the Vhalf value calculated from the previous step. The voltage is then stepped to 0 mV for 20 msec, which is used for analysis, then to −120 mV for 2 sec (see Critical Parameters), and finally back to −120 mV or Vhalf . The sweep interval is 10 sec. The online analysis ‘NaPharm’ is run simultaneously during the recording, with the peak amplitude plotted against time and the peak amplitude automatically exported to an Excel spreadsheet in the format needed for loading into Igor for analysis. 29. The pipet of the Patchliner adds increasing concentrations of tetracaine as defined in the Joblist. The pipet is washed between each addition. 30. An optional wash step involving the addition of NSExtSNa can be integrated in the tree to occur after all concentrations of teteracaine are added. If so, this wash step is performed now. 31. The experiment is complete. The chip wagon will move out of the recording chamber and the pipet will be washed at the wash station.

Analyze data 32. Open Igor. Go to the Patchliner tab and choose ‘Load Experiment’–‘Load single experiment’. Browse the computer folders to locate the relevant Excel file. By default the file is saved in a folder of the day’s date in the format YYMMDD_00x_x.xls. Each experiment that is performed on a single day contained within the same experiment in PatchMaster will have the same date and a sequential experiment number i.e., YYMMDD_001_1, YYMMDD_001_2 . . . etc. If a new experiment is created in PatchMaster, the data will be stored as YYMMDD_002_1, YYMMDD_002_2 etc. Results from the entire day can be loaded by selecting ‘All experiments in folder’ rather than just ‘single experiment’.

33. Choose the relevant analysis type from the drop-down menus. In this case, the analysis should be set as blocker (Fig. 11.13.8). 34. Data will be loaded into Igor. After pressing Display in the data browser window, the data will be displayed showing all successful concentration response curves for each cell for that experiment. Press Raw to display the raw data traces and Time to display the time courses. If upon visual inspection any cells look faulty, i.e., peak amplitude is very small (less than about 30 pA) or cannot be distinguished from noise, the peak amplitude is positive (under the experimental conditions the peak amplitude should be negative), or a spike in the data has been used as the peak amplitude by mistake, unclick the channel to remove these cells from the analysis. Electrophysiological Techniques


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Figure 11.13.8 Screenshot for setting analysis type in Igor when loading data for TTX or tetracaine block of hNaV 1.7 on the Patchliner. As TTX and tetracaine block the current response, the analysis type chosen is ‘Blocker’. When multiple online analysis values are saved into the Excel spreadsheet, the average current amplitude over several points can be calculated and used for the analysis. In order to do this, ‘statistics over max last 3 points’ is chosen in the second drop-down menu. A leak subtraction protocol was used during collection of the data and, therefore, the zero offset is subtracted automatically from the data by selecting ‘Do zero subtract’. The peak amplitude recorded will always be negative under the experimental conditions and thus, polarity of online results is selected as ‘Negative’.

35. Press ‘Average result’ in the Data Browser window to display the averaged concentration-response curves for all active channels. The average IC50 value ±S.E.M taken from the individual IC50 values is shown, along with the IC50 value of the average plot. The results from several compounds can be loaded into Igor. Each compound is sorted by name, and an average concentration-response curve is generated for each agent. The results from several days can also be loaded into Igor. As long as the compound name is spelled correctly, the results will be averaged according to compound.

36. Click ‘show summary’ in the Data Browser window to generate a table of results showing average IC50 values for each of the compounds loaded into Igor. The data can be saved as an Igor Experiment. ALTERNATE PROTOCOL 2

ACTIVATION OF nAChα7R USING THE PATCHLINER Because the nAChα7R exhibits fast desensitization kinetics, a rapid solution exchange and low compound exposure time are needed to reliably record the channel. Solution exchange times as low as 10 msec can be achieved on the Patchliner, and a ligand exposure time of less than 200 msec can be achieved by using small sample volumes and rapid application. Described in this protocol is the procedure for a control experiment for repeated applications of nicotine to nAChα7R using the Patchliner. This assay can be used for investigating allosteric modulators.

Additional Materials (see also Basic Protocol 2)


HEK cells expressing nAChα7R (Induced; Galantos Pharma) NSExtSnAChα7R (see recipe) 100 μM nicotine (Sigma) prepared in NSExtSnAChα7R. The stock 10 mM nicotine solution is prepared in DMSO. Store aliquots of the stock solution at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the required nicotine concentrations in NSExtSnAChα7R.



General experimental set-up 1. Load the pre-programmed tree Ligand_DoubleStacked_1212.xml. Select the Edit mode to browse the experiment steps and to make any modifications, such as changing the parameters of the stacked solutions function (see below). 2. Follow steps 4 to 9 of Basic Protocol 2.

Perform cell capture and sealing procedure 3. Follow steps 10 to 15 of Basic Protocol 2. 4. NSExtSnAChα7R is pipetted automatically into all eight channels. 5. Follow steps 17 to 19 of Basic Protocol 2. 6. External solution does not need to be exchanged as the solution used in step 4 is the solution used for the recordings.

Activation of nAChα7R by nicotine 7. The ‘stacked solutions’ function is employed. In the stacked solutions function, the volume of ligand, volume of wash solution, and speed of application are defined. Within the stacked solutions function, the solutions are aspirated into the pipet (first wash solution then ligand), the recording is initiated (continuous holding potential −80 mV), the solutions pipetted into the chip (first ligand followed by wash), and the online analysis results exported to Excel (peak amplitude). This is performed for each cell individually for the first application of nicotine. The parameters for the stacked solutions function are: VolWash: 280 (μl); VolCompound: 30 (μl); VolFast: 120 (μl); SpeedFast: 12; SpeedSlow: 22; OnlineResult: 3; ChannelOffset: 4. The positions of the wash solution and compound are defined in the Joblist. Two speeds are entered. The SpeedFast is 171 μl/sec (in the software this is denoted 12). This is the speed at which the first 120 μl solution (VolFast) is pipetted. The remaining solution (wash buffer) is added with a slower speed of 21 μl/sec (number 22 in the software).

8. The pipet adds the 100 μM nicotine eight times sequentially until all solutions have been added to all cells. In this experiment, the control involves adding nicotine eight times to ensure that the nAChα7R can be reproducibly activated before adding an allosteric modulator. In an alternative experiment, after three control additions of nicotine, a concentration-response curve to an allosteric modulator is generated.

9. Complete step 27 of Basic Protocol 2.

Analyze data 10. Follow step 32 of Basic Protocol 2. 11. Choose the relevant analysis type from the drop-down menu. In this case, the analysis should be set as activator or potentiator. The rest of the parameters can be set as shown in Figure 11.13.8. Because in this experiment a control was performed, no concentration-response curve is generated. An alternative experiment using an activator, or an allosteric modulator after the control applications, would generate a concentration-response curve to the activator or modulator. In this case, activator or potentiator can be chosen from the drop-down menu, respectively. When ‘statistics over last 3 points’ is chosen, the mean of the last three results of each concentration is calculated and used for analysis. If only one online result is taken, this will be used for the online analysis. Alternatively, ‘no statistics – take last point’ can be chosen. Electrophysiological Techniques


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12. Follow steps 34 to 36 of Basic Protocol 2. If an activator or allosteric modulator was used in the experiment the IC50 , which is actually the EC50 value, is shown. ALTERNATE PROTOCOL 3

TEMPERATURE EFFECTS ON PHARMACOLOGY OF nAChα7R USING THE PATCHLINER The allosteric modulation of nAChα7R by PNU120596 is influenced by temperature (Sitzia et al., 2011). This property could have profound effects on how nAChα7R allosteric modulators are investigated. Described in this protocol is the use of the temperature control feature of the Patchliner to record and compare the allosteric modulatory effects of PNU120596 on nAChα7R at different temperatures.

Additional Materials (see also Basic Protocol 2) GH4C1 cells expressing nAChα7R (cells kindly provided by Thomas Seeger, Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany) NSExtSnAChα7R (see recipe) 10 μM PNU120596 (Tocris) prepared in NSExtSnAChα7R (see recipe). The stock 10 mM solution of PNU120596 is prepared in DMSO. Store aliquots at –20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the required PNU120596 concentrations in NSExtSnAChα7R. 100 μM nicotine (Sigma) + 10 μM PNU120596 (Tocris) prepared in NSExtSnAChα7R (see recipe) Temperature-control add-on for the Patchliner General experimental set-up 1. Load the pre-programmed tree Ligand_DoubleStacked_Temperature.xml. Select the Edit mode to browse the experiment steps and to make any modifications. 2. Follow steps 4 to 9 of Basic Protocol 2.

Perform cell capture and sealing procedure 3. Follow steps 10 to 15 of Basic Protocol 2. 4. NSExtSnAChα7R is added automatically by pipet to all eight channels. 5. Follow steps 17 to 19 of Basic Protocol 2. The external solution does not need to be exchanged, as the solution used in step 4 is the solution used for the recordings.

Allosteric modulation of nAChα7R by PNU120596 at different temperatures 6. A triple stack function is employed. The pipet first aspirates wash solution, then PNU120596 + nicotine (pre-mixed), and finally PNU120596. As described in step 7 of Alternate Protocol 2, the pipet first aspirates wash solution, followed by PNU120596 + nicotine, and then PNU120596. At this stage, the pipet heats to the desired temperature, 20°C (room temperature), for the first step, the solutions are added to the chip (first PNU120596 followed by PNU120596 + nicotine and then wash solution) while the recording is initiated, and the online analysis is exported into an Excel file.


The parameters for the triple stack are: VolWash: 125 (μl); VolCompound: 30 (μl); VolFast: 70 (μl); SpeedFast: 12; VolTip: 20 (μl); SpeedSlow: 20; OnlineResult: 3; ChannelOffset: 4. The positions of the wash solution and compound are defined in the Joblist. Two speeds are entered. The FastSpeed 12 (171 μl/sec) is the speed at which the first 70 μl solution (VolFast) is pipetted, with the remaining solution (wash buffer) added



at a slower speed of 20 (24 μl/sec). VolTip is the extra volume aspirated by the pipet to account for any loss of solution during heating.

7. The set temperature of the pipet is increased by 5°C and step 6 is repeated. 8. Step 7 is repeated until the set temperature is 40°C. 9. Perform step 27 of Basic Protocol 2.

Analyze data 10. Perform step 32 of Basic Protocol 2. 11. Perform step 33 of Basic Protocol 2. In this experiment, the temperature is used as the concentration in the Excel file.

12. Perform step 34 of Basic Protocol 2 to display the raw traces and time courses.

HIGH-THROUGHPUT ELECTROPHYSIOLOGICAL ANALYSIS OF NaV 1.7 ON THE SyncroPatch 96

BASIC PROTOCOL 3

The SyncroPatch 96 (Fig. 11.13.1D) is a high-throughput device offering GΩ seals at a throughput of 96 cells recorded in parallel. The recording software, PatchControl 96, is flexible and user-friendly. Like the PatchControlHT software for the Patchliner, the experiments are designed in a ‘tree’ format. Detailed in this protocol are data acquisition and analysis for hNaV 1.7 using the SyncroPatch 96. The acquisition and analysis of activation and inactivation current-voltage plots are described.

Materials Nanion standard internal solution for Na+ channels (SyncroPatch; NSIntSNaSyncro; see recipe) NSExtSNa (see recipe) Nanion standard external solution for Na+ channels (SyncroPatch; NSExtSNaSyncro; see recipe) SES (see recipe) CHO cells expressing hNaV 1.7 (Anaxon AG) or HEK cells expressing hNaV 1.7 (EMD Millipore) SyncroPatch 96 recording station (Nanion Technologies) Computer running the following software: PatchControl 96 software (Nanion Technologies) DataControl 96 software (Nanion Technologies) R

NPC -96 chips, resistance 2-3.5 MΩ (Nanion Technologies) General experimental set-up 1. Turn on the computer and the SyncroPatch 96 by pressing the On button. 2. Double click the PatchControl 96 icon on the desktop of the computer and wait until all three software windows (Settings, Acquisition and Experiment Design Window) open. The amplifier is integrated in the SyncroPatch 96 and is activated when the device is switched on. At this stage, make certain that there is distilled water in the wash bottle and that the waste bottle is empty.

3. Load the tree NaV17_Activation_Inactivation.xml. 4. Highlight the INIT_Hardware folder in the tree by clicking on it, and press ‘play’.



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At this point the pipetting arm, chip tray, measure head, and amplifier of the SyncroPatch 96 are initialized.

5. Place a used chip in the chip tray and all tubes for internal solution intake (on the left hand side of the SyncroPatch 96) into a bottle of distilled water. Highlight the function ‘System Flush – Startup/Shutdown’ in the tree and press ‘play’. This function will fully flush the tubing of the SyncroPatch 96, after which the chip tray is moved back out. The software will stop and wait for further instructions from the user because of a stop function.

6. Make any changes to the tree, adding functions by drag and drop. 7. Open the Compound Dialog (Setup > Compound) and edit the compound positions. In the Compound Dialog, the positions and compositions of all solutions (including internal and external recording solutions) and cell type are defined and saved with the recorded data.

8. Prepare solutions and place them at the appropriate positions. In this case, the NSIntSNaSyncro should be placed in the internal solution rack (position 1); the NSExtSNa, NSExtSNaSyncro, and SES are placed in external positions A to L, according to the positions defined in the Compound Dialog.

9. Place tube 1 for the internal solution intake into the bottle containing the NSIntSNaSyncro. 10. Prepare cells, equip the cell hotel with blue pipet tips, and place the cells in the cell hotel. 11. Start the cell hotel. As a safety precaution, the cell hotel stops automatically whenever the arm of the cell hotel is lifted. When the arm of the cell hotel is replaced into the cell hotel, the cell hotel must be restarted manually. Also, whenever the arm of the cell hotel is lifted, movement of all moving parts of the robot, such as the pipetting arm and chip tray, is prohibited. Remember to lower the cell hotel arm to continue the experiment.

12. Place a new chip in the chip tray. 13. Choose how many rows of the chip should be activated. The user can choose to record the whole chip or a double row at a time. In this case, three double rows, i.e., half a chip, 48 wells in total, are used. When the robot has finished the first half of the chip, it automatically fills the second half of the chip and completes the experiment. Performing the experiments this way results in a greater success rate for completed experiments.

14. Highlight the INIT_Settings folder and press ‘play’ to begin the experiment. The software will run through each of the tree commands in turn. Below is a list of the steps performed. Although the robot will automatically run through the steps in turn, it is possible to stop the robot at any time during the experiment to change functions in the tree. Highlight the desired restart step and then press ‘play’ to resume the experiment.

Perform cell capture and sealing procedure 15. The chip is first filled with NSIntSNaSyncro. This occurs independently of the pipet, as the internal solution is filled via tubing lines underneath the chip. Automated Patch Clamp Analysis of Ion Channels

16. The pipet collects NSExtSNa for all 48 wells that are to be recorded and fills the chip 16 wells at a time (via 16 pipets).



NSExtSNa is used to fill the chip and for cell capture. If NSExtSNaSyncro is used for filling the chip, cell capture does not occur and the experiment is unsuccessful (reason unknown).

17. In the acquisition window of PatchControl 96, the 96 wells show the square current pulse and the corresponding resistance of the aperture. The wells are color coded; the thresholds are set using the slider bar. At the beginning of an experiment, thresholds are set by default: light blue indicates resistance 6 MΩ. 18. Holding potential is reduced to −20 mV. 19. The pipet then collects the cell suspension for the first 16 channels. The cells are added and suction is applied simultaneously to attract a cell to each aperture (default value = −150 mbar). The pipet collects the cell suspension for the next 16 channels and suction is applied. This is repeated until cells have been added to all wells used in this measurement. The pressure is reset to −50 mbar at the end of the cell catch. Color-coding of the wells is not changed for this step. When a cell is caught, the well background turns green.

20. Holding potential is reduced to −40 mV. 21. SES is added by the pipet as described in step 16. The pipet simultaneously adds the SES and removes the waste via a separate channel in the pipet. 22. Voltage is reduced to −80 mV. 23. Suction is increased for a few seconds to −200 mbar to achieve whole cell configuration. Color-coding of wells indicates resistances: green >100 MΩ; blue 50 to 100 MΩ, light blue 500 MΩ; blue 100 to 500 MΩ, light blue 500 MΩ; gray wells indicate wells that are turned off.


derived from the IV data after normalization for the driving force, followed by a fit to the Boltzmann equation using a macro in Igor. The Vhalf of activation was −11 mV. Raw traces using an inactivation voltage protocol from an exemplar cell are shown with the corresponding inactivation plot from n = 5 cells (Fig. 11.13.9C,D). The Vhalf of inactivation was −73 mV, which is in good agreement with values reported in the literature (Klugbauer et al., 1995; Sangameswaran et al., 1997; Vijayaragavan et al., 2001; Cummins et al., 2004). Displayed in Figure 11.13.10 are typical activation and inactivation curves of hNaV 1.7 recorded on the Patchliner. Shown on panel A are raw traces from an exemplar cell in response to an activation voltage protocol, with the display on panel B being the corresponding IV curve from an average of n = 8 cells. The Boltzmann equation-derived Vhalf of activation calculated in Igor was –19 mV,

which is in good agreement with literature values (Klugbauer et al., 1995; Sangameswaran et al., 1997; Vijayaragavan et al., 2001; Cummins et al., 2004). Raw traces from an example cell in response to an inactivation voltage protocol are shown in Figure 11.13.10C, with the corresponding inactivation plot for an average of n = 8 cells displayed in panel D. The Vhalf of inactivation was −74 mV, which is in good agreement with values reported in the literature (Klugbauer et al., 1995; Sangameswaran et al., 1997; Vijayaragavan et al., 2001; Cummins et al., 2004). Displayed in Figures 11.13.11 and 11.13.12 are screenshots of the SyncroPatch 96 software (PatchControl 96) during a recording of hNaV 1.7. Shown in Figure 11.13.11 are the raw current traces of the 96 cells, and in Figure 11.13.12 the online analysis of the activation curves. Displayed in Figure 11.13.13 are the average IV and inactivation



Figure 11.13.12 Screenshot of PatchControl 96 showing the online analysis during a recording of hNaV 1.7. Shown are the corresponding IV plots for the cells shown in Figure 11.13.11. The user can easily toggle between raw traces and online analysis during the course of the experiment.

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Figure 11.13.13 (A) Current-voltage plot for an average of 82 CHO cells expressing hNaV 1.7 recorded on the SyncroPatch 96. Vhalf of activation was −5 mV. (B) Inactivation plot for an average of 82 cells recorded on the SyncroPatch 96. Vhalf of inactivation was −50 mV.


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Figure 11.13.14 (A) Raw current traces of hNaV 1.7 and subsequent block by TTX recorded on the Port-aPatch. Shown are raw current traces from an exemplar CHO cell expressing hNaV 1.7, elicited using a voltage step protocol from a holding potential of −120 mV to 0 mV for 20 msec, and increasing block by increasing concentrations of TTX. (B) Concentration-response curve for block of hNaV 1.7 by TTX for an average of 5 cells. IC50 = 43 ± 7 nM (n = 5). (C) Raw current traces of hNaV 1.7 and subsequent block by TTX recorded on the Patchliner. Shown are raw current traces from an exemplar CHO cell expressing hNaV 1.7, elicited using a voltage step protocol from a holding potential of the Vhalf to 0 mV for 20 msec, and increasing block by increasing concentrations of TTX. (D) Concentration-response curve for block of hNaV 1.7 by TTX for an average of 6 cells. IC50 = 20 ± 9 nM (n = 6).

curves for an average of n = 82 cells on the SyncroPatch 96. The Vhalf of activation was −5 mV and inactivation −50 mV. This is slightly more depolarized than values seen on the Port-a-Patch and Patchliner, which is most likely due to the different solutions used for the measurements.


TTX block of hNaV 1.7 Displayed in Figure 11.13.14A are the raw traces of hNaV 1.7 in a single voltage step protocol and increasing inhibition of peak current by increasing concentrations of TTX recorded on the Port-a-Patch. The TTX acts quickly, with the effect evident within a few sweeps, and steady state attained in less than 2 min. The average concentration-response curve for TTX was fitted in Igor using a

Hill equation (Fig. 11.13.14B). The IC50 value for TTX blockade of hNaV 1.7 was 43 ± 7 nM (n = 5), which is in good agreement with literature reports (Klugbauer et al., 1995). Shown on Figure 11.13.14C are the raw traces of hNaV 1.7 in a single voltage step protocol and increasing inhibition of peak current by increasing concentrations of TTX recorded on the Patchliner. In this case, the TTX concentration-response curve was generated using a voltage protocol where half of the receptors are in the inactivated state, i.e., the cells were held at the Vhalf , which was calculated for each cell individually. The average concentration-response curve for TTX was fitted automatically in Igor using a Hill equation (Fig. 11.13.14D). The IC50 value



for TTX blockade of hNaV 1.7 (at Vhalf ) was 20 ± 9 nM (n = 6), which is similar to what is reported in the literature (Klugbauer et al., 1995). The IC50 value was little affected by holding potential, although holding potential is known to affect the potency of other compounds, such as tetracaine (see below). Effect of holding potential on tetracaine IC50 of hNaV 1.7 The hNaV 1.7 was recorded on the Patchliner using two different holding potentials in order to compare the IC50 values in the closed state (holding potential −120 mV) and in the inactivated state (holding potential = Vhalf ) of the receptor. To measure the potency of tetracaine on the inactivated receptor, the Vhalf of inactivation, i.e., the potential required to inactivate 50% of the receptors, was calculated using an inactivation voltage protocol. In PatchMaster, the Vhalf value was then calculated for each individual cell and then entered into the pgf for the subsequent pharmacology analysis. Displayed in Figure 11.13.15 are the raw traces from an exemplar cell using a closed state (panel A) or an inactivated state (panel C) protocol. The time courses for the closed and inactivated state protocols are shown on panels B and D, respectively. On panel E are the concentration-response curves for tetracaine using a holding potential of –120 mV (blue triangles) and Vhalf (black circles) overlaid. There is an approximately 10-fold shift in the potency of tetracaine, with the IC50 value at −120 mV = 11 ± 2 μM (n = 6), while it is 1.2 ± 0.1 μM (n = 7) at Vhalf . nAChα7R activation using nicotine or acetylcholine Displayed in Figure 11.13.16A is the response of an exemplar HEK cell expressing nAChα7R in the presence of 70 μM nicotine using the Fast Perfusion System for the Port-a-Patch. Shown in the inset is an expansion of the activation portion of the trace. Nicotine was perfused for 100 msec to minimize desensitization of the channel. Shown in Figure 11.13.16B are multiple responses of this cell to four applications of nicotine. A small amount of current decrease occurs between the first and fourth responses. Displayed in Figure 11.13.16C are raw traces of a HEK cell induced to express nAChα7R and currents elicited by application of 100 μM nicotine on the Patchliner using the stacked solutions approach. Wash buffer (280 μl) was first aspirated into the Patchliner pipet, followed by 30 μl nicotine. The solution was then added to the cell at a speed of 171 μl/sec,

resulting in an exposure time of 175 msec. Nicotine was removed quickly by the wash buffer. With this approach several applications of nicotine could be made on each individual cell with highly reproducible results. In this experiment, eight repeat applications of nicotine were made on a single cell. Allosteric modulators can be tested in this way after performing three control applications. Shown in Figure 11.13.17 is a screenshot of the SyncroPatch 96 software during a recording of nAChα7R expressed in HEK cells (inducible cell line). Displayed are current traces of 96 cells in response to 100 μM ACh. The experiments were performed as described above for the Patchliner, with the solutions stacked inside the pipet of the SyncroPatch 96 and applied rapidly to the cells to minimize ligand exposure time. Exposure time for the SyncroPatch 96 was 350 msec. The cell-to-cell variation seen in the responses to ACh is due mostly to differences in cell size and expression levels. If a modulator, such as PNU120596, is used in combination with ACh, the degree of allosteric modulation will be similar across the cells even if the actual peak amplitude varies (data not shown). Effect of temperature on allosteric modulation of nAChα7R Temperature plays a critical role in the allosteric modulation of nAChα7R (Sitzia et al., 2011). The temperature control function of the Patchliner makes it possible to record allosteric responses at different temperatures (above room temperature). Displayed in Figure 11.13.18 is the effect of the allosteric modulator, PNU120596 (in combination with 100 μM nicotine), on nAChα7R expressed in GH4C1 cells at 20°, 35°, and 40°C (Sitzia et al., 2011). The control was co-application of PNU120596 with nicotine at room temperature (20°C). The experiment was conducted on the same cell at 35°C and 40°C. The current elicited by PNU120596 when it was coapplied with nicotine at 35°C and 40°C was considerably less than that elicited at 20°C (room temperature).

Time Considerations A single cell line requires approximately 10 min for cell passaging. Following passaging, allow 1 to 3 days for cells to recover and grow before using them for experiments. Cell harvesting for Port-a-Patch, Patchliner, and SyncroPatch 96 experiments takes approximately 10 min, whereas preparation of standard recording solutions takes about 30 min



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Figure 11.13.15 Effect of holding potential on tetracaine block of hNaV 1.7. (A) Raw current traces from an exemplar CHO cell expressing hNaV 1.7 recorded on the Patchliner, elicited using a voltage step protocol from a holding potential of −120 mV to 0 mV for 20 msec, and increasing block with increasing concentrations of tetracaine. (B) Time course of the experiment showing application of tetracaine at the indicated concentrations. Each point represents peak amplitude at the given time point. (C) Raw current traces from an exemplar cell recorded on the Patchliner, elicited using a voltage step protocol from a holding potential of Vhalf to 0 mV for 20 msec, and increasing block by increasing concentrations of tetracaine. (D) Time course of the experiment showing application of tetracaine at the indicated concentrations. Each point represents peak amplitude at the given time point. (E) Concentration-response curve for tetracaine at a holding potential of −120 mV and Vhalf are shown overlaid for an average of 6 and 7 cells, respectively. Using a more depolarized potential as the holding potential (Vhalf ) causes an almost 10-fold decrease in the IC50 . IC50 at −120 mV = 11 ± 2 μM (n = 6), IC50 at Vhalf = 1.2 ± 0.1 μM (n = 7).



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Figure 11.13.16 (A) Raw trace of an exemplar cell showing nAChα7R-mediated response elicited by perfusing 70 μM nicotine briefly (100 msec) using the Fast Perfusion System for the Port-a-Patch. Holding potential remained constant at –80 mV. Inset shows activation of nAChα7R, which occurred in 50 MΩ, blue wells indicate Rmemb of 5 to 50 MΩ and gray wells indicate wells which are turned off.


per solution. As large quantities can be stored in the refrigerator or freezer, solutions do not need to be prepared fresh daily. Preparation of test compound solutions takes between 10 min and 1 hr, depending on the number of compounds to be tested. One Port-a-Patch experiment on NaV 1.7, including a TTX concentration-response curve, takes about 20 min. Approximately 2 to 3 min of this is cell capture and sealing, with 18 to 20 min needed for the actual experiment. Because compounds such as TTX act quickly, a long wait time between additions is not necessary. One experiment on the Port-a-Patch using nAChα7R and the Fast Perfusion Kit takes approximately 30 min. A long wash time after nicotine addition is needed for the channels to recover from desensitization. Data analysis for one experiment on the Porta-Patch takes approximately 1 hr.

A single experiment on the Patchliner to study NaV 1.7, including a TTX or tetracaine concentration-response curve, requires approximately 30 min. Of this, 10 min is needed for the cell capture and sealing procedure and 20 min for the actual experiment. As with the Port-a-Patch, long wait times between additions are not necessary because the compounds act so quickly. Data analysis for each experiment requires 15 to 30 min. For nAChα7Rs, a concentration-response curve to ACh takes approximately 30 min to 1 hr, depending on the number of cells recorded. Of this, 10 min are needed for cell capture and sealing, and 30 to 50 min to conduct the experiment. As the pipet collects solutions separately for each channel, the number of channels activated affects the time needed to conduct the experiment. Data analysis takes approximately 30 min for each experiment.



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Figure 11.13.18 Effect of temperature on allosteric modulation of nAChα7R expressed in GH4C1 cells recorded on the Patchliner. (A) Shown are raw traces of an exemplar cell showing currents elicited by co-application of PNU120596 and nicotine at 20°C, 35°C, and 40°C. Solutions were stacked inside the pipet of the Patchliner and heated to the indicated temperature before they were rapidly applied to the cell. Cells were held at a constant holding potential of −80 mV. The current response elicited at 20°C was much larger than that elicited at the higher temperatures. (B) Time course of the experiment where peak amplitude is plotted against temperature. As temperature is increased, peak amplitude of the current elicited by co-application of PNU120596 and nicotine is decreased.

For temperature experiments on the Patchliner, one experiment requires approximately 30 to 50 min, and data analysis an additional 15 to 30 min. One experiment on the SyncroPatch 96 for NaV 1.7 (activation and inactivation curves), or nAChα7R (activation by ACh), takes approximately 30 min. Of this, 15-min is cell capture and sealing with an additional 15 min needed to complete the experiment. Data analysis takes approximately 10 min for each experiment.

Acknowledgements We thank Thomas Seeger of the Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany, for GH4C1 cells expressing nAChα7R, Galantos Pharma for the inducible HEK-nAChα7R cell line, EMD Millipore for HEK cells expressing NaV 1.7, and Anaxon AG for CHO cells expressing NaV 1.7.

Literature Cited Balansa, W., Islam, R., Fontaine, F., Piggott, A.M., Zhang, H., Webb, T.I., Gilbert, D.F., Lynch, J.W., and Capon, R.J. 2010. Ircinialactams: Subunit-selective glycine receptor modulators

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Studying mechanosensitive ion channels with an automated patch clamp.

Patch clamp studies of microbial ion channels.

Electrophysiological Studies of Voltage-Gated Sodium Channels Using QPatch HT, an Automated Patch-Clamp System.

Ion channels. Ten years of patch-clamp studies.

Patch clamp techniques to study ion channels from organelles.

The development of automated patch clamp assays for canonical transient receptor potential channels TRPC3, 6, and 7.

Electrophysiological analysis of mammalian cells expressing hERG using automated 384-well-patch-clamp.

Dissociation and culture of mechanosensory neurons for patch clamp analysis.

The patch clamp technique.

Nonstationary noise analysis and application to patch clamp recordings.

Automated and manual patch clamp data of human induced pluripotent stem cell-derived dopaminergic neurons.

Neuropeptide modulation of single calcium and potassium channels detected with a new patch clamp configuration.

Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording.

Ion Channels in Native Chloroplast Membranes: Challenges and Potential for Direct Patch-Clamp Studies.

Constructing a patch clamp setup.

Early identification of hERG liability in drug discovery programs by automated patch clamp.

Investigation of miscellaneous hERG inhibition in large diverse compound collection using automated patch-clamp assay.

Development of Automated Patch Clamp Technique to Investigate CFTR Chloride Channel Function.

Patch-Clamp Study of Hepatitis C p7 Channels Reveals Genotype-Specific Sensitivity to Inhibitors.

Patch clamp techniques: an overview.

Development of Automated Patch Clamp Assay for Evaluation of α7 Nicotinic Acetylcholine Receptor Agonists in Automated QPatch-16.

Priming and testing silicon patch-clamp neurochips.

Closed-Loop Real-Time Imaging Enables Fully Automated Cell-Targeted Patch-Clamp Neural Recording In Vivo.

The extracellular patch clamp: a method for resolving currents through individual open channels in biological membranes.