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

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

UNIT 11.14

Yi Liu1 1

Neuroscience Discovery, Janssen Research & Development, LLC, San Diego, California

ABSTRACT Voltage-gated sodium (Nav ) channels are highly sensitive to membrane potential and have fast gating kinetics. Patch clamp electrophysiology has long been the gold standard for studying these channels. Combining high throughput with high information content/ accuracy, automated patch clamp technologies have emerged as critical tools in ion channel drug discovery. Described in this unit is the use of QPatch, one of the automated patch clamp systems, to study Nav channel function and pharmacology. Curr. Protoc. C 2014 by John Wiley & Sons, Inc. Pharmacol. 65:11.14.1-11.14.45. Keywords: voltage-gated sodium channel r Nav channel r Nav 1.7 r QPatch r patch clamp r HTS

INTRODUCTION Voltage-gated sodium (Nav ) channels are essential for the initiation and propagation of action potentials. They are highly sensitive to the membrane potential and display fast gating kinetics. Given their outstanding voltage control and high temporal resolution, conventional (a.k.a. manual) patch clamp electrophysiology has for decades been the technique of choice for studying their function and pharmacology. The emergence of automated patch clamp technologies have made it possible to increase throughput in studying these channels, a welcome development for ion-channel drug-discovery programs. One automated patch clamp system, the QPatch HT (Sophion Biosciences) is capable of performing 48 independent whole-cell recordings simultaneously. Along with its laminar flow fluidics and the ability to make gigaohm seals and perform unattended operations, QPatch HT offers medium throughput and high information content/accuracy, making it well suited for studying Nav channels (Mathes et al., 2009). Described in this unit is the use of QPatch HT in whole-cell patch-clamp studies of the functional properties and effects of pharmacological agents on Nav channels. Examination of Nav 1.7, a Nav channel subtype, is described in order to exemplify the use of this technique, which can be applied to study other Nav subtypes as well. The essential elements of the QPatch system are described under Strategic Planning, after which the Basic Protocol for whole-cell recordings is detailed. Detailed in Support Protocols 1, 2, and 4 are the whole-cell, voltage and application protocols, respectively, used in the QPatch experiments (jobs) detailed in this unit. Provided in Support Protocol 3 are compound plate lists/maps and formats. Provided in Support Protocol 5 are directions for creating QPatch jobs using the individual components detailed in Support Protocols 1 to 4. Described in Support Protocol 6 is a cell preparation procedure suitable for QPatch experiments, with QPatch data analysis discussed in Support Protocol 7. The Commentary is devoted to a discussion of Critical Parameters, Anticipated Results, and Time Considerations. Although this unit is designed to enable the reader to conduct QPatch Electrophysiological Techniques Current Protocols in Pharmacology 11.14.1-11.14.45, June 2014 Published online June 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/0471141755.ph1114s65 C 2014 John Wiley & Sons, Inc. Copyright

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experiments, it is not a substitute for the user manual provided with this equipment. The user manual must be consulted as well, as it gives additional critical information on the proper operation of the instrument.

STRATEGIC PLANNING The aim of this section is to familiarize the reader with the essential features of QPatch. In subsequent sections, specific protocols are provided to illustrate how the QPatch system is used to study Nav channels. The QPatch system consists of assay software, a workstation, and a screening station.

Assay Software The QPatch assay software serves two functions: (1) to create a job, i.e., to set up a job to be run on the screening station by combining individual components of instructions (e.g., voltage and application protocols, etc.), which are themselves created using the assay software, and (2) to process/analyze the resultant data and/or to export them to third-party software for analysis (see Support Protocol 7). Initiation of a QPatch job involves the assembly of four components, namely Assay, Compound list, Solutions and Cells, and Experiments. The assay component contains three separate protocols, each of which is composed of a set of related parameters. 1. Whole-cell protocol. This protocol governs a series of events that occur before initiating the experiment, from cell positioning (guiding cells to recording sites), to gigaseal formation, to the establishment of the whole-cell configuration. 2. Application protocol. Parameters established by this protocol control compound application procedures, such as the order, volume, frequency, and number of compound applications. 3. Voltage protocol. This is a waveform (versus time) of the voltage applied at predetermined intervals. It is usually executed shortly after liquid application.

These three protocols are discussed in detail in Support Protocols 1, 2, and 4, respectively. A compound list (See Support Protocol 3) displays the agents contained in a 96-well microtiter plate (MTP), indicating the location of each, as well as other information (e.g., name, concentration, etc.). Once the assay and compound list are generated, a job can be assembled from these two components, along with Solutions/Cells and Experiments, which are not discussed in detail.

Workstation The workstation is a server computer that hosts a database for data storage and on which the assay software is installed and run.

QPatch HT, an Automated Patch-Clamp System

Screening Station The QPatch screening station (supplied by Sophion, http://www.sophion.com/; Fig. 11.14.1A) houses the essential components for executing jobs. These include the electronics (amplifiers/digitizers), QPlates, containers for solutions/drugs and for cell storage and preparation, pressure system, robotic liquid handling system, and other hardware elements. It has a separate, built-in computer that executes jobs and communicates with the workstation (e.g., data transfer). It has a touch-screen user interface. Shown in Figure 11.14.1B is a close-up view of most of the major components with which the user interacts while executing QPatch jobs. Once all the consumables (e.g., cells, saline


Current Protocols in Pharmacology

A

B wash station

pipets saline reservoir

robotic arm

measurement position grabber arm barcode reader cell storage tank and stirrer

used QPlates new QPlates new compound plates

centrifuge

used compound plates

active compound plate manifold

Figure 11.14.1 QPatch HT, an automated patch clamp system. (A) The screening station. (B) Close-up view of the screening station work platform. Hidden from view underneath the work platform are the electronics and pressure system, as well as, in a drawer that can be pulled out from the left-hand side, a water tank (used for pipet washing) and a waste container (connected to the wash station). A separate, stand-alone vacuum pump (not shown) is used for maintaining negative pressures during cell positioning and formation of the whole-cell configuration, etc.

reservoir, QPlates, MTPs, etc.) are in place and the Start button on the touch screen is pressed, a job is completed in a fully automated fashion, as summarized below. 1. Cells are transferred manually or by pipets connected to the robotic arm on the left from the cell storage tank (“hotel”) to the centrifuge, where they are washed using a built-in, multi-step spinning/resuspension protocol. Following this washing, the cells are ready to be applied to the QPlate. 2. A 96-well MTP containing test compounds is moved by the grabber connected to the robotic arm on the right from the “New compound plates” location to the “Active compound plate” location. There are three slots for the “Active compound plate” for use when multiple MTPs are involved in a job. The plate is scanned in transit by the barcode reader for identification purposes. 3. A QPlate is transferred by the grabber from the “New QPlates” location to the ”Measurement position” location. It too is scanned in transit by the barcode reader for serial number identification. The QPlate is loaded with intracellular and extracellular solutions from the saline reservoir via the pipets, and is primed (meaning that a QPlate is loaded with extra- and intracellular solutions and pressure is applied to push the solutions through their respective channels and through the recording site where the two solutions meet to complete the electric circuit). 4. At the measurement position, initial quality control (e.g., “open electrode” resistance measurement) is conducted on all 48 QPlate measurement sites followed by pipet delivery of cells in suspension from the cell reservoir in the centrifuge to those sites that have passed QC. Cells are then positioned to the measurement sites, followed by the formation of gigaseal and subsequent whole-cell configuration using a pressure controller. 5. Whole-cell currents evoked by predefined voltages are recorded. Solutions from the saline reservoir and/or the active compound plate are applied to each cell during recording as defined by the application protocol. Pipets are washed at the “Wash station” after each liquid application. Current Protocols in Pharmacology

Electrophysiological Techniques


6. At the end of the assay procedure, the QPlate is moved to the “Used plates” slot and a new QPlate selected for priming, for loading with new cells from the centrifuge that are freshly transferred from the hotel, and for recording using the same assay protocol. Once all of the compounds in the active compound plate are tested, the old MTP is moved to the “Used compound plate” position and a new MTP transferred to the “Active compound plate” position. This process continues until either the job is completed (i.e., all compounds in all the MTPs are tested) or the screening station needs fresh supplies, such as QPlates, cells or saline solutions. 7. The recorded data are transferred to the workstation for storage and analysis. BASIC PROTOCOL

PATCH CLAMP STUDIES OF NAV 1.7 SODIUM CHANNELS USING QPATCH HT Provided in this protocol is the step-by-step procedure for conducting patch clamp experiments on hNav 1.7 using the QPatch system. Unlike manual patch clamping, with the automated QPatch virtually all of the manual tasks are executed before and after the actual recording. Four steps are detailed in subsequent support protocols. These are: (1) creating components of a job (step 1), which involves preparing whole-cell, application, and voltage protocols (Support Protocols 1, 2, and 4, respectively) and generating compound list(s) (Support Protocol 3); (2) creating a job, which involves assembling the above components into a job (step 2; Support Protocol 5); (3) preparing cells for QPatch recording (step 9; Support Protocol 6); and (4), data analysis (Support Protocol 7). Four separate jobs are described in this Basic Protocol, ranging from functional/biophysical characterization (jobs #1 and #2) to pharmacological studies of the channel (jobs #3 and #4). While the basic workflow for all these jobs, and indeed for all QPatch jobs in general, is the same, some steps and materials vary as a function of the study objective. To minimize confusion, the Basic Protocol is divided into four separate stand-alone subsections, each of which is associated with only one of the four jobs. Each job may be performed independently, without necessarily following the order of presentation. When possible, identical materials and steps are cross-referenced to minimize repetition.

Materials


For job #1: Whole-cell protocol #1 (Support Protocol 1) Voltage protocols #1, #2, #3, and #5 (Support Protocol 2) Compound list #1 (Support Protocol 3) Application protocol #1 (Support Protocol 4) Screening station (see Strategic Planning) Workstation (see Strategic Planning) Vacuum pump (Sophion, cat. no. SB3020) Extracellular solution (see recipe) Intracellular solution (see recipe) 0.5 ml glass inserts (Sophion, cat. no. SB2052) 96-well MTP holder, with barcode (00000) attached to the left side of the holder (Sophion, cat. no. 2053) QPlate (stored at 4°C) (Sophion, cat. no. 2040) 150-cm2 (T-150) flask of HEK293 cells stably expressing hNav 1.7 Cell storage tank (Sophion, cat. no. 2050) Additional reagents and equipment for harvesting cells (Support Protocol 6) Stir bar (Sophion, cat. no. 3070) Plastic tubes for on-station centrifuge (Sophion, cat. no. 2051) For job #2:



Whole cell protocol #1 (Support Protocol 1) Voltage protocol #4 ( Support Protocol 2) Compound list #1 (Support Protocol 3) Application protocol #2 (Support Protocol 4) Additional materials as for job #1 (see above) For job #3: Whole-cell protocol #1 (Support Protocol 1) Voltage protocol #6 (Support Protocol 2) Compound list #2 (Support Protocol 3) Application protocol #3 (Support Protocol 4) 1 M lidocaine stock in 100% DMSO (Sigma, cat. no. L7757; store at room temperature) Frozen (−20°C) aliquot (10 μl) of 1 mM TTX stock (Alomone Labs, cat. no. T-550) in vendor-provided/recommended buffer (store in 10-μl aliquots at −20°C) Additional materials as for job #1 (see above) For job #4: Whole-cell protocol #2 (Support Protocol 1) Voltage protocol #7 (Support Protocol 2) Compound list #3 (Support Protocol 3) Application protocol #4 (Support Protocol 4) Frozen (−20°C) aliquot (10 μl) of 100 μM synthetic huwentoxin-IV stock (Alomone Labs, cat. no. STH-100) in deionized water containing 0.1% BSA (store at −20°C) Frozen (−20°C) aliquot (10 μl) of 1 mM TTX stock (Alomone Labs, cat. no. T-550) in vendor-provided/recommended buffer (store in 10-μl aliquots at −20°C) Additional materials as for job #1 (see above) Additional reagents and equipment for assembling jobs from individual components (Support Protocol 5) Job #1: Characterization of (1) the voltage dependence of activation and inactivation and (2) recovery from fast inactivation 1a. Create whole-cell protocol #1 (in Support Protocol 1), voltage protocols #1, #2, #3 and #5 (in Support Protocol 2), compound list #1 (in Support Protocol 3), and application protocol #1 (in Support Protocol 4) using the assay software. 2a. Create job #1 in Support Protocol 5 using the assay software and forward to the screening station. It is preferable to send the job to the screening station well in advance so that you can focus on the procedures that must be performed on the day of experiment.

3a. Take one QPlate, intracellular solution (20 ml), and extracellular (30 ml) solutions from the refrigerator. Allow sufficient time for the QPlate (30 min) and solutions to warm to room temperature before initiating the experiment. Do not warm more QPlates than are needed for that day, as it is best to keep them stored at 5°C in the protective bag for as long as possible.

4a. Activate the workstation. The assay software need not be running for a job to be executed on the workstation.

5a. Power up the screening station and vacuum pump.


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The pump should be actively pumping for a few seconds initially, but only briefly on occasion after that. If it is overly active, there is likely a leak in the system that must be repaired before resuming the experiment.

6a. Open the drawer on the left-hand side of the screening station. Add deionized water to the water tank to 70% capacity and then place the tank on the block with the cap facing down. 7a. Make certain the waste container in the same drawer is empty. Tighten the cap and close the drawer. 8a. Log on to the screening station using the touch screen. 9a. Press the Status button, followed by the Flush Pipettes button, and then OK. Use the default setting (20 ml). Pressing this button also raises the elevators to the platform level.

10a. Harvest a 150-cm2 flask of HEK293 cells stably expressing hNav 1.7. See Support Protocol 6 for details.

11a. Load the cell suspension into the cell storage tank. 12a. When pipet flushing is complete (as it should be by now), press the Back button on the touch screen to return to the previous window. 13a. Press the Unlock Cabinet button to unlock the cabinet door. Open the cabinet door. Pressing this button also lowers the elevators to the ground level.

14a. Put a stir bar in the cell storage tank and place the tank on the stirring unit of the screening station. Allow the cell suspension to recover on the stirring unit for 15 min before initiating the experiment.

15a. Add 30 ml extracellular solution and 20 ml intracellular solution to the respective compartments in the saline reservoir (right slot for extracellular solution and middle slot for intracellular solution). The amount of intra- and extracellular solutions needed for a particular job depends on multiple factors, including the success rate and job size. Monitor the consumption rate of these solutions while the job is ongoing. Add more of these solutions as needed.

16a. Place the loaded saline reservoir in the saline reservoir slot (labeled AUX1). 17a. Load MTP with solutions according to compound list #1 (Support Protocol 3). In general, if it takes longer than 15 min to accomplish this, consider moving this step to before cell harvest (step 10a).

18a. Open the drawer on the right-hand side of the screening station and place the MTP in the New compound plates slot. Ensure that the barcode on the MTP matches that of the compound list (see step 9a in compound list #1, Support Protocol 3), or the job will not begin.

19a. Place the QPlate in the New QPlates slot in the same drawer. Close the drawer. 20a. On the touch screen, select the job with the name: “Nav1.7 biophysics_1_CP” (i.e., job #1, Support Protocol 5). QPatch HT, an Automated Patch-Clamp System

21a. Press Next to bring up the next screen. 22a. Under QPlate Section, check the boxes next to 1 and 2.



This instructs the QPatch to use only one third (i.e., the first two columns or 16 measurement sites) of a full 48-site QPlate.

23a. Under Suspend, check the Suspend Job after each QPlate box. Then press Next to bring up the next screen. 24a. Under Automatic Cell Wash, check the No QStirrer radio button. This informs the QPatch that cells will be manually added to the tube in the centrifuge. In this job, and in job #2, cells are manually loaded to the tube in the centrifuge, as this is best for small or more customized jobs, such as those described here.

25a. Under QFuge Parameters, select the following numbers for lines 1 to 4: 350 (Final volume in QFuge in μl); 1 (Number of washes); 150 (Centrifugation time in sec); and 150 (Centrifugation speed in G). Press Next to bring up the next screen. 26a. Insert a new plastic test tube into the centrifuge and manually transfer 1 ml of the cell suspension from the cell storage tank to the tube in the centrifuge. Cover the centrifuge unit with the protective cylinder. 27a. Close the cabinet door. It locks automatically upon closing.

28a. Carefully read the checklist (the four lines of reminders in red) on the touch screen and verify that all tasks are completed. 29a. Press the Start Job button. 30a. After the job is complete, flush the pipets again as in step 9a. 31a. Press the Back button to return to the previous screen. 32a. Press the Logout button on the touch screen. 33a. Press the Shutdown button. 34a. Power down the screening station. 35a. Open the right-hand drawer to (i) retrieve the partially used QPlate from the Used QPlates slot for a later job and (ii) to remove the used MTP. Close the drawer. 36a. Open the left-hand drawer to (i) empty the water tank and (ii) empty and rinse the waste container. Close the drawer. 37a. Analyze the QPatch data.

Job #2: Characterization of recovery kinetics from total inactivation 1b. Create whole-cell protocol #1 (Support Protocol 1), voltage protocol #4 (Support Protocol 2), compound list #1 (Support Protocol 3), and application protocol #2 (Support Protocol 4) using the assay software. 2b. Create job #2 in Support Protocol 5 using the assay software and forward to the screening station. 3b. Perform steps 3a to 19a. 4b. On the touch screen, select the job with the name: “Nav1.7 biophysics_2_CP” (i.e., job #2 in Support Protocol 5). 5b. Press Next to bring up the next screen. 6b. Under QPlate Section, check the boxes next to 1 and 2.



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As with job #1, only one third of a QPlate is needed for this job. If the boxes next to 1 and 2 are checked, the system assumes you are using a new QPlate. Alternatively, you could “reuse” the partially used QPlate from job #1. If so, then check the boxes next to 3 and 4, rather than 1 and 2.

7b. Perform steps 23a to 37a.

Job #3: Screening for Nav 1.7 small molecule blockers 1c. Create whole-cell protocol #1 (Support Protocol 1), voltage protocol #6 (Support Protocol 2), compound list #2 (Support Protocol 3), and application protocol #3 (Support Protocol 4) using the assay software. 2c. Create job #3 in Support Protocol 5 using the assay software and forward to the screening station. 3c. Take three QPlates, and intracellular (20 ml), and extracellular (50 ml) solutions from the refrigerator. Remove an aliquot (10 μl) of 1 mM TTX stock from the freezer. 4c. Perform steps 4a to 14a. 5c. Add 50 ml extracellular solution, 20 ml intracellular solution, and 10 ml of extracellular solution containing 1 μM TTX to the respective compartments in the saline reservoir. Use the left slot for TTX.

6c. Place the loaded saline reservoir in the saline reservoir slot (labeled AUX1). 7c. Load MTP with compound solutions according to compound list #2 (Support Protocol 3). 8c. Open the drawer on the right-hand side of the screening station and place the QPlates in the New QPlates slot. 9c. Place the MTP in the New compound plates slot in the same drawer. Close the drawer. 10c. On the touch screen, select: “Nav1.7 small molecule blockers_CP” (i.e., job #3 in Support Protocol 5). 11c. Press Next to bring up the next screen. 12c. Under QPlate Section, check all boxes next to 1 to 6. This instructs the QPatch to use the entire (all 6 columns) of the QPlate.

13c. Under Suspend, leave the Suspend Job after each QPlate box unchecked. Then press Next to bring up the next screen. This directs the QPatch to automatically load a new QPlate (i.e., without suspending the job) if the job is not completed after the previous QPlate has been used. This is unless the number of cells yet to be tested is fewer than that specified in “Min. experiments to continue.”

14c. Under Automatic Cell Wash, check the User defined radio button. This directs the QPatch to automatically transfer cells from the cell storage tank to the centrifuge tube. QPatch HT, an Automated Patch-Clamp System

15c. Under QStirrer, select 10 for Cell volume in QStirrer in ml. The number selected should roughly correspond to the actual volume of the cell suspension (in ml) in the cell storage tank. Do not select a number that is larger.



16c. Under QFuge parameters, select the following numbers for lines 1 to 4: 350 (Final volume in QFuge in μl); 1 (Number of washes); 150 (Centrifugation time in sec); and 150 (Centrifugation speed in G). Press Next to bring up the next screen. 17c. Insert a new plastic test tube into the centrifuge. Then cover the centrifuge unit with the protective cylinder. Transfer of cells from the cell storage tank to the tube in the centrifuge will be done automatically.

18c. Perform steps 27a to 34a. 19c. Open the right drawer to (i) retrieve any unused and partially used QPlates from the New QPlates slot, (ii) remove the used QPlate(s) from the Used QPlates slot, and (iii) remove the used MTP from the Used compound plates slot. Close the drawer. 20c. Open the left drawer to (i) empty the water tank and (ii) empty and rinse the waste container. Close the drawer. 21c. Analyze the QPatch data.

Job #4: Screening for Nav 1.7 peptide blockers 1d. Create whole-cell protocol #2 (Support Protocol 1), voltage protocol #7 (Support Protocol 2), compound list #3 (Support Protocol 3), and application protocol #4 (Support Protocol 4) using the assay software. 2d. Create job #4 (Support Protocol 5) using the assay software and forward it to the screening station. 3d. Remove from the refrigerator three QPlates, and pre-made intracellular (20 ml) and extracellular (50 ml) solutions, as well as one aliquot (10 μl) of 100 μM synthetic huwentoxin-IV stock and one aliquot (10 μl) of 1 mM TTX stock. 4d. Perform steps 4c to 6c. 5d. Load MTP with compound solutions in accord with compound list #3 (Support Protocol 3). 6d. Perform steps 8c to 9c. 7d. Select on the touch screen the job titled “Nav1.7 peptide blockers_CP” (i.e., job #4, Support Protocol 5). 8d. Perform steps 11c to 21c.

PREPARING WHOLE-CELL PROTOCOLS A whole-cell protocol in QPatch defines a number of parameters in a set of related processes that range from chip validation (ensuring that the “open electrode” resistance is in the proper range), to cell positioning (guiding cells to find recording sites), to gigaseal and whole-cell formation. An optimal whole-cell protocol should result in relatively high success rates, which are defined as a high percentage of cells finding a recording site, obtaining good seals (particularly gigaseals), and achieving the whole-cell configuration. Ultimately, the protocol should positively affect the number of cells that complete an experiment, although this end point may be difficult to evaluate inasmuch as many factors other than those being optimized influence the fate of the cell. Included among these other factors are assay duration, voltage and application protocols, and the nature of the applied compounds. Nonetheless, it should be possible to achieve reasonable

SUPPORT PROTOCOL 1



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optimization using percent completed cells as readout by maintaining these factors. This procedure is particularly useful for screening assays because, in this case, most of the factors, except for test agents, do not change from job to job. Optimal parameter values in a whole-cell protocol may vary not only with cell type (e.g., CHO, HEK293, etc.), but also with the type of channel expressed. Thus, creating a good whole-cell protocol for a particular, stable cell line is, in many ways, empirical. Each QPatch system comes with a few vendor-established whole-cell protocols that are, at least to a certain extent, optimized for standard cell lines such as CHO and HEK293. Further optimization for a specific cell type expressing a particular ion channel can usually be achieved by slight modifications of the “built-in” protocols. Generally speaking, the success rate tends to be higher for CHO cells than for HEK293 cells. This (support) protocol does not require empirical testing, with only optimized values provided for HEK293 cells stably expressing hNav 1.7. A quick way to prepare a new whole-cell protocol is to begin with an existing one. Two slightly different whole-cell protocols are detailed in the steps below, with the only difference being the holding potential. One is for jobs #1 to 3 (whole-cell protocol #1) and the other for job #4 (whole-cell protocol #2). Refer to the user manual for a description of the parameters shown in the figures for this and other support protocols.

Whole-cell protocol #1 1a. Click on the Assay tab and then on Whole-cell Protocols. 2a. Select any protocol from the existing list shown in the right panel to use as a template. 3a. Double click on the selected protocol and click Copy at the bottom of the window to prepare a duplicate. 4a. Click on Advanced at the bottom of the duplicated protocol. 5a. Rename the protocol “Nav1.7_HEK_1_CP”. 6a. For each parameter, input the value (or value range) as displayed in Figure 11.14.2A. The values displayed under the five tabs (Chip validation, Cell positioning, etc.) on Figure 11.14.2A are for Chip validation. If the selection is not on Chip validation, click on Chip validation and enter the values as shown on Figure 11.14.2A.

7a. Click on Cell positioning. Input values as indicated for the parameters in Figure 11.14.2B. 8a. Click on Gigaseal method. Input values as shown in Figure 11.14.2C. 9a. Click on Whole-cell method. Input values as shown in Figure 11.14.2D. 10a. Click on Rseries suction. Leave the Enable Rseries suction box unchecked (Fig. 11.14.2E). 11a. Click Apply to save.

Whole-cell protocol #2 1b. Copy “Nav1.7_HEK_1_CP” (i.e., whole-cell protocol #1 above). 2b. Rename the copy “Nav1.7_HEK_2_CP.” QPatch HT, an Automated Patch-Clamp System

3b. Change the holding potential under Whole-cell requirements to –75 mV. 4b. Click Apply to save.



A

B

C

D

E

Figure 11.14.2

Illustrations of the optimized parameters for whole-cell protocol: Nav1.7_HEK_1_CP.

MAKING VOLTAGE PROTOCOLS A voltage protocol in QPatch defines a number of parameters in a set of related procedures that includes the voltage waveform, holding potential (in the General tab that also specifies several other parameters), filtering/sampling rates (Filtering tab), leak subtraction options (Leak protocol tab), and series resistance compensation (Rseries tab). The voltage waveform specifies the time dependence of the applied membrane potential during whole-cell recording. For voltage-gated channels, it is usually defined by several segments. For ligand-activated channels, it usually consists of a single (time-independent) holding potential. Each segment consists of a start time, duration, a start voltage, an end voltage, and, sometimes, a start and end voltage and/or duration step. The complete voltage protocol is applied to the cell for each addition of a control or test agent solution, as specified in the application protocol (Support Protocol 4).

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As with whole-cell protocols, a new voltage protocol is created by copying an existing voltage protocol. It can also be created from scratch using default values provided in the assay software, as described below. 1a. Click on the Assay tab and then Voltage Protocols. 2a. Click on New at the lower left corner in the right panel. A default protocol appears in the right panel.

3a. Select the Voltage gated radio button under Gating type. 4a. Name the protocol at the top. If desired, briefly describe the protocol in the Description box.

5a. Edit each of the five tabs named General, Filtering, Leak protocol, Online cursor, and Rseries. 6a. Compose the voltage waveform using the buttons near the lower-right corner of the voltage protocol window (Add, Edit, etc.). 7a. Click Apply to save the new protocol. Detailed instructions are provided for steps 4a to 7a above for each of the eight voltage protocols detailed below. These voltage protocols are used when the four jobs in Support Protocol 5 are assembled.

Voltage protocol #1: Voltage dependence of activation and fast inactivation This protocol is employed to examine the voltage dependence of activation (e.g., the peak I-V/G-V relationship) and fast steady-state inactivation. These aspects of the channel gating are combined in one voltage protocol because they can be performed together. 1b. Name the protocol “Nav1.7_act & fast SSI_CP” (top section of Fig. 11.14.3E). 2b. Enter Vh = −120 mV; leak subtraction; 100 msec depolarizations in the Description box (top section of Fig. 11.14.3E). 3b. Click on the Rseries tab (circled in red in Fig. 11.14.3A), enter the values, and select the radio button, as shown in Figure 11.14.3A. Ignore the grayed-out box for Feedback cut-off frequency. 4b. Click the Online cursor tab and enter the values as shown in Figure 11.14.3B. 5b. Click the Leak protocol tab and enter the values as shown in Figure 11.14.3C. 6b. Click the Filtering tab and enter the values as shown in Figure 11.14.3D. 7b. Click the General tab and enter the values and select the boxes as shown in Figure 11.14.3E (lower section). 8b. Click the Add button near the lower-right corner of the voltage protocol window, enter the values, and select the check box/radio buttons as indicated in Figure 11.14.3F. After clicking OK, a line will appear to the left of the Add button showing the selections of the parameter values performed in this step (see Fig. 11.14.3I for the completed voltage protocol).


9b. Click on Add again, enter the values, and select the check box/radio buttons as indicated in Figure 11.14.3G. Click OK. A second line will appear to the left of the Add button, reflecting the actions taken in this step.



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B F

C

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G

H D

Figure 11.14.3

Illustrations of the parameters for voltage protocol #1: Nav1.7_act and fast SSI_CP.

10b. Click on Add once again, enter the values, and select the check box/radio buttons as indicated in Figure 11.14.3H. Click OK. A third line will appear, reflecting the actions taken in this step. 11b. Click Apply to save the protocol. The full right panel should now look like Figure 11.14.3I. The square waveforms in the middle section of the display are a graphic representation of the voltage protocol. The three lines below the waveforms are a numerical representation of the same voltage protocol.

Voltage protocol #2: Rate of recovery from fast inactivation This protocol is used to examine the rate of recovery, at −120 mV, from fast inactivation induced by a 10 msec depolarization to 0 mV. 1c. Name the protocol “Nav1.7_rec_fast inactivation_CP.” 2c. Enter 10 msec prepulse to 0 mV; rec @ −120 mV in the Description box.



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Figure 11.14.4

Illustrations of the parameters for voltage protocol #2: Nav1.7_rec_fast inactivation_CP.

3c. Click the Rseries tab and enter the same values as shown in Figure 11.14.3A. 4c. Click the Online cursor button and enter the same values as shown in Figure 11.14.3B. 5c. Click the Leak protocol button and uncheck the Enable leak subtraction protocol box. 6c. Click the Filtering button and enter the same values as shown in Figure 11.14.3D. 7c. Click the General tab, enter the values, and select the boxes as shown on Figure 11.14.4A. 8c. Click on Add near the lower-right corner of the voltage protocol window and enter the values and select the check box/radio buttons that produce the first line in Figure 11.14.4B. Click OK. This line automatically appears after the values are entered as in step 8b (voltage protocol #1).

9c. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.4B. Click OK. 10c. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.4B. Click OK. 11c. Click Add and enter the values and select the check box/radio buttons that produce the 4th line in Figure 11.14.4B. Click OK. 12c. Click Add and enter the values and select the check box/radio buttons that produce the 5th line in Figure 11.14.4B. Click OK. 13c. Click Apply to save the protocol.


Voltage protocol #3: Voltage dependence of slow and total inactivation This protocol is used to examine the voltage dependence of slow and total inactivation induced by long, 20-sec depolarizations. For total inactivation, non-inactivated currents are accessed immediately after each 20-sec depolarization. For slow inactivation, a



A

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Figure 11.14.5

Illustrations of the parameters for voltage protocol #3: Nav1.7_slow and total SSI_CP.

300-msec gap at −120 mV is introduced after each 20-sec depolarization to allow for recovery from fast inactivation before significant recovery from slow inactivation occurs. 1d. Name the protocol “Nav1.7_slow and total SSI_CP.” 2d. Enter in the Description box Vh = −120 mV, 20 sec depolarizations, 0 or 300 msec gap at −120 mV. 3d. Click the Rseries tab and enter the same values as in Figure 11.14.3A. 4d. Click the Online cursor button and enter the same values as in Figure 11.14.3B. 5d. Click the Leak protocol button and uncheck the Enable leak subtraction protocol box. 6d. Click the Filtering button and enter the same values as in Figure 11.14.3D. 7d. Click the General tab and enter the values and select the boxes as in Figure 11.14.5A. 8d. Click Add near the lower-right corner of the voltage protocol window and enter the values and select the check box/radio buttons that produce the first line in Figure 11.14.5B. Click OK. 9d. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.5B. Click OK. 10d. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.5B. Click OK. 11d. Click Add and enter the values and select the check box/radio buttons that produce the 4th line in Figure 11.14.5B. Click OK. 12d. Click Add and enter the values and select the check box/radio buttons that produce the 5th line in Figure 11.14.5B. Click OK. 13d. Click Add and enter the values and select the check box/radio buttons that produce the 6th line in Figure 11.14.5B. Click OK.



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Figure 11.14.6

Illustrations of the parameters for voltage protocol #4: Nav1.7_rec_total inactivation_CP.

14d. Click Add and enter the values and select the check box/radio buttons that produce the 7th line in Figure 11.14.5B. Click OK. 15d. Click Apply to save the protocol.

Voltage protocol #4: Rate of recovery from total inactivation This protocol is employed for examining the rate of recovery, at −120 mV, from total inactivation induced by a 10-sec depolarization to 0 mV. 1e. Name the protocol “Nav1.7_rec_total inactivation_CP.” 2e. Enter in the Description box 10 sec prepulse @ 0 mV; rec @ −120 mV. 3e. Click the Rseries tab and enter the same values as in Figure 11.14.3A. 4e. Click the Online cursor button and enter the same values as in Figure 11.14.3B. 5e. Click the Leak protocol button and uncheck the Enable leak subtraction protocol box. 6e. Click the Filtering button and enter the same values as in Figure 11.14.3D. 7e. Click the General tab and enter the values and select the boxes as shown in Figure 11.14.6A. 8e. Click Add near the lower-right corner of the voltage protocol window and enter the values and select the check box/radio buttons that produce the first line in Figure 11.14.6B. Click OK. 9e. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.6B. Click OK. QPatch HT, an Automated Patch-Clamp System

10e. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.6B. Click OK. 11e. Click Add and enter the values and select the check box/radio buttons that produce the 4th line in Figure 11.14.6B. Click OK.



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Figure 11.14.7

Illustrations of the parameters for voltage protocol #5: Nav1.7_steady pulsing_CP.

12e. Click Add and enter the values and select the check box/radio buttons that produce the 5th line in Figure 11.14.6B. Click OK. 13e. Click Add and enter the values and select the check box/radio buttons that produce the 6th line in Figure 11.14.6B. Click OK. 14e. Click Apply to save the protocol.

Voltage protocol #5: Tonic stimulation (steady pulsing) protocol This protocol is used to measure the time dependence of the amplitude of Nav 1.7 currents induced by a brief, 5-msec depolarization to 0 mV once every 20 sec. 1f. Name the protocol “Nav1.7_steady pulsing_CP.” 2f. Enter in the Description box Vh = −120 mV; leak subtraction; depolarize to 0 mV for 5 msec once every 20 sec. 3f. Use the same values in the Rseries, Online cursor, Leak protocol, and Filtering tabs as in Figure 11.14.3A-D, respectively. 4f. Click the General tab and enter the values and select the boxes as shown in Figure 11.14.7A. 5f. Click Add near the lower-right corner of the voltage protocol window, enter the values, and select the check box/radio buttons that produce the first line in Figure 11.14.7B. Click OK. 6f. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.7B. Click OK. 7f. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.7B. Click OK. 8f. Click Apply to save the protocol.

Voltage protocol #6: Screening protocol for small molecule Nav 1.7 blockers This is an “all-in-one” screening protocol used to simultaneously determine the effects of compounds, particularly small molecules, in multiple states of Nav 1.7.



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Figure 11.14.8

Illustrations of the parameters for voltage protocol #6: Nav1.7_small molecule_CP.

1g. Name the protocol “Nav1.7_small molecule_CP.” 2g. Enter in the Description box 10 sec depolarization to −50 mV; 10 msec gap @ −120 mV; 100 msec gap @ −160 mV. 3g. Click the Rseries tab and enter the same values as in Figure 11.14.3A. 4g. Click the Online cursor button and enter the same values as in Figure 11.14.3B. 5g. Click the Leak protocol button and uncheck the Enable leak subtraction protocol box. 6g. Click the Filtering button and enter the same values as in Figure 11.14.3D. 7g. Click the General tab and enter the values and select the boxes as shown in Figure 11.14.8A. 8g. Click Add near the lower-right corner of the voltage protocol window and enter the values and select the check box/radio buttons that produce the first line in Figure 11.14.8B. Click OK. 9g. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.8B. Click OK. 10g. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.8B. Click OK. 11g. Click Add and enter the values and select the check box/radio buttons that produce the 4th line in Figure 11.14.8B. Click OK. 12g. Click Add and enter the values and select the check box/radio buttons that produce the 5th line in Figure 11.14.8B. Click OK. 13g. Click Add and enter the values and select the check box/radio buttons that produce the 6th line in Figure 11.14.8B. Click OK. QPatch HT, an Automated Patch-Clamp System

14g. Click Add and enter the values and select the check box/radio buttons that produce the 7th line in Figure 11.14.8B. Click OK.



15g. Click Add and enter the values and select the check box/radio buttons that produce the 8th line in Figure 11.14.8B. Click OK. 16g. Click Add and enter the values and select the check box/radio buttons that produce the 9th line in Figure 11.14.8B. Click OK. 17g. Click Add and enter the values and select the check box/radio buttons that produce the 10th line in Figure 11.14.8B. Click OK. 18g. Click Add and enter the values and select the check box/radio buttons that produce the 11th line in Figure 11.14.8B. Click OK. 19g. Click Add and enter the values and select the check box/radio buttons that produce the 12th line in Figure 11.14.8B. Click OK. 20g. Click Apply to save the protocol.

Voltage protocol #7: Protocol for testing peptide blockers of Nav 1.7 This protocol, along with voltage protocol #8, is useful for studying the modulation of Nav 1.7 currents by peptides. 1h. Name the protocol “Nav1.7_peptide_CP.” 2h. Enter in the Description box Vh = −75 mV; 2 sec gap @ −120 mV; 60 sec between test pulses. 3h. Click the Rseries tab and enter the same values as in Figure 11.14.3A. 4h. Click the Online cursor button and enter the same values as in Figure 11.14.3B. 5h. Click the Leak protocol button and uncheck the Enable leak subtraction protocol box. 6h. Click the Filtering button and enter the same values here as in Figure 11.14.3D. 7h. Click the General tab and enter the values and select the boxes as shown in Figure 11.14.9A. 8h. Click Add near the lower-right corner of the voltage protocol window and enter the values and select the check box/radio buttons that produce the first line in Figure 11.14.9B. Click OK. 9h. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.9B. Click OK. 10h. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.9B. Click OK. 11h. Click Add and enter the values and select the check box/radio buttons that produce the 4th line in Figure 11.14.9B. Click OK. 12h. Click Add and enter the values and select the check box/radio buttons that produce the 5th line in Figure 11.14.9B. Click OK. 13h. Click Apply to save the protocol.

Voltage protocol #8: Alternative protocol for testing peptide blockers of Nav 1.7 This protocol is simpler than voltage protocol #7 in that it involves only one brief test pulse. In this case, 5 msec to 0 mV is given once every 60 sec, with the cell held at −100 mV for the rest of the time, as there is no need for a period of recovery from inactivation. The method also potentially simplifies mechanistic interpretation of an effect, such as



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C

Figure 11.14.9 Illustrations of the parameters for voltage protocol #7: Nav1.7_peptide_CP (A and B) and #8: Nav1.7_peptide_1_CP (C).

closed state versus mixed closed/inactivated states. In addition, this protocol also makes it easier to perform leak subtraction. However, because of the long duration of such experiments (1 hr from start to finish—see application protocol #4 in Support Protocol 4), fewer cells survive at the relatively hyperpolarized holding potential used in this protocol (−100 mV here versus −75 mV in voltage protocol #7). 1i. Name the protocol “Nav1.7_peptide_1_CP.” 2i. Enter in the Description box Vh = −100 mV; 60 sec between test pulses. 3i. Click the Rseries tab and enter the same values as in Figure 11.14.3A. 4i. Click the Online cursor button and enter the same values as in Figure 11.14.3B. 5i. Click the Leak protocol button and uncheck the Enable leak subtraction protocol box. 6i. Click the Filtering button and enter the same values as in Figure 11.14.3D. 7i. Click the General tab and enter the values and select the boxes as shown in Figure 11.14.9A, except for the Holding potential (Vhold) box, which is now −100. 8i. Click Add near the lower-right corner of the voltage protocol window and enter the values and select the check box/radio buttons that produce the first line in Figure 11.14.9C. Click OK. QPatch HT, an Automated Patch-Clamp System

9i. Click Add and enter the values and select the check box/radio buttons that produce the second line in Figure 11.14.9C. Click OK.



10i. Click Add and enter the values and select the check box/radio buttons that produce the third line in Figure 11.14.9C. Click OK. 11i. Click Apply to save the protocol.

PREPARING COMPOUND LISTS A compound list designates the location of each test solution in the MTP. Depending on the specifics of an application protocol, an MTP can adopt any one of the six QPatchsupported formats: 96×1, 8×(6×2), 8×(4×3), 8×(3×4), 8×(2×6), and 8×12. Two of these formats, 96×1 and 8×(6×2), are used in the jobs described in this unit. In the 96×1, the simplest format, each MTP well is an independent unit (i.e., test condition). In the 8×(6×2) format, each pair of solutions in two adjacent and horizontal MTP wells (e.g., A1 and A2) forms a unit. The number of times each solution in the pair is applied, as well as the order in which each of them is applied, is specified by the associated application protocol. An MTP in this format can contain up to six pairs of solutions per row, and up to 8×6 = 48 pairs of solutions in all. These pairs may be the same or may differ from one another.

SUPPORT PROTOCOL 3

Information about the identity, concentration, and position in the MTP of each compound must be specified in the Compounds table. With the QPatch software, the Compounds table can be filled manually or automatically using the Auto Fill function. An alternative and efficient way to generate the Compounds table, particularly when the number of compounds is large, is to prepare a compound list in an Excel template, copy the list and paste it into the Compounds table. This approach is described below wherein three compound lists are generated for the four jobs in Support Protocol 5.

Compound list #1: Biophysical characterization of Nav 1.7 Follow the steps below to generate a compound list using an Excel template to study the functional/biophysical properties of Nav 1.7 (for jobs #1 and #2 in Support Protocol 5). 1a. Click Assay and then Compound Lists. 2a. Click New at the bottom-left corner of the content pane. 3a. Name the Compound list: “Nav1.7_biophysics_CP.” 4a. Type biophysics in the Description box. 5a. Select the MTP-96 96×1 format. 6a. Type 500 in the box next to Well volume. 7a. Click the Add button below Compound plates. 8a. Double click on the space shaded blue under the MTP barcode. 9a. Type 00000 (i.e., five zeros) in the box and hit return. 10a. Create a compound table in an Excel sheet as that shown in Figure 11.14.10A. 11a. Copy the area shaded yellow. 12a. Click the Paste button in the content pane of the Assay software. The Compounds table is now generated in the assay software (the four lines next to the Add, Edit and Delete buttons in Fig. 11.14.10B).

13a. Click Apply to save the Compound list. Electrophysiological Techniques


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Figure 11.14.10

Compound list #1: Nav1.7_biophysics_CP.

Compound list #2: Lidocaine concentration responses Follow the steps below to generate a compound list using an Excel template to study the concentration responses of lidocaine at Nav 1.7 (for job #3 in Support Protocol 5). 1b. Click Assay and then Compound Lists. 2b. Click New at the bottom-left corner of the content pane. 3b. Name the Compound list: “Nav1.7_small molecule_CP.” 4b. Type Lidocaine concentration response in the Description box. 5b. Select the MTP-96 8×(6×2) format. 6b. Type 500 in the box next to Well volume. 7b. Click the Add button below Compound plates. 8b. Double click on the space shaded blue under the MTP barcode. 9b. Type 00000 in the box and hit return. 10b. Create a compound list in an Excel sheet as that shown in Figure 11.14.11A. 11b. Copy the area shaded yellow. 12b. Click the Paste button in the content pane of the Assay software. 13b. Click Apply to save the Compound list (partially displayed in Fig. 11.14.11B).


Compound list #3: Huwentoxin-IV concentration responses Follow the steps below to generate a compound list using an Excel template to study the concentration responses of huwentoxin-IV at Nav 1.7 (for job #4 in Support Protocol 5). 1c. Click Assay and then Compound Lists.



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Figure 11.14.11

Compound list #2: Nav1.7_small molecule_CP.

2c. Click New at the bottom-left corner of the content pane. 3c. Name the Compound list Nav1.7_peptide_CP. 4c. Type Huwentoxin-IV concentration response in the Description box. 5c. Select the MTP-96 8×(6×2) format. 6c. Type 500 in the box next to Well volume. 7c. Click the Add button below Compound plates. 8c. Double click the space shaded blue under the MTP barcode. 9c. Type 00000 in the box and hit return. 10c. Create a compound list in an Excel sheet as that shown in Figure 11.14.12. All solutions in this MTP should contain 0.1% BSA.

11c. Copy the area shaded yellow. 12c. Click the Paste button in the content pane of the Assay software. 13c. Click Apply to save the Compound list.

PREPARING APPLICATION PROTOCOLS A QPatch application protocol specifies the sequence, timing, and volume of all the liquid applications in a job. In addition, an application protocol also specifies what voltage protocol to execute and how many times it is executed after each liquid application. In

SUPPORT PROTOCOL 4 Electrophysiological Techniques


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Figure 11.14.12


Compound list #3: Nav1.7_peptide_CP.

other words, an application protocol contains line-by-line instructions for the QPatch on when to collect how much of what solution, from where, to apply to a cell using which voltage protocol. For example, a job may begin with the application of 10 μl control solution from an MTP well, followed 5 voltage protocol repeats later by the application of 10 μl of a test compound from another well in the MTP, and terminate following 5 voltage protocol repeats after the compound application with the application of 10 μl of a reference compound from the reference slot in the reservoir. The QPatch dispenses discrete, small (up to 15 μl each time) puffs of solution. All liquid applications and voltage protocols in a particular job must be specified in the Experiment cycles table (see application protocols below and Figs. 11.14.13B and 11.14.14 to 11.14.16). The investigator must ensure that the MTP format specified in the application protocol matches the MTP format in the compound list used in the same job. Otherwise, a job cannot be assembled. Four application protocols are shown below for the four respective jobs in Support Protocol 5.

Application protocol #1: Nav 1.7 biophysics_1 1a. Click the Assay tab and then Application Protocols.



2a. Click New at the bottom-left corner in the content pane to generate a template. 3a. Name the protocol: “Nav1.7_biophysics_1_CP.” 4a. Type in the Description box activation, fast, slow & total inactivation & rec from fast inactivation. 5a. Select the “voltage gated” radio button under Gating type. 6a. Check the Single experiment per cell box in the lower-left corner of the content pane. 7a. Click Edit and select MTP-96 96×1 from the MTP format drop-down menu. This should appear as the default format.

8a. Click the Add button in the lower-right corner. One line now appears in the Experiment cycles table under Liquid periods.

9a. Click Edit at the bottom to display a dialog box. 10a. Fill in the box as shown in Figure 11.14.13A. Click OK. MTP: Compound means a compound solution in an MTP well. Volume of 5 μl and Repetitions of 1 mean 5 μl of the MTP compound solution is applied to the cell once. “Voltage protocol runs” specifies the number of times a voltage protocol is executed following this liquid application. When clicking the Select button to choose a voltage protocol, select “Nav1.7_rec_fast inactivation_CP.”

11a. Click Add to add a second line and then click Edit. 12a. Use the same values as on Figure 11.14.13A, except select “Nav1.7_act & fast SSI_CP” as the voltage protocol. Click OK. 13a. Click Add to add a third line and then click Edit. 14a. Use the same values as on Figure 11.14.13A except i. Enter 3 in the voltage protocol runs box. ii. Select “Nav1.7_steady pulsing_CP” as the voltage protocol. Click OK. 15a. Click Add to insert a 4th line and then click Edit. 16a. Use the same values as on Figure 11.14.13A except to select “Nav1.7_slow and total SSI_CP” as the voltage protocol. Click OK. 17a. Repeat steps 8a to 16a twice more. 18a. Click Apply to save the protocol. The Experiment cycles table now should look like Figure 11.14.13B. A set of four voltage protocols is executed three times to evaluate if/how channel properties change over time.

Application protocol #2: Nav 1.7 biophysics_2 1b. Click the Assay tab and then Application Protocols. 2b. Click New at the bottom-left corner in the content pane to generate a template. 3b. Name the protocol: “Nav1.7_biophysics_2_CP.” 4c. Type rec from total inactivation in the Description box. 5c. Select the “voltage gated” radio button under Gating type.



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Figure 11.14.13

Illustrations of the parameters for application protocol #1: Nav1.7_biophysics_1_CP.

6c. Check the Single experiment per cell box at the lower-left corner of the content pane. 7c. Click Edit and select MTP-96 96×1 from the MTP format drop-down menu. 8c. Click the Add button in the lower-right corner. 9c. Click Edit to display a dialog box. 10c. Fill in the box as shown in Figure 11.14.13A, except for the following: i. Enter 5 in the box for voltage protocol runs. ii. Select “Nav1.7_steady pulsing_CP” as the voltage protocol. Click OK. 11c. Click Add to insert a second line and then click Edit. 12c. Use the same values as in Figure 11.14.13A except for the following: select “Nav1.7_rec_total inactivation_CP” as the voltage protocol. Click OK. QPatch HT, an Automated Patch-Clamp System

13c. Repeat steps 11c and 12c one more time. 14c. Click Apply to save the protocol. The Experiment cycles table now should look like Figure 11.14.14.



Figure 11.14.14

Illustrations of the parameters for application protocol #2: Nav1.7_biophysics_2_CP.

Application protocol #3: Nav 1.7 small molecule blockers 1d. Click the Assay tab and then Application Protocols. 2d. Click New at the bottom-left corner in the content pane to generate a template. 3d. Name the protocol: “Nav1.7_small molecule_CP.” 4d. Type small molecule blockers in the Description box. 5d. Select the “voltage gated” radio button under Gating type. 6d. Check the Single experiment per cell box at the lower-left corner of the content pane. 7d. Click Edit and select MTP-96 8×(6×2) from the MTP format drop-down menu. 8d. Click the Add button in the lower-right corner. 9d. Click Edit to display a dialog box. 10d. Fill in the box as shown in Figure 11.14.13A, except for the following: i. Select MTP: Concentration 1 in the drop-down box next to Liquid. ii. Enter 10 in the volume box. iii. Select “Nav1.7_small molecule_CP” as the voltage protocol. Click OK. 11d. Click Add to insert a second line and then click Edit. 12d. Use the same values as in step 10d except for the following: i. Check the Use default voltage protocol box. ii. Do not click on the Select button. Click OK. Checking the Use default voltage protocol box instructs for use of the same voltage protocol as the default one, which in this case means to use the voltage protocol named “Nav1.7_small molecule_CP,” since only one voltage protocol is associated with this application protocol. Alternatively, it is always permissible to click on the Select button and explicitly choose a voltage protocol each time without checking the Use default voltage protocol box.

13d. Repeat steps 11d and 12d once more. 14d. Click Add to insert a 4th line and then click Edit. 15d. Use the same values as those in step 10d except for the following: i. ii. iii. iv. v.

Select MTP:Concentration 2 in the drop-down box next to Liquid. Enter 2 in the Repetitions box. Enter 2.0 in the Delay box Check the Use default voltage protocol box. Do not click on the Select button. Click OK.



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Figure 11.14.15

Illustrations of the parameters for application protocol #3: Nav1.7_small molecule_CP.

Steps ii and iii indicate that two 10-μl applications from MTP:Concentration 2 are made to the cell, with the second application being 2 sec after the first.

16d. Repeat steps 14d to 15d twice more. 17d. Click Add to insert a 7th line and then click Edit. 18d. Use the same values as those in step 10 except for the following: i. ii. iii. iv. v. vi.

Select Res: Reference in the drop-down box next to Liquid. Enter 2 in the Repetitions box. Enter 2.0 in the Delay box. Enter 2 in the box next to voltage protocols runs. Check the Use default voltage protocol box. Do not click on the Select button. Click OK. Res: Reference is the left-most slot in the reservoir for reference compound or for vehicle. It cannot be used for both in the same job.

19d. Click Apply to save the protocol. The Experiment cycles table should now look like Figure 11.14.15.

Application protocol #4: Nav 1.7 peptide blockers 1e. Click the Assay tab and then Application Protocols. 2e. Click New at the bottom-left corner in the content pane to generate a template. 3e. Name the protocol: “Nav1.7_peptide_CP.” 4e. Type in the Description box peptide blockers; 10 VP runs/app.; 1 ctrl app., 3 drug apps. & 1 ref app. 5e. Select the “voltage gated” radio button under Gating type. 6e. Check the Single experiment per cell box at the lower-left corner of the content pane. 7e. Click Edit and select MTP-96 8×(6×2) from the MTP format drop-down menu. 8e. Click the Add button in the lower-right corner. 9e. Click Edit to display a dialog box. 10e. Fill in the box as shown in Figure 11.14.13A, except for the following: QPatch HT, an Automated Patch-Clamp System

i. Select MTP: Concentration 1 in the drop-down box next to Liquid. ii. Enter 10 in the box next to Voltage protocol runs. iii. Click on Select and choose “Nav1.7_peptide_CP” as the voltage protocol. Click OK.



Figure 11.14.16

Illustrations of the parameters for application protocol #4: Nav1.7_peptide_CP.

11e. Click Add to insert a second line and then click Edit. 12e. Use the same values as step 10e except for the following: i. Select MTP: Concentration 2 in the drop-down box next to Liquid. ii. Check the Use default voltage protocol box. iii. Do not click on the Select button. Click OK. 13e. Repeat steps 11e and 12e twice more. 14e. Click Add to insert a 5th line and then click Edit. 15e. Use the same values as shown in step 10e except for the following: i. ii. iii. iv. v. vi. vii.

Select Res: Reference in the drop-down box next to Liquid. Enter 10 in the Volume box. Enter 3 in the Repetitions box. Enter 2.0 in the Delay box. Enter 2 in the Voltage protocol runs box. Check the Use default voltage protocol box. Do not click on the Select button. Click OK.

16e. Click Apply to save the protocol. The Experiment cycles table now should look like Figure 11.14.16.

ASSEMBLING JOBS FROM INDIVIDUAL COMPONENTS Four jobs are created in this support protocol by combining the individual components from Support Protocols 1 to 4, above. These jobs can then be executed on the QPatch by following the steps in the respective subsections in the basic protocols.

SUPPORT PROTOCOL 5

Job #1: Characterization of (1) the voltage dependence of activation and inactivation and (2) recovery kinetics from fast inactivation 1a. Click the Assay tab and then Assays. 2a. Click New in the lower-left corner of the content pane. 3a. Name the assay “Nav1.7 biophysics_1_CP.” 4a. Type in the Description box activation, fast, slow & total inactivation & rec from fast inactivation. 5a. Select “HEK293” in the Cell type box and “hNav1.7” in the “ion channel” box. These values need to be entered in advance via the Administration tab.

6a. Select “Nav1.7_HEK_1_CP” under Whole-cell protocol. 7a. Select “Nav1.7_biophysics_1_CP” under Application protocol.



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8a. Select “Nav1.7_rec_fast inactivation_CP” as the default voltage protocol. 9a. Click Apply. 10a. Click Prepared Assays. 11a. Click New in the new window. 12a. Click the Select button and select “Nav1.7_biophysics_CP” from the Compound list. 13a. Select “CsCL-CsF/2 Ca, 5.4K, 1 Mg” in the Single IC/EC solution pairs box and leave the other boxes under Solutions and Cells unchanged. These include Disabled for the Multiple IC/EC solution pairs box and the “Cell cloning” box, and Unspecified for the Reference and Vehicle boxes. The IC/EC pair must be inserted in advance via the Administration tab.

14a. Enter 1, 4, and 4, respectively, for the three boxes under Experiments. These values represent the Min. experiments to continue, the Min. successful repetitions, and the Max. repetitions, respectively. 15a. Select the QPlate usage radio button. 16a. Click Apply. 17a. Click Execute.

Job #2: Characterization of recovery kinetics from total inactivation 1b. Click the Assay tab and then Assays. 2b. Click New in the lower-left corner of the content pane. 3b. Name the assay “Nav1.7 biophysics_2_CP.” 4b. Type rec from total inactivation in the Description box. 5b. Select “HEK293” in the Cell type box and “hNav1.7” in the “ion channel” box. 6b. Select “Nav1.7_HEK_1_CP” under Whole-cell protocol. 7b. Select “Nav1.7_biophysics_2_CP” under Application protocol. 8b. Select “Nav1.7_rec_total inactivation_CP” as the default voltage protocol. 9b. Click Apply. 10b. Click Prepared Assays. 11b. Click New in the new window. 12b. Click the Select button and select “Nav1.7_biophysics_CP” from the Compound list. 13b. Select “CsCL-CsF/2 Ca, 5.4K, 1 Mg” in the Single IC/EC solution pairs box and leave unchanged the other boxes under Solutions and cells. 14b. Enter 1, 4, and 4, respectively, for the three boxes under Experiments. These values represent the Min. experiments to continue, the Min. successful repetitions, and the Max. repetitions, respectively. QPatch HT, an Automated Patch-Clamp System

15b. Select the QPlate usage radio button. 16b. Click Apply.



Click “yes” if a warning box appears stating that “Data acquisition exceeds recommended max value, etc.”

17b. Click Execute.

Job #3: Screening for Nav 1.7 small molecule blockers 1c. Click the Assay tab and then Assays. 2c. Click New in the lower-left corner of the content pane. 3c. Name the assay “Nav1.7 small molecule blockers_CP.” 4c. Type “Nav1.7 small molecule blockers” in the Description box. 5c. Select “HEK293” in the Cell type box and “hNav1.7” in the ion channel box. 6c. Select “Nav1.7_HEK_1_CP” under Whole-cell protocol. 7c. Select “Nav1.7_small molecule_CP” under Application protocol. 8c. Select “Nav1.7_small molecule_CP” as the default voltage protocol. 9c. Click Apply. 10c. Click Prepared Assays. 11c. Click New in the new window. 12c. Click the Select button and select “Nav1.7_small molecule_CP” from the Compound list. 13c. Select “CsCL-CsF/2 Ca, 5.4K, 1 Mg” in the Single IC/EC solution pairs box and leave the other boxes under Solutions and Cells unchanged. 14c. Enter 1, 2, and 2, respectively, for the three boxes under Experiments. These values represent the Min. experiments to continue, the Min. successful repetitions, and the Max. repetitions, respectively. 15c. Select the QPlate usage radio button. 16c. Click Apply. 17c. Click Execute.

Job #4: Screening for Nav 1.7 peptide blockers 1d. Click the Assay tab and then Assays. 2d. Click New in the lower-left corner of the content pane. 3d. Name the assay “Nav1.7 peptide blockers_CP.” 4d. Type “Nav1.7 peptide blockers” in the Description box. 5d. Select “HEK293” in the Cell type box and “hNav1.7” in the ion channel box. 6d. Select “Nav1.7_HEK_2_CP” under Whole-cell protocol. 7d. Select “Nav1.7_peptide_CP” under Application protocol. 8d. Select “Nav1.7_peptide_CP” as the default voltage protocol. 9d. Click Apply. 10d. Click Prepared Assays. 11d. Click New in the new window.



Supplement 65

12d. Click the Select button and select “Nav1.7_peptide_CP” from the Compound list. 13d. Select “CsCL-CsF/2 Ca, 5.4K, 1 Mg” in the Single IC/EC solution pairs box and leave unchanged the other boxes under Solutions and cells. 14d. Enter 1, 6, and 8, respectively, for the three boxes under Experiments. These values represent the Min. experiments to continue, the Min. successful repetitions, and the Max. repetitions, respectively. 15d. Select the QPlate usage radio button. 16d. Click Apply. 17d. Click Execute. SUPPORT PROTOCOL 6

HARVESTING CELLS FOR QPATCH EXPERIMENTS This is arguably the most critical procedure in the entire experiment, because the quality of the dissociated cells directly impacts the quality of the recordings. The key to achieving high quality of dissociated cells is obtaining fully individualized cells without compromising their integrity, by using an appropriate amount of cell trypsinization and trituration.

Materials HEPES (Sigma, cat. no. H3375; store at room temperature) CHO-S-SFM medium (Invitrogen; 500 ml; store at 4°C) 150-cm2 (T-150) flask of HEK293 cells (ATCC #CRL-1573) stably expressing hNav 1.7 (α subunit) cultured in cell culture medium (see recipe) Cell culture medium (see recipe; 500 ml; store at 4°C) Dulbecco’s phosphate-buffered saline (DPBS), Ca2+ - and Mg2+ -free (e.g., Invitrogen; 500 ml; store at room temperature) 0.05% trypsin/EDTA (Sigma, cat. no. 59417C; 100 ml; store at 4°C) Syringe filter (0.2-μm pore size) Manual or automated cell counter Cell storage tank with stirring bar (Sophion, cat. nos. SB2050 and SB3070) 1. Add 25 mM HEPES to 20 ml CHO-S-SFM medium that has been prewarmed to room temperature. Filter the medium through a 0.2-μm filter after the HEPES is fully dissolved. The HEPES-supplemented CHO-S-SFM medium should be prepared fresh daily.

2. On the day of the experiment, remove a 150-cm2 flask of cells cultured to reach 80% confluency from the incubator. This should provide 20 × 106 cells. The number of cells needed for each full QPlate for the experiment is 1–2 × 106 . If a job is smaller, use a proportionally smaller flask of cells. If a job is larger, use either a larger flask of cells, such as a 225-cm2 flask (T-225), or multiple flasks of cells.

3. Remove culture medium by aspiration. 4. Gently rinse cells with 20 ml DPBS without Ca or Mg to remove leftover medium and debris. QPatch HT, an Automated Patch-Clamp System

5. Remove DPBS by aspiration. 6. Add 7 ml room temperature 0.05% trypsin/EDTA to the flask. Once all of the cells are covered with trypsin, remove the trypsin with an aspirating pipet.



7. Leave the flask at 37°C for 5 min. 8. Add 7 ml HEPES-supplemented CHO-S-SFM medium (from step 1) to the flask and gently triturate the cells with a 5-ml serological pipet to break cell clumps. This step is crucial. Triturate just enough to individualize cells without damaging them. If the cells are over-triturated, they become unhealthy and debris accumulates, interfering with seal formation.

9. Determine the cell density with an automated cell counter. Add appropriate amount of CHO-S-SFM medium to adjust the cell density to 2 × 106 /ml. 10. Transfer the cells to the cell storage tank into which a stirring bar has been inserted. 11. Place the cell storage tank on the stirring unit of the screening station. Gently stir the cells for 15 min before use.

DATA ANALYSIS The QPatch assay software comes with data analysis capabilities. For complex or customized analyses, however, it may be better to export the data to Excel, or other third-party applications, where data processing can be handled be customized, such as with the use of templates, macros and/or other analytical tools. Described below is a procedure for exporting QPatch data to Excel. Regardless of whether the data are ultimately analyzed using the assay software or a third-party application, the assay software is needed for initial processing. This includes creating a project from a job to be analyzed, and, within the project, selecting usable cells for analysis and defining the signal(s) to be analyzed, whether they be, for example, peak current or mean current over a certain period of time. Job #3 is used as an example to illustrate these steps.

SUPPORT PROTOCOL 7

Exporting data from QPatch 1. Click the Jobs tab and then the Jobs folder below the tabs in the navigation pane. 2. In the content pane, select the job to be analyzed. This is job 2430 in this example (Fig. 11.14.17A). 3. Click the New Project button at the bottom of the window. 4. A pop-up window appears with a request to choose between selecting an existing project as a template or creating a new project. Click on Create project from scratch at the lower-left corner of the pop-up window. Click OK. This creates a project for the job with a 3-way split window display. The upper-left window contains several lines, each of which represents a layer of analysis performed by the assay software. The lower-left window displays the ID of all the cells recorded in this job. For example, Exp 2430.1.1 (ctrl) is a cell tested with control buffer whereas Exp 2430.7.1 (30 μM lidocaine) is a cell tested with 30 μM lidocaine. The window on the right side displays different information depending on which lines in the upper-left and lower-left windows are selected.

5. Select Sweep Plot in the upper-left window and a cell ID, such as Exp 2430.25.2 (1000 μM lidocaine) in the lower-left window. The right-hand side window now displays the current traces recorded from this cell, along with thin, solid red lines representing the voltage waveform (Fig. 11.14.17A). The current traces in Figure 11.14.17A are compressed and difficult to discern because the voltage waveform is 13 sec long. The most relevant information in this job is the peak current in response to each of the four brief (5 msec) depolarizing pulses to 0 mV. To visualize a peak, click and drag the mouse on the plot and zoom in to the area surrounding the peak.



Supplement 65

A

peak 3

peak 2 peak 4

peak 1

B

C

Sweep plot 0

1 M TTX

20

0

Current [pA]

60 1000

80

1 mM lidocaine

100

1500 peak 1

2000

Voltage [mV]

40

500

120 140

control

2500

160 5.0

5.5

6.0

6.5

7.0

7.5

8.0

8.5

Time (msec)

Figure 11.14.17 Initial data processing using the QPatch software. (A) A 3-way split window display of a project. Displayed in the right-hand side window are currents recorded from the selected cell in the lower-left window, Exp 2430.22.1. (B) Zoomed-in view of peak 1 in (A). (C) Setup of parameters to measure the amplitude of peak 1 in (B).

6. Zoom in to peak 1, which is near the 5-msec mark, as shown in Figure 11.14.17B. Right click with the cursor placed inside the window and select “Keep zoom” from the pop-up box. Three control traces, three 1000 μM lidocaine traces, and two 1 μM TTX traces are displayed in this expanded plot.

7. Click the Cursor Intervals and Voltage Protocols tab at the top-left corner of the window. A new window appears, replacing the previous window.



8. In the new window, click the Add Cursor Interval tab at the bottom. A pop-up window now appears, as shown in Figure 11.14.17C. 9. Fill in the information as shown in Figure 11.14.17C. Click OK. This returns the user to the window in step 7, with an additional line about peak 1 in the lower middle region of the window, just below Cursor intervals. This line defines Current Protocols in Pharmacology

a narrow region of 0.36 msec in duration, encompassing peak 1, and determines the minimum current amplitude in this region. This is the value of peak 1 in this case.

10. Click the Plot tab at the top-left corner of the window to return to the window in step 6. A vertical region shaded green now appears in the plot, as shown in Figure 11.14.17B. Examine the shaded area to make certain the peaks of all the current traces, except for those in the presence of TTX, lie inside the shaded area.

11. Repeat steps 6 through 10 for the other three peaks: peak 2, which is near the 115msec mark, peak 3, which is near the 13,130-msec mark, and peak 4, which is near the 13,232 msec mark. After completing these steps, there should be a shaded region around each of the four peaks which may be viewable when zoomed-in individually to each peak. Also, the peak current amplitude of each of the four peaks is determined. Note that steps 6 to 11 need only be performed for one cell. The other cells in the job automatically receive the same treatment.

12. Zoom in to each of the four peaks for each cell and make certain that all of the peaks for all the cells fall within the respective shaded areas. If some peaks are outside the shaded region, adjust the width of the shaded area to include them. Note that if this is performed to a particular peak, say peak 1, for a particular cell, then the width of the shaded area around peak 1 will be changed in the same way for all other cells. The widths of the shaded areas around the other peaks are unaffected. Do not widen the shaded region too much, to avoid the inclusion of capacitance spikes as peaks.

13. To exclude a cell from analysis, right click on the cell ID in the lower-left window of the 3-way split display and select “bad” in the dialog box. A red cross will take the place of the green check mark to the left of the cell ID to indicate that the cell has been excluded from further analysis.

14. Click Sweep Results in the upper-left window to display, in the right-hand-side window, the default parameters for each cell to be exported, such as Experiment, Liquid Period, Sweet Time, as well as Min(peak1), Min(peak2), Min(peak3), and Min(peak4). It is possible to include additional parameters to be exported if desired. Refer to the user manual for instructions on how to accomplish this.

15. Select “All data” and “Passed all filters” in the two pull-down menus at the bottom of the window. 16. Click the Export tab at the bottom left of the window. 17. In the dialog box that appears next, select the Store file and Whole table radio buttons, check the Use prefix box, and select Excel in the drop-down menu for File format. Click OK. 18. Select a destination for the file to be exported to when prompted. 19. Name the file and click Save. 20. Open the saved file in Excel and analyze the data.

Analyzing QPatch data Described below are the methods/procedures employed in the analysis of the data acquired from the four jobs described above after data exportation to Excel. Data analysis is performed using Microsoft Excel in combination with Origin 7.0 (OriginLab). Results are expressed as mean±SEM.



Supplement 65

A

B 1

2

20 mV 0 mV

1

120 mV

129.5 130.0 130.5 131.0 131.5 132.0 132.5 133.0 133.5 134.0 134.5

5 msec 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5

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60 40 20 0 120 100 80

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Figure 11.14.18 Voltage dependence of steady-state fast inactivation of hNav 1.7. (A) Voltage protocol. The 100-msec pulses were delivered with increasing depolarization from –120 to 20 mV in 10-mV increments. The time interval between the start of consecutive pulses was 2 sec. (B) Whole-cell current traces in response to the voltage protocol in (A). Leak-current subtraction was performed using a P/3 protocol. Data were sampled and filtered at 25 and 5 kHz, respectively. (C) Average I-V relationship constructed from peak currents like those shown in the left half of (B) (labeled 1) for 25 cells. The protocol was run twice: once shortly after whole-cell formation (labeled 0 min) and a second time 12 min later (labeled 12 min). (D) Dependence of steady-state fast inactivation on the voltage of 100-msec preconditioning pulses. Data are normalized peak currents (to that at –120 mV) as those shown in the right half of (B) (labeled 2), averaged over 25 cells. Curves are best fits of a Boltzmann function to the data. The voltage protocol was run twice: once shortly after whole-cell formation (labeled 0 min) and a second time 12 min later (labeled 12 min).

21. Nav 1.7 channel biophysics (jobs #1 and #2).


For I-V curves, peak currents elicited by the voltage steps (labeled 1 in Fig. 11.14.18A and B) are plotted as a function of the command voltage. For voltage dependence of steady-state fast inactivation, the amplitude of the peak current in response to depolarization to 0 mV (labeled 2 in Fig. 11.14.18A and B) for a preconditioning voltage is plotted against the preconditioning voltage after being normalized to that for the preconditioning voltage of –120 mV. For voltage dependence of steady-state total and slow inactivation, the amplitude of the peak currents elicited by the second and third depolarizations to 0 mV (labeled 2 and 3, respectively, in Fig. 11.14.19A and B) for a



A

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20 0 120 100

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Preconditioning voltage (mV)

Figure 11.14.19 Voltage dependence of steady-state slow and total inactivation of hNav 1.7. (A) Voltage protocol. The 20-sec pulses were given with increasing depolarization from −120 to −20 mV in 10-mV increments. The time interval between the start of consecutive pulses was 50 sec. (B) Whole-cell current traces in response to the voltage protocol in (A). Data were sampled and filtered at 25 and 5 kHz, respectively. (C) Dependence of steady-state slow and total inactivation on the voltage of 20-sec preconditioning pulses. Data are normalized peak currents (i.e., peaks 2 and 3, labeled 2 and 3, respectively, normalized to the first peak, labeled 1, for each trace) as those shown in (B), averaged over 4 cells. Curves are best fits of the data to a Boltzmann function. The voltage protocol was run twice: once shortly after whole-cell formation (labeled 0 min) and a second time 12 min later (labeled 12 min).

preconditioning voltage is plotted against the preconditioning voltage after being normalized to that elicited by the first depolarization to 0 mV (labeled 1 in Figs. 11.14.19A and B) for the same preconditioning voltage. The voltage dependence of inactivation is fitted to a Boltzmann function to determine V1/2 , the voltage at which 50% of the channels are inactivated. For recovery from fast and slow inactivation, the amplitude of the peak current elicited by the second depolarization to 0 mV (labeled 2 in Fig. 11.14.20A and B) for a recovery time interval is plotted against the recovery time interval after being normalized to that elicited by the first depolarization to 0 mV (labeled 1 in Fig. 11.14.20A and B) for the same recovery time interval. The recovery data are fitted to a double exponential function to determine the time constants and relative fractions for each component.

22. Small molecule data (job #3). For each cell, the amplitude of all peak currents elicited by each of the four depolarizations to 0 mV (labeled 1 to 4 in Fig. 11.14.21A and B) in the presence of control or compound solutions is first subtracted from that in the presence of 1 μM TTX for the corresponding depolarization. The TTX-sensitive amplitude of the four peak currents in the presence of a compound (compound cells) or the control solution (control cells) after the fourth, fifth, and sixth liquid applications is normalized to the value of the



Supplement 65

B A 1

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10 or 10,000 msec Current (pA)

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1000 2000 3000

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t

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80 60 10-msec prepulse (0 mV) 0 min (1 3.0 0.6 msec (92.5%)2 63.4 92.0 msec)

40

12 min ( 1 3.9 0.8 msec (92.0%)2 62.6 88.6 sec) 10,000-msec prepulse (0 mV) 10 min (1 86.0 17.4 msec (65.0%)2 1.3 0.4 sec)

20

22 min (1 89.1 18.1 msec (65.5%)2 1.2 0.4 sec)

0 1

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1000 10,000 100,000

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Figure 11.14.20 Recovery from inactivation of hNav 1.7. (A) Voltage protocol, where t = 2m msec (m = 0, 1, 2, . . . , 11) for 10-msec prepulses to 0 mV and t = 75 × 2m msec (m = 0, 1, 2, . . . , 8) for 10,000-msec prepulses to 0 mV. The time interval was 3 sec between the start of the mth and (m + 1)th 10-msec prepulses and 45 sec between the start of the mth and (m + 1)th 10,000-msec prepulses. (B) Representative current responses (12 superimposed traces) of a cell to the 10-msec prepulse voltage protocol in (A). For clarity, only a 5-msec segment is shown for each of the current traces that encompass the peak current in response to the second depolarization to 0 mV. (C) Time courses of recovery from fast and total inactivation obtained from the protocols in (A). The time courses of recovery (n = 4 and 3, respectively, for fast and slow/total inactivation) were each approximated by a double-exponential function. Data were sampled and filtered at 25 and 5 kHz, respectively. The voltage protocols were run twice: once about 0 min (fast inactivation) or 10 min after whole-cell formation (slow/total inactivation), and a second time 12 min (fast inactivation) or 22 min (slow/total inactivation) later.

corresponding peaks in the third (or, for compound cells, the last control) liquid application. To correct for current rundown, the normalized peak values for each compound cell are further normalized to the temporally-corresponding average values of the control cells in the same MTP. The twice-normalized value from the sixth liquid application, f, is used to calculate the % inhibition value (R), as follows: R = 100×(1 – f). The IC50 value, the concentration at which 50% inhibition occurs, is determined from the best fit of the concentration-response data to a logistic function of the form: R = 100/(1+c/IC50 )p , where p is the Hill coefficient and c is the compound concentration.

23. Peptide data (job #4). QPatch HT, an Automated Patch-Clamp System

For each cell, the peak amplitude of the last current trace obtained in the presence of TTX is subtracted from that of each of the current traces obtained during the preceding four solution applications to obtain the peak values of TTX-sensitive currents. These values



A

1 M TTX 1

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50 mV

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100 M lidocaine

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120 mV

160 mV

control 1

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10 5 3000

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5 msec

D peak1 (1478.6 123 M) peak2 (98.1 7.0 M)

100

peak1 (73.5 5.4 nM) peak2 (63.5 3.9 nM)

peak3 (77.2 18.6 M) peak4 (1621.5 242.6 M)

80 % Inhibition

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peak3 (37.0 5.5 nM) peak4 (37.4 1.2 nM)

100

60 40

60 40

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[Tetrodotoxin](nM)

Figure 11.14.21 A patch clamp assay for screening small molecule blockers simultaneously in multiple states of hNav 1.7. (A) An “all-in-one” voltage protocol that simultaneously probes compound effects in the closed/open (peak 1), fast inactivated [peak 2 after a relatively short duration (100 msec) of compound exposure and peak 3 after a long duration (10 sec) of compound exposure], and slow inactivated (peak 4) states of the channel. (B) Whole-cell current traces in response to the four depolarizations to 0 mV in (A) in the presence of control buffer, 100 μM lidocaine, and 1 μM TTX. (C) Concentration-response relationships for lidocaine (n = 4 to 8 for the concentrations tested) as measured by the four peak currents indicated in (B). Numbers in the parentheses are IC50 values. The dashed lines are the best fits to a logistic function. (D) Concentration-response relationships for TTX (n = 5 to 7 for the concentrations tested) studied the same way as in (C). Numbers in the parentheses are IC50 values. The dashed lines are the best fits to a logistic function. Note the 2 fold increase in potency for peaks 3 and 4 over peaks 1 and 2.

are normalized to that of the last trace in the first, i.e., control buffer application. To correct for current rundown, the values for each cell in the presence of a test compound are further normalized to the temporally-corresponding average values for control cells in the same experiment. The mean of the last two such twice-normalized values in the last test-compound application (i.e., the normalized and rundown-corrected values obtained 30 min following the initial application of a test compound), f, is used to calculate the % inhibition value for each cell at the particular test-compound concentration tested, as follows: R = 100×(1 – f). The average of the % inhibition values for all cells tested at each test-compound concentration is used in concentration-response calculations. The IC50 values are determined from the best fit of the concentration-response data to a logistic function as described above.



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REAGENTS AND SOLUTIONS Use deionized, distilled water to make extracellular and intracellular solutions. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Cell culture medium DMEM/F-12 (with L-glutamine) supplemented with: 10% fetal bovine serum 1× nonessential amino acids (add from concentrated stock; e.g., Invitrogen) 400 μg/ml G418 Store up to 1 month at 4ºC Extracellular solution (mM) 137 mM NaCl 5.4 mM KCl 1 mM MgCl2 2 mM CaCl2 5 mM glucose 10 mM HEPES Adjust pH to 7.4 with NaOH Adjust osmolarity to 315 mOsm with sucrose or deionized water Filter through 0.2-μm pore size filter Store up to several months at 4°C Intracellular solution (mM) 135 mM CsF 10 mM CsCl 5 mM EGTA 5 mM NaCl 10 mM HEPES Adjust pH to 7.3 with CsOH Adjust osmolarity to 290 mOsm with sucrose or deionized water Filter through 0.2-μm pore size filter Store up to several months at 4°C COMMENTARY Background Information



Voltage-gated sodium channels consist of a pore-forming α subunit that can form a functional channel and auxiliary β subunits. The voltage sensitivity of Nav channels largely comes from the positively charged fourth transmembrane segment, S4, in each of the four homologous domains of the α subunit, which moves to open or close the channel in response to changes in membrane potential. Given this property, S4 is commonly referred to as the voltage sensor. Nav channels exist in one of three classes of conformational states: closed, open, or inactivated. The channel is in the closed state at hyperpolarized membrane potentials, such as −100 mV, and opens within a millisecond upon membrane depolarization. Continued depolarization causes the channel to enter nonconducting, inactivated states. Channel inactivation can occur from both open and pre-open, nonconducting states.

Many peptides and small molecules bind to one or more of these states, thereby modulating Nav channel function. For example, local anesthetics block Nav channels by preferentially binding to the inactivated state(s) at an intracellular site of the channel pore (Catteral, 2000). Tetrodotoxin (TTX), a smallmolecular-weight neurotoxin, blocks the pore of an open channel from the extracellular side. Other agents, such as huwentoxin-IV, a 35-amino-acid peptidyl toxin, block several subtypes of Nav channels by preferentially binding to the voltage sensor in the closed state (Xiao et al., 2008). To date, nine functional α subunits, Nav 1.1 to Nav 1.9, have been identified from mammalian species (Catterall et al., 2003). They are often grouped into two classes: TTX-sensitive (Nav 1.1 to 1.4, 1.6, and 1.7) and TTX-resistant (Nav 1.5, 1.8, and 1.9). One of the TTX-sensitive and peripherally expressing subtypes, Nav 1.7, is a prime Current Protocols in Pharmacology

target for analgesic drug discovery after human genetic studies strongly linked this channel to pain signaling. Patients with Nav 1.7 gain-offunction mutations develop extremely painful disorders (Cummins et al., 2004; Fertleman et al., 2006), whereas those with Nav 1.7 lossof-function mutations experience no pain (Cox et al., 2006). It has been proposed that the relatively slow channel inactivation kinetics of Nav 1.7 may help boost threshold signals during slowly depolarizing inputs to neurons, amplifying pain signals (Cummins et al., 1998). The human genetics data suggest that drugs selectively inhibiting Nav 1.7 may be excellent analgesics. The voltage control and temporal resolution afforded by patch clamp electrophysiology makes it an ideal technique for measuring the fast kinetics and steep voltage sensitivity of Nav channels. The accurate and direct nature of these measurements, combined with the high information content they provide and the flexibility to design/perform experiments rapidly, has resulted in this becoming the “gold standard” for studying ion channel function and pharmacology. However, conventional patch clamp has low throughput. Thus, a well-trained patch clamper can only test a small number of compounds per day, greatly limiting the utility of manual patch clamp in drug discovery. Before the advent of medium- and highthroughput automated patch clamp electrophysiology, screening of compounds against ion-channel targets typically involved higherthroughput binding and/or fluorescence-based assays. However, these and other HTS methods lack voltage control, which is particularly important for voltage-gated ion channels, and high information content, such as measurement of fast channel kinetics. In addition, fluorescence-based assays provide only indirect measurements of channel function via fluorescent dyes, and binding assays are nonfunctional in nature, requiring a known highaffinity, radiolabeled ligand. In general, the binding assays identify only hits that compete for binding with the known ligand, which can yield both false positive and false negative findings. Automated patch clamp systems bridge the gap between traditional HTS methods and manual patch clamp. They offer greatly increased throughput and ease of operation over manual patch clamp while at the same time preserving the high information content/accuracy that characterizes manual patch clamp. For these reasons, automated patch

clamp is well suited for screening a large number of compounds for drug discovery. The medium-throughput QPatch HT is particularly suitable for studying voltage-gated ion channels, given its ability to make gigaohm seals and to produce high-quality recordings comparable to manual patch clamp. A notable feature of the QPatch system is its unattended mode of operation, whereby recordings are made for hours without operator intervention, greatly increasing the efficiency of operation and extending the throughput. Although most of the procedures described in this unit are specific to QPatch, the voltage protocols can be adopted readily in other patch clamp platforms, including manual patch clamp.

Critical Parameters and Troubleshooting Preparation of cells for QPatch recording is the single most critical procedure in the overall yield and quality of QPatch data. The confluency of cells in culture should not exceed 90% before splitting or harvesting. The confluency must be maintained at 100 msec,



it will not be detected in peak 2 despite the high-affinity state because there is insufficient time for the compound to bind in that state. Rather, it is more likely to be detected by peak 3, since this allows binding of the blocker in the state to develop for a much longer period. Phenytoin is a good example of a Nav blocker that belongs to this category (Kuo and Bean, 1994; Liu et al., 2011). Lidocaine binds to the fast inactivated state with higher affinity than in the closed/open state (Ragsdale et al., 1996). This is consistent with the results shown on Figure 11.14.21C. It is also a fast binder, at least at 0 mV, at which fast desensitization develops in

Electrophysiological studies of the auditory system.

Electrophysiological studies on the effect of long-term 5-HT reuptake inhibition on the function of 5-HT neurons.

Potentiation of 5-HT neurotransmission by short-term lithium: in vivo electrophysiological studies.

N-acetylcysteine modulates hallucinogenic 5-HT(2A) receptor agonist-mediated responses: behavioral, molecular, and electrophysiological studies.

Distribution of sodium channels in chronically demyelinated spinal cord axons: immuno-ultrastructural localization and electrophysiological observations.

Theoretical and simulation studies on voltage-gated sodium channels.

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

Studying mechanosensitive ion channels with an automated patch clamp.

Development of a QPatch automated electrophysiology assay for identifying KCa3.1 inhibitors and activators.

Effect of drugs influencing 5-HT function on ethanol drinking and feeding behaviour in rats: studies using a drinkometer system.

An evaluative paradigm for community mental health centers using an automated data system.

An automated ultrasound transducer beam profiling system.

An automated system for ECG monitoring.

Botulism: electrophysiological studies.

Outpatient electrophysiological studies.

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

Development of an Automated Medical Diagnosis System for Classifying Thyroid Tumor Cells using Multiple Classifier Fusion.

Successful novice's training in obtaining accurate assessment of carotid IMT using an automated ultrasound system.

[Respiratory viral diagnosis by using an automated system of multiplex PCR (FilmArray) compared to conventional methods].

Sodium channels in ocular epithelia.

Trial prospector: matching patients with cancer research studies using an automated and scalable approach.

The cerebellopontine system: an electrophysiological study in the rat.

An automated microfluidic system for screening Caenorhabditis elegans behaviors using electrotaxis.

Monitoring polio supplementary immunization activities using an automated short text messaging system in Karachi, Pakistan.