Automated Patch Clamp Analysis of nAChα7 and NaV1.7 Channels

Alison Obergrussberger1, Claudia Haarmann1, Ilka Rinke1, Nadine Becker1, David Guinot1, Andrea Brueggemann1, Sonja Stoelzle‐Feix1, Michael George1, Niels Fertig1

1 Nanion Technologies, Munich
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.13
DOI:  10.1002/0471141755.ph1113s65
Online Posting Date:  June, 2014
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Abstract

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high‐quality and information‐rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage‐gated sodium subtype 1.7 (NaV1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of NaV1.7, and activation and allosteric modulation of nAChα7R. Curr. Protoc. Pharmacol. 65:11.13.1‐11.13.48. © 2014 by John Wiley & Sons, Inc.

Keywords: automated patch clamp; NaV1.7; nicotinic acetylcholine receptors

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Activation and Inactivation Kinetics of NaV1.7 Using the Port‐a‐Patch
  • Alternate Protocol 1: Activation of nAChα7R on the Port‐a‐Patch using the Fast Perfusion Chamber
  • Support Protocol 1: Cell Culture for Use on the Port‐a‐Patch, Patchliner, and SyncroPatch 96
  • Support Protocol 2: Induction of HEK nAChα7R Cell Line
  • Support Protocol 3: Cell Harvesting for Use on the Port‐a‐Patch, Patchliner, and SyncroPatch 96
  • Basic Protocol 2: Electrophysiological Measurements of NaV1.7 AT Vhalf Inactivation on the Patchliner
  • Alternate Protocol 2: Activation of nAChα7R Using the Patchliner
  • Alternate Protocol 3: Temperature Effects on Pharmacology of nAChα7R Using the Patchliner
  • Basic Protocol 3: High‐Throughput Electrophysiological Analysis of NaV1.7 on the SyncroPatch 96
  • Alternate Protocol 4: High‐Throughput Electrophysiological Recordings of nAChα7R on the SyncroPatch 96
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Activation and Inactivation Kinetics of NaV1.7 Using the Port‐a‐Patch

  Materials
  • Nanion standard internal solution for Na+ channels (NSIntSNa; see recipe)
  • CHO cells expressing hNa V1.7 (Anaxon AG) or HEK cells expressing hNa V1.7 (EMD Millipore)
  • Nanion standard external solution for Na+ channels (NSExtSNa; see recipe)
  • Seal enhancing solution (SES; see recipe)
  • 3 nM, 10 nM, 30 nM, 100 nM, 300 nM and 1 µM TTX (Tocris) prepared in NSExtSNa. Prepare the 1 nM TTX stock solution in NSExtSNa. Store aliquots at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment for preparing the TTX concentrations by serial dilution.
  • Amplifier (e.g., EPC10 USB, HEKA Elektronik)
  • Computer running the following software:
    • PatchControl software (Nanion Technologies)
    • PatchMaster software (HEKA Elektronik)
    • Igor software (Wavemetrics)
    • Excel (Microsoft)
  • NPC®‐1 chips, resistance 2 to 3.5 MΩ (Nanion Technologies)
  • Port‐a‐Patch recording station (Nanion Technologies)
  • Port‐a‐Patch suction control (Nanion Technologies)
  • External perfusion system for the Port‐a‐Patch, including a laminar flow chamber (Nanion Technologies; optional)

Alternate Protocol 1: Activation of nAChα7R on the Port‐a‐Patch using the Fast Perfusion Chamber

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

Support Protocol 1: Cell Culture for Use on the Port‐a‐Patch, Patchliner, and SyncroPatch 96

  Materials
  • Ion‐channel overexpressing cell lines, e.g.:
    • CHO cells expressing hNa V1.7 (Anaxon AG)
    • HEK cells expressing hNa V1.7 (EMD Millipore)
    • GH4C1 cells expressing nAChα7R (cells kindly provided by Thomas Seeger, Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany)
    • HEK cells expressing nAChα7R (inducible cell line; Galantos Pharma)
  • Cell culture medium (see recipe)
  • PBS‐EDTA: PBS (PAA Laboratories, GmbH) containing 2 mM EDTA
  • 1× trypsin‐EDTA (PAA Laboratories, GmbH)
  • 100‐mm culture dishes (PAA Laboratories, GmbH)
  • 75‐cm2 (T‐75) culture flasks (PAA Laboratories, GmbH)
  • Light microscope
  • 15‐ml conical tubes (Falcon‐style tubes; PAA Laboratories, GmbH)
  • Tabletop centrifuge

Support Protocol 2: Induction of HEK nAChα7R Cell Line

  Materials
  • HEK cells expressing nAChα7R (inducible cell line; Galantos Pharma)
  • Culture medium (see recipe)
  • Inducer Supplemented Media (Galantos Pharma)

Support Protocol 3: Cell Harvesting for Use on the Port‐a‐Patch, Patchliner, and SyncroPatch 96

  Materials
  • Ion‐channel overexpressing cell lines, e.g.:
    • CHO cells expressing hNa V1.7 (Anaxon AG)
    • HEK cells expressing hNa V1.7 (EMD Millipore)
    • GH4C1 cells expressing nAChα7R (cells kindly provided by Thomas Seeger, Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany)
    • HEK cells expressing nAChα7R (induced; Galantos Pharma)
  • DMEM/Ham's F12 medium including glutamine (PAA Laboratories, GmbH)
  • PBS‐EDTA: PBS (PAA Laboratories, GmbH) containing 2 mM EDTA
  • 1× trypsin‐EDTA (PAA Laboratories, GmbH)
  • Nanion standard external solution for Na+ channels (NSExtSNa; see recipe)
  • Light microscope
  • 15‐ml Falcon tube
  • Small centrifuge

Basic Protocol 2: Electrophysiological Measurements of NaV1.7 AT Vhalf Inactivation on the Patchliner

  Materials
  • NSIntSNa (see recipe)
  • NSExtSNa (see recipe)
  • SES (see recipe)
  • TTX (Tocris) 1 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 µM prepared in NSExtSNa (see recipe), with the highest concentration being the stock solution. Store aliquots at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the various TTX concentrations by serial dilution.
  • Tetracaine (Sigma) 10 nM, 100 nM, 1 µM, 10 µM, 100 µM, 1 mM made up in NSExtSNa. The 10 mM tetracaine stock solution is prepared in NSExtSNa (see recipe). Store aliquots at −20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the needed tetracaine concentrations by serial dilution
  • CHO cells expressing hNa V1.7 (Anaxon AG) or HEK cells expressing hNa V1.7 (EMD Millipore)
  • Patchliner recording station (Nanion Technologies)
  • Two EPC10 Quadro amplifiers (HEKA Elektronik)
  • Computer running the following software:
    • PatchControlHT software (Nanion Technologies)
    • PatchMaster software (HEKA Elektronik)
    • Igor software (Wavemetrics)
  • NPC®‐16 chips, resistance 2‐3.5 MΩ (Nanion Technologies)

Alternate Protocol 2: Activation of nAChα7R Using the Patchliner

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

Alternate Protocol 3: Temperature Effects on Pharmacology of nAChα7R Using the Patchliner

  Additional Materials
  • GH4C1 cells expressing nAChα7R (cells kindly provided by Thomas Seeger, Bundeswehr, Institute for Pharmacology and Toxicology, Munich, Germany)
  • NSExtSnAChα7R (see recipe)
  • 10 µM PNU120596 (Tocris) prepared in NSExtSnAChα7R (see recipe). The stock 10 mM solution of PNU120596 is prepared in DMSO. Store aliquots at –20°C. Remove a fresh aliquot from the freezer on the day of the experiment and prepare the required PNU120596 concentrations in NSExtSnAChα7R.
  • 100 µM nicotine (Sigma) + 10 µM PNU120596 (Tocris) prepared in NSExtSnAChα7R (see recipe)
  • Temperature‐control add‐on for the Patchliner

Basic Protocol 3: High‐Throughput Electrophysiological Analysis of NaV1.7 on the SyncroPatch 96

  Materials
  • Nanion standard internal solution for Na+ channels (SyncroPatch; NSIntSNaSyncro; see recipe)
  • NSExtSNa (see recipe)
  • Nanion standard external solution for Na+ channels (SyncroPatch; NSExtSNaSyncro; see recipe)
  • SES (see recipe)
  • CHO cells expressing hNa V1.7 (Anaxon AG) or HEK cells expressing hNa V1.7 (EMD Millipore)
  • SyncroPatch 96 recording station (Nanion Technologies)
  • Computer running the following software:
    • PatchControl 96 software (Nanion Technologies)
    • DataControl 96 software (Nanion Technologies)
  • NPC®‐96 chips, resistance 2‐3.5 MΩ (Nanion Technologies)

Alternate Protocol 4: High‐Throughput Electrophysiological Recordings of nAChα7R on the SyncroPatch 96

  Additional Materials
  • HEK cells expressing nAChα7R (induced; Galantos Pharma)
  • NSIntNa (see recipe)
  • NSExtSnAChα7R (see recipe)
  • 100 µM ACh (Sigma) in NSExtSnAChα7R. The 10 mM ACh stock solution is prepared in NSExtSNa (see recipe). Store aliquots at −20°C. Remove a fresh aliquot from the freezer and prepare the required ACh concentrations in NSExtSnAChα7R.
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Figures

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Literature Cited

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