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

Yi Liu1

1 Janssen Research & Development, LLC, San Diego, California
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.14
DOI:  10.1002/0471141755.ph1114s65
Online Posting Date:  June, 2014
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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. Pharmacol. 65:11.14.1‐11.14.45. © 2014 by John Wiley & Sons, Inc.

Keywords: voltage‐gated sodium channel; Nav channel; Nav1.7; QPatch; patch clamp; HTS

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Patch Clamp Studies of NAv1.7 Sodium Channels Using QPatch HT
  • Support Protocol 1: Preparing Whole‐Cell Protocols
  • Support Protocol 2: Making Voltage Protocols
  • Support Protocol 3: Preparing Compound Lists
  • Support Protocol 4: Preparing Application Protocols
  • Support Protocol 5: Assembling Jobs from Individual Components
  • Support Protocol 6: Harvesting Cells for QPatch Experiments
  • Support Protocol 7: Data Analysis
  • Reagents and Solutions
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Patch Clamp Studies of NAv1.7 Sodium Channels Using QPatch HT

  Materials
  • For job #1:
    • Whole‐cell protocol #1 ( protocol 2)
    • Voltage protocols #1, #2, #3, and #5 ( protocol 3)
    • Compound list #1 ( protocol 4)
    • Application protocol #1 ( protocol 5)
    • 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 hNa v1.7
    • Cell storage tank (Sophion, cat. no. 2050)
    • Additional reagents and equipment for harvesting cells ( protocol 7)
    • Stir bar (Sophion, cat. no. 3070)
    • Plastic tubes for on‐station centrifuge (Sophion, cat. no. 2051)
  • For job #2:
    • Whole cell protocol #1 ( protocol 2)
    • Voltage protocol #4 ( protocol 3)
    • Compound list #1 ( protocol 4)
    • Application protocol #2 ( protocol 5)
    • Additional materials as for job #1 (see above)
  • For job #3:
    • Whole‐cell protocol #1 ( protocol 2)
    • Voltage protocol #6 ( protocol 3)
    • Compound list #2 ( protocol 4)
    • Application protocol #3 ( protocol 5)
    • 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 ( protocol 2)
    • Voltage protocol #7 ( protocol 3)
    • Compound list #3 ( protocol 4)
    • Application protocol #4 ( protocol 5)
    • 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 ( protocol 6)

Support Protocol 1: Preparing Whole‐Cell Protocols

  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 hNa v1.7 (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)
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Figures

Videos

Literature Cited

Literature Cited
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  Cummins, T.R., Howe, J.R., and Waxman, S.G. 1998. Slow closed‐state inactivation: A novel mechanism underlying ramp currents in cells expressing the hNE/PN1 sodium channel. J. Neurosci. 18:9607‐9619.
  Cummins, T.R., Dib‐Hajj, S.D., and Waxman, S.G. 2004. Electrophysiological properties of mutant Nav1.7 sodium channels in painful inherited neuropathy. J. Neurosci. 24:8232‐8236.
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  Mathes, C., Friis, S., Finley, M., and Liu, Y. 2009. QPatch: The missing link between HTS and ion channel drug discovery. Comb. Chem. High Thr. Screen. 12:78‐95.
  Patton, D.E. and Goldin, A.L.A. 1991. Voltage‐dependent gating transition induces use‐dependent block by tetrodotoxin of rat IIA sodium channels expressed in Xenopus oocytes. Neuron 7:637‐647.
  Ragsdale, D.S., Mcphee, J.C., Scheuer, T., and Catterall, W.A. 1996. Common molecular determinants of local anesthetic, antiarrhythmic, and anticonvulsant block of voltage‐gated Na+ channels. Proc. Natl. Acad. Sci. U.S.A. 93:9270‐9275.
  Sheets, P.L., Jarecki, B.W., and Cummins, T.R. 2011. Lidocaine reduces the transition to slow inactivation in Na(v)1.7 voltage‐gated sodium channels. Br. J. Pharmacol. 164:719‐730.
  Xiao, Y., Bingham, J.P., Zhu, W., Moczydlowski, E., Liang, S., and Cummins, T.R. 2008. Tarantula huwentoxin‐IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration. J. Biol. Chem. 283:27300‐27313.
Internet Resources
  http://www.sophion.com/
  Web site of Sophion, supplier of QPatch.
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