Electrophysiological Analysis of Heterologously Expressed Kv and SK/IK Potassium Channels

Neil A. Castle1, Alan D. Wickenden1, Anruo Zou1

1 Icagen, Durham, North Carolina
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.5
DOI:  10.1002/0471141755.ph1105s20
Online Posting Date:  May, 2003
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Abstract

This unit describes protocols to aid investigators in determining the electrophysiological and pharmacological profile of heterologously expressed voltage or calcium‐activated potassium channels belonging to the Kv1.x and SK/IK gene families. Protocols for data acquisition as well as analysis are provided.

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Analysis of Kv1.x Voltage‐Dependent Potassium Channels by Patch‐Clamp Recording
  • Alternate Protocol 1: Use of Voltage Ramps to Elicit Currents in Analysis of Kv Channels
  • Support Protocol 1: Data Analysis of Kv1.x Potassium Channels
  • Support Protocol 2: Preparation and Maintenance of Cells
  • Basic Protocol 2: Analysis of IK/SK Calcium‐Activated Potassium Channels by Patch‐Clamp Recording
  • Alternate Protocol 2: Measuring SK/IK Channel Activity by Monitoring Holding Current
  • Support Protocol 3: Measuring Calcium Content of a Buffer
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Analysis of Kv1.x Voltage‐Dependent Potassium Channels by Patch‐Clamp Recording

  Materials
  • Internal patch pipet solution for voltage‐dependent potassium channels (see recipe)
  • Extracellular buffer for voltage‐dependent potassium channels (see recipe)
  • Cells stably or transiently expressing heterologous Kv potassium channel, grown on glass coverslips (see protocol 4)
  • Test compounds in extracellular buffer: add compounds from 10 to 100 mM stocks in DMSO; dilute in recipeextracellular buffer so that DMSO is ≤0.1% final
  • Patch microelectrodes (see )
  • Electrophysiology setup including recording chamber and drug delivery manifold (see ) with data analysis software
  • Inverted microscope

Alternate Protocol 1: Use of Voltage Ramps to Elicit Currents in Analysis of Kv Channels

  Materials
  • Inhibitor (see entry for Test Compounds under protocol 1 materials)
  • Software packages such as Clampfit (Axon Instruments) or PulseFit (HEKA) with supporting analysis and graphing achieved with Excel and Origin (OriginLab) or Sigmaplot
  • Additional reagents and equipment for analysis of voltage‐dependent potassium channels (see protocol 1)

Support Protocol 1: Data Analysis of Kv1.x Potassium Channels

  Materials
  • LMTK cell line (ATCC# CCL‐1.3)
  • Dulbecco's modified Eagle medium (DMEM; Life Technologies or Hyclone), high‐glucose formulation, supplemented with 10% heat‐inactivated (1 hr at 57°C) fetal bovine serum (FBS; appendix 2A)
  • Dulbecco's calcium and magnesium free phosphate buffered saline (CMF‐PBS; Life Technologies or Hyclone)
  • Trypsin/EDTA (Life Technologies or Hyclone)
  • 25‐cm2 tissue culture flasks
  • 10% CO 2 /90% O 2 incubator
  • 35‐mm tissue culture dishes
  • Round glass coverslips (optionally coated with poly‐D‐lysine; see recipe)
NOTE: All solutions and equipment coming into contact with live cells must be sterile, and proper aseptic technique should be used accordingly.

Support Protocol 2: Preparation and Maintenance of Cells

  Materials
  • Internal patch pipet solution for calcium‐dependent potassium channels (see recipe): INT buffer 1 for studying inhibitory pharmacology and INT buffer 2 for studying channel activator pharmacology
  • Extracellular buffer for calcium‐dependent potassium channels (see recipe): EX buffer 1 or EX buffer 2 (see recipe)
  • CHO cells stably expressing human IK1 or SK or calcium activated potassium channels grown on glass coverslips (see protocol 4).
  • Test compounds (including reference inhibitor; see step below) in extracellular buffer: add compounds from 10 to 100 mM stocks in DMSO; dilute in extracellular buffer so that DMSO is ≤0.1% final
  • Patch microelectrodes (see Strategic Planning)
  • Electrophysiology setup including recording chamber and drug delivery manifold (see Strategic Planning) with data analysis software
  • Inverted microscope

Basic Protocol 2: Analysis of IK/SK Calcium‐Activated Potassium Channels by Patch‐Clamp Recording

  Materials
  • Oregon Green 488 BAPTA‐2 “cell impermeant” (Molecular Probes)
  • Dimethyl sulfoxide (DMSO; Sigma)
  • Calcium‐ and magnesium‐free phosphate‐buffered saline (CMF‐PBS; Life Technologies or Hyclone)
  • Calcium buffer calibration standards: 11 premade buffers of known free calcium concentration, ranging from 10−8 to 10−4 M (CALBUF‐2; World Precision Instruments)
  • 96‐well microtiter plate
  • Fluorescence plate reader (e.g., FLIPR; unit 9.2)
  • Data analysis software (Origin from OriginLab, or Sigmaplot)
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Figures

Videos

Literature Cited

   Axon Instruments. 1993 The Axon Guide. Axon Instruments, Union City, Calif.
   Beeton, C., Wulff, H., Barbaria, J., Clot‐Faybesse, O., Pennington, M., Bernard, D., Cahalan, M.D., Chandy, K.G., and Beraud, E. 2001. Selective blockade of T lymphocyte K+ channels ameliorates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. Proc. Natl. Acad. Sci. U.S.A. 98:13942.
   Cahalan, M.D. and Chandy, K.G. 1997. Ion channels in the immune system as targets for immunosuppression. Curr. Opin. Biotechnol. 8:749‐756.
   Castle, N.A. 1999. Recent advances in the biology of small conductance calcium‐activated potassium channels. Perspect. Drug Discov. Des. 15/16:131‐154.
   Castle, N.A., Fadous, S., Logothetis, D.E., and Wang, G.K. 1994. Aminopyridine block of Kv1.1 potassium channels expressed in mammalian cells and Xenopus oocytes. Mol Pharmacol. 46:1175‐1181.
   Chen, J.Q., Galanakis, D., Ganellin, C.R., Dunn, P.M., and Jenkinson, D.H. 2000. bis‐Quinolinium cyclophanes: 8,14‐diaza‐1,7(1, 4)‐diquinolinacyclotetradecaphane (UCL 1848), a highly potent and selective, nonpeptidic blocker of the apamin‐sensitive Ca2+‐activated K+ channel. J. Med. Chem. 43:3478‐3481.
   Coleman, S.K., Newcombe, J., Pryke, J., and Dolly, J.O. 1999. Subunit composition of Kv1 channels in human CNS. J. Neurochem. 73:849‐858.
   Courtemanche, M., Ramirez, R.J., and Nattel, S. 1999. Ionic targets for drug therapy and atrial fibrillation‐induced electrical remodeling: Insights from a mathematical model. Cardiovasc. Res. 42:477‐489.
   Devor, D.C., Singh, A.K., Gerlach, A.C., Frizzell, R.A., and Bridges, R.J. 1997. Inhibition of intestinal Cl– secretion by clotrimazole: Direct effect on basolateral membrane K+ channels. Am. J. Physiol. 273:C531‐40.
   Grissmer, S., Nguyen, A.N., Aiyar, J., Hanson, D.C., Mather, R.J., Gutman, G.A., Karmilowicz, M.J., Auperin, D.D., and Chandy, K.G. 1994. Pharmacological characterization of five cloned voltage‐gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5, and 3.1, stably expressed in mammalian cell lines. Mol. Pharmacol. 45:1227‐1234.
   Grunnet, M., Jensen, B.S., Olesen, S.P., and Klaerke, D.A. 2001. Apamin interacts with all subtypes of cloned small‐conductance Ca2+‐activated K+ channels. Pflugers Arch. 441:544‐550.
  Haugland, R.P. (ed.) 2002. Handbook of Fluorescent Probes and Research Products, 9th ed. Molecular Probes, Eugene, Oreg.
   Heinemann, S.H., Rettig, J., Graack, H.R., and Pongs, O. 1996. Functional characterization of Kv channel beta‐subunits from rat brain. J. Physiol. 493:625‐633.
   Hopkins, W.F. 1998. Toxin and subunit specificity of blocking affinity of three peptide toxins for heteromultimeric, voltage‐gated potassium channels expressed in Xenopus oocytes. J. Pharmacol. Exp. Ther. 285:1051‐1060.
   Ishii, T.M., Silvia, C., Hirschberg, B., Bond, C.T., Adelman, J.P., and Maylie, J. 1997. A human intermediate conductance calcium‐activated potassium channel. Proc. Natl. Acad. Sci. U.S.A. 94:11651‐11656.
   Jensen, B.S., Strobaek, D., Olesen, S.P., and Christophersen, P. 2001. The Ca2+‐activated K+ channel of intermediate conductance: A molecular target for novel treatments? Curr. Drug Targets. 2:401‐422.
   Khanna, R., Chang, M.C., Joiner, W.J., Kaczmarek, L.K., and Schlichter, L.C. 1999. hSK4/hIK1, a calmodulin‐binding KCa channel in human T lymphocytes: Roles in proliferation and volume regulation. J. Biol. Chem. 274:14838‐14849.
   Koren, G., Liman, E.R., Logothetis, D.E., Nadal‐Ginard, B., and Hess, P. 1990. Gating mechanism of a cloned potassium channel expressed in frog oocytes and mammalian cells. Neuron 4:39‐51.
   Nashmi, R. and Fehlings, M.G. 2001. Mechanisms of axonal dysfunction after spinal cord injury: With an emphasis on the role of voltage‐gated potassium channels. Brain Res. Brain Res. Rev. 38:165‐191.
   Rasband, M.N. and Trimmer, J.S. 2001. Subunit composition and novel localization of K+ channels in spinal cord. J. Comp. Neurol. 429:166‐176.
   Snyders, D.J., Tamkun, M.M., and Bennett, P.B. 1993. A rapidly activating and slowly inactivating potassium channel cloned from human heart: Functional analysis after stable mammalian cell culture expression. J. Gen. Physiol. 101:513‐543.
   Strobaek, D., Jorgensen, T.D., Christophersen, P., Ahring, P.K., and Olesen, S.P. 2000. Pharmacological characterization of small‐conductance Ca2+‐activated K+ channels stably expressed in HEK 293 cells. Br. J. Pharmacol. 129:991‐999.
   Syme, C.A., Gerlach, A.C., Singh, A.K., and Devor, D.C. 2000. Pharmacological activation of cloned intermediate‐ and small‐conductance Ca2+‐activated K+ channels. Am. J. Physiol. Cell Physiol. 278:570‐581.
   Wang, Z., Fermini, B., and Nattel, S. 1993. Sustained depolarization‐induced outward current in human atrial myocytes: Evidence for a novel delayed rectifier K+ current similar to Kv1.5 cloned channel currents. Circ. Res. 73:1061‐1076.
   Werkman, T.R., Gustafson, T.A., Rogowski, R.S., Blaustein, M.P., and Rogawski, M.A. 1993. Tityustoxin‐K alpha, a structurally novel and highly potent K+ channel peptide toxin, interacts with the alpha‐dendrotoxin binding site on the cloned Kv1.2 K+ channel. Mol. Pharmacol. 44:430‐436.
Internet Resources
  http://www.axon.com
  The Axon Instruments Web site provides information on their product line of electrophysiology equipment as well as a comprehensive guide for conducting electrophysiological experiments.
  http://www.axon.com/MR_Axon_Guide.html
  Additional information about patch clamp electrophysiological protocols can be found at the following Web sites
  http://usa.biologists.com/Micro/index.html
  Web site of the Microelectrode Techniques Handbook. Contains additional information about patch clamp electrophysiological protocols.
  http://web.ukonline.co.uk/a.hughes/patchwork/patchwork.htm
  “Patch Works” Web site containing the online booklet: Patch Clamping, A Guide. Contains additional information about patch clamp electrophysiological protocols.
  http://www.atcc.org/home.cfm
  Web site of American Type Culture Collection (ATCC). Comprehensive supplier of cell lines.
  http://www.stanford.edu/∼cpatton/webmaxc2.htm
  Web site of online calculator for estimating free calcium concentration in solutions containing calcium chelators.
  http://www.gene.ucl.ac.uk/nomenclature/genefamily/KCN.shtml
  HUGO Gene Nomenclature Committee Web site provides information/updates on the gene families and nomenclature for potassium channels.
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