Electrophysiological Analysis of ATP‐Sensitive Potassium Channels in Mammalian Cells and Xenopus Oocytes

Char‐Chang Shieh1, Murali Gopalakrishnan1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 11.6
DOI:  10.1002/0471141755.ph1106s21
Online Posting Date:  July, 2003
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Abstract

This unit describes general methodologies for the characterization of ATP‐sensitive K+ channels and the study of ligand‐channel interactions in native tissues and clonal cell lines by electrophysiological techniques. Detailed protocols on how to establish patch‐clamp single‐channel and whole‐cell current recording are presented. Two‐electrode voltage clamp techniques for studying ATP‐sensitive K+ channels expressed in Xenopus oocytes are also included.

Keywords: ATP‐sensitive K+ channels; patch clamp; single‐channel recording; whole‐cell current recording; perforated patch; two‐electrode voltage‐clamp technique; Xenopus oocytes; electrophysiology; potassium channels; K+ channels

     
 
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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: Patch‐Clamp Single‐Channel Recording for KATP Channels
  • Alternate Protocol 1: Inside‐Out Macropatch Recording for KATP Channels
  • Alternate Protocol 2: Whole‐Cell Patch Clamp Recording of KATP Currents
  • Alternate Protocol 3: Perforated‐Patch Recording of KATP Channels
  • Basic Protocol 2: Two‐Electrode Voltage Clamp in Xenopus Oocytes
  • Basic Protocol 3: Constant Holding Potential Voltage‐Clamp Recording for KATP Currents
  • Alternate Protocol 4: Step Voltage Pulse Protocol for KATP Current Recording
  • Alternate Protocol 5: Ramp Protocols for KATP Current Recording
  • Support Protocol 1: Analysis of Data from KATP Channel Recordings
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Patch‐Clamp Single‐Channel Recording for KATP Channels

  • Sylgard (184 silicone elastomer; Dow Corning)
  • Single‐channel pipet solution (see recipe)
  • Single‐channel bath solution (see recipe)
  • HEK293 cells expressing channel of interest, (for example, Gopalakrishnan et al. ), for human SUR1‐Kir6.2
  • Test compound(s) appropriate for channel of interest
  • Corning 7056 glass capillary tubing: outer diameter (o.d.), 1.65 mm; inner diameter (i.d.), 1.1 mm; 100 mm long (Warner Instrument)

Alternate Protocol 1: Inside‐Out Macropatch Recording for KATP Channels

  • Inside‐out pipet solution (see recipe)
  • Inside‐out bath solution (see recipe)

Alternate Protocol 2: Whole‐Cell Patch Clamp Recording of KATP Currents

  • Whole‐cell pipet solution (see recipe)
  • Whole‐cell bath solution (see recipe)

Alternate Protocol 3: Perforated‐Patch Recording of KATP Channels

  • 20 mg/ml amphotericin B (Sigma) in DMSO, prepared fresh on day of experiment
  • Perforated‐patch pipet solution (see recipe)
  • Perforated‐patch bath solution (see recipe)
  • Ultrasonicator (e.g., Bransonic Ultrasonic Cleaner 1510; Branson Ultrasonics)

Basic Protocol 2: Two‐Electrode Voltage Clamp in Xenopus Oocytes

  • Female Xenopus laevis frogs (NASCO)
  • 1‐liter beaker containing 500 ml water and 1.4 g tricane (3‐aminobenzoic acid ethyl ester; Sigma)
  • Ca2+‐free Barth's solution (see recipe)
  • 15‐ml centrifuge tube containing 10 ml of 2 mg/ml type 1A collagenase (Sigma) in Ca2+‐free Barth's solution (see recipe)
  • 2 mg/ml type 1A collagenase (Sigma) in Ca2+‐free Barth's solution (see recipe)
  • Barth's solution (see recipe)
  • 0.01 µg/µl cRNA encoding Kir6.2 or Kir6.1
  • 0.05 µg/µl cRNA encoding SUR1, SUR2A, or SUR2B
  • 3 M KCl
  • Two‐electrode bath solution (see recipe)
  • Test compound(s) appropriate for channel of interest
  • Dissection board, ice cold
  • Sterile dissection instruments, including scissors and forceps
  • 35‐mm and 100 × 15–mm petri dishes
  • Rocking platform (e.g., model no. 100; VWR Scientific)
  • Dissection microscope
  • Borosilicate glass capillary tubes: outer diameter (o.d.), 1.0 mm; inner diameter (i.d.), 0.5 mm (Sutter Instruments), for cRNA injection
  • Fine forceps
  • Drummond Nanoject injector (Drummond Scientific) attached to micromanipulator
  • Incubator, 14° to 19°C
  • Borosilicate glass capillary tubes with filaments: o.d., 1.5 mm; i.d., 1.1 mm (Sutter Instruments), for two‐electrode whole‐cell recordings

Basic Protocol 3: Constant Holding Potential Voltage‐Clamp Recording for KATP Currents

  Materials
  • Test compound(s) appropriate for channel of interest, such as K ATP channel openers (KCOs) and K ATP channel blockers
  • Electrophysiology setup, including cells or oocytes expressing channel of interest, for inside‐out macropatch recording, whole‐cell patch clamp, or two‐electrode voltage clamp (see Strategic Analysis, see protocol 5, and see protocol 2Alternate Protocols 1 and protocol 32)
  • pClamp software (Axon Instruments), including Clampex

Alternate Protocol 4: Step Voltage Pulse Protocol for KATP Current Recording

  Materials
  • Electrophysiology setup, including cell or oocyte expressing channel of interest (see protocol 5, and see protocol 2Alternate Protocols1 and protocol 32)
  • Test compound(s) appropriate for channel of interest, such as K ATP channel openers (KCOs) and sulfonylurea K ATP channel blockers glyburide and tolbutamide
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Figures

Videos

Literature Cited

Literature Cited
   Aguilar‐Bryan, L., Clement, J.P. IV, Gonzalez, G., Kunjilwar, K., Babenko, A., and Bryan, J. 1998. Toward understanding the assembly and structure of KATP channels. Physiol. Rev. 78:227‐245.
   Ashcroft, F.M. and Rorsman, P. 1990. ATP‐sensitive K+ channels: A link between β‐cell metabolism and insulin secretion. Biochem. Soc. Trans. 18:109‐111.
   Asmild, M., Oswald, N., Korsgaard, M.P.G., Krzywkowski, K.M., Friis, S., Jacobsen, R.B., Reuter, D., Taboryski, R., Kutchinsky, J., Vestergaard, R.K., Schroder, R.L., Sorensen, C.B., Bech, M., and Willumsen, N.J. 2003. Upscaling and automation of electrophysiology: Toward high throughput screening in ion channels drug discovery. Receptors Channels 9:49‐58.
   Babenko, A.P., Gonzalez, G., Aguilar‐Bryan, L., and Bryan, J. 1998. Reconstituted human cardiac KATP channels: Functional identity with the native channels from the sarcolemma of human ventricular cells. Circ. Res. 83:1132‐1143.
   Bonev, A.D. and Nelson, M.T. 1993. ATP‐sensitive potassium channels in smooth muscle cells from guinea pig urinary bladder. Am. J. Physiol. 264:C1190‐C1200.
   Coghlan, M., Carroll, W.A., and Gopalakrishnan, M. 2001. Recent developments in the biology and medicinal chemistry of potassium channel modulators: Update from a decade of progress. J. Med. Chem. 44:1627‐1653.
   Gopalakrishnan, M., Whiteaker, K.L., Molinari, E.J., Davis‐Taber, R., Scott, V.E., Shieh, C.C., Buckner, S.A., Milicic, I., Cain, J.C., Postl, S., Sullivan, J.P., and Brioni, J.D. 1999. Characterization of the ATP‐sensitive potassium channels (KATP) expressed in guinea pig bladder smooth muscle cells. J. Pharmacol. Exp. Ther. 289:551‐558.
   Gopalakrishnan, M., Molinari, E.J., Shieh, C.C., Monteggia, L.M., Roch, J.M., Whiteaker, K.L., Scott, V.E., Sullivan, J.P., and Brioni, J.D. 2000. Pharmacology of human sulphonylurea receptor SUR1 and inward rectifier K+ channel Kir6.2 combination expressed in HEK‐293 cells. Br. J. Pharmacol. 129:1323‐1332.
   Gopalakrishnan, M., Davis‐Taber, R., Molinari, E.J., Whiteaker, K.L., Buckner, S.A., Shieh, C.C., Scott, V.E.S., Rotert, G., Altenbach, R., Coghlan, M., and Carroll, W.A. 2003. [125I]A‐312110: A novel high affinity 1,4‐dihydropyridine ATP‐sensitive K+ channel opener: Characterization and pharmacology of binding. Mol. Pharm. (in press).
   Gribble, F.M., Ashfield, R., Ammala, C., and Ashcroft, F.M. 1997. Properties of cloned ATP‐sensitive K+ currents expressed in Xenopus oocytes. J. Physiol. 498:87‐98.
   Grover, G.J. and Garlid, K.D. 2000. ATP‐sensitive potassium channels: A review of their cardioprotective pharmacology. J. Mol. Cell. Cardiol. 32:677‐695.
   Gundersen, C.B., Miledi, R., and Parker, I. 1983. Voltage‐operated channels induced by foreign messenger RNA in Xenopus oocytes. Proc. R. Soc. Lond. B 220:131‐140.
   Hamill, O.P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F.J. 1981. Improved patch‐clamp techniques for high‐resolution current recording from cells and cell‐free membrane patches. Pflugers Arch. 391:85‐100.
   Hilgemann, D.W. 1995. The giant membrane patch. In Single‐Channel Recording, 2nd ed. (B. Sakmann and E. Neher, eds.) pp. 307‐327. Plenum Press, New York.
   Hille, B. 2001. Ion Channels of Excitable Membranes, 3rd ed. Sinauer Associates, Sunderland, Mass.
   Hogg, R.C. and Adams, D.J. 2001. An ATP‐sensitive K+ conductance in dissociated neurons from adult rat intracardiac ganglia. J. Physiol. 534:713‐720.
   Horn, R. and Korn, S.J. 1992. Prevention of rundown in electrophysiological recording. Methods Enzymol. 207:149‐155.
   Horn, R. and Marty, A. 1988. Muscarinic activation of ionic currents measured by a new whole‐cell recording method. J. Gen. Physiol. 92:145‐159.
   Inagaki, N., Gonoi, T., Clement, J.P. IV, Namba, N., Inazawa, J., Gonzalez, G., Aguilar‐Bryan, L., Seino, S., and Bryan, J. 1995. Reconstitution of IKATP: An inward rectifier subunit plus the sulfonylurea receptor. Science 270:1166‐1170.
   Inagaki, N., Gonoi, T., Clement, J.P., Wang, C.Z., Aguilar‐Bryan, L., Bryan, J., and Seino, S. 1996. A family of sulfonylurea receptors determines the pharmacological properties of ATP‐sensitive K+ channels. Neuron 16:1011‐1017.
   Isomoto, S., Kondo, C., Yamada, M., Matsumoto, S., Higashiguchi, O., Horio, Y., Matsuzawa, Y., and Kurachi, Y. 1996. A novel sulfonylurea receptor forms with BIR (Kir6.2) a smooth muscle type ATP‐sensitive K+ channel. J. Biol. Chem. 271:24321‐24324.
   Kiss, L., Bennett, P.B., Uebele, V.N., Koblan, K.S., Kane, S.A., Neagle, B., and Schroeder, K. 2003. High throughput ion‐channel pharmacology: Planar‐array‐based voltage clamp. Assay Drug Dev. Technol. 1:127‐135.
   Klemic, K.G., Klemic, J.F., Reed, M.A., and Sigworth, F.J. 2002. Micromolded PDMS planar electrode allows patch clamp electrical recordings from cells. Biosens. Bioelectron. 17:597‐604.
   Noma, A. 1983. ATP‐regulated K+ channels in cardiac muscle. Nature 305:147‐148.
   Nuttle, L.C. and Farley, J.M. 1997. Muscarinic receptors inhibit ATP‐sensitive K+ channels in swine tracheal smooth muscle. Am. J. Physiol. 273:L478‐L484.
   Quayle, J.M., Nelson, M.T., and Standen, N.B. 1997. ATP‐sensitive and inwardly rectifying potassium channels in smooth muscle. Physiol. Rev. 77:1165‐1232.
   Rae, J., Cooper, K., Gates, P., and Watsky, M. 1991. Low access resistance perforated patch recordings using amphotericin B. J. Neurosci. Methods 37:15‐26.
   Sakmann, B. and Neher, E. 1995. Single‐Channel Recording, 2nd ed. Plenum Press, New York.
   Schnizler, K., Fejtl, M., Kuster, M., and Methfessel, C. 2003. The Roboocyte: Automated cDNA/mRNA injection and subsequent TEVC recording on Xenopus oocytes in 96‐well microtiter plates. Receptors Channels 9:41‐48.
   Seino, S. 1999. ATP‐sensitive potassium channels: A model of heteromultimeric potassium channel/receptor assemblies. Annu. Rev. Physiol. 1:337‐362.
   Shieh, C.C., Coghlan, M., Sullivan, J.P., and Gopalakrishnan, M. 2000. Potassium channels: Molecular defects, diseases, and therapeutic opportunities. Pharmacol. Rev. 52:557‐594.
   Shieh, C.C., Feng, J., Buckner, S.A., Brioni, J.D., Coghlan, M.J., Sullivan, J.P., and Gopalakrishnan, M. 2001. Functional implication of spare ATP‐sensitive K+ channels in bladder smooth muscle cells. J. Pharmacol. Exp. Ther. 296:669‐675.
   Shih, T.M., Smith, R.D., Toro, L., and Goldin, A.L. 1998. High‐level expression and detection of ion channels in Xenopus oocytes. Methods Enzymol. 293:529‐556.
   Shindo, T., Yamada, M., Isomoto, S., Horio, Y., and Kurachi, Y. 1998. SUR2 subtype (A and B)‐dependent differential activation of the cloned ATP‐sensitive K+ channels by pinacidil and nicorandil. Br. J. Pharmacol. 124:985‐991.
   Shyng, S., Ferrigni, T., and Nichols, C.G. 1997. Control of rectification and gating of cloned KATP channels by the Kir6.2 subunit. J. Gen. Physiol. 110:141‐153.
   Spanswick, D., Smith, M.A., Groppi, V.E., Logan, S.D., and Ashford, M.L. 1997. Leptin inhibits hypothalamic neurons by activation of ATP‐sensitive potassium channels. Nature 390:521‐525.
   Terstappen, G.C. 1999. Functional analysis of native and recombinant ion channels using a high‐capacity nonradioactive rubidium efflux assay. Anal. Biochem. 272:149‐155.
   Trumbull, J.D., Bertrand, D., Maslana, E.S., McKenna, D.G., Nemcek, T.A., Niforatos, W., Pan, J.Y., Parihar, A.S., Shieh, C.C., Wilkins, J.A., and Briggs, C.A. 2003. High throughput electrophysiology using a fully automated, multiplexed recording system. Receptors Channels 9:19‐28.
   Whiteaker, K.L., Gopalakrishnan, S.M., Groebe, D., Shieh, C.C., Warrior, U., Burns, D.J., Coghlan, M.J., Scott, V.E., and Gopalakrishnan, M. 2001. Validation of FLIPR membrane potential dye for high throughput screening of potassium channel modulators. J. Biomol. Screen. 6:305‐312.
   Yagupolskii, L.M., Antepohl, W., Artunc, F., Handrock, R., Klebanov, B.M., Maletina, I.I., Marxen, B., Petko, K.I., Quast, U., Vogt, A., Weiss, C., Zibold, J., and Herzig, S. 1999. Vasorelaxation by new hybrid compounds containing dihydropyridine and pinacidil‐like moieties. J. Med. Chem. 42:5266‐5271.
   Yamada, M., Isomoto, S., Matsumoto, S., Kondo, C., Shindo, T., Horio, Y., and Kurachi, Y. 1997. Sulphonylurea receptor 2B and Kir6.1 form a sulphonylurea‐sensitive but ATP‐insensitive K+ channel. J. Physiol. 499:715‐720.
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