Characterization of Potassium Channel Binding

Victoria E.S. Scott1

1 Abbott Laboratories, Abbott Park, Illinois
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 1.17
DOI:  10.1002/0471141755.ph0117s06
Online Posting Date:  May, 2001
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Abstract

This unit provides an introduction to the binding of specific ligands to K+ channels that are expressed in a number of different preparations. The assays described can be used for screening a large number of compounds for interaction at specific binding sites and provides, in selected cases, details of expected results for unlabeled ligands that are known to bind to certain K+ channels.

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Measurement of the Binding of [125I]Iberiotoxin to Rat Brain Membranes
  • Support Protocol 1: Preparation of Synaptic Plasma Membranes from Rat Cerebral Cortex
  • Basic Protocol 2: Displacement Analysis of [125I]-DTX to Solubilized Rat Brain Synaptic Membranes
  • Basic Protocol 3: Displacement Analysis of [3H]P 1075 Binding to Cultured A10 Cells
  • Support Protocol 2: Culturing A10 Cells
  • Basic Protocol 4: Displacement Binding of [3H]Glyburide to RINm5F Cell Membranes
  • Support Protocol 3: Culturing RINm5F Cells
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Measurement of the Binding of [125I]Iberiotoxin to Rat Brain Membranes

 Materials
  • Rat brain synaptic plasma membranes (see Support Protocol 1)
  • Unlabeled test compounds
  • IbTX assay buffer (see recipe)
  • [125I]IbTX-D19Y/Y36F, (2200 Ci/mmol; NEN Life Sciences)
  • IbTX (Research Biochemicals)
  • 0.5% (w/v) polyethyleneimine (PEI; Sigma) in H2O
  • Wash buffer: 50 mM Tris×Cl (pH 7.2; appendix 2A), ice-cold (store up to 2 months at 4°C)
  • counter (Wallac or equivalent)
  • Glass fiber filters (Whatman GF/B)
  • 96-well cell harvester with tubes (Skatron)
  • Four-parameter logistics software program (e.g., GraphPad Prism)
  • Additional reagents and equipment for protein assay (appendix 3A) and calculation of IC50 and Ki (unit 1.3)

Support Protocol 1: Preparation of Synaptic Plasma Membranes from Rat Cerebral Cortex

 Materials
  • Sprague-Dawley rats (180 to 200 g; 12 rats needed)
  • Membrane homogenization buffer (see recipe), ice-cold
  • Membrane lysis buffer (see recipe), ice-cold
  • 24% and 48% sucrose solutions (see recipe)
  • 5 mM Tris×Cl, pH 8.2 (appendix 2A)
  • Dissecting instruments
  • Polytron homogenizer (Brinkmann)
  • Beckman ultracentrifuge and 45Ti rotor with two 45Ti ultracentrifuge tubes (or equivalent centrifuge, rotor, and tubes)
  • Beckman SW 25Ti rotor with four SW 25Ti centrifuge tubes (or equivalents)
  • Sorvall RC-5 centrifuge and SS-34 rotor with two SS-34 centrifuge tubes (or equivalents)

Basic Protocol 2: Displacement Analysis of [125I]-DTX to Solubilized Rat Brain Synaptic Membranes

 Materials
  • Rat brain synaptic plasma membranes (see Support Protocol 1)
  • 1× -DTX receptor solubilization buffer (see recipe), ice-cold
  • [125I]-DTX (2000 Ci/mmol; Amersham Life Sciences)
  • Unlabeled -DTX (Alomone Labs)
  • Unlabeled test compounds
  • -DTX wash buffer (see recipe)
  • 1.2 mg/ml cytochrome c in H2O
  • Sephadex G-100 resin (Amersham Pharmacia Biotech; allow to swell in H2O for a minimum of 24 hr prior to use)
  • Beckman ultracentrifuge and 50Ti rotor with two 50Ti ultracentrifuge tubes (or equivalent centrifuge, rotor, and tubes)
  • 1.5-ml silanized microcentrifuge tubes (see recipe)
  • 3.0-ml polypropylene disposable syringes
  • Whatman filter paper (cut into small circles using a hole puncher)
  • 15-ml polypropylene centrifuge tubes (Falcon)
  • Sorvall RT-6000 centrifuge (or equivalent)
  • 5-ml plastic -counting tubes
  • counter (Wallac or equivalent)
  • Four-parameter logistics software program (e.g., GraphPad Prism)
  • Additional reagents and equipment for protein assay (appendix 3A) and calculation of IC50 and Ki (unit 1.3)

Basic Protocol 3: Displacement Analysis of [3H]P 1075 Binding to Cultured A10 Cells

 Materials
  • Rat aortic cell line (A10 cells) growing in 162-cm2 flasks (see Support Protocol 2)
  • A10 cell growth medium (see recipe)
  • Unlabeled test compound
  • Unlabeled P 1075 or pinacidil (Research Biochemicals)
  • P 1075 assay buffer: 0.1 M HEPES×HCl, pH 7.4 (store at 4°C)
  • 50 µM [3H]P 1075 in ethanol (80 to 120 Ci/mmol; Amersham)
  • Wash buffer: 50 mM HEPES×HCl, pH 7.4 (store at 4°C)
  • 0.1 N NaOH
  • 0.1 N HCl
  • Ecolume scintillation cocktail (ICN Biomedicals)
  • 3.5-ml Pyrex tubes (VWR)
  • 12-well tissue culture plates (Costar)
  • 20-ml glass scintillation vials
  • scintillation counter (Beckman)

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Support Protocol 2: Culturing A10 Cells

 Materials
  • Rat aortic cell line A10 (ATCC #CRL 1476)
  • A10 cell growth medium (see recipe), 37°C
  • Cell dissociation buffer (see recipe)
  • Tabletop centrifuge
  • 25- and 162-cm2 tissue culture flasks

NOTE: All culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise specified.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.

Basic Protocol 4: Displacement Binding of [3H]Glyburide to RINm5F Cell Membranes

 Materials
  • Rat pancreatic insulinoma cell line (RINm5F cells) growing in 162-cm2 flasks (see Support Protocol 3) at 90% confluency
  • Assay buffer: 50 mM Tris×Cl, pH 7.2 (appendix 2A), ice-cold
  • Unlabeled test compounds
  • 5 nM [3H]glyburide (80 to 120 Ci/mmol; NEN Life Sciences)
  • 100 µM glyburide (unlabeled; Research Biochemicals)
  • Ecolume scintillation cocktail (ICN Biomedicals)
  • Cell scraper III (Costar)
  • Polytron homogenizer (Brinkmann)
  • Sorvall RC-5 centrifuge and SM-24 rotor with two SM-24 centrifuge tubes
  • 96-well cell harvester with tubes (Skatron)
  • Glass scintillation vials
  • scintillation counter (LS 5000 TD, Beckman)
  • Additional reagents and equipment for protein assay (appendix 3A) and calculation of KD and Bmax (unit 1.3)

Support Protocol 3: Culturing RINm5F Cells

 Materials
  • RIN5mF cells (ATCC #CRL 11605; supplied frozen in a 1-ml ampule)
  • RINm5F cell growth medium (see recipe); 37°C
  • Cell dissociation buffer (see recipe)
  • Tabletop centrifuge
  • 25- and 162-cm2 tissue culture flasks

NOTE: All tissue culture incubations are performed in a humidified 37°C, 5% CO2 incubator unless otherwise noted.

NOTE: All solutions and equipment coming into contact with living cells must be sterile, and aseptic technique should be used accordingly.
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Figures

  •  FigureFigure 1.17.1 Displacement of [3H]P 1075 whole-cell binding to KATP channels in rat A10 cells using P 1075 and two potassium channel–opener compounds. The data was fitted using GraphPad Prism program. Figure kindly contributed by R. Davis Taber.
    View Image
  •  FigureFigure 1.17.2 Inhibition of the binding of [3H]glyburide to KATP channels in isolated RINm5F membranes by a selection of sulfonylureas. The data was fitted using GraphPad Prism. Figure kindly contributed by E. Molinari.
    View Image

Videos

Literature Cited

 Literature Cited
    Aguilar-Bryan, L., Clement, J.P., Gonzalez, G., Kunjilwar, K., Babenko, A., and Bryan, J. 1998. Towards understanding the assembly and structure of KATP channels. Physiol. Rev. 78:227-245.
    Black, A.R., Denny, B.J., Donegan, C.M., and Dolly, J.O. 1988. Solubilization and physical characterization of acceptors for dendrotoxin and -bungarotoxin from synaptic membranes from rat brain. Biochemistry 27:6814-6820.
    Cheng, Y.C. and Prusoff, W.H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition of an enzymatic reaction. Biochem. Pharmacol. 22:3099-3108.
    Dolly, J.O. and Parcej, D.N. 1996. Molecular properties of voltage-gated K+ channels. J. Bioenerg. Biomembr. 28:231-253.
    Halliwell, J.V., Othman, I.B., Pelchen-Matthews, A., and Dolly, J.O. 1986. Central action of dendrotoxin: Selective reduction of transient K+ conductance in hippocampus and binding to localized acceptors. Proc. Natl. Acad. Sci. U.S.A. 83:493-497.
    Hulme, E.C. and Buckley, N.J. 1992. Receptor preparation for binding studies. In A Practical Approach to Receptor-Ligand Interactions (E.C. Hulme, ed.) pp. 177-212. Oxford University Press, New York.
    Isomoto, S., Kondo, C., and Kurachi, Y. 1997. Inwardly rectifying potassium channels: Their molecular heterogeneity and function. Jpn. J. Physiol. 47:11-19.
    Jones, D.H. and Matus, A.A. 1974. Isolation of synaptic plasma membranes from brain by combined floatation-sedimentation density gradient centrifugation. Biochem. Biophys. Acta 356:276-287.
    Koschak, A., Koch, R.O., Liu, J., Kaczorowski, G.J., Reinhart, P.H., Garcia, M.L., and Knaus, H.G. 1997. [125I]Iberiotoxin-D19Y/Y36F, the first selective, high specific activity radioligand for high-conductance calcium-activated potassium channels. Biochemistry 36:1943-1952.
    Luzi, L. and Pozza, G. 1997. Glibenclamide: An old drug with a novel mechanism of action? Acta Diabetol. 34:239-244.
    Mehraban, F., Breeze, A.L., and Dolly, J.O. 1984. Identification by cross-linking of a neuronal acceptor protein for dendrotoxin, a convulsant polypeptide. FEBS Lett. 174:116-122.
    Miller, T.J., Taber, R.D., Molinari, E.J., Whiteaker, K.L., Monteggia, L.M., Scott, V.E.S., Brioni, J.D., Sullivan, J.P., and Gopalakrishnan, M. 1999. Pharmacological and molecular characterization of ATP sensitive potassium channels in the TE671 human medulloblastoma cell line. Eur. J. Pharmacol. 370:179-185.
    Muller, G. Hartz, D., Punter, J., Okonomopulos, R., and Kramer, W. 1994. Differential interaction of glimepiride and glibenclamide with the beta cell sulfonylurea receptor. I Binding characteristics. Biochim. Biophys. Acta 1191:267-277.
    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.
    Russ, U., Metzger, F., Kickenweiz, E., Hambrock, A., Krippeit-Drews, P., and Quast, U. 1997. Binding and effects of KATP channel openers in the vascular smooth muscle cell line, A10. Br. J. Pharmacol. 122:1110-1126.
 Key References
    Black et al., 1988. See above.

[125I]-DTX binding: Describes in detail the binding of -DTX to solubilized rat brain synaptosomes and gives details of the conditions that are required for optimum receptor solubilization and binding.

    Koschak et al., 1997. See above.

[125I]Iberiotoxin-D19Y/Y36F binding: Provides the first clear and comprehensive description of the binding properties of [125I]-Iberiotoxin-D19Y/Y36F to rat brain membranes.

    Muller et al., 1994. See above.

[3H]Glyburide binding: Describes the binding parameters of [3H]glyburide to the KATP channels in pancreatic -cells.

    Russ et al., 1997. See above.

[3H]P 1075 binding: Details conditions for obtaining good specific binding of [3H]P 1075 to whole cells.

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