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

  • 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]α‐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
  • 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 protocol 2)
  • 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 H 2O
  • Wash buffer: 50 mM Tris⋅Cl (pH 7.2; appendix 2A), ice‐cold (store up to 2 months at 4°C)
  • γ 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 IC 50 and K i (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]α‐DTX to Solubilized Rat Brain Synaptic Membranes

  Materials
  • Rat brain synaptic plasma membranes (see protocol 2)
  • 1× α‐DTX receptor solubilization buffer (see recipe), ice‐cold
  • [125I]α‐DTX (2000 Ci/mmol; Amersham Life Sciences)
  • Unlabeled α‐DTX (Alomone Labs)
  • Unlabeled test compounds
  • α‐DTX wash buffer (see recipe)
  • 1.2 mg/ml cytochrome c in H 2O
  • Sephadex G‐100 resin (Amersham Pharmacia Biotech; allow to swell in H 2O 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 γ‐counting tubes
  • γ 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 IC 50 and K i (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 protocol 5)
  • 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
  • β scintillation counter (Beckman)
NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 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% CO 2 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 protocol 7) 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
  • β scintillation counter (LS 5000 TD, Beckman)
  • Additional reagents and equipment for protein assay ( appendix 3A) and calculation of K D and B max (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% CO 2 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

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 β‐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]α‐DTX binding: Describes in detail the binding of α‐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 β‐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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