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Probing Subunits Interactions in KATP Channels Using Photo-Crosslinking via Genetically Encoded p-Azido-l-phenylalanine

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Book cover Potassium Channels

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1684))

Abstract

Potassium channels are multimeric protein complexes regulated by diverse physiological and pharmacological ligands. The key to understanding mechanisms of channel regulation is the ability to detect structural changes associated with ligand binding. While high-resolution structural methods such as X-ray crystallography and single-particle cryo-electron microscopy offer direct visualization of channel structures, these methods do have limitations and may not be suitable for the question of interest. In this chapter, we describe the use of a photo-cross-linker unnatural amino acid, p-azido-l-phenylalanine, to probe interactions between two proteins, the sulfonylurea receptor 1 and the inwardly rectifying potassium channel Kir6.2, that form the ATP-sensitive potassium (KATP) channel complex in the absence or presence of ligands. The difference in the extent of crosslinking between a liganded state and unliganded state can be used as a readout of ligand-induced structural changes. We anticipate that the protocol described here will also be applicable for other potassium channels and protein complexes.

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References

  1. Clement JP, Kunjilwar K, Gonzalez G, Schwanstecher M, Panten U, Aguilar-Bryan L, Bryan J (1997) Association and stoichiometry of K(ATP) channel subunits. Neuron 18(5):827–838

    Article  CAS  PubMed  Google Scholar 

  2. Inagaki N, Gonoi T, Clement JP, Namba N, Inazawa J, Gonzalez G, Aguilar-Bryan L, Seino S, Bryan J (1995) Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 270(5239):1166–1170

    Article  CAS  PubMed  Google Scholar 

  3. Inagaki N, Gonoi T, Seino S (1997) Subunit stoichiometry of the pancreatic beta-cell ATP-sensitive K+ channel. FEBS Lett 409(2):232–236

    Article  CAS  PubMed  Google Scholar 

  4. Martin GM, Yoshioka C, Rex EA, Fay JF, Xie Q, Whorton MR, Chen JZ, Shyng SL (2017) Cryo-EM structure of the ATP-sensitive potassium channel illuminates mechanisms of assembly and gating. eLife 6. doi:10.7554/eLife.24149

  5. Shyng S, Nichols CG (1997) Octameric stoichiometry of the KATP channel complex. J Gen Physiol 110(6):655–664

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Aguilar-Bryan L, Bryan J (1999) Molecular biology of adenosine triphosphate-sensitive potassium channels. Endocr Rev 20(2):101–135. doi:10.1210/edrv.20.2.0361

    CAS  PubMed  Google Scholar 

  7. Martin GM, Chen PC, Devaraneni P, Shyng SL (2013) Pharmacological rescue of trafficking-impaired ATP-sensitive potassium channels. Front Physiol 4:386. doi:10.3389/fphys.2013.00386

    PubMed  PubMed Central  Google Scholar 

  8. Martin GM, Rex EA, Devaraneni P, Denton JS, Boodhansingh KE, DeLeon DD, Stanley CA, Shyng SL (2016) Pharmacological correction of trafficking defects in ATP-sensitive potassium channels caused by sulfonylurea receptor 1 mutations. J Biol Chem 291(42):21971–21983. doi:10.1074/jbc.M116.749366

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Yan F, Lin CW, Weisiger E, Cartier EA, Taschenberger G, Shyng SL (2004) Sulfonylureas correct trafficking defects of ATP-sensitive potassium channels caused by mutations in the sulfonylurea receptor. J Biol Chem 279(12):11096–11105. doi:10.1074/jbc.M312810200

    Article  CAS  PubMed  Google Scholar 

  10. Yan FF, Lin YW, MacMullen C, Ganguly A, Stanley CA, Shyng SL (2007) Congenital hyperinsulinism associated ABCC8 mutations that cause defective trafficking of ATP-sensitive K+ channels: identification and rescue. Diabetes 56(9):2339–2348. doi:10.2337/db07-0150

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lin YW, Jia T, Weinsoft AM, Shyng SL (2003) Stabilization of the activity of ATP-sensitive potassium channels by ion pairs formed between adjacent Kir6.2 subunits. J Gen Physiol 122(2):225–237. doi:10.1085/jgp.200308822

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Pratt EB, Zhou Q, Gay JW, Shyng SL (2012) Engineered interaction between SUR1 and Kir6.2 that enhances ATP sensitivity in KATP channels. J Gen Physiol 140(2):175–187. doi:10.1085/jgp.201210803

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Rappsilber J (2011) The beginning of a beautiful friendship: cross-linking/mass spectrometry and modelling of proteins and multi-protein complexes. J Struct Biol 173(3):530–540. doi:10.1016/j.jsb.2010.10.014

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Weisbrod CR, Chavez JD, Eng JK, Yang L, Zheng C, Bruce JE (2013) In vivo protein interaction network identified with a novel real-time cross-linked peptide identification strategy. J Proteome Res 12(4):1569–1579. doi:10.1021/pr3011638

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Huber T, Naganathan S, Tian H, Ye S, Sakmar TP (2013) Unnatural amino acid mutagenesis of GPCRs using amber codon suppression and bioorthogonal labeling. Methods Enzymol 520:281–305. doi:10.1016/B978-0-12-391861-1.00013-7

    Article  CAS  PubMed  Google Scholar 

  16. Grunbeck A, Huber T, Abrol R, Trzaskowski B, Goddard WA 3rd, Sakmar TP (2012) Genetically encoded photo-cross-linkers map the binding site of an allosteric drug on a G protein-coupled receptor. ACS Chem Biol 7(6):967–972. doi:10.1021/cb300059z

    Article  CAS  PubMed  Google Scholar 

  17. Grunbeck A, Huber T, Sachdev P, Sakmar TP (2011) Mapping the ligand-binding site on a G protein-coupled receptor (GPCR) using genetically encoded photocrosslinkers. Biochemistry 50(17):3411–3413. doi:10.1021/bi200214r

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Grunbeck A, Sakmar TP (2013) Probing G protein-coupled receptor-ligand interactions with targeted photoactivatable cross-linkers. Biochemistry 52(48):8625–8632. doi:10.1021/bi401300y

    Article  CAS  PubMed  Google Scholar 

  19. Koole C, Reynolds CA, Mobarec JC, Hick C, Sexton PM, Sakmar TP (2017) Genetically-encoded photocrosslinkers determine the biological binding site of exendin-4 in the N-terminal domain of the intact human glucagon-like peptide-1 receptor (GLP-1R). J Biol Chem. doi:10.1074/jbc.M117.779496

  20. Park M, Sivertsen BB, Els-Heindl S, Huber T, Holst B, Beck-Sickinger AG, Schwartz TW, Sakmar TP (2015) Bioorthogonal labeling of ghrelin receptor to facilitate studies of ligand-dependent conformational dynamics. Chem Biol 22(11):1431–1436. doi:10.1016/j.chembiol.2015.09.014

    Article  CAS  PubMed  Google Scholar 

  21. Periole X, Knepp AM, Sakmar TP, Marrink SJ, Huber T (2012) Structural determinants of the supramolecular organization of G protein-coupled receptors in bilayers. J Am Chem Soc 134(26):10959–10965. doi:10.1021/ja303286e

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Rannversson H, Andersen J, Sorensen L, Bang-Andersen B, Park M, Huber T, Sakmar TP, Stromgaard K (2016) Genetically encoded photocrosslinkers locate the high-affinity binding site of antidepressant drugs in the human serotonin transporter. Nat Commun 7:11261. doi:10.1038/ncomms11261

    Article  PubMed  PubMed Central  Google Scholar 

  23. Valentin-Hansen L, Park M, Huber T, Grunbeck A, Naganathan S, Schwartz TW, Sakmar TP (2014) Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a photoreactive amino acid. J Biol Chem 289(26):18045–18054. doi:10.1074/jbc.M113.527085

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Grunbeck A, Huber T, Sakmar TP (2013) Mapping a ligand binding site using genetically encoded photoactivatable crosslinkers. Methods Enzymol 520:307–322. doi:10.1016/B978-0-12-391861-1.00014-9

    Article  CAS  PubMed  Google Scholar 

  25. Naganathan S, Grunbeck A, Tian H, Huber T, Sakmar TP (2013) Genetically-encoded molecular probes to study G protein-coupled receptors. J Vis Exp:79. doi:10.3791/50588

  26. Devaraneni PK, Martin GM, Olson EM, Zhou Q, Shyng SL (2015) Structurally distinct ligands rescue biogenesis defects of the KATP channel complex via a converging mechanism. J Biol Chem 290(12):7980–7991. doi:10.1074/jbc.M114.634576

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgments

We are grateful to Dr. Thomas Sakmar for the suppressor tRNA plasmid and the plasmid of tRNA synthetase specific for p-azido-l-phenylalanine, and for valuable advice. This work was supported by National Institutes of Health grant R01DK066485.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR, 97239, USA

    Prasanna Devaraneni & Emily A. Rex

  2. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, OR, 97239, USA

    Show-Ling Shyng

Authors
  1. Prasanna Devaraneni
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  2. Emily A. Rex
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  3. Show-Ling Shyng
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Corresponding author

Correspondence to Show-Ling Shyng .

Editor information

Editors and Affiliations

  1. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA

    Show-Ling Shyng

  2. Department of Physiology and Pharmacology, Oregon Health and Science University, Portland, Oregon, USA

    Francis I. Valiyaveetil

  3. The Vollum Institute, Oregon Health and Science University, Portland, Oregon, USA

    Matt Whorton

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Devaraneni, P., Rex, E.A., Shyng, SL. (2018). Probing Subunits Interactions in KATP Channels Using Photo-Crosslinking via Genetically Encoded p-Azido-l-phenylalanine. In: Shyng, SL., Valiyaveetil, F., Whorton, M. (eds) Potassium Channels. Methods in Molecular Biology, vol 1684. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7362-0_5

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  • DOI: https://doi.org/10.1007/978-1-4939-7362-0_5

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7361-3

  • Online ISBN: 978-1-4939-7362-0

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