Molecular Probes for Visualization of Nicotinic Acetylcholine Receptors Based on Snake Three-Finger Toxins and a Red Fluorescent Protein

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Abstract

The visualization of macromolecular complexes is an important task in modern bioorganic chemistry, which can be addressed using various methodological approaches. The most widely used techniques involve radioactive- and fluorescent-labeled ligands. In this study, molecular probes were developed in the form of hybrid constructs combining one of three snake toxins (α-bungarotoxin, α-cobratoxin, or neurotoxin NT-II) with the red fluorescent protein mKate2. These chimeric proteins were produced in a bacterial expression system and purified via gel filtration. Using competitive radioligand binding assay with radiolabeled α-bungarotoxin, it was demonstrated that the obtained probes exhibit high affinity for the nicotinic acetylcholine receptor of the electric organ from orpedo californicaT, with half-maximal inhibitory concentration values in the nanomolar range. The fluorescent probes were successfully employed for the visualization of acetylcholine receptors on the surface of SH-SYSY cells.

About the authors

A. I Kuzmenkov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: aleksey.kuzmenkov@gmail.com
Moscow, Russia

I. S Chudetsky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

D. S Kudryavtsev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

I. E Kasheverov

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

V. I Tsetlin

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

A. A Vassilevski

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Moscow, Russia

References

  1. Corringer P.J., Novère N.L., Changeux J.P. // Annu. Rev. Pharmacol. Toxicol. 2000. V. 40. P. 431–458. https://doi.org/10.1146/annurev.pharmtox.40.1.431
  2. Karlin A. // Nat. Rev. Neurosci. 2002. V. 3. P. 102–114. https://doi.org/10.1038/nrn731
  3. Cecchini M., Corringer P.-J., Changeux J.-P. // Annu. Rev. Biochem. 2024. V. 93. P. 339–366. https://doi.org/10.1146/annurev-biochem-030122-033116
  4. Lukas R.J., Changeux J.P., Le Novère N., Albuquerque E.X., Balfour D.J., Berg D.K., Bertrand D., Chiappinelli V.A., Clarke P.B., Collins A.C., Dani J.A., Grady S.R., Kellar K.J., Lindstrom J.M., Marks M.J., Quik M., Taylor P.W., Wonnacott S. // Pharmacol. Rev. 1999. V. 51. P. 397–401. https://doi.org/10.1016/S0031-6997(24)01406-6
  5. Hone A.J., McIntosh J.M. // Pharmacol. Res. 2023. V. 190. P. 106715. https://doi.org/10.1016/j.phrs.2023.106715
  6. Dutertre S., Lewis R.J. // Biochem. Pharmacol. 2006. V. 72. P. 661–670. https://doi.org/10.1016/j.bcp.2006.03.027
  7. Chang C., Lee C. // Arch. Int. Pharmacodyn. Ther. 1963. V. 144. P. 241–257.
  8. Karlsson E., Eaker D., Ponterius G. // Biochim. Biophys. Acta. 1972. V. 257. P. 235–248. https://doi.org/10.1016/0005-2795(72)90275-9
  9. Surin A.M., Pluzhnikov K.A., Utkin Y.N., Karlsson E., Tsetlin V.I. // Bioorg. Khim. 1983. V. 9. P. 756–767.
  10. Utkin Y.N. // Toxicon. 2013. V. 62. P. 50–55. https://doi.org/10.4331/wjbc.v10.i1.17
  11. Kini R.M., Doley R. // Toxicon. 2010. V. 56. P. 855–867. https://doi.org/10.1016/j.toxicon.2010.07.010
  12. Kini R.M., Koh C.Y. // Biochem. Pharmacol. 2020. V. 181. P. 114105. https://doi.org/10.1016/j.bcp.2020.114105
  13. Kuzmenkov A.I., Vassilevski A.A. // Neurosci. Lett. 2018. V. 679. P. 15–23. https://doi.org/10.1016/j.neulet.2017.10.050
  14. O’Brien R.D., Eldefrawi M.E., Eldefrawi A.T. // Annu. Rev. Pharmacol. 1972. V. 12. P. 19–34. https://doi.org/10.1146/annurev.pa.12.040172.000315
  15. Крюкова Е.В., Иванов Д.А., Копылова Н.В., Старков В.Г., Андреева Т.В., Иванов И.А., Цетлин В.И., Уткин Ю.Н. // Биоорг. химия. 2023. Т. 49. С. 296–305. https://doi.org/10.31857/S0132342323030156
  16. Sahoo H. // RSC Adv. 2012. V. 2. P. 7017. https://doi.org/10.1039/C2RA20389H
  17. Axelrod D. // Proc. Natl. Acad. Sci. USA. 1980. V. 77. P. 4823–4827. https://doi.org/10.1073/pnas.77.8.4823
  18. Anderson M.J., Cohen M.W. // J. Physiol. 1974. V. 237. P. 385–400. https://doi.org/10.1113/jphysiol.1974.sp010487
  19. Tsetlin V.I., Karlsson E., Arseniev A.S., Utkin Y.N., Surin A.M., Pashkov V.S., Pluzhnikov K.A., Ivanov V.T., Bystrov V.F., Ovchinnikov Y.A. // FEBS Lett. 1979. V. 106. P. 47–52. https://doi.org/10.1016/0014-5793(79)80692-4
  20. Kuzmenkov A.I., Nekrasova O.V., Kudryashova K.S., Peigneur S., Tytgat J., Stepanov A.V., Kirpichnikov M.P., Grishin E.V., Feofanov A.V., Vassilevski A.A. // Sci. Rep. 2016. V. 6. P. 33314. https://doi.org/10.1038/srep33314
  21. Chudakov D.M., Matz M.V., Lukyanov S.A., Lukyanov K.A. // Physiol. Rev. 2010. V. 90. P. 1103–1163. https://doi.org/10.1152/physrev.00038.2009
  22. Mishin A.S., Belousov V.V., Solntsev K.M., Lukyanov K.A. // Curr. Opin. Chem. Biol. 2015. V. 27. P. 1–9. https://doi.org/10.1016/j.cbpa.2015.05.002
  23. Kasheverov I.E., Kuzmenkov A.I., Kudryavtsev D.S., Chudetskiy I.S., Shelukhina I.V., Barykin E.P., Ivanov I.A., Siniavin A.E., Ziganshin R.H., Baranov M.S., Tsetlin V.I., Vassilevski A.A., Utkin Y.N. // Front. Mol. Biosci. 2021. V. 8. P. 753283. https://doi.org/10.3389/fmolb.2021.753283
  24. Shcherbo D., Murphy C.S., Ermakova G.V., Solovieva E.A., Chepurnykh T.V., Shcheglov A.S., Verkhusha V.V., Pletnev V.Z., Hazelwood K.L., Roche P.M., Lukyanov S., Zaraisky A.G., Davidson M.W., Chudakov D.M. // Biochem. J. 2009. V. 418. P. 567. https://doi.org/10.1042/BJ20081949
  25. Cormack B.P., Valdivia R.H., Falkow S. // Gene. 1996. V. 173. P. 33–38. https://doi.org/10.1016/0378-1119(95)00685-0
  26. Chandna R., Tae H., Seymour V.A., Chathrath S., Adams D.J., Kini R.M. // FASEB Bioadv. 2019. V. 1. P. 115–131. https://doi.org/10.1096/fba.1027
  27. Utkin Y.N., Kukhtina V.V., Kryukova E.V., Chiodini F., Bertrand D., Methfessel C., Tsetlin V.I. // J. Biol. Chem. 2001. V. 276. P. 15810–15815. https://doi.org/10.1074/jbc.M100788200
  28. Tsetlin V.I., Kasheverov I.E., Utkin Y.N. // J. Neuro-chem. 2021. V. 158. P. 1223–1235. https://doi.org/10.1111/jnc.15123
  29. Nirthanan S., Gwee M.C.E. // J. Pharmacol. Sci. 2004. V. 94. P. 1–17. https://doi.org/10.1254/jphs.94.1
  30. Rahman M., Teng J., Worrell B.T., Noviello C.M., Lee M., Karlin A., Stowell M.H., Hibbs R.E. // Neuron. 2020. V. 106. P. 952–962.e5. https://doi.org/10.1016/j.neuron.2020.03.012
  31. Shelukhina I.V., Kryukova E.V., Lips K.S., Tsetlin V.I., Kummer W. // J. Neurochem. 2009. V. 109. P. 1087–1095. https://doi.org/10.1111/j.1471-4159.2009.06033.x
  32. Lukas R.J., Norman S.A., Lucero L. // Mol. Cell. Neurosci. 1993. V. 4. P. 1–12. https://doi.org/10.1006/mcne.1993.1001
  33. Ravdin P., Axelrod D. // Anal. Biochem. 1977. V. 80. P. 585–592. https://doi.org/10.1016/0003-2697(77)90682-0
  34. Yang Y., Tan Y., Zhangsun D., Zhu X., Luo S. // ACS Chem. Neurosci. 2021. V. 12. P. 3662–3671. https://doi.org/10.1021/acschemneuro.1c00392
  35. Muttenthaler M., Nevin S.T., Inserra M., Lewis R.J., Adams D.J., Alewood P. // Aust. J. Chem. 2020. V. 73. P. 327–333. https://doi.org/10.1071/ch19456
  36. Lebedev D.S., Kryukova E.V., Ivanov I.A., Egorova N.S., Timofeev N.D., Spirova E.N., Tufanova E.Y., Siniavin A.E., Kudryavtsev D.S., Kasheverov I.E., Zouridakis M., Katsarava R., Zavradashvili N., Iagorshvili I., Tzartos S.J., Tsetlin V.I. // Mol. Pharmacol. 2019. V. 96. P. 664–673. https://doi.org/10.1124/mol.119.117713

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