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Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential

Abstract

Voltage-gated ion channels regulate many physiological functions and are targets for a number of drugs. Patch-clamp electrophysiology is the standard method for measuring channel activity because it fulfils the requirements for voltage control, repetitive stimulation and high temporal resolution, but it is laborious and costly. Here we report an electro-optical technology and automated instrument, called the electrical stimulation voltage ion probe reader (E-VIPR), that measures the activity of voltage-gated ion channels using extracellular electrical field stimulation and voltage-sensitive fluorescent probes. We demonstrate that E-VIPR can sensitively detect drug potency and mechanism of block on the neuronal human type III voltage-gated sodium channel expressed in human embryonic kidney cells. Results are compared with voltage-clamp and show that E-VIPR provides sensitive and information-rich compound blocking activity. Furthermore, we screened 400 drugs and observed sodium channel–blocking activity for 25% of them, including the antidepressants sertraline (Zoloft) and paroxetine (Paxil).

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Figure 1: Measurements of Nav channel–dependent voltage changes with E-VIPR.
Figure 2: E-VIPR signals and membrane-potential changes.
Figure 3: Detection of use-dependent lidocaine block of hNav1.3 with E-VIPR and voltage clamp.
Figure 4: Blocking properties of Nav-channel drugs in E-VIPR and voltage clamp.
Figure 5: Screen of 400 known drugs and hNav1.3-blocking activity of antidepressants.

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References

  1. Brau, M.E., Dreimann, M., Olschewski, A., Vogel, W. & Hempelmann, G. Effect of drugs used for neuropathic pain management on tetrodotoxin-resistant Na(+) currents in rat sensory neurons. Anesthesiology 94, 137–144 (2001).

    Article  CAS  Google Scholar 

  2. Carmeliet, E. & Mubagwa, K. Antiarrhythmic drugs and cardiac ion channels: mechanisms of action. Prog. Biophys. Mol. Biol. 70, 1–72 (1998).

    Article  CAS  Google Scholar 

  3. White, H.S. Comparative anticonvulsant and mechanistic profile of the established and newer antiepileptic drugs. Epilepsia 40 suppl. Suppl 5, S2–10 (1999).

    Article  CAS  Google Scholar 

  4. Courtney, K.R. Mechanism of frequency-dependent inhibition of sodium currents in frog myelinated nerve by the lidocaine derivative GEA. J. Pharmacol. Exp. Ther. 195, 225–236 (1975).

    CAS  PubMed  Google Scholar 

  5. Scholz, A. Mechanisms of (local) anaesthetics on voltage-gated sodium and other ion channels. Br. J. Anaesth. 89, 52–61 (2002).

    Article  CAS  Google Scholar 

  6. Clare, J.J., Tate, S.N., Nobbs, M. & Romanos, M.A. Voltage-gated sodium channels as therapeutic targets. Drug Discov. Today 5, 506–520 (2000).

    Article  CAS  Google Scholar 

  7. Schroeder, K., Neagle, B., Trezise, D.J. & Worley, J. Ionworks HT: a new high-throughput electrophysiology measurement platform. J. Biomol. Screen. 8, 50–64 (2003).

    Article  CAS  Google Scholar 

  8. Wood, C., Williams, C. & Waldron, G.J. Patch clamping by numbers. Drug Discov. Today 9, 434–441 (2004).

    Article  CAS  Google Scholar 

  9. Bennett, P.B. & Guthrie, H.R. Trends in ion channel drug discovery: advances in screening technologies. Trends Biotechnol. 21, 563–569 (2003).

    Article  CAS  Google Scholar 

  10. Worley, J.F., III & Main, M.J. An industrial perspective on utilizing functional ion channel assays for high throughput screening. Receptors Channels 8, 269–282 (2002).

    Article  CAS  Google Scholar 

  11. Zheng, W., Spencer, R.H. & Kiss, L. High throughput assay technologies for ion channel drug discovery. Assay Drug Dev. Technol. 2, 543–552 (2004).

    Article  CAS  Google Scholar 

  12. Felix, J.P. et al. Functional assay of voltage-gated sodium channels using membrane potential-sensitive dyes. Assay Drug Dev. Technol. 2, 260–268 (2004).

    Article  CAS  Google Scholar 

  13. Maher, M.P. & Gonzalez, J.E. Multi-well plate and electrode assemblies for ion channel assays. US patent 6,969,449 B2 (2005).

  14. Maher, M.P. & Gonzalez, J.E. High throughput method and system for screening candidate compounds for activity against ion channels. US patent 6,686,193 B2 (2004).

  15. Kim, C.H., Oh, Y., Chung, J.M. & Chung, K. The changes in expression of three subtypes of TTX sensitive sodium channels in sensory neurons after spinal nerve ligation. Brain Res. Mol. Brain Res. 95, 153–161 (2001).

    Article  CAS  Google Scholar 

  16. Black, J.A., Liu, S., Tanaka, M., Cummins, T.R. & Waxman, S.G. Changes in the expression of tetrodotoxin-sensitive sodium channels within dorsal root ganglia neurons in inflammatory pain. Pain 108, 237–247 (2004).

    Article  CAS  Google Scholar 

  17. Gonzalez, J.E. & Tsien, R.Y. Voltage sensing by fluorescence resonance energy transfer in single cells. Biophys. J. 69, 1272–1280 (1995).

    Article  CAS  Google Scholar 

  18. Gonzalez, J.E. & Tsien, R.Y. Improved indicators of cell membrane potential that use fluorescence resonance energy transfer. Chem. Biol. 4, 269–277 (1997).

    Article  CAS  Google Scholar 

  19. Gonzalez, J.E., Oades, K., Leychkis, Y., Harootunian, A. & Negulescu, P.A. Cell-based assays and instrumentation for screening ion-channel targets. Drug Discov. Today 4, 431–439 (1999).

    Article  CAS  Google Scholar 

  20. Mao, J. & Chen, L.L. Systemic lidocaine for neuropathic pain relief. Pain 87, 7–17 (2000).

    Article  CAS  Google Scholar 

  21. Lenkowski, P.W., Shah, B.S., Dinn, A.E., Lee, K. & Patel, M.K. Lidocaine block of neonatal Nav1.3 is differentially modulated by co-expression of beta1 and beta3 subunits. Eur. J. Pharmacol. 467, 23–30 (2003).

    Article  CAS  Google Scholar 

  22. Postma, S.W. & Catterall, W.A. Inhibition of binding of [3H]batrachotoxinin A 20-alpha-benzoate to sodium channels by local anesthetics. Mol. Pharmacol. 25, 219–227 (1984).

    CAS  PubMed  Google Scholar 

  23. Conti, F., Gheri, A., Pusch, M. & Moran, O. Use dependence of tetrodotoxin block of sodium channels: a revival of the trapped-ion mechanism. Biophys. J. 71, 1295–1312 (1996).

    Article  CAS  Google Scholar 

  24. Eickhorn, R., Weirich, J., Hornung, D. & Antoni, H. Use dependence of sodium current inhibition by tetrodotoxin in rat cardiac muscle: influence of channel state. Pflugers Arch. 416, 398–405 (1990).

    Article  CAS  Google Scholar 

  25. John, V.H. et al. Heterologous expression and functional analysis of rat Na(V)1.8 (SNS) voltage-gated sodium channels in the dorsal root ganglion neuroblastoma cell line ND7–23. Neuropharmacology 46, 425–438 (2004).

    Article  CAS  Google Scholar 

  26. Song, J.H., Huang, C.S., Nagata, K., Yeh, J.Z. & Narahashi, T. Differential action of riluzole on tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels. J. Pharmacol. Exp. Ther. 282, 707–714 (1997).

    CAS  PubMed  Google Scholar 

  27. Wang, G.K., Russell, C. & Wang, S.Y. State-dependent block of voltage-gated Na+ channels by amitriptyline via the local anesthetic receptor and its implication for neuropathic pain. Pain 110, 166–174 (2004).

    Article  CAS  Google Scholar 

  28. Nau, C., Seaver, M., Wang, S.Y. & Wang, G.K. Block of human heart hH1 sodium channels by amitriptyline. J. Pharmacol. Exp. Ther. 292, 1015–1023 (2000).

    CAS  PubMed  Google Scholar 

  29. Bielefeldt, K., Ozaki, N., Whiteis, C. & Gebhart, G.F. Amitriptyline inhibits voltage-sensitive sodium currents in rat gastric sensory neurons. Dig. Dis. Sci. 47, 959–966 (2002).

    Article  CAS  Google Scholar 

  30. Ragsdale, D.S., McPhee, J.C., Scheuer, T. & Catterall, W.A. Molecular determinants of state-dependent block of Na+ channels by local anesthetics. Science 265, 1724–1728 (1994).

    Article  CAS  Google Scholar 

  31. Akiba, I. et al. Stable expression and characterization of human PN1 and PN3 sodium channels. Receptors Channels 9, 291–299 (2003).

    Article  CAS  Google Scholar 

  32. Kuo, C.C. & Lu, L. Characterization of lamotrigine inhibition of Na+ channels in rat hippocampal neurones. Br. J. Pharmacol. 121, 1231–1238 (1997).

    Article  CAS  Google Scholar 

  33. Lang, D.G., Wang, C.M. & Cooper, B.R. Lamotrigine, phenytoin and carbamazepine interactions on the sodium current present in N4TG1 mouse neuroblastoma cells. J. Pharmacol. Exp. Ther. 266, 829–835 (1993).

    CAS  PubMed  Google Scholar 

  34. Xie, X., Lancaster, B., Peakman, T. & Garthwaite, J. Interaction of the antiepileptic drug lamotrigine with recombinant rat brain type IIA Na+ channels and with native Na+ channels in rat hippocampal neurones. Pflugers Arch. 430, 437–446 (1995).

    Article  CAS  Google Scholar 

  35. Hebert, T., Drapeau, P., Pradier, L. & Dunn, R.J. Block of the rat brain IIA sodium channel alpha subunit by the neuroprotective drug riluzole. Mol. Pharmacol. 45, 1055–1060 (1994).

    CAS  PubMed  Google Scholar 

  36. Zhang, J.H., Chung, T.D. & Oldenburg, K.R. A Simple statistical parameter for use in evaluation and validation of high throughput screening assays. J. Biomol. Screen. 4, 67–73 (1999).

    Article  CAS  Google Scholar 

  37. Pera, M. The Ambiguous Frog: The Galvani-Volta Controversy on Animal Electricity (Princeton University Press, Princeton, N.J., 1992).

  38. Basser, P.J. & Roth, B.J. New currents in electrical stimulation of excitable tissues. Annu. Rev. Biomed. Eng. 2, 377–397 (2000).

    Article  CAS  Google Scholar 

  39. Roth, B.J. Mechanisms for electrical stimulation of excitable tissue. Crit. Rev. Biomed. Eng. 22, 253–305 (1994).

    CAS  PubMed  Google Scholar 

  40. Benabid, A.L. et al. Therapeutic electrical stimulation of the central nervous system. C. R. Biol. 328, 177–186 (2005).

    Article  Google Scholar 

  41. Burnett, P. et al. Fluorescence imaging of electrically stimulated cells. J. Biomol. Screen. 8, 660–667 (2003).

    Article  CAS  Google Scholar 

  42. Baxter, D.F. et al. A novel membrane potential-sensitive fluorescent dye improves cell-based assays for ion channels. J. Biomol. Screen. 7, 79–85 (2002).

    Article  CAS  Google Scholar 

  43. Leffler, A., Herzog, R.I., Dib-Hajj, S.D., Waxman, S.G. & Cummins, T.R. Pharmacological properties of neuronal TTX-resistant sodium channels and the role of a critical serine pore residue. Pflugers Arch. 451, 454–463 (2005).

    Article  CAS  Google Scholar 

  44. Tomiko, S.A., Rosenberg, R.L., Emerick, M.C. & Agnew, W.S. Fluorescence assay for neurotoxin-modulated ion transport by the reconstituted voltage-activated sodium channel isolated from eel electric organ. Biochemistry 25, 2162–2174 (1986).

    Article  CAS  Google Scholar 

  45. Vickery, R.G. et al. Comparison of the pharmacological properties of rat Na(V)1.8 with rat Na(V)1.2a and human Na(V)1.5 voltage-gated sodium channel subtypes using a membrane potential sensitive dye and FLIPR. Receptors Channels 10, 11–23 (2004).

    Article  CAS  Google Scholar 

  46. Yang, Y.C. & Kuo, C.C. Inhibition of Na(+) current by imipramine and related compounds: different binding kinetics as an inactivation stabilizer and as an open channel blocker. Mol. Pharmacol. 62, 1228–1237 (2002).

    Article  CAS  Google Scholar 

  47. Abdi, S., Lee, D.H. & Chung, J.M. The anti-allodynic effects of amitriptyline, gabapentin, and lidocaine in a rat model of neuropathic pain. Anesth. Analg. 87, 1360–1366 (1998).

    CAS  PubMed  Google Scholar 

  48. Carter, G.T. & Sullivan, M.D. Antidepressants in pain management. Curr. Opin. Investig. Drugs 3, 454–458 (2002).

    CAS  PubMed  Google Scholar 

  49. Maizels, M. & McCarberg, B. Antidepressants and antiepileptic drugs for chronic non-cancer pain. Am. Fam. Physician 71, 483–490 (2005).

    PubMed  Google Scholar 

  50. Schulz, M. & Schmoldt, A. Therapeutic and toxic blood concentrations of more than 800 drugs and other xenobiotics. Pharmazie 58, 447–474 (2003).

    CAS  PubMed  Google Scholar 

  51. Doran, A. et al. The impact of P-glycoprotein on the disposition of drugs targeted for indications of the central nervous system: evaluation using the MDR1A/1B knockout mouse model. Drug Metab. Dispos. 33, 165–174 (2005).

    Article  CAS  Google Scholar 

  52. Chen, Y.H. et al. Cloning, distribution and functional analysis of the type III sodium channel from human brain. Eur. J. Neurosci. 12, 4281–4289 (2000).

    CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Charlie Cohen, Jennings Worley, Jeff Stack and Kevin Beaumont for review of manuscript and providing useful suggestions.

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Correspondence to Jesús E González.

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Huang, CJ., Harootunian, A., Maher, M. et al. Characterization of voltage-gated sodium-channel blockers by electrical stimulation and fluorescence detection of membrane potential. Nat Biotechnol 24, 439–446 (2006). https://doi.org/10.1038/nbt1194

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