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Controlling epithelial sodium channels with light using photoswitchable amilorides

Abstract

Amiloride is a widely used diuretic that blocks epithelial sodium channels (ENaCs). These heterotrimeric transmembrane proteins, assembled from β, γ and α or δ subunits, effectively control water transport across epithelia and sodium influx into non-epithelial cells. The functional role of δβγENaC in various organs, including the human brain, is still poorly understood and no pharmacological tools are available for the functional differentiation between α- and δ-containing ENaCs. Here we report several photoswitchable versions of amiloride. One compound, termed PA1, enables the optical control of ENaC channels, in particular the δβγ isoform, by switching between blue and green light, or by turning on and off blue light. PA1 was used to modify functionally δβγENaC in amphibian and mammalian cells. We also show that PA1 can be used to differentiate between δβγENaC and αβγENaC in a model for the human lung epithelium.

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Figure 1: ENaCs and their blockers.
Figure 2: PA synthesis and characterization.
Figure 3: PA1 photoswitching in Xenopus oocytes.
Figure 4: Switching δβγENaC in HEK cells using 10 μM PA1.
Figure 5: Photocontrol of transepithelial potential in H441 monolayers.

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References

  1. Alvarez de la Rosa, D., Canessa, C. M., Fyfe, G. K. & Zhang, P. Structure and regulation of amiloride-sensitive sodium channels. Ann. Rev. Physiol. 62, 573–594 (2000).

    Article  CAS  Google Scholar 

  2. Canessa, C. M., Merillat, A. M. & Rossier, B. C. Membrane topology of the epithelial sodium channel in intact cells. Am. J. Physiol. 267, C1682–C1690 (1994).

    Article  CAS  Google Scholar 

  3. Canessa, C. M. et al. Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367, 463–467 (1994).

    Article  CAS  Google Scholar 

  4. Bubien, J. K. Epithelial Na+ channel (ENaC), hormones, and hypertension. J. Biol. Chem. 285, 23527–23531 (2010).

    Article  CAS  Google Scholar 

  5. Chang, S. S. et al. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nature Genet. 12, 248–253 (1996).

    Article  CAS  Google Scholar 

  6. Boucher, R. C. New concepts of the pathogenesis of cystic fibrosis lung disease. Eur. Respir. J. 23, 146–158 (2004).

    Article  CAS  Google Scholar 

  7. Hummler, E. et al. Early death due to defective neonatal lung liquid clearance in αENaC-deficient mice. Nature Genet. 12, 325–328 (1996).

    Article  CAS  Google Scholar 

  8. Scherrer, U. et al. High-altitude pulmonary edema: from exaggerated pulmonary hypertension to a defect in transepithelial sodium transport. Adv. Exp. Med. Biol. 474, 93–107 (1999).

    Article  CAS  Google Scholar 

  9. Althaus, M., Clauss, W. G. & Fronius, M. Amiloride-sensitive sodium channels and pulmonary edema. Pulm. Med. 2011, 830320 (2011).

    Article  Google Scholar 

  10. Althaus, M. ENaC inhibitors and airway re-hydration in cystic fibrosis: state of the art. Curr. Mol. Pharmacol. 6, 3–12 (2013).

    Article  CAS  Google Scholar 

  11. Fronius, M. Treatment of pulmonary edema by ENaC activators/stimulators. Curr. Mol. Pharmacol. 6, 13–27 (2013).

    Article  CAS  Google Scholar 

  12. Bull, M. B. & Laragh, J. H. Amiloride: a potassium-sparing natriuretic agent. Circulation 37, 45–53 (1968).

    Article  CAS  Google Scholar 

  13. Schoenberger, M. & Althaus, M. Novel small molecule epithelial sodium channel inhibitors as potential therapeutics in cystic fibrosis – a patent evaluation. Expert Opin. Therapeut. Patents 23, 1383–1389 (2013).

    Article  CAS  Google Scholar 

  14. Waldmann, R., Champigny, G., Bassilana, F., Voilley, N. & Lazdunski, M. Molecular cloning and functional expression of a novel amiloride-sensitive Na+ channel. J. Biol. Chem. 270, 27411–27414 (1995).

    Article  CAS  Google Scholar 

  15. Giraldez, T. et al. Cloning and functional expression of a new epithelial sodium channel delta subunit isoform differentially expressed in neurons of the human and monkey telencephalon. J. Neurochem. 102, 1304–1315 (2007).

    Article  CAS  Google Scholar 

  16. Wesch, D. et al. Differential N termini in epithelial Na+ channel delta-subunit isoforms modulate channel trafficking to the membrane. Am. J. Physiol. Cell Physiol. 302, C868–879 (2012).

    Article  CAS  Google Scholar 

  17. Teruyama, R., Sakuraba, M., Wilson, L. L., Wandrey, N. E. & Armstrong, W. E. Epithelial Na+ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei. Am. J. Physiol. Endocrinol. Metab. 302, E273–E285 (2012).

    Article  CAS  Google Scholar 

  18. Ji, H.-L. et al. δENaC: a novel divergent amiloride-inhibitable sodium channel. Am. J. Physiol. Lung Cell. Mol. Physiol. 303, L1013–L1026 (2012).

    Article  CAS  Google Scholar 

  19. Yamamura, H., Ugawa, S., Ueda, T., Nagao, M. & Shimada, S. A novel spliced variant of the epithelial Na+ channel delta-subunit in the human brain. Biochem. Biophys. Res. Commun. 349, 317–321 (2006).

    Article  CAS  Google Scholar 

  20. Miller, R. L., Wang, M. H., Gray, P. A., Salkoff, L. B. & Loewy, A. D. ENaC-expressing neurons in the sensory circumventricular organs become c-Fos activated following systemic sodium changes. Am. J. Physiol. Regul. Integr. Comp. Physiol. 305, R1141–R1152 (2013).

    Article  CAS  Google Scholar 

  21. Giraldez, T., Dominguez, J. & Alvarez de la Rosa, D. ENaC in the brain – future perspectives and pharmacological implications. Curr. Mol. Pharmacol. 6, 44–49 (2013).

    Article  CAS  Google Scholar 

  22. Giraldez, T., Rojas, P., Jou, J., Flores, C. & Alvarez de la Rosa, D. The epithelial sodium channel δ-subunit: new notes for an old song. Am. J. Physiol. Renal Physiol. 303, F328–F338 (2012).

    Article  CAS  Google Scholar 

  23. Kellenberger, S. & Schild, L. Epithelial sodium channel/degenerin family of ion channels: a variety of functions for a shared structure. Physiol. Rev. 82, 735–767 (2002).

    Article  CAS  Google Scholar 

  24. Jasti, J., Furukawa, H., Gonzales, E. B. & Gouaux, E. Structure of acid-sensing ion channel 1 at 1.9 Å resolution and low pH. Nature 449, 316–23 (2007).

    Article  CAS  Google Scholar 

  25. Fehrentz, T., Schonberger, M. & Trauner, D. Optochemical genetics. Angew. Chem. Int. Ed. 50, 12156–12182 (2011).

    Article  CAS  Google Scholar 

  26. Banghart, M., Borges, K., Isacoff, E., Trauner, D. & Kramer, R. H. Light-activated ion channels for remote control of neuronal firing. Nature Neurosci. 7, 1381–1386 (2004).

    Article  CAS  Google Scholar 

  27. Mourot, A. et al. Rapid optical control of nociception with an ion-channel photoswitch. Nature Methods 9, 396–402 (2011).

    Article  Google Scholar 

  28. Volgraf, M. et al. Allosteric control of an ionotropic glutamate receptor with an optical switch. Nature Chem. Biol. 2, 47–52 (2006).

    Article  CAS  Google Scholar 

  29. Levitz, J. et al. Optical control of metabotropic glutamate receptors. Nature Neurosci. 16, 507–516 (2013).

    Article  CAS  Google Scholar 

  30. Tochitsky, I. et al. Optochemical control of genetically engineered neuronal nicotinic acetylcholine receptors. Nature Chem. 4, 105–111 (2012).

    Article  CAS  Google Scholar 

  31. Cragoe, E. J., Woltersd. O. W., Bicking, J. B., Kwong, S. F. & Jones, J. H. Pyrazine diuretics. 2. N-amidino-3-amino-5-substituted L-halopyrazinecarboxamides. J. Med. Chem. 10, 66–75 (1967).

    Article  CAS  Google Scholar 

  32. Kleyman, T. R. & Cragoe, E. J. Jr. Amiloride and its analogs as tools in the study of ion transport. J. Membr. Biol. 105, 1–21 (1988).

    Article  CAS  Google Scholar 

  33. Cuthbert, A. W., Fanelli, G. M. & Scriabine, A. Amiloride and Epithelial Sodium Transport (Urban & Schwarzenberg, 1979).

    Google Scholar 

  34. Li, J. H., Cragoe, E. J. Jr & Lindemann, B. Structure–activity relationship of amiloride analogs as blockers of epithelial Na channels: II. Side-chain modifications. J. Membr. Biol. 95, 171–185 (1987).

    Article  CAS  Google Scholar 

  35. Garvin, J. L., Simon, S. A., Cragoe, E. J. Jr & Mandel, L. J. Phenamil: an irreversible inhibitor of sodium channels in the toad urinary bladder. J. Membr. Biol. 87, 45–54 (1985).

    Article  CAS  Google Scholar 

  36. Hirsh, A. J. et al. Design, synthesis, and structure–activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: drugs for cystic fibrosis and chronic bronchitis. J. Med. Chem. 49, 4098–4115 (2006).

    Article  CAS  Google Scholar 

  37. Sadovski, O., Beharry, A. A., Zhang, F. & Woolley, G. A. Spectral tuning of azobenzene photoswitches for biological applications. Angew. Chem. Int. Ed. 48, 1484–1486 (2009).

    Article  CAS  Google Scholar 

  38. Mourot, A. et al. Tuning photochromic ion channel blockers. ACS Chem. Neurosci. 2, 536–543 (2011).

    Article  CAS  Google Scholar 

  39. Pulgarin, J. A. M., Molina, A. A. & Lopez, P. F. Direct analysis of amiloride and triamterene mixtures by fluorescence spectrometry using partial-least squares calibration. Anal. Chim. Acta 449, 179–187 (2001).

    Article  CAS  Google Scholar 

  40. Althaus, M., Bogdan, R., Clauss, W. G. & Fronius, M. Mechano-sensitivity of epithelial sodium channels (ENaCs): laminar shear stress increases ion channel open probability. FASEB J. 21, 2389–2399 (2007).

    Article  CAS  Google Scholar 

  41. Fronius, M., Bogdan, R., Althaus, M., Morty, R. E. & Clauss, W. G. Epithelial Na+ channels derived from human lung are activated by shear force. Respir. Physiol. Neurobiol. 170, 113–119 (2010).

    Article  CAS  Google Scholar 

  42. Geffeney, S. L. et al. DEG/ENaC but not TRP channels are the major mechanoelectrical transduction channels in a C. elegans nociceptor. Neuron 71, 845–857 (2011).

    Article  CAS  Google Scholar 

  43. Kim, E. C., Choi, S. K., Lim, M., Yeon, S. I. & Lee, Y. H. Role of endogenous ENaC and TRP channels in the myogenic response of rat posterior cerebral arteries. PLoS One 8, e84194 (2013).

    Article  Google Scholar 

  44. 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 

  45. Althaus, M. et al. Nitric oxide inhibits highly selective sodium channels and the Na+/K+-ATPase in H441 cells. Am. J. Respir. Cell Mol. Biol. 44, 53–65 (2011).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank the European Research Commission for an ERC Advanced Grant (Grant No. 268795 to D.T.). M.A. is supported by grants from the German Research Foundation (AL1453/1-1 and AL1453/1-2), M.F. and W.C. acknowledge a grant provided by the Federal State of Hesse (LOEWE Research Focus, Non-neuronal cholinergic systems). M.S. was supported by a grant from the German Study Foundation and the International Max Planck Research School (IMPRS-LS). We acknowledge the support of K. Hüll (chemical synthesis) and L. de la Osa de la Rosa (cell-culture work), and we thank D. Barber for helpful comments. We also thank D. Alvarez de la Rosa for providing constructs of all ENaC subunits in pcDNA3.1.

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D.T., M.A., M.S. and M.F. conceived the study and designed the experiments. M.S. performed the chemical synthesis and UV/vis characterization. M.S. and M.A. performed the electrophysiological characterization. D.T. and W.C. supervised the study and wrote the paper, together with M.S., M.F. and M.A.

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Correspondence to Dirk Trauner.

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The authors declare no competing financial interests.

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Schönberger, M., Althaus, M., Fronius, M. et al. Controlling epithelial sodium channels with light using photoswitchable amilorides. Nature Chem 6, 712–719 (2014). https://doi.org/10.1038/nchem.2004

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