Controlling epithelial sodium channels with light using photoswitchable amilorides

Journal name:
Nature Chemistry
Volume:
6,
Pages:
712–719
Year published:
DOI:
doi:10.1038/nchem.2004
Received
Accepted
Published online

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.

At a glance

Figures

  1. ENaCs and their blockers.
    Figure 1: ENaCs and their blockers.

    a, Schematic drawing of heterotrimeric αβγENaC and δβγENaC. Each subunit contains two transmembrane helices TM1 and TM2, relatively short C and N termini at the intracellular (in) site and a large extracellular (ex) loop. Under physiological conditions, ENaCs allow for a constant influx of sodium into the cell (indicated by blue arrows). b, Chemical structure of ​amiloride and its more potent derivatives ​phenamil, ​benzamil and compound 1. c, Structures of photoswitchable ​amiloride derivatives that contain an ​azobenzene functional group. d, Photoisomerization of PA1. Illumination with 400 nm induces isomerization to cis-PA1, which is the thermodynamically less-stable form. The cis isomer can relax back to the trans state thermally, or, more rapidly, by illumination using 500 nm radiation.

  2. PA synthesis and characterization.
    Figure 2: PA synthesis and characterization.

    a, Synthesis of PA1 and PA2 starts with 4-aminoazobenzenes 2 and 3, respectively. Reaction with ​cyanamide installs ​guanidine functional groups, which can be coupled with an active ester of 4 to yield PA1 and PA2. b, Synthesis of PA3 and PA4 starts from the protected phenethylamine 5, which can be either condensed with ​nitrosobenzene or oxidized to a ​nitrosobenzene and condensed with ​p-(diethylamino)aniline. After acidic deprotection, primary amine building blocks 6 and 7 are functionalized to guanidines, which can react with ester 9 to yield PA3 and PA4, respectively. c, X-ray structure of PA1 indicates an extended hydrogen-bond network (dotted lines). d, Optimal wavelengths for photoswitching were determined using UV/vis spectroscopy. Generally, the more conjugated candidates PA1 and PA2 are more red-shifted than their corresponding homologues PA3 and PA4. Substitution with the electron-donating ​diethylaniline functionality leads to a red shift compared to the unsubstituted candidates (PA2 and PA4 versus PA1 and PA3). e, IC50 values of PAs in their dark-adapted trans form. PA4 is the most-potent and PA2 is the least-potent ENaC blocker. All PA molecules are more potent on the αβγ isoform. PA2 discriminates the most between αβγENaC and δβγENaC.

  3. PA1 photoswitching in Xenopus oocytes.
    Figure 3: PA1 photoswitching in Xenopus oocytes.

    a, Reversible block of δβγENaC currents evoked by photoisomerization of PA1 (10 µM) in 10 s and 5 s intervals. b, Action spectrum of PA1. Wavelengths were switched between 500 nm and 400 + x nm (x = 0, 20, 40, 60). The largest photoswitchable currents are observed when 500 and 400 nm are used as illumination wavelengths for cis and trans isomerization. c, Photoswitching of PA1 tested on an ​amiloride-sensitive current (IA) of αβγENaC-expressing (left) and δβγENaC-expressing (right) Xenopus oocytes by two-electrode voltage clamp recordings. Perfusion was stopped and illumination wavelengths were switched between 500 and 400 nm in one minute intervals. Current values were taken at the end of each interval and averaged (n = 12 and 10, respectively). Error bars indicate s.e.m., and significance refers to switching from 400 nm to 500 nm (*p < 0.05). There is a reversible photoswitching effect on both αβγENaC and δβγENaC. However, the amplitude of PA1-evoked photoswitching is higher on δβγENaC.

  4. Switching δβγENaC in HEK cells using 10 μM PA1.
    Figure 4: Switching δβγENaC in HEK cells using 10 μM PA1.

    a, By switching between 400 and 500 nm both large currents (upwards deflection = inhibition) and potentials (downwards deflection = hyperpolarization) can be controlled by light. b, The average current amplitude of photoswitching (ΔIP) is 182 pA for δβγENaC and 9 pA for αβγENaC. The average amplitude of the light-mediated membrane potential (ΔVP) is 57 mV for δβγENaC and 4 mV for αβγENaC (currents and potentials were recorded from the same cell, n = 10 biological repeats from three independent transfections and holding potential was –60 mV). c, Comparison between light-induced and thermal relaxation. d, Application of light with wavelengths between 400 nm and 500 nm reveals the physiological action spectrum of PA1 on δβγENaC-expressing HEK cells. Graded effects can be installed for both current (top) and membrane potential (bottom). e, Light-induced hyperpolarization and depolarization in the current-clamp mode. When the starting potential is adjusted to –70 mV under green light (left), photoswitching leads to hyperpolarization. When the starting potential is adjusted to –70 mV under blue light (right), photoswitching leads to depolarization. f, PA1 can be loaded into HEK cells by a short incubation. After one minute incubation at room temperature (r.t.) in 10 µM PA1 (yellow Petri dish) and subsequent wash (blue Petri dish), cells are ready for photoexperiments using the patch–clamp technique.

  5. Photocontrol of transepithelial potential in H441 monolayers.
    Figure 5: Photocontrol of transepithelial potential in H441 monolayers.

    a, Experimental configuration with an H441 monolayer (ML) on a filter inlet. The apical compartment contained 10 µM PA1 in sodium- or lithium-based buffer and was illuminated with a monochromator. The transepithelial potential VE between the apical and basolateral compartment was measured by a voltmeter (apical electrode is the reference). b, VE can be switched by toggling between 500 nm and 400 nm radiation (top), as well as between 694 nm and 400 nm (bottom). The 400 nm radiation was applied for ten seconds, the longer wavelengths for 20 seconds and the voltage was recorded at the end of each illumination period. Photoeffects are robust and reversible for both protocols. c, Comparison of the entire ​amiloride-sensitive VE and PA1-mediated photosensitive VE with apical sodium or lithium using both 400/500 nm and 400 nm/dark switching (n = 7). d, Comparison of the effect of extracellular lithium on αβγENaC and δβγENaC photocurrents in Xenopus oocytes (n ≥ 7). Error bars indicate s.e.m., *p-value < 0.05.

Compounds

16 compounds View all compounds
  1. 3,5-Diamino-N-carbamimidoyl-6-chloropyrazine-2-carboxamide
    Compound amiloride 3,5-Diamino-N-carbamimidoyl-6-chloropyrazine-2-carboxamide
  2. 3,5-Diamino-6-chloro-N-(N-phenylcarbamimidoyl)pyrazine-2-carboxamide
    Compound phenamil 3,5-Diamino-6-chloro-N-(N-phenylcarbamimidoyl)pyrazine-2-carboxamide
  3. 3,5-Diamino-N-(N-benzylcarbamimidoyl)-6-chloropyrazine-2-carboxamide
    Compound benzamil 3,5-Diamino-N-(N-benzylcarbamimidoyl)-6-chloropyrazine-2-carboxamide
  4. (E)-3,5-Diamino-6-chloro-N-(N-(4-(phenyldiazenyl)phenyl)carbamimidoyl)pyrazine-2-carboxamide
    Compound PA1 (E)-3,5-Diamino-6-chloro-N-(N-(4-(phenyldiazenyl)phenyl)carbamimidoyl)pyrazine-2-carboxamide
  5. (E)-3,5-Diamino-6-chloro-N-(N-(4-((4-(diethylamino)phenyl)diazenyl)phenyl)carbamimidoyl)pyrazine-2-carboxamide
    Compound PA2 (E)-3,5-Diamino-6-chloro-N-(N-(4-((4-(diethylamino)phenyl)diazenyl)phenyl)carbamimidoyl)pyrazine-2-carboxamide
  6. (E)-3,5-Diamino-6-chloro-N-(N-(4-(phenyldiazenyl)phenethyl)carbamimidoyl)pyrazine-2-carboxamide
    Compound PA3 (E)-3,5-Diamino-6-chloro-N-(N-(4-(phenyldiazenyl)phenethyl)carbamimidoyl)pyrazine-2-carboxamide
  7. (E)-3,5-Diamino-6-chloro-N-(N-(4-((4-(diethylamino)phenyl)diazenyl)phenethyl)carbamimidoyl)pyrazine-2-carboxamide
    Compound PA4 (E)-3,5-Diamino-6-chloro-N-(N-(4-((4-(diethylamino)phenyl)diazenyl)phenethyl)carbamimidoyl)pyrazine-2-carboxamide
  8. 3,5-Diamino-6-chloro-N-(N-phenethylcarbamimidoyl)pyrazine-2-carboxamide
    Compound 1 3,5-Diamino-6-chloro-N-(N-phenethylcarbamimidoyl)pyrazine-2-carboxamide
  9. (E)-4-(Phenyldiazenyl)aniline
    Compound 2 (E)-4-(Phenyldiazenyl)aniline
  10. (E)-4-((4-Aminophenyl)diazenyl)-N,N-diethylaniline
    Compound 3 (E)-4-((4-Aminophenyl)diazenyl)-N,N-diethylaniline
  11. 3,5-Diamino-6-chloropyrazine-2-carboxylic acid
    Compound 4 3,5-Diamino-6-chloropyrazine-2-carboxylic acid
  12. tert-Butyl 4-aminophenethylcarbamate
    Compound 5 tert-Butyl 4-aminophenethylcarbamate
  13. (E)-2-(4-(Phenyldiazenyl)phenyl)ethanamine
    Compound 6 (E)-2-(4-(Phenyldiazenyl)phenyl)ethanamine
  14. (E)-4-((4-(2-Aminoethyl)phenyl)diazenyl)-N,N-diethylaniline
    Compound 7 (E)-4-((4-(2-Aminoethyl)phenyl)diazenyl)-N,N-diethylaniline
  15. N-Chloro-1H-pyrazole-1-carboximidamide
    Compound 8 N-Chloro-1H-pyrazole-1-carboximidamide
  16. Methyl 3,5-diamino-6-chloropyrazine-2-carboxylate
    Compound 9 Methyl 3,5-diamino-6-chloropyrazine-2-carboxylate

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Author information

Affiliations

  1. Department of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians-Universität München, Butenandtstraße 5–13 (F4.086), 81377 Munich, Germany

    • Matthias Schönberger &
    • Dirk Trauner
  2. Institute of Animal Physiology, Justus Liebig University Giessen, Heinrich-Buff-Ring 26, 35392 Giessen, Germany

    • Mike Althaus,
    • Martin Fronius &
    • Wolfgang Clauss
  3. Department of Physiology, University of Otago, PO Box 913, Dunedin 9054, New Zealand

    • Martin Fronius

Contributions

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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The authors declare no competing financial interests.

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