2. Schematic drawing of heterotrimeric αβγENaC and δβγENaC
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.

3. Synthetic route:

4. 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.
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.
Photoisomerization of PA1- Illumination with 400 nm induces isomerization to cis-PA1, The cis isomer can relax back to the trans state thermally, or more rapidly, by illumination using 500 nm radiation.

5. Photocurrents in Xenopus oocytes.
Two electrode voltage clamp recordings from ENaC expressing Xenopus oocytes – PAx Screen. Photoswitchable amiloride derivatives PA1-PA4 (left column) were tested on ENaC (middle column) and ENaC (right column).

6. Reversible block of δβγENaC currents evoked
Wavelengths were switched between 500 nm and 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
Reversible block of δβγENaC currents evoked
by photoisomerization of PA1 (10 μM) in 10s
and 5s intervals.
Control experiment. ENaC expressing Xenopus oocytes do not show innate sensitivity to light in both unblocked and blocked state.

7. Switching δβγENaC in HEK cells using 10 μM PA1.
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 57mV for δβγENaC and 4 mV for αβγENaC
Comparison between light-induced and thermal relaxation. This indicates the novel optical tool can be operated by turning on and off a simple light source.
By switching between 400 and 500 nm both large currents (upwards deflection = inhibition) and potentials (downwards deflection = hyperpolarization) can be controlled by light

8. 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.

9. Conclusions:
The photoswitchable ENaC blocker PA1 has been designed, synthesized and functionally characterized using electrophysiology with Xenopus oocytes, HEK293t cells and H441 cell monolayers.
results provide new insights into the structure–activity relationships of the amiloride class of diuretics andoutline a strategy to address selectively the αβγ and δβγ isoforms of ENaC channels.
PA1 can be used to control δβγENaCs optically by switching between blue and green light, or by turning on and off blue light. This therefore allows ENaC to be investigated without mechanical stress.
The combination of photoswitchable PA1 and the voltage-insensitive δβγENaC is a powerful tool to control precisely the membrane potential with light, allowing for both hyper- and depolarization.
With its unique functional profile, PA1 could enable new insights into the δβγENaC function, for instance in the primate brain.