1. Dirk Trauner
Richard H. Kramer
Department of Molecular and Cellular Biology
University of California, Berkeley
Department of Chemistry, University of Munich, Germany

2. Voltage gated ion channels
Ion channels are pore-forming proteins that help establish and control the small voltage gradient across the plasma membrane of all living cells by allowing the flow of ions down their electrochemical gradient.
They are present in the membranes that surround all biological cells.
Voltage gated ion channels
Voltage gated ion channels are activated by changes in electrical potential difference near the channel.
Voltage gated ion channels are especially critical in neurons, but are common in many types of cells.
Potassium channels
Potassium channels are the most widely distributed type of ion channel.
Potassium channels form potassium-selective pores that span cell membranes.
Furthermore potassium channels are found in most cell types and control a wide variety of cell functions.

3. Potassium channel blockers
Potassium channel blockers are agents which interfere with conduction through potassium channels.

4. Photochromic ligands (PCLs)
Photochromic ligands (PCLs) can be optically switched between isomers that show different biological activities.
Photochromic ligands convert ligand-actuated pathways into light-actuated pathways, thus making it possible to control a wide range of biological processes with light.
PCL’s have been used in various biological systems nicotinic acetylcholine receptor, a ligand-gated ion channel, were described more than thirty years ago.

5. Molecules which are used in this study
These molecules target the tetrameric voltage gated ion channels
These molecules acts on the intracellular tetraethyl ammonium binding site of the potassium channels

6. Internal and external tetraethylammonium binding sites of the Potassium channels

7. The studies carried out in HEK293 cells which expressing Sh
The studies carried out in HEK293 cells which expressing Sh.IR and SPARK Channels
The electrical characterization of the channels
Voltage clamp
Patch clamp
Whole-cell current responses to 200 ms depolarizing voltage steps from 70 to +40 mV under irradiation at 380 nm (gray line) or 500 nm (black line)

8. Concentration dependence of extra cellular AAQ
Increasing the concentration of the AAQ the current block is in creased
The data recorded at the photo stationary states of the cis and trans isomer
Dose–response Curves generated by irradiating 380 and 500 nm light in the presence of different concentrations of AAQ.
The measurements reveals a 30-fold difference in block potency (IC50-(500 nm)=(2.00.2) μm, IC50(380 nm)=(64  2.1) μm.
The photoswitchable block was observed without any indication of covalent modification, these experiments demonstrate that AAQ can act as a PCL to block the internal TEA binding site.

9. AAQ contains an electrophilic functional group
To facilitate partitioning into the membrane, several analogues contain aliphatic “tails” of increasing hydrophobicity (3–7).
UV/Vis spectroscopy confirmed that irradiation under 380 nm and 500 nm
light produced nearly identical photostationary states, which consisted of at least 80% cis and trans isomers

10. The doubly charged analogue 8 did not block channels after extracellular treatment (tested at concentrations up to 2 mm).

11. A general trend was observed in which potency correlates with tail length and hydrophobicity
Table 1 shows the lowest concentrations required to produce more than 95% block of Iss at +40 mV.
compound 9 exhibited comparable block in both the cis and trans forms
Interactions between the tail and channel protein might account for the differences in affinity between the isomers.

12. The dose–response curve indicates that trans-BzAQ has a similar affinity to trans-AAQ when applied directly to the internal TEA binding site.
The enhanced potency of trans-BzAQ is most likely due to better membrane
partitioning rather than increased affinity for the blocking site.

13. Studies in neurons
Tested the most potent analogue BzAQ (7) in dissociated hippocampal cultures
Iss value at +40 mV was compared after irradiation at 380 and 500 nm, and revealed that on average, ( )% of Iss is blocked by BzAQ at 20 μM
BzAQ was found to depolarize the cellular membrane potential when switched from cis to trans, which was sufficient to induce action potential firing
BzAQ did not affect depolarizing Na+ currents in neurons
Did not photosensitize voltage-gated Na+ or Ca2+ channels

14. The data indicate that BzAQ is an effective photomodulator of neural activity.
BzAQ has features similar to AAQ, but has the advantage of increased potency and avoids the potential toxicity and immunogenicity associated with reactive reagents.
Given the many structural, functional, and pharmacological similarities of voltage-gated ion channels, the principles established herein should also enable the development of additional PCLs for voltage-gated Na+ and Ca2+ channels.
Our photochromic neuromodulators have already proven themselves as useful tools in neurobiology and could have therapeutic value, for instance in attempts to restore vision.