87The first example of a photoswitchable ion channel was demonstrated with wild-
type nicotinic acetylcholine receptors (nAChR) in living cells. Proof of principle
experiments using a non-covalent, symmetric molecule: Bis-Q (Bartels et al. 1971 )
demonstrated that the trans isomer activates the receptor; however, its cis isomer was
inactive. The authors then synthesized an electrophilic derivative, Q-Br (Fig. 3b),
which alkylated reduced nAChRs. Similar to the free diffusing agonist, Bis-Q, Q-Br
only activated the receptor in the trans conformation (Bartels et al. 1971 ; Lester
et al. 1979 ). The similarities between free-diffusing and tethered agonist suggested
that formation of the agonist-receptor complex was not the rate-limiting step that
governs channel opening and closing (Lester et al. 1980 ). Later, the tethered ago-
nist, BrACh (Fig. 3b) was used to probe the kinetics of receptor activation (Chabala
and Lester 1986 ). Because these tethered reagents eliminate the diffusion-limited
formation of the initial ligand-channel complex, Lester and colleagues could con-
vincingly show that receptor antagonists bound an order of magnitude slower than
agonists, providing critical insight into ligand-gated ion channel modulation.
The solution of a high-resolution structure of a Shaker-like voltage-gated K+
channel enabled this strategy to be readily applied to the voltage-gated K+ channel
family. Kramer and co-workers developed a “light gate” to confer sensitivity to the
Shaker K+ channel using an azobenzene tether connecting a quaternary ammoni-
um blocker and cysteine-reactive maleimide (MAL-AZO-QA) (Fig. 3a) (Banghart
et al. 2004 ). Application of MAL-AZO-QA to Xenopus oocytes expressing a Shaker
Fig. 3 Photoswitchable ligands control channel function. a Generic structure of cysteine-reactive
photoisomerizable azobenzene linker. Purple and green spheres represent a channel ligand or
blocker in the azobenzene cis or trans state, respectively. Upon exposure to different wavelengths
of light, the effective tether length changes by ~ 7 Å. b Structures of the earliest tethered agonists
used to probe the nicotinic acetylcholine receptor. c K+ channels modified with MAL-AZO-QA
exhibit large current in 380 nm light and block in 500 nm light. d Neuronal firing in hippocampal
neurons can be silenced with open K+ channels tethered with cis MAL-AZO-QA, which is revers-
ible by blocking these constitutively open channels with exposure to 500 nm light
Bioreactive Tethers