88 K. Mruk and W. R. Kobertz
mutant containing an extracellular cysteine, blocked channel current that could be
reversed after ultraviolet light consistent with trans to cis isomerization. To photo
manipulate neuronal excitability, hippocampal neurons were transfected with an ex-
tracellular cysteine mutant of inactivation-removed Shaker ( Shaker-IR) and labeled
with MAL-AZO-QA (Banghart et al. 2004 ). Exposure to ultraviolet light silenced
spontaneous action potentials in transfected cells, which was restored upon visible-
light illumination. To photo stimulate action potentials, a Shaker pore mutation was
used to convert the K+ channel into a non-selective cation channel (Chambers et al.
2006 ). Later, MAL-AZO-QA was used to control the two-pore TREK1 channel in
transfected hippocampal neurons (Sandoz et al. 2012 ). Cysteines were engineered
into homologous sites on each pore loop. Modification of the first pore loop (P1)
resulted in channel block in the trans state whereas modification of the second pore
loop (P2) resulted in block in the cis state. This differential block - suggested that
there was structural asymmetry between the two loops—a hypothesis supported by
the subsequent crystal structures of this class of potassium leak channels (Brohawn
et al. 2012 , 2013 ; Miller and Long 2012 ). Although these studies laid the foun-
dation for spatiotemporally controlling neuronal excitability and probing channel
structure with light, they were limited because overexpression of non-native exog-
enous channels was required, which can result in hyperexcitability and formation
of heteromultimers with native channel subunits. Furthermore, this technique was
not readily generalizable to ligand-gated channels responsible for the generation of
most neuronal action potentials.
To target ligand-gated channels, the Kramer lab developed a tethered analogue
of glutamate containing the azobenzene moiety called MAG (Volgraf et al. 2006 ).
Cysteine scanning mutagenesis identified several positions within the ionotropic
glutamate receptor (iGLuR), which could be modified with MAG and activated
in a light-dependent manner when expressed in HEK293 cells. Chemically modi-
fied channels were activated by both free glutamate and ultraviolet light indicating
that MAG may only permit partial closure of the ligand-binding domain. Further
characterization of this system revealed that (i) MAG controls only the cysteine-
engineered receptor, (ii) the photoswitch kinetics are fast (milliseconds), and (iii)
upon photoswitching the effective local concentration of the tethered glutamate is
in the millimolar range (Gorostiza et al. 2007 ). Because ligand-binding domains are
present on a large number of proteins, these studies demonstrated the overall appli-
cability of the azobenzene tether. To demonstrate the generality of a ligand-binding
domain approach, Kramer and co-workers redesigned the original photorevers-
ible Q-Br reagent (Fig. 3b) and targeted neuronal nicotinic acetylcholine receptors
(nAChRs) with photoswitchable tethered agonists and antagonists (Tochitsky et al.
2012 ).
Azobenzene-containing tethers have also been utilized to dissect complex neural
networks. One technical limitation of the light-gated K+ channel or glutamate re-
ceptor is the inability to control activity within densely packed neural circuits. To
precisely stimulate a subset of neurons, Zhang and colleagues coupled two LEDs to
a digital micromirror device to produce patterned light. Expression of a light-gated
glutamate receptor (LiGluR) in combination with with calcium imaging permitted