Photochromic blockers of voltage-gated potassium channels

Article abstract of DOI:10.1002/anie.200904504

Light work: Studies into the mechanism of AAQ, a photoswitchable blocker of voltage-gated K⁺ channels, led to the discovery and development of photochromic ligands that act at the internal tetraethylammonium binding site (see picture). These mo

Full text of DOI:10.1002/anie.200904504

Angewandte
Chemie
DOI: 10.1002/anie.200904504
Photopharmacology
Photochromic Blockers of Voltage-Gated Potassium Channels**
Matthew R. Banghart, Alexandre Mourot, Doris L. Fortin, Jennifer Z. Yao, Richard H. Kramer,*
and Dirk Trauner*
Photochromic ligands (PCLs) can be optically switched
between isomers that show different biological activities. As
such, they offer an opportunity to convert ligand-actuated
pathways into light-actuated pathways, thus making it possi-
ble to control a wide range of biological processes with light.
PCLs have been explored for various classes of target
proteins, including enzymes,[1–3] ligand-gated ion channels,[4–6]
and G-protein-coupled receptors.[7] For instance, photochro-
mic agonists[5] and antagonists[8] for the nicotinic acetylcho-
line receptor, a ligand-gated ion channel, were described
more than thirty years ago. More recently, we have introduced
a photochromic version of glutamate that acts as a PCL on
kainate receptors and can be used to trigger neuronal firing.[6]
The PCL approach can be particularly effective in neural
systems, where the nonlinear nature of cellular excitability
can accentuate relatively small changes in efficacy or
incomplete photoconversion between isomers.
We report herein a family of amphiphilic azobenzene
molecules that target tetrameric voltage-gated ion channels
(Figure 1a). Channels of this type are not gated by extra-
cellular ligands but can be blocked by small molecules such as
lipophilic cations.[9,10] Our molecules function as photochro-
mic blockers of voltage-gated K+ channels and act on the
intracellular tetraethylammonium (TEA) binding site (Fig-
Figure 1. Photocontrol of K+ channels. a) Structures and photoisomeri-
zation of photoswitchable K+ channel blockers. b) A PCL for the
internal TEA binding site. The linear trans isomer is a better blocker
than the bent cis isomer. c) A PTL for the external TEA binding site.
The extended trans isomer presents the blocking particle to the pore
[*] Dr. A. Mourot,[+] Dr. D. L. Fortin, Prof. R. H. Kramer
Department of Molecular and Cellular Biology
University of California, Berkeley
Berkeley, CA 94720 (USA)
Fax: (+1)510-643-6791
while the shorter cis isomer is unable to reach.
E-mail: rhkramer@berkeley.edu
Prof. D. Trauner
Department of Chemistry, University of Munich
Butenandtstrasse 5-13 (F4.086), 81377 Munich (Germany)
have long-lasting effects in cells after a single, transient
Fax: (+49)89-2180-77972
E-mail: dirk.trauner@cup.uni-muenchen.de
Dr. M. R. Banghart,[+] J. Z. Yao, Prof. D. Trauner
ure 1b). They can be applied from the extracellular side and
application. In excitable cells, they function as photochromic
neuromodulators and can be used to optically control action
potential firing.
Department of Chemistry, University of California, Berkeley
We have previously reported the molecule AAQ (Acryl-
amide-Azo-Quaternary ammonium, 1), which was shown to
photosensitize wild-type K+ channels.[11] We had hypothesized
that AAQ functions as a photoswitchable tethered ligand
(PTL) at the external tetraethylammonium (TEA) binding
site and that it would attach to native residues through affinity
labeling (Figure 1c). However, our attempts to verify an
interaction at this site were inconclusive (Figures S1 and S2 in
the Supporting Information). Instead, our mechanistic studies
indicate that AAQ is not covalently bound and acts as a PCL
at the internal TEA binding site (Figure 1b).
Berkeley, CA 94720 (USA)
[+] These authors contributed equally to this work.
[**] This work was supported by a Laboratory Directed Research
Development Award from the Lawrence Berkeley National Labo-
ratory (R.H.K.), NSF-DFG grant CHE0724214 (D.T.), the National
Institutes of Health Nanomedicine Development Center for the
Optical Control of Biological Function (5PN2EY018241; R.H.K. and
D.T.), and by a grant from the Human Frontier Science Program
(RGP23-2005; R.H.K. and D.T.). We thank F. Tombola and E. Isacoff
for insightful discussions, the Isacoff and Yellen laboratories for
Shaker clones, and T. Fehrentz for additional experiments (not
included).
Potassium channels are not only blocked by alkyl
ammonium ions at the external tetraethylammonium (TEA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200904504.
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binding site, but also at the internal entrance to the selectivity
The current–voltage (I/V) curve further indicates that
AAQ acts at the internal TEA binding site and shows that
block by AAQ is distinctly voltage-dependent, so that Iss is
blocked more effectively at more depolarized membrane
potentials (Figure 2c). Under irradiation at 380 nm, the
current increases linearly with voltage once the channels are
fully activated (gray line). Under irradiation at 500 nm, the
current is predominantly blocked at all membrane potentials
(black line). However, irradiation at 420 nm, which produces
only partial conversion to the cis isomer, reveals voltage-
dependent block (dashed black line), as indicated by the
decline in Iss at potentials more positive than + 10 mV
(Figure S4a in the Supporting Information shows the raw
current responses). This result is typical of positively charged
intracellular K+ channel blockers.[10,22] Just as depolarization
provides a driving force for positively charged K+ ions to flow
in the outward direction, internal alkyl ammonium ions are
driven into their binding site within the membrane electric
field and block more effectively as membrane depolarization
is increased.
filter.[12–15] Charged blockers of the internal but not external
TEA binding site exhibit “open-channel block”, wherein pore
occupancy does not occur until after the voltage gate has
opened.[9,10,16] This effect is most easily observed in Shaker IR
(Sh-IR) and other channels that lack the fast-inactivating
N-terminal peptide.[17,18] In response to step depolarization,
some ionic current initially flows through the unbound
channel, but this flow quickly decays over tens of milliseconds
as the blocking molecules bind to the pore.
Figure 2a shows the current responses to depolarizing
voltage steps of a HEK293 cell expressing Sh-IR that has been
treated with 400 mm AAQ. Under irradiation at 380 nm, the
As K+ ions move through the permeation pathway of K+
channels in a single file, high concentrations of external K+
([K+]o) electrostatically repel intracellular charged blockers
to accelerate their exit rate from the channel and thereby
reduce their blocking potency.[23,24] Accordingly, the extent of
AAQ block correlates inversely with the extracellular potas-
sium concentration [K+]o , as revealed by the current values
shown in Figure 2d. After establishing a voltage clamp in
standard external buffer ([K+]o = 1.5 mm) and measuring Iss
under irradiation at 380 and 500 nm, cells were locally
perfused with solutions containing 0.3 mm and 20 mm [K+]o .
We controlled for the change in maximal current, which
results from the altered K+ driving force, by measuring
currents at 380 nm, which completely unblocks the channels.
This trend of lower efficiency was consistent across the range
of voltages that activate Sh-IR (Figure S4b in the Supporting
Information).
Consistent with this mode of action, direct application of
AAQ to the internal TEA binding site in both the inside-out
patch (Figure 3) and whole-cell recordings (Figure S5a in the
Supporting Information) also produced photoswitchable
open-channel block. Because inclusion in the patch pipette
does not permit solution exchange at the cytosolic interface,
inside-out patches were pulled from HEK293 cells expressing
Sh-IR to allow AAQ application, followed by washout. In this
Figure 2. AAQ is an open-channel blocker of the Sh-IR internal TEA-
binding site. a,b) Whole-cell current responses to 200 ms depolarizing
voltage steps from À70 to +40 mV under irradiation at 380 nm (gray
line) or 500 nm (black line). a) Open-channel block is apparent in
AAQ-treated Sh-IR channels (scale bar: 6 nA, 25 ms). b) Open-channel
block is absent in MAQ-treated SPARK channels (scale bar: 0.5 nA,
25 ms). c) Steady-state I/V curves under 380 nm (gray line), 420 nm
(black dashes) and 500 nm (black line) irradiation. I/V curves from
each of four cells were normalized to the current measured at 380 nm
and +60 mV. d) Dependence of AAQ block on [K+]o . Whole-cell
current responses of a cell to depolarizing voltage steps from À70 to
+40 mV under irradiation at 380 nm (gray line) or 500 nm (black line)
at the indicated [K+]o value. Similar results were obtained in two other
cells (Scale bar: 5 nA, 50 ms.)
channels are not blocked by AAQ (gray trace). However,
when the channels are blocked by AAQ under irradiation at
500 nm, an initial transient current remains (Ipk), which
rapidly decays so that nearly all of the steady-state current
(Iss) is blocked (black trace). This effect is not observed during
blockade of SPARK channels,[19] which contain an extracel-
lular cysteine residue for covalent attachment of the male-
imide analogue MAQ (2; Figure 2b).
The data obtained suggest that AAQ acts predominantly
at the internal TEA binding site of Sh-IR, while MAQ acts on
the external TEA binding site of the SPARK channels.[20,21]
A
more extensive characterization of the open-channel block
observed with AAQ is provided in Figure S3 in the Support-
ing Information. These experiments directly demonstrate the
requirement for channel opening to occur before AAQ is able
to block the pore, and reveal that pore occupancy is
correlated with the frequency of channel opening.
Figure 3. AAQ is a PCL for the internal-TEA binding site of Sh-IR. a) Iss
recorded from inside-out patches as AAQ was applied at the indicated
concentrations (mm). b) Dose-response relationships for AAQ applied
to inside-out patches under 500 nm (black line) and 380 nm (gray line)
irradiation.
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case, current block by AAQ was relieved within several
seconds of washout (Figure 3a), which indicates that covalent
binding did not occur under these conditions. Dose–response
curves could therefore be generated by irradiating patches
under 380 and 500 nm light in the presence of different
concentrations of AAQ. Summary data for photostationary
states enriched in trans- and cis-AAQ are shown in Figure 3b,
which reveals a 30-fold difference in block potency (IC50-
(500 nm) = (2.0 Æ 0.2) mm, IC50(380 nm) = (64 Æ 2.1) mm, n =
3–5 patches). Because 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.
AAQ is an acrylamide and therefore contains an electro-
philic functional group, yet our data indicated that analogues
that lack an electrophile would function in a similar manner.
To test this model and identify photochromic blockers with
improved biophysical properties, we prepared a series of
analogues (Figure 1a, 3–10). To facilitate partitioning into the
membrane, several analogues contain aliphatic “tails” of
increasing hydrophobicity (3–7). In contrast, the doubly
charged, symmetric analogue 8 was expected to exhibit poor
membrane penetration. In other members of the series, the
amide tail was either removed completely (9) or replaced with
a propyl group (10) to mimic the length of 3, which was the
shortest amide examined. Prior to screening, UV/Vis spec-
troscopy 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 for 380 and
500 nm irradiation, respectively (data not shown, details for
chemical synthesis are given in the Supporting Information)
Strikingly, after a brief extracellular application followed
by washout, all of the new compounds that contain a
hydrophobic tail (3–7, 9, 10) were found to function as
open-channel blockers and persistently blocked Sh-IR cur-
rents for the duration of a typical recording (5 min). Iss
measurements from cells treated with either AAQ, 7, or 10
under repeated 380 nm and 500 nm irradiation are shown in
Figure 4a–c. Step responses to depolarization for each new
compound are shown in Figure S5 in the Supporting Infor-
mation.
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. The doubly charged analogue 8 did not block
channels after extracellular treatment (tested at concentra-
tions up to 2 mm), although inclusion in the patch pipette did
afford photoswitchable block (results not shown). Benzoyl-
Azo-QA (BzAQ, 7), was found to block more than 95% of Iss
at a concentration of only 25 mm. A dose–response curve
obtained from inside-out patches (Figure 4d) indicates that
trans-BzAQ has a similar affinity to trans-AAQ when applied
directly to the internal TEA binding site (IC50(500 nm) =
(1.3 Æ 0.2) mm, IC50(380 nm) = (83 Æ 20) mm, n = 4). Therefore,
its enhanced potency is most likely due to better membrane
partitioning rather than increased affinity for the blocking
site.
Figure 4. PCLs persistently photosensitize Sh-IR. Iss recorded over time
from a cell treated with a) 400 mm AAQ, b) 20 mm BzAQ (7), and
c) 40 mm PrAQ (10) under 380 nm (gray bars) and 500 nm (black bars)
irradiation. d) Dose–response relationships for BzAQ (7) applied to
inside-out patches under 500 nm (black line) and 380 nm (gray line)
irradiation.
Table 1: Structure–activity relationships.
Compound
mm[a]
% Block[b]
Active isomer
1 (AAQ)
2 (MAQ)
3
4
5
6
7 (BzAQ)
8
9
400
400[c]
1000
800
300
200
25
2000
1000
40
>95
0
trans
–
>95
>95
>95
>95
>95
0
trans
trans
trans
trans
trans
trans
trans=cis
cis
ND[d]
ꢀ50
10 (PrAQ)
[a] Concentrations reported for 1–6 and 8 were determined by first
identifying a dose that produced greater than 95% block. The concen-
tration was reduced in 100 mm increments until less than 95% block was
observed. For compounds 7 and 10, 5 mm increments were used to
determine the reported values. At each reported concentration, deter-
minations were repeated in three to five cells from two separately treated
coverslips. [b] For compounds 1–9, the block percentage was calculated
as the ratio between the Iss values measured under irradiation at 500 nm
(block) and 380 nm (reference). For compound 10, the wavelengths were
reversed. In each case, unblock was estimated as complete by the lack of
open-channel block, as shown in Figure S5 in the Supporting Informa-
tion. [c] Treatment of HEK293 cells with MAQ at concentrations greater
than 400 mm did not result in stable recordings. [d] Although substantial
open-channel block was observed, the lack of unblock at either
wavelength prevented calculation of the block percentage. ND=not
determined.
the Supporting Information). This result suggests that inter-
actions between the tail and channel protein might account
for the differences in affinity between the isomers. Indeed,
Propyl-Azo-QA (PrAQ, 10), preferentially blocks when it is
in the cis form (Figure 4c). External treatment with 40 mm of
compound 10 resulted in approximately 50% block of Iss with
the cis form, but no obvious block by the trans isomer was
observed, as judged by the lack of rapid current decay upon
In contrast to the amides 3–7, compound 9 exhibited
comparable block in both the cis and trans forms (Figure S5 in
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irradiation at 500 nm. However, at higher concentrations,
43[25] and many voltage-sensitive dyes.[26] It has been proposed
that classic local anesthetics access the internal binding site of
voltage-gated sodium channels through hydrophobic path-
ways.[27] Regardless of the exact mechanism of retention, the
phenomenon of long-lasting block is not unique to our
photochromic blockers. An array of hydrophobic ammonium
ions such as the lidocaine derivative Tonocaine, and tetra-
pentylammonium (TPeA), have been shown to become
trapped after crossing the cell membrane and block voltage-
gated sodium and potassium channels for many minutes after
washout.[28,29]
AAQ was originally designed to covalently bind at the
external TEA binding site. Although the results presented
here do not rule out covalent modification of Sh-IR alto-
gether, they do suggest that AAQ predominantly acts as a
PCL at the internal TEA binding site instead. This result has
allowed us to define an entire class of photochromic ligands
that have a new mode of function and explore their structure-
activity relationships in detail. 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 neuro-
biology and could have therapeutic value, for instance in
attempts to restore vision.
blocking by the trans isomer was observed (data not shown).
As the thermally stable trans isomer is inactive, optimized cis-
blocking analogues of PrAQ will reduce cellular exposure to
UV light and avoid unintentional channel blocking during
PCL application in the dark.
To determine if these novel PCLs function in neurons in
the same way as AAQ, we examined the most potent
analogue BzAQ (7) in dissociated hippocampal cultures, a
preparation in which AAQ was previously found to photo-
sensitize endogenous K+ after preincubation at 300 mm.[11]
Current–voltage curves from neurons recorded in the
whole-cell voltage clamp configuration showed that the K+
channels that contribute to Iss in neurons are modulated after
extracellular treatment with 20 mm BzAQ (Figure 5a). From
Figure 5. BzAQ photoregulates endogenous K+ channels in dissociated
hippocampal neurons to modulate neural activity. a) Steady-state I/V
curves under 380 nm (gray line) and 500 nm (black line) irradiation
recorded from neurons treated with 20 mm BzAQ. Recordings from
four individual cells were normalized to the current measured at
380 nm and +70 mV. b) Current clamp recording from a neuron
showing the induction of action potential firing in response to 500 nm
light (black line). Similar results were obtained in at least three
additional cells. Scale bar: 10 mV, 10 s.
Received: August 12, 2009
Published online: October 30, 2009
Keywords: azo compounds · ion channels · open-channel block ·
.
photochromism · structure–activity relationships
this data, the Iss value at + 40 mV was compared after
irradiation at 380 and 500 nm, and revealed that on average,
(35.5 Æ 4.7)% (n = 10) of Iss is blocked by BzAQ under these
conditions. In the current clamp mode, which allows the
recording of action potentials, BzAQ was found to depolarize
the cellular membrane potential when switched from cis to
trans, which was sufficient to induce action potential firing
(Figure 5b). Furthermore, similarly to AAQ,[11] BzAQ did not
affect depolarizing Na+ currents in neurons (data not shown)
and did not photosensitize voltage-gated Na+ or Ca2+
channels expressed in GH3 cells (Figure S6 in the Supporting
Information). Taken together, these data indicate that BzAQ
is an effective photomodulator of neural activity, which has
features similar to AAQ,[11] but has the advantage of
increased potency and avoids the potential toxicity and
immunogenicity associated with reactive reagents.
The results presented here do not explain how the blocker
molecules are retained by cells for long periods of time. While
AAQ can effectively photosensitize Sh-IR in HEK293T cells
for just over one hour, dissociated hippocampal neurons
respond to light at least 24 hours after exposure to AAQ
(Figure S7 in the Supporting Information). It is likely that
retention occurs by tight association with the plasma mem-
brane, as is the case with a structurally related styryl class of
fluorophores, which includes the endosomal marker FM1-
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