Julie B.
Trads
ab,
Jessica
Burgstaller
c,
Laura
Laprell
a,
David B.
Konrad
a,
Luis
de la Osa de la Rosa
a,
C. David
Weaver
d,
Herwig
Baier
c,
Dirk
Trauner
*a and
David M.
Barber
*a
aDepartment of Chemistry and Center for Integrated Protein Science, Ludwig Maximilians University Munich, Butenandtstraße 5-13, 81377 Munich, Germany. E-mail: dirk.trauner@lmu.de; david.barber@cup.uni-muenchen.de
bCenter for DNA Nanotechnology, Department of Chemistry and iNANO, Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark
cMax Planck Institute of Neurobiology, Am Klopferspitz 18, 82152 Martinsried, Germany
dDepartment of Pharmacology and Institute of Chemical Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA
First published on 30th November 2016
G-protein coupled inwardly rectifying potassium (GIRK) channels are an integral part of inhibitory signal transduction pathways, reducing the activity of excitable cells via hyperpolarization. They play crucial roles in processes such as cardiac output, cognition and the coordination of movement. Therefore, the precision control of GIRK channels is of critical importance. Here, we describe the development of the azobenzene containing molecule VLOGO (Visible Light Operated GIRK channel Opener), which activates GIRK channels in the dark and is promptly deactivated when illuminated with green light. VLOGO is a valuable addition to the existing tools for the optical control of GIRK channels as it circumvents the need to use potentially harmful UV irradiation. We therefore believe that VLOGO will be a useful research tool for studying GIRK channels in biological systems.
GIRK channels exist as either homo- or heterotetramers comprised of the four subunits GIRK1-4, which exhibit distinct expression patterns throughout the body.4 For example, GIRK1/2 channels are typically found in the central nervous system (CNS) and GIRK1/4 channels are characteristic of the cardiovascular system.5 It has been shown that GIRK channels are activated by a variety of small molecules, albeit with varying degrees of selectivity.6 The compounds ML297 and VU0259369 are exceptions as they are potent and selective activators of GIRK channels bearing the GIRK1 subunit (Fig. 1a).7
We recently demonstrated that GIRK channels can be endowed with light control using photopharmacology.8 Our freely diffusible photoswitch LOGO, is a derivative of VU0259369 that contains an azobenzene moiety as the photoresponsive element (Fig. 1a).9 It harnesses the precision of light to accurately control the spontaneous action potential firing of dissociated hippocampal neurons and the swimming behaviour of zebrafish larvae. However, a significant drawback of LOGO is that UV and blue light are required for its cis/trans photoisomerisation. This results in increased levels of phototoxicity10 and limits the amount of tissue penetration.11 By employing photoswitches that respond to red-shifted wavelengths of light we would be able to improve these properties.12
Herein, we present VLOGO (Visible Light Operated GIRK channel Opener), which is an ortho-fluorinated azobenzene photoswitch that enables the optical control of GIRK channels using violet and green light. The red-shifted properties of VLOGO improve its suitability for experiments in vivo and we therefore envisage that VLOGO will be a valuable tool for GIRK channel research.
With our small library of compounds in hand, we determined their optimum photoswitching properties using UV-Vis spectroscopy (Fig. S2†). We then applied this information to our initial compound analysis using patch clamp electrophysiology of GIRK1/2 channels expressed in HEK293T cells (Fig. 2a). Starting with the 4′-substituted azobenzenes 1–4 (Fig. 2a), we observed small amounts of GIRK1/2 channel opening and moderate levels of current modulation upon photoswitching with the 4′-amino azobenzene 2 and the 4′-N,N-diethyl azobenzene 3. In contrast, almost no change in current was observed upon photoswitching using azobenzenes 1 and 4. We next evaluated VLOGO and found it to be an excellent photoswitchable opener of GIRK1/2 channels using 400 and 500 nm light (Fig. 2a). Spurred on by this result we assessed ortho-chloro azobenzene 6. However, we discovered that this photoswitch could not endow GIRK1/2 with significant amounts of light control. This was an unfortunate result, as azobenzene 6 would have allowed longer wavelengths of light to be used for the optical control of GIRK channels. When examining all of the results, it becomes clear that only small modifications to the azobenzene photoswitch are tolerated in order to retain both potent agonism and good photoswitching behaviour at GIRK1/2 channels.
With ortho-fluoro azobenzene VOLOGO identified as our optimal red-shifted photoswitch for GIRK1/2 channels, we further investigated its properties using patch clamp electrophysiology. Firstly, we investigated the action spectrum of VLOGO by switching the wavelength between violet light (400 nm) and different wavelengths of blue/green light (440–540 nm), we observed large differences in current (Fig. 2b). The minimal inward current was exhibited when illuminating at 500 nm. In contrast, 440, 460, 480, 520 and 540 nm resulted in more inward current (Fig. S3†). We then established that reversible photoactivation of VLOGO (10 μM) is very robust, with almost no loss of photocurrent over many switching cycles (Fig. 2c). The trans-isomer of VLOGO, which predominates in the dark and under violet light (400 nm) illumination, is the most active form of VLOGO. Upon photoisomerisation using green light (500 nm), the considerably less active cis-isomer of VLOGO is formed, causing a reduction in the observed current. When operating in current clamp mode the photoswitching of VLOGO was also found to be highly reproducible (Fig. S4†). Lastly, we examined the stability of cis-VLOGO in the dark (Fig. 2d). After the full cis-isomer content had been reached under 500 nm light illumination, VLOGO was exposed to dark conditions, quickly resulting in increased inward current. The minimal inward current was achieved again using 500 nm light. This result demonstrates that constant illumination of VLOGO is required to maintain the maximum cis-isomer content.
Having characterised VLOGO using patch-clamp electrophysiology in HEK293T cells, we subsequently evaluated the activity of VLOGO towards different GIRK channel subtypes using the thallium flux assay technique (Fig. 3).17 We found that VLOGO activates GIRK channels containing the GIRK1 subunit, whilst not affecting homodimeric GIRK2 channels. VLOGO exhibits similar potency at GIRK channels (GIRK1/2: EC50 = 2.0 ± 0.11 μM; GIRK1/4: EC50 = 1.9 ± 0.06 μM) as the non-substituted photoswitchable GIRK channel agonist LOGO.9 However, the efficacy of VLOGO is significantly reduced (GIRK1/2: %Emax = 49.4 ± 4.7; GIRK1/4: %Emax = 30.9 ± 5.2).
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Fig. 3 Potency, efficacy and selectivity profile of VLOGO. Shown are fits to representative data obtained from testing multiple concentrations of VLOGO on cell lines stably expressing GIRK1/2 (blue circles), GIRK1/4 (green triangles) and GIRK2 (red squares). The measured potencies for GIRK1/2 and GIRK1/4 were 2.0 ± 0.11 μM and 1.9 ± 0.06 μM, respectively. Efficacy values were normalised to the maximum activity observed using ML297 (10 μM) on GIRK1/2-expressing cells. Error bars represent mean ± SEM obtained from triplicate wells. Reported potency values represent the mean ± SEM obtained from three independent experiments.18 |
After demonstrating that VLOGO can optically control cellular currents in HEK293T cells heterologously expressing GIRK1/2 channels, we next investigated if it could be used to control GIRK channels in excitable cells.19 For this endeavour we conducted electrophysiological experiments on CA1 hippocampal neurons found in acute brain slice preparations from wild type mice. Firstly, we recorded current–voltage (I–V) relationships comparing the control traces with those obtained in the presence of VLOGO (Fig. 4a). Due to the inwardly rectifying nature of GIRK channels, we would expect to see increasing amounts of inward current as the voltage becomes more negative. After the application of VLOGO (100 μM) and subsequent wash out, the hippocampal neuron exhibited increasing inward current at voltages lower than −90 mV during a −120 mV to +30 mV voltage ramp. This is indicative of GIRK channel opening.7 Most importantly VLOGO was able to reversibly silence spontaneous action potential firing under violet light (400 nm) illumination when the hippocampal neurons were held at depolarised potentials (Fig. 4b). Changing the illumination wavelength to 500 nm then efficiently restored the action potential firing.
After accomplishing the control of native GIRK channels in mouse hippocampal neurons using VLOGO, we investigated if it could be used to optically control the movement of living animals. For these experiments we selected zebrafish larvae (Danio rerio) as light can easily be delivered to them due to their transparency.20 Accordingly, zebrafish larvae 5–7 days post fertilisation were exposed to 2 minute intervals of alternating violet light (420 nm) and green light (520 nm) (Fig. S5†). After the first cycle of violet and green light, the distance (mm) that the zebrafish larvae moved in a 10 second time period within a representative section of each 2 minute interval was measured to give the background swimming behaviour for each condition. The zebrafish larvae were then incubated with VLOGO (100 μM) for 1 hour and the same protocol was used to determine its effect by calculating the change in swimming distance. Pleasingly, the zebrafish larvae showed significantly different swimming distances in the presence of VLOGO, which could be modulated by changing the illumination from violet to green light (Fig. 5). The zebrafish larvae exhibited reduced swimming distances when illuminated with violet light compared to the control zebrafish. After illuminating with green light for a total of 2 minutes, the swimming distance of the zebrafish was not significantly different from the control experiment. We then performed additional experiments using the non-photoswitchable GIRK activator ML297 in an effort to dissect the effect of VLOGO from the native responses to violet and green light. These experiments confirmed that there is almost no change in the swimming distances of zebrafish larvae when illumination is changed between violet and green light. This is significantly different from the results obtained using VLOGO in conjunction with violet and green light.
Footnote |
† Electronic supplementary information (ESI) available: Experimental procedures and characterisation data. See DOI: 10.1039/c6ob02153k |
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