Article pubs.acs.org/ac
Protein-Coupled Fluorescent Probe To Visualize Potassium Ion Transition on Cellular Membranes Tomoya Hirata,† Takuya Terai,*,†,⊥ Hisao Yamamura,⊗ Manabu Shimonishi,‡,○ Toru Komatsu,†,# Kenjiro Hanaoka,† Tasuku Ueno,†,⊥ Yuji Imaizumi,⊗ Tetsuo Nagano,§ and Yasuteru Urano*,†,⊥,∥ †
Graduate School of Pharmaceutical Sciences, ‡GCOE Program, §Drug Discovery Initiative (DDI), and ∥Graduate School of Medicine, The University of Tokyo, Tokyo 113-0033, Japan ⊥ CREST and #PRESTO, Japan Science and Technology Agency, Tokyo 102-0076, Japan ⊗ Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan S Supporting Information *
ABSTRACT: K+ is the most abundant metal ion in cells, and changes of [K+] around cell membranes play important roles in physiological events. However, there is no practical method to selectively visualize [K+] at the surface of cells. To address this issue, we have developed a protein-coupled fluorescent probe for K+, TLSHalo. TLSHalo is responsive to [K+] in the physiological range, with good selectivity over Na+ and retains its K+-sensing properties after covalent conjugation with HaloTag protein. By using cells expressing HaloTag on the plasma membrane, we successfully directed TLSHalo specifically to the outer surface of target cells. This enabled us to visualize localized extracellular [K+] change with TLSHalo under a fluorescence microscope in real time. To confirm the experimental value of this system, we used TLSHalo to monitor extracellular [K+] change induced by K+ ionophores or by activation of a native Ca2+-dependent K+ channel (BK channel). Further, we show that K+ efflux via BK channel induced by electrical stimulation at the bottom surface of the cells can be visualized with TLSHalo by means of total internal reflection fluorescence microscope (TIRFM) imaging. Our methodology should be useful to analyze physiological K+ dynamics with high spatiotemporal resolution. contrast, fluorescence imaging using functional probes readily provides spatiotemporal information about the target molecules,9−11 and fluorescent K+ probes have the potential to detect physiological [K+] transition in a simpler way. However, classical 18-crown-6 based K+ probes such as PBFI12 are not suitable for monitoring [K+]o, because they suffer from relatively low fluorescence, poor selectivity for K+ over Na+ (the ion that predominantly exists in extracellular medium), and suboptimal cellular localization. Recently, a K+-selective chelator, 2-triazacryptand [2,2,3]-1-(2-methoxyethoxy)benzene (TAC) was developed,13 and several fluorescent K+ probes based on TAC have been reported.14−17 Among these K+ probes, TAC-Red14 and TAC-Limedex16 were reported as extracellular [K+] probes, and they were applied for measurement of K+ waves in mouse brain cortex14 and [K+] in airway surface liquid in ex vivo and in vivo trachea,18 respectively. However, although these K+ probes are capable of detecting bulk [K+]o change in tissues, there is room for improvement in
P
otassium ion (K+) is the most abundant metal cation in cells and plays essential roles in physiological events such as cardiac and neuronal excitability, cellular ionic homeostasis, etc.1 K+ is highly localized inside cells in the resting state (extracellular concentrations of potassium ([K+]o) = 5−10 mM and intracellular concentrations of potassium ([K+]i) = 150− 200 mM),2 but when the cells are stimulated, it effuses through K+ channels, resulting in hyperpolarization of the cell membrane. Also, [K+]o around cells has recently been regarded as an active participant in cellular signaling transduction in the brain3−5 and blood vessels.6 Although values of [K+]o near activated cells in vivo are not precisely known, simulations suggest [K+]o is transiently increased to about 20 mM,3 and this change relative to the resting state should be larger than that in cytosol or than the average change in the extracellular medium. So, selective measurement of [K+]o near the cell surface will be a sensitive tool to analyze [K+] dynamics induced by K+ channels and other proteins. Currently, [K+]o is usually measured with a K+-sensitive, double-barreled microelectrode, but the use of microelectrodes at the single cell level is technically challenging and invasive, despite its accuracy.7,8 Also, it is not possible to get comprehensive spatial information with a microelectrode. In © XXXX American Chemical Society
Received: October 24, 2015 Accepted: February 4, 2016
A
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
from Dr. Takahiro Nagase (Kazusa DNA Research Institute, Japan). General chemicals were from Tokyo Chemical Industries, Wako Pure Chemical, Aldrich Chemical Co., and Invitrogen. Fluorescence Spectroscopy. Fluorescence measurements were performed on an F4500 (Hitachi) in HEPES buffer (5 mM, pH 7.0), sodium phosphate buffer (100 mM, various pH), or PBS containing less than 2% (v/v) DMSO as a cosolvent. Specific conditions are described in each figure legend. Methods of ΦFL calculation and Kd fitting are described in the Supporting Information. Protein Labeling in Vitro. To 40 μL of PBS containing recombinant protein (45−50 μM) and BSA (90−100 μM) was added a developed K+ probe (as 1 mM stock solution, 1 equiv to recombinant protein). The reaction mixture was incubated at 37 °C for 1 h and then purified on a PD-10 desalting column (GE Healthcare). Cell Imaging. HeLa cells and HT29 cells (RIKEN BioResource Center, Japan) were cultured in DMEM and McCoy’s 5a Medium (Invitrogen), respectively. Transient transfection was done with Lipofectamine LTX (Invitrogen). Cells seeded on 35 mm glass-bottomed dishes were washed with 1 mL of medium, then incubated in medium containing a probe for 10−30 min at 25 or 37 °C. Fluorescence images were captured using a TCS SP5 confocal microscope (Leica) and a 63× objective lens. Detailed protocols are described in the Supporting Information. K+ Diffusion Experiment. FemtoJet (Eppendorf) was used as a microinjector, and InjectMan (Eppendorf) was used as a manipulator. An IX 71 inverted microscope (Olympus) equipped with a cooled CCD camera (Coolsnap HQ, Olympus), xenon lamp (AH2RX-T; Olympus), and 40× objective lens (UplanFL, N.A. 1.30 Oil; Olympus) was used obtain images. The system was controlled by Metamorph software (Molecular Devices Inc.). TIRF Imaging. TIRF imaging was performed using a fluorescence microscope (ECLIPSE TE2000-U, Nikon), an objective lens (CFI Plan Apo TIRF 60×/1.45, Nikon), an EMCCD camera (C9100-12, Hamamatsu Photonics), and Aquacosmos software (Hamamatsu Photonics). HEK293 cells (Health Science Research Resources Bank, Japan) were transiently transfected with cDNAs coding sp-HT-Rhod and hBKα using LipofectAmine 2000 (Invitrogen). Electrophysiological experiments were performed using a whole-cell patchclamp configuration with a CEZ-2400 amplifier (Nihon Kohden, Japan), an analog-digital converter (Digidata 1440A; Molecular Devices-Axon Instruments), and pCLAMP software (Molecular Devices-Axon Instruments). For detailed protocols, see the Supporting Information.
regards to imaging at the single-cell level. First, currently reported extracellular K+ probes localize uniformly in the outer medium, which results in substantial background fluorescence from excess probe molecules located far from the cell surface, where the signal does not change much. In the case of visualizing [K+]o, this situation would particularly impact the S/ N of imaging, because the fluorescence increase of the probe upon activation is expected to be only 2−3-fold at most. As shown below, these probes provide only blurred fluorescence images at the microscopic level. In addition, because the probes can move outside the cells, the obtained image does not precisely reflect local [K+]o at high spatiotemporal resolution, i.e., spatial diffusion of probe has to be taken into account. To address these issues, we have designed, synthesized, and evaluated a new type of K+ fluorescent probe: a membranelocalized probe (Figure 1a). By stably accumulating probe
Figure 1. Development of protein-coupled K+ probes: (a) cartoon of a K+ probe that detects K+ channel activity at the cell surface, (b) structures of the K+ probes, (c, d) absorption (c) and emission (d) (λex = 522 nm) spectra of 1 μM TLSHalo measured in 5 mM HEPES buffer (pH 7.0) containing the indicated [K+], with 1% DMSO as a cosolvent. Ionic strength was maintained at 150 mM by addition of NaCl.
■
molecules on the cell membrane, we aimed to reduce background fluorescence from the bulk medium and to visualize biologically relevant [K+]o at the cell surface with a higher S/N. Specifically, we covalently tethered a water-soluble [K+]-selective fluorescent probe on the cell surface with the help of tag protein technology. This approach enabled us to image local [K+]o change at the cell surface after stimulation of endogenous or transiently expressed K+ channels for the first time.
RESULTS AND DISCUSSION Strategy to Localize K+ Fluorescent Probe on the Cell Surface. To localize fluorescent probe molecules on the surface of cells, conjugation of the probes with lipid moieties would be a reasonable strategy. Indeed, this approach has been applied to develop probes for various physiological metal ions, such as Ca2+19 and Zn2+.20,21 However, passive internalization of lipid-based probes is often inadequate for prolonged observation,22 and it is difficult to localize the probes only on the cells of interest. In addition, technical difficulty in probe loading often arises due to the poor water solubility of lipids. So, we adopted a tag protein as an alternative platform to localize K+ probes on the cellular membrane. A tag protein is
■
EXPERIMENTAL SECTION Materials. K+ probes were synthesized and characterized as described in the Supporting Information. Plasmids coding HaloTag protein were purchased from Promega or were gifts B
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry Table 1. Optical Properties of the K+ Probes
artificially modified to bind covalently to a specific, nonendogenous chemical structure.23−26 By using a fluorophore conjugated with a suitable reactive structure, researchers can visualize proteins of interest that are genetically fused to the tag protein. Tag proteins available for cell-based experiments include SNAP-tag,23 CLIP-tag,24 HaloTag,25 and PYP-tag.26 In addition, small-molecular fluorescent sensors that bind to such protein tags and detect their target molecules in specific organella of cells have been developed. For example, Kamiya et al. reported a Ca2+ probe that can be linked to SNAP-tag fusion proteins and used it for nucleus-specific Ca2+ imaging.27 Also, a few H2O2 probes have been reported that can bind to SNAPtag fusion proteins localized to various organelles.28,29 Also notably, during preparation of this manuscript, Li et al. reported a Zn2+ probe, ZIMIR HaloTag that covalently and specifically binds to HaloTag fusion proteins on membranes; they successfully used it to visualize Zn2+/insulin secretion from beta cells.30 Here, we selected HaloTag as the tag protein because of its high reactivity and ease of ligand handling.25 Design, Synthesis, and Optical Properties of K+ Probes with Halo Ligand Moiety. We then designed and synthesized K+ probes for labeling HaloTag, based on the following three requirements. First, the probe should respond selectively to K+ over Na+ and emit increased fluorescence. This was achieved by adopting the TAC chelator13 as the recognition moiety and PeT (photoinduced electron transfer) as the regulation mechanism.31,32 After preliminary synthesis of several TAC-conjugated fluorophores,33 we chose TAC-Lime (Table S1, Figure S1), previously reported by Verkman as an synthetic intermediate of TAC-Limedex,16 as a core structure of our probe. TAC-Lime, composed of TAC chelator and borondipyrromethene (BODIPY) fluorophore bearing two propionic acid moieties, has various advantageous properties, including high fluorescence quantum yield in water, a suitable dissociation constant (Kd) to detect physiological [K+], and many chemically modifiable positions to introduce functional moieties, including HaloTag ligand. Second, the probe should bind to HaloTag protein selectively and rapidly, while maintaining the ability to respond to K+. To meet this requirement, we introduced the HaloTag ligand at the propionic acid moiety of TAC-Lime, with or without a spacer. Third, the probe should not be cellular membrane-permeable, because if even a small proportion of the probe enters cells, it would emit strong fluorescence in cytoplasm, resulting in an increase of the background signal. To exclude the K+ probe from cells, we aimed to increase the hydrophilicity of the probe by introducing an ether chain (PEG) moiety or sulfonic acid groups as a spacer structure between BODIPY and the HaloTag ligand. Consequently, we synthesized three K+responsive fluorescent HaloTag ligands: TLHalo, TLPegHalo, and TLSHalo (Figure 1b). We then performed a titration experiment with each K+ probe against KCl in HEPES buffer, while maintaining the total ionic strength at 150 mM with NaCl (Figure 1c,d, Figure S2). All the probes showed 10- to 20-fold fluorescence increase upon addition of K+ (0−150 mM, Table 1), the most likely interfering ion in biological quantification of K+. Interestingly, the probes we synthesized were much brighter than TACLimedex, the dextran-conjugate of TAC-Lime previously developed as an extracellular K+ probe.16 We measured the fluorescence quantum yield (ΦFL) of TAC-Limedex as 0.086 at [K+] = 150 mM (Table S1), which is about 20% of that of TLSHalo. Conjugation of TLSHalo with dextran in the same
Abs.max (nm) TLHaloa HaloTag-TLHalob TLPegHaloa HaloTagTLPegHalob TLSHaloa HaloTag- TLSHalob
Em.max (nm)e
522 522 522 522
538 538 537 538
(535) (535) (535) (535)
522 522
538 (535) 537 (535)
ΦFLc,e 0.015 0.033 0.049 0.029
Kd (mM)d
(0.28) (0.22) (0.41) (0.33)
22 19 20 25
0.040 (0.41) 0.031 (0.36)
18 19
a Measured in 5 mM HEPES buffer (pH 7.0) containing the indicated [K+], with 1% DMSO as a cosolvent (λex= 522 nm). Ionic strength was maintained at 150 mM by addition of NaCl. bMeasured in PBS (pH 7.4) containing the indicated [K+] (λex= 522 nm). cRelative fluorescence quantum yield determined by using fluorescein (ΦFL= 0.85 in 0.1 N NaOH (aq) as a reference. dEstimated according to the method described in the Supporting Information. eValues in parentheses were measured in the presence of K+ (150 mM).
way, to afford TLSHalodex, resulted in a similar decrease of ΦFL (Table S1). Decrease of quantum yield and change of absorption spectra are often observed when BODIPY-based probes are linked to proteins, probably due to π-stacking and/ or electron transfer with aromatic amino acids.34,35 However, there was no significant change of the absorption spectra of our dextran-conjugated probe (Figure S1). Although the quenching mechanism of dextran-conjugated probes remains unclear, our newly developed K+ probes were promising in terms of brightness, K+-selective activation, and water solubility, and therefore we further evaluated their properties after conjugation with HaloTag protein. Conjugation of K+ Probes to HaloTag Protein. Since the environment near a protein surface is usually somewhat hydrophobic, the properties of fluorescent probes may change after conjugation. Hence, it is important to check the optical properties of the synthesized probes after linking them to the tag protein. To study this point, we expressed and purified recombinant HaloTag protein from E. coli and incubated the protein with the probes in vitro. After 1 h (by which time the labeling reaction is completed),36 the protein fraction was separated and fluorescence spectra were measured. Overall, the K+ probes retained their optical properties and showed sufficient performance to detect physiological [K+] changes, even after conjugation to HaloTag protein (Table 1, Figure S3). Nevertheless, the activation ratio of TLHalo in response to K+ was lowered from 18.7-fold to 6.7-fold because of the increase of background ΦFL and decrease of maximum ΦFL. In contrast, TLPegHalo and TLSHalo, having a hydrophilic “spacer” moiety between TAC-Lime and the HaloTag ligand, showed slight improvements of the activation ratio (TLPegHalo: 8.4-fold to 11.4-fold, TLSHalo: 10.3-fold to 11.6-fold), mainly because of decrease of background ΦFL. It seems likely that the hydrophilic spacer moieties prevented undesirable interaction between the fluorophore and the surface of HaloTag protein. When proteins such as antibodies are nonspecifically labeled with fluorescent dyes, ΦFL is usually decreased by surrounding amino acids35 and by other dyes labeled on the same protein. In the case of labeling tag proteins, such effects can be minimized, because the binding site is strictly determined and formation of a 1:1 complex between protein and dye is guaranteed. Labeling of HaloTag Protein on Cell Membrane. Next, we examined if our K+ probes could selectively label HaloTag C
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
such as Na+, Ca2+, Mg2+, Zn2+, Fe3+, and Cu2+. TLSHalo responded to K+ selectively even in the presence of these metal ions. On the other hand, TLSHalo responded to Rb+ and Cs+, which are alkali metal ions with larger radius than K+. These ions are chelated by TAC but are normally not present in the physiological environment, and so do not affect K+ detection. Also, glucose did not affect the fluorescence response. Next, we tested the pH dependence of TLSHalo (Figure S7b). The fluorescence intensity of TLSHalo in the absence of K+ was gradually increased as the pH was decreased below 7.0, due to cancellation of PeT via protonation of the amine moiety of TAC, but we believe this is not a serious problem for measuring [K]o. Lastly, we confirmed covalent binding and selectivity of TLSHalo with HaloTag protein by gel electrophoresis (SDSPAGE). We incubated purified HaloTag protein with 1 equiv of TLSHalo in PBS at 37 °C in the presence of an excess amount of BSA for 1 h. Then, the mixture was analyzed by SDS-PAGE. Only the band of HaloTag (33 kDa) showed green fluorescence, indicating that TLSHalo bound covalently to HaloTag protein with selectivity (Figure S7c). Quantitative Detection of [K+] on Cells. To demonstrate the response of TLSHalo to K+ at the cell surface, a titration experiment of membrane-bound TLSHalo was performed using confocal microscopy. First, we simply plotted the change of fluorescence intensity of TLSHalo as a function of [K+]o (Figure S8a). Although a general increase was observed, the intensity varied greatly from cell to cell, mainly because the expression levels of HaloTag were not identical. Therefore, we decided to use the K+-insensitive red fluorescence of HaloTag Alexa Fluor 660 Ligand as an internal reference to calibrate expression level. Since HaloTag protein binds covalently to a single ligand, TLSHalo and HaloTag Alexa Fluor 660 Ligand would be bound to different HaloTag protein molecules. However, at standard microscopy resolution, the plasma membrane of these cells was well costained with the two fluorophores. As a result, the fluorescence intensity ratio between TLSHalo and HaloTag Alexa Fluor 660 Ligand could be used to monitor [K+]o with much higher accuracy (Figure 3). This method also allowed us to easily identify transfected cells based on the bright red fluorescence of Alexa Fluor 660. The apparent dissociation constant for K+ was estimated as 7.9 mM, lower than the value measured in HEPES buffer (Table 1). When we calculated the Kd value in vitro in HBSS (the medium used for cell imaging), Kd was 8.3 mM, similar to that of Figure 3 (Figure S8b). The observed difference may be mainly due to the different buffer composition. Detection of K+ Diffusing near the Cell Surface. Although we have not determined association/dissociation rate constants of TLSHalo to K+, the reported data of TACbased K+ probes37 suggest that it should rapidly respond to an increase of [K+] of the order of 10−3 mM−1 ms−1 and to the release of K+ of the order of 10−1 ms−1 when the surrounding [K+] is decreased. Also, as mentioned earlier, probes present in the medium can be easily washed away in our method. Hence, TLSHalo would be able to detect local [K+] transition at a small area of the target cell, with sufficiently low background fluorescence. To demonstrate these advantages, we artificially induced local [K+] transition just above the cell surface and performed fluorescence imaging (Figure 4a). Specifically, we used a microinjection apparatus to transiently release a small amount of buffer containing high [K+] around the cell of interest. We prepared HeLa cells expressing sp-HT-Rhod and compared TLSHalo and TAC-Limedex.16 In this experiment, we
protein on cell surface. To control the localization of HaloTag protein, we transfected a plasmid that encodes HaloTag fused with an engineered GPCR (human rhodopsin), bearing an IL6-derived signaling sequence at the N-terminal (Figure S4). The whole protein is called sp-HT-Rhod. We confirmed that sp-HT-Rhod provides extracellular topology of HaloTag on the cytoplasmic membrane by using membrane-impermeable HaloTag Alexa Fluor 488 Ligand (Figure S4). We then applied our probes to HeLa cells expressing sp-HTRhod. Red fluorescence of cotransfected mCherry was used as a marker of HaloTag expression. However, contrary to our expectation, after incubation of 10 μM K+ probe for 30 min, TLHalo and TLPegHalo passed through the cell membrane and strong green fluorescence was observed from inner membrane structures of all cells (Figure 2a−f). Because the
Figure 2. Confocal microscopic images of HeLa cells transfected with sp-HT-Rhod and labeled with 10 μM K+ probe. (a, d, g) Fluorescence images of TLHalo, TLPegHalo, TLSHalo, respectively. (b, e, h) Fluorescence images of mCherry, a marker of transfected cells. (c, f, i) Merged images of probes and mCherry. Scale bar: 50 μm.
green fluorescence disappeared after fixing and washing the cells (Figure S5), these probes were not covalently bound to sp-HT-Rhod on the cell surface or in cells but were simply localized at lipophilic structures in the cells. This can be attributed to the relatively high lipophilicity of these probes. On the other hand, TLSHalo did not penetrate the cell membrane and stained only the surface of transfected cells (Figure 2g−i). Its fluorescence was still observed after fixation and washing (Figure S5), indicating that it was covalently bound to sp-HTRhod expressed on the surface of transfected cells. The labeling of sp-HT-Rhod by TLSHalo was also confirmed by costaining with membrane-impermeable HaloTag Alexa Fluor 660 Ligand (Figure S6). The results indicated that although all the probes work well in vitro, only TLSHalo is promising for cell imaging of [K+]o. Detailed Evaluation of TLSHalo. As TLSHalo was the most promising candidate, we characterized its properties in detail before proceeding to more advanced imaging experiments. First, we checked the metal selectivity of TLSHalo (Figure S7a). Like previous probes bearing TAC chelator,13,14 TLSHalo did not respond to biologically abundant metal ions D
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
Figure 3. Confocal microscopic images of HeLa cells transfected with sp-HT-Rhod and labeled with 2 μM TLSHalo and 10 nM HaloTag Alexa Fluor 660 Ligand. The images were taken in HBSS containing the indicated concentration of K+. (a−c) Fluorescence images of TLSHalo. (d−f) Fluorescence images of HaloTag Alexa Fluor 660 Ligand. (g) The ratio of fluorescence intensity (FTLSHalo/FAlexa660) as a function of [K+]. The values were calculated by averaging three ROIs. Error bar: SD. Green channel: λex = 522 nm, λem = 535−570 nm. Red channel: λex = 660 nm, λem= 680−800 nm. Scale bar: 100 μm.
Figure 4. K+ diffusion imaging using a microinjector. (a) Schematic illustration of the experiment. (b) DIC image of labeled HeLa cells and the microinjector needle. (c) Epifluorescence microscopic image of cells labeled with 2 μM TLSHalo at time 0. (d) Normalized fluorescence intensity (F/F0) of TLSHalo in each ROI as a function of time. During the experiment, 150 mM KCl aq was effused every 2.5 s from the needle for 300 ms each at 1500 hPa. ROI 5 is the background. (e) Epifluorescence images of cells labeled with 10 nM HaloTag Alexa Fluor 660 Ligand at time 0. (f) F/F0 of HaloTag Alexa Fluor 660 Ligand in each ROI as a function of time. Scale bar: 50 μm.
plotted the percentage fluorescence intensity change (F/F0, single color measurement) to increase time resolution. Immediately after each pulse-like K+ release, a transient signal increase of TLSHalo propagated from the position just around the head of the injection needle to the periphery (Figure 4b−d, Supporting Movie 1; the gradual and slight decrease of fluorescence intensity with time is due to photobleaching of fluorophore). When the needle head was moved to another site, the starting point of TLSHalo fluorescence increase moved correspondingly to the needle location (Figure S9). TSLHalo did not respond to solution containing Na+ (Figure S10), and the fluorescence of a K+-insensitive HaloTag Alexa Fluor 660 Ligand was unchanged after K+ injection (Figure 4e,f, Figure S9). These results indicate that the TLSHalo fluorescence at each point reflects local [K+] in real time, and the movie represents the spatiotemporal diffusion of K+ from the region near the injection needle to more distant regions. In contrast, the [K+] transition around cells of interest was not clearly visualized by TAC-Limedex, mainly because of high background fluorescence throughout the medium (Figure S11). Hence, TLSHalo is more suitable to monitor local [K+] changes near the surface of target cells. Detection of [K+] Efflux from a Cell. We next examined whether TLSHalo could detect a physiological level of K+ efflux from cells. In tissues such as brain and heart, where K+ plays important roles, cells are in close contact in three dimensions. So, diffusion of extracellular K+ is limited compared with conventional single-layer cultured cells. To mimic this environment, we mounted a cover glass onto 2D-cultured cells that had been labeled with TLSHalo and HaloTag Alexa Fluor 660 Ligand, just before imaging16,38 (Figure 5a). First, we used a K+-selective ionophore, valinomycin, and a K+/H+ ionophore, nigericin, to induce a nonphysiological, large K+ efflux from cells. After valinomycin or nigericin had been added to the
medium, the fluorescence of TLSHalo increased within a minute (Figure 5b-e, Figure S12). TLSHalo fluorescence decreased to baseline level after removal of the cover glass. In control experiments where medium alone was added, the increase of TLSHalo fluorescence was very slight and slow (Figure 5e, Figure S12). These results indicated that TLSHalo can readily visualize the increase of [K+]o around cells induced by K+ ionophores. Also, by using the calibration curve (Figure 3g), we could estimate the value of [K+]o around each cell (Figure 5e). It is noteworthy that when we performed the same experiment using TAC-Limedex,16 the obtained images were not suitable for analysis of [K+]o near the cell surface (Figure S13).39 We then examined whether TLSHalo could detect a more physiological K + transition. We focused on the large conductance Ca2+-dependent K+ channel (BK channel) and set out to image K+ released through this channel. BK channels are activated by cellular membrane depolarization and/or by increase of intracellular [Ca 2+]. They regulate various physiological events, including smooth muscle tone and neuronal excitability.40,41 Because the HT29 cell line, derived from human colon adenocarcinoma, expresses BK channel on the cell membrane,42 we used this cell line to examine whether TLSHalo had sufficient sensitivity to detect K+ efflux through endogenous BK channel. To trigger [Ca 2+] elevation, ionomycin (a Ca2+-selective ionophore) was used. First, we confirmed that intracellular Ca2+ transition occurred after E
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry
Figure 5. Fluorescence imaging of K+ efflux from cells. A cover glass was mounted just after addition of (b−e) 10 μM valinomycin or (f) 3 μM ionomycin to induce K+ efflux. (a) Schematic representation of the experiment. (b−d) Fluorescence intensity ratio images of HeLa cells dual-labeled with TLSHalo and HaloTag Alexa Fluor 660 Ligand. The images were taken (b) before, (c) 3 min after addition of valinomycin, or (d) after removal of the cover glass. Scale bar: 50 μm. (e) [K+]o during valinomycin stimulation was estimated from 3 ROIs in confocal microscopic images of dual-labeled HeLa cells. Error bar: SD. (f) [K+]o during ionomycin stimulation was similarly estimated from 3 ROIs in confocal microscopic images. Error bar: SD.
Figure 6. TIRFM imaging of TLSHalo. (a) Schematic illustration of [K+] imaging at the bottom of a cell using TIRF microscopy. The cell, which expresses sp-HT-Rhod and hBKα, was labeled with TLSHalo, and BK channel current was observed with a patch-clamp. (b) TIRF microscopic images of HEK293 cells. (c) Changes of TLSHalo fluorescence and electric current. (d) Fluorescence changes of HaloTag Alexa Fluor 488 Ligand (Figure S18) and TLSHalo during depolarization by whole-cell patch-clamp (n = 4). Error bar: S.E. Asterisk: p = 0.002 vs HaloTag Alexa Fluor 488 Ligand.
stimulation with ionomycin by using a fluorescence probe for Ca2+, Fluo-4 AM. Cytosolic [Ca2+] was immediately elevated after addition of ionomycin-Ca2+ salt (Figure S14). Then we transfected sp-HT-Rhod, stained the cells with both TLSHalo and HaloTag Alexa Fluor 660 Ligand, and stimulated them with ionomycin. TLSHalo fluorescence immediately increased in the presence of ionomycin (Figure 5f, Figure S15). On the other hand, when the cells were preincubated with a cytosolic Ca2+ chelator BAPTA-AM or a selective BK channel inhibitor iberiotoxin (IBTX),43 the fluorescence elevation of TLSHalo after ionomycin stimulation was suppressed (Figure 5f, Figure S15), indicating that TLSHalo detected the change of [K+]o induced by stimulation of the native BK channel. This result showed that TLSHalo is sufficiently sensitive to monitor physiological [K+] change at the cell surface, at least when the diffusion of K+ is controlled in some way. Detection of K+ Efflux by TIRFM Imaging. The results of the local K+ addition experiment and K+ efflux imaging with a cover glass showed that TLSHalo has the potential to visualize K+ efflux from a single cell in real time if an appropriate setup is used. To demonstrate such an application, we used total internal reflection fluorescence microscopy (TIRFM) to observe K+ efflux at the bottom of the cell (Figure 6a). Because only probes within the evanescent field (∼100 nm from the surface of the slide glass) are excited in TIRFM, we expected to specifically observe the fluorescence of TLSHalo at the bottom of the cell, which should reflect [K+] fluctuation in the small space between the cell bottom and the glass surface. We transfected sp-HT-Rhod and human BK channel α subunit (hBKα) into HEK293 cells and stained the cells with TLSHalo or HaloTag Alexa Fluor 488 Ligand as a K+insensitive control. Initially we checked whether TLSHalo could detect [K+]o transition at the bottom of the cells, by perfusing medium containing 40 mM K+ (Figure S16).
Fluorescence of TLSHalo increased and decreased with a small time lag, which may reflect gradual permeation of K+ into the narrow interspace where the probe exists. Such fluorescence intensity change was not observed for cells stained with HaloTag Alexa Fluor 488 Ligand (Figure S16). Next, we electrically stimulated the cells by means of the whole-cell patch clamp method to induce depolarization and K+ efflux through hBKα during TIRFM imaging. At the bottom of the cells expressing hBKα, the fluorescence intensity of TLSHalo gradually increased, and when the stimulation was stopped, the fluorescence intensity started to decrease, reaching the initial level within 30 s (Figure 6b,c, Supporting Movie 2). These gradual fluorescence changes were assumed to reflect continuous release and slow diffusion/uptake of K+ at the interspace. In the case of well-adherent cells, the fluorescence increased by around 7% during electrical stimulation for 10 s at 100 mV (Figure 6d). The fluorescence intensity did not change at the bottom of cells that did not express hBKα (Figure S17). Also, the fluorescence intensity of HaloTag Alexa Fluor 488 Ligand did not change during stimulation even in the presence of hBKα (Figure 6d, Figure S18, Supporting Movie 3). These results show that TLSHalo can detect transient [K+]o change triggered by electrical stimulation at the bottom surface of adherent cells. Using this setup, it should be possible to evaluate the effects of inhibitors, activators, and regulatory proteins on K+ channel activity in live cells. Although electrophysiological detection is the gold standard, efflux of K+ would be a more direct and universal index to evaluate K+ channel activity. Further, the interspace between the bottom of F
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry the cell and slide glass may mimic intercellular spaces in tissue samples containing closely packed cells, so TLSHalo may be useful to examine [K+] transition in tissues with high resolution and sensitivity in the future.
■
■
CONCLUSION We synthesized three K+-sensitive, TAC-based fluorescent K+ probes that react with HaloTag protein, which could selectively detect [K+] in the physiological range, and bind covalently to HaloTag protein while retaining favorable optical properties. Among them, TLSHalo did not pass through the cell membrane, and we showed that TLSHalo, in combination with membrane-localized HaloTag protein, could monitor transient local [K+]o changes on the surface of target cells at subcellular resolution. We also visualized [K+]o change near cells induced by K+ ionophore or by stimulation of endogenous BK channel with ionomycin. Finally, we used TIRFM imaging to visualize K+ efflux through hBKα channel induced by electrical stimulation at the bottom surface of cells in real time. Several K+ probes have been developed to visualize intracellular and extracellular [K+] change,13−18,37,44−47 but to our knowledge, TLSHalo is the first membrane-localized K+ fluorescent probe suitable for measuring biologically important local [K+]o and visualizing K+ dynamics near the cellular membrane. Practically, it offers three advantages over existing probes. First, it increases the S/N of [K+]o imaging near the surface of cells (see Figure 4), due to the absence of background signal from the bulk solution. Second, we can label only cells of interest by genetically controlling HaloTag expression. This provides great flexibility; for example, it might be possible to distinguish [K+]o dynamics around neurons and astrocytes. Third, stable anchoring of TLSHalo on the cell surface simplifies interpretation of experimental findings. For example, if we had used a conventional extracellular probe in high [K+] TIRF experiment (Figure S16), it would have been necessary to take into account diffusion of both the probe and K+. Our strategy has some drawbacks, such as moderate photobleaching of the probe, but this may be improved by modifying the fluorophore. Washout of probe molecules that have not reacted with HaloTag proteins may be a hurdle for use in vivo, but this might be circumvented by masking the fluorescence of unlabeled probes through sophisticated chemical design, as has been done for some other tag proteins.48,49 Also, the use of microfluidics would circumvent the necessity of applying the cover glass manually. We are planning to further modify TLSHalo and apply it to 3Dcultured cells and tissues.
■
TIRF images of a HEK293 cell expressing BK channel using HaloTag Alexa Fluor 488 Ligand during depolarization by whole-cell patch-clamp (AVI)
AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. Present Address ○
M.S.: Medical Innovation Center, Graduate School of Medicine, Kyoto University, 53 Shogoin Kawaharacho, Sakyoku, Kyoto 606-8507, Japan.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS This research was supported by the Ministry of Education, Culture, Sports, Science and Technology [KAKENHI (Grant 15K05529 to T.T, Grant 22000006 to T.N.) and Platform for Drug Discovery, Informatics, and Structural Life Science], as well as NEDO and The Mochida Memorial Foundation for Medical and Pharmaceutical Research (to T.T.). We thank Dr. Takahiro Nagase for the plasmid encoding sp-HT-Rhod.
■
REFERENCES
(1) Shieh, C.; Coghlan, M.; Sullivan, J.; Gopalakrishnan, M. Pharmacol. Rev. 2000, 52, 557−594. (2) Bear, M. F.; Connors, B. W.; Paradiso, M. A. Neuroscience: Exploring the Brain, 3rd ed.; Lippincott Williams and Wilkins: Philadelphia, PA, 2007. (3) Durand, D. M.; Park, E. H.; Jensen, A. L. Philos. Trans. R. Soc., B 2010, 365, 2347−2362. (4) Wallraff, A.; Köhling, R.; Heinemann, U.; Theis, M.; Willecke, K.; Steinhäuser, C. J. Neurosci. 2006, 26, 5438−5447. (5) de Lanerolle, N. C.; Lee, T.-S.; Spencer, D. D. Neurotherapeutics 2010, 7, 424−438. (6) Edwards, G.; Dora, K. A.; Gardener, M. J.; Garland, C. J.; Weston, A. H. Nature 1998, 396, 269−272. (7) Danker, T.; Gassner, B.; Oberleithner, H.; Schwab, A. Pflügers Arch. 1996, 433, 71−76. (8) Messerli, M. A.; Collis, L. P.; Smith, P. J. Biophys. J. 2009, 96, 1597−1605. (9) Terai, T.; Nagano, T. Curr. Opin. Chem. Biol. 2008, 12, 515−521. (10) Yuan, L.; Lin, W.; Zheng, K.; He, L.; Huang, W. Chem. Soc. Rev. 2013, 42, 622−661. (11) Yin, J.; Hu, Y.; Yoon, J. Chem. Soc. Rev. 2015, 44, 4619−4644. (12) Minta, A.; Tsien, R. Y. J. Biol. Chem. 1989, 264, 19449−19457. (13) He, H.; Mortellaro, M. A.; Leiner, M. J.; Fraatz, R. J.; Tusa, J. K. J. Am. Chem. Soc. 2003, 125, 1468−1469. (14) Padmawar, P.; Yao, X.; Bloch, O.; Manley, G. T.; Verkman, A. S. Nat. Methods 2005, 2, 825−827. (15) Sui, B.; Yue, X.; Tichy, M. G.; Liu, T.; Belfield, K. D. Eur. J. Org. Chem. 2015, 6, 1189−1192. (16) Namkung, W.; Padmawar, P.; Mills, A. D.; Verkman, A. S. J. Am. Chem. Soc. 2008, 130, 7794−7795. (17) Kong, X.; Su, F.; Zhang, L.; Yaron, J.; Lee, F.; Shi, Z.; Tian, Y.; Meldrum, D. R. Angew. Chem., Int. Ed. 2015, 54, 12053−12057. (18) Namkung, W.; Song, Y.; Mills, A. D.; Padmawar, P.; Finkbeiner, W. E.; Verkman, A. S. J. Biol. Chem. 2009, 284, 15916−15926. (19) Etter, E. F.; Kuhn, M. A.; Fay, F. S. J. Biol. Chem. 1994, 269, 10141−10149. (20) Li, D.; Chen, S.; Bellomo, E. A.; Tarasov, A. I.; Kaut, C.; Rutter, G. A.; Li, W. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 21063−21068. (21) Iyoshi, S.; Taki, M.; Yamamoto, Y. Org. Lett. 2011, 13, 4558− 4561.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03970. Experimental procedures, synthesis and characterization of compounds, and supplementary figures (PDF) Epifluorescence imaging of K+ diffusion using TLSHalolabelled HeLa cells and a microinjection apparatus (AVI) TIRF images of a HEK293 cell expressing BK channel using TLSHalo during depolarization by whole-cell patch-clamp (AVI) G
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
Article
Analytical Chemistry (22) Hao, M.; Maxfield, F. R. J. Biol. Chem. 2000, 275, 15279−15286. (23) Juillerat, A.; Gronemeyer, T.; Keppler, A.; Gendreizig, S.; Pick, H.; Vogel, H.; Johnsson, K. Chem. Biol. 2003, 10, 313−317. (24) Gautier, A.; Juillerat, A.; Heinis, C.; Correa, I. R., Jr.; Kindermann, M.; Beaufils, F.; Johnsson, K. Chem. Biol. 2008, 15, 128−136. (25) Los, G.; Encell, L.; McDougall, M.; Hartzell, D.; Karassina, N.; Zimprich, C.; Wood, M.; Learish, R.; Ohana, R.; Urh, M.; Simpson, D.; Mendez, J.; Zimmerman, K.; Otto, P.; Vidugiris, G.; Zhu, J.; Darzins, A.; Klaubert, D.; Bulleit, R.; Wood, K. ACS Chem. Biol. 2008, 3, 373− 382. (26) Hori, Y.; Ueno, H.; Mizukami, S.; Kikuchi, K. J. Am. Chem. Soc. 2009, 131, 16610−16611. (27) Kamiya, M.; Johnsson, K. Anal. Chem. 2010, 82, 6472−6479. (28) Abo, M.; Minakami, R.; Miyano, K.; Kamiya, M.; Nagano, T.; Urano, Y.; Sumimoto, H. Anal. Chem. 2014, 86, 5983−5990. (29) Srikun, D.; Albers, A. E.; Nam, C. I.; Iavarone, A. T.; Chang, C. J. J. Am. Chem. Soc. 2010, 132, 4455−4465. (30) Li, D.; Liu, L.; Li, W.-H. ACS Chem. Biol. 2015, 10, 1054−1063. (31) Ueno, T.; Urano, Y.; Setsukinai, K.; Takakusa, H.; Kojima, H.; Kikuchi, K.; Ohkubo, K.; Fukuzumi, S.; Nagano, T. J. Am. Chem. Soc. 2004, 126, 14079−14085. (32) Urano, Y.; Kamiya, M.; Kanda, K.; Ueno, T.; Hirose, K.; Nagano, T. J. Am. Chem. Soc. 2005, 127, 4888−4894. (33) We have tried fluorescein derivatives, but they aggregated and lost responsiveness to [K+] in aqueous buffer, presumably due to the hydrophobic nature of the TAC chelator. In addition, we examined 2,6-carboxy BODIPY, which is more resistant to photobleaching.50 Although this compound was soluble in buffer and responded to K+, it showed a much lower quantum yield than TAC-Lime and was not suitable for K+ imaging. (34) Vaiana, A. C.; Neuweiler, H.; Schulz, A.; Wolfrum, J.; Sauer, M.; Smith, J. C. J. Am. Chem. Soc. 2003, 125, 14564−14572. (35) Chen, H.; Ahsan, S. S.; Santiago-Berrios, M. E. B.; Abruña, H. D.; Webb, W. W. J. Am. Chem. Soc. 2010, 132, 7244−7245. (36) Reaction rates for the three probes were not determined precisely, but no apparent change was observed among them. (37) Magzoub, M.; Padmawar, P.; Dix, J. A.; Verkman, A. S. J. Phys. Chem. B 2006, 110, 21216−21221. (38) Piao, W.; Tsuda, S.; Tanaka, Y.; Maeda, S.; Liu, F.; Takahashi, S.; Kushida, Y.; Komatsu, T.; Ueno, T.; Terai, T.; Nakazawa, T.; Uchiyama, M.; Morokuma, K.; Nagano, T.; Hanaoka, K. Angew. Chem., Int. Ed. 2013, 52, 13028−13032. (39) In our experiments, there was always a physiological concentration of K+ in the outer medium unlike previously reported experiments in reference 16, where extracellular K+ was removed to increase the S/N ratio of detection. (40) Ghatta, S.; Nimmagadda, D.; Xu, X.; O’Rourke, S. T. Pharmacol. Ther. 2006, 110, 103−116. (41) Félétou, M. Br. J. Pharmacol. 2009, 156, 545−562. (42) Wu, H.; Franklin, C.; Kim, H.; Turner, J. Am. J. Physiol. 1991, 260, C35−C42. (43) Galvez, A.; Gimenez-Gallego, G.; Reuben, J. P.; Roy-Contancin, L.; Feigenbaum, P.; Kaczorowski, G. J.; Garcia, M. L. J. Biol. Chem. 1990, 265, 11083−11090. (44) Hirata, T.; Terai, T.; Komatsu, T.; Hanaoka, K.; Nagano, T. Bioorg. Med. Chem. Lett. 2011, 21, 6090−6093. (45) Kong, D.-M.; Ma, Y.-E.; Guo, J.-H.; Yang, W.; Shen, H.-X. Anal. Chem. 2009, 81, 2678−2684. (46) Zhou, X.; Su, F.; Tian, Y.; Youngbull, C.; Johnson, R. H.; Meldrum, D. R. J. Am. Chem. Soc. 2011, 133, 18530−18533. (47) Zhou, X.; Su, F.; Gao, W.; Tian, Y.; Youngbull, C.; Johnson, R. H.; Meldrum, D. R. Biomaterials 2011, 32, 8574−8583. (48) Komatsu, T.; Johnsson, K.; Okuno, H.; Bito, H.; Inoue, T.; Nagano, T.; Urano, Y. J. Am. Chem. Soc. 2011, 133, 6745−6751. (49) Hori, Y.; Norinobu, T.; Sato, M.; Arita, K.; Shirakawa, M.; Kikuchi, K. J. Am. Chem. Soc. 2013, 135, 12360−12365.
(50) Komatsu, T.; Oushiki, D.; Takeda, A.; Miyamura, M.; Ueno, T.; Terai, T.; Hanaoka, K.; Urano, Y.; Mineno, T.; Nagano, T. Chem. Commun. 2011, 47, 10055−10057.
H
DOI: 10.1021/acs.analchem.5b03970 Anal. Chem. XXXX, XXX, XXX−XXX
