In Situ Visualization of hERG Potassium Channel via Dual Signal

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In Situ Visualization of hERG Potassium Channel via Dual Signal Amplification Xue-Jiao Yang, Kai Zhang, Hong-Yuan Chen, and Jing-Juan Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00725 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 19, 2018

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Analytical Chemistry

In Situ Visualization of hERG Potassium Channel via Dual Signal Amplification Xue-Jiao Yang1, Kai Zhang1, Jing-Juan Xu1,2,* Hong-Yuan Chen1

1

State Key Laboratory of Analytical Chemistry for Life Science and Collaborative

Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China 2

Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College

of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China *Phone/fax: +86-25-89687294. E-mail: [email protected] .

1

ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT Dysfunction of the human ether-a-go-go related gene (hERG)-encoded potassium channel is identified as a major cause of the long Q-T syndrome, a marker for the lethal cardiac arrhythmia. Furthermore, recent studies revealed that hERG K+ channel is a regulator of tumor cell apoptosis and proliferation. Herein, an ultrasensitive fluorescence assay combining DNA-functionalized gold nanoparticles and rolling circle amplification (RCA) is first attempted to visualize hERG channels in living cells. The spherical nucleic acids gold nanoparticles which can anchor on hERG channels in the cell membrane, not only act as the primary amplification elements, but also trigger the subsequent RCA reaction to achieve the secondary amplification. Within 30 minutes, the ratio of reporter to target can reach up to 104, realizing the detection of hERG channels in cells with low-level expression. Therefore, the strategy provides a valuable tool for hERG-related studies. More importantly, it opens a new horizon for imaging various membrane proteins which possess specific aptamer or antibody.

KEYWORDS: In situ visualization, hERG Potassium Channel, RCA, Dual-amplification, Gold nanoparticles

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INTRODUCTION The human ether-a-go-go related gene (hERG) encodes the pore-forming alpha subunit of a voltage-gated potassium (K+) channel, KV11.1, expressed in the heart and in nervous tissue.1 In the heart, hERG makes up most part, if not all, of the channel that conducts the rapid component of the delayed rectifier current, which plays a critical role in cardiac action potential repolarization.2-4 Genetic mutations in the hERG channel result in chromosome 7-associated long QT syndrome (LQTS type 2), a disorder in which the patient has a substantial risk of sudden death due to an arrhythmia, and blockade of KV11.1 causes drug-induced QT prolongation.5 Most strikingly, hERG expression also has been found in a variety of tumor cell lines such as human breast cancer (MCF-7) and neuroblastoma (SH-SY5Y), but absent from the healthy cells from which the respective tumor cells are derived.6-10 Moreover, hERG potassium channels may work in regulating tumor cell apoptosis and proliferation, and provide a potential new target for cancer therapy.11-13 Electrophysiological properties of hERG potassium channel have been best characterized by patch-clamp recordings.3,14-16 To better understand the role of hERG channels in cell regulation, several imaging methods have been developed in recent years, mainly including fluorescence labeling method, immunofluorescence assay and small molecule fluorescent probes.17-20 Though fluorescence labeling possesses high accuracy, there will be vast difference between endogenous protein and fusion protein in molecular size and properties, which may affect the function of hERG channel protein.17 Conventional immunofluorescence technique and fluorescent probes also can do very little for imaging hERG channels in tumor cells with relatively low-level expression. Hence, it is of great importance to develop novel amplification strategies to improve the detection sensitivity. Rolling circle amplification (RCA) is a powerful but simple isothermal DNA replication technique, in which a short DNA primer is elongated to generate a long single-stranded DNA (ssDNA) containing thousands of repeat units with the assistance of a circle DNA template and special polymerases.21,22 Because it is high 3



efficient, programmable and easy to combine with fluorescence labeling technique, RCA has been widely used as a signal-amplification tool for sensitive biosensing,22-26 drug delivery,24,27 and so on. However, until now, there is no report on fluorescent imaging of membrane protein using RCA method. Here, we develop a cascade signal amplification strategy to image hERG channels. This is the first report on in situ imaging membrane proteins by performing RCA on spherical nucleic acids (SNA) gold nanoparticles.28 The method is easy to conduct and possesses good biocompatibility. As illustrated in Scheme 1 and Figure 1c, the developed probe named primer@AuNP@Ab2 is composed of a gold nanoparticle assembled with the duplexes of RCA primer sequence and circle DNA template sequence, and the recognition unit, secondary antibody (Ab2). When visualizing the hERG channels, two amplification steps are included. First, using gold nanoparticles as carriers, the primer@AuNP@Ab2 conjugates are synthesized for the primary signal amplification by increasing the ratio of primer to channel protein. After anchoring on hERG channels, primers on the AuNPs initiate the subsequent RCA reaction to accomplish the secondary amplification. Alexa Fluor 488 labeled dUTP, unit of RNA, acts as the reporter and is conjugated to the extended ssDNA sequence during RCA. In the first 30 minutes, the signal-to-target ratio can reach up to 104, ensuring the extreme sensitivity in bio-imaging. Besides, by selecting the appropriate RCA buffer, the problem that the primer sequence will be detached from the central AuNP during RCA is solved, thereby ensuring signal amplification efficiency. Then the proposed strategy successfully makes hERG proteins visible in MCF-7 cells, in which hERG channels are expressed at a very low level. So the work offers a powerful tool for hERG-related biological studies.

EXPERIMENTAL SECTION Chemicals and Materials. Trisodium citrate, chloroauric acid (HAuCl4·4H2O), mercaptoethanol and N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochlorid (EDC) were purchased from Sigma-Aldrich. Anti-hERG (rabbit, polyclonal) was 4


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purchased from Alomone Labs (Israel). Tris (2-carboxyethyl) phosphine hydrochloride (TCEP),

adenosine

5’-triphosphate

2’-deoxyguanosine-5’-triphosphate

disodium solution

salt

hydrate, (dGTP),

2’-deoxyadenosine-5’-triphosphate solution (dATP), 2’-deoxycytidine-5’-triphosphate solution (dCTP), Alexa Fluor 488 labeled goat anti-rabbit IgG, goat anti-rabbit IgG (Ab2), DEPC-treated water, dithiothreitol (DTT) and N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) were from Sangon Biotechnology Co. Ltd. (Shanghai, China; DEPC = diethyl pyrocarbonate). T4 DNA ligase, phi29 DNA polymerase, exonuclease I and exonuclease III were obtained from New England Biolabs. Zeocin, Alexa Fluor 488-5-dUTP, Dulbecco’s Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased from ThermoFisher. The hERG transfected HEK293 cell line was purchased from Cobioer Biotechnology Co. Ltd. (Nanjing, China). The human breast cancer cell (MCF-7), human embryonic kidney cell (HEK293) and RPMI-1640 were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China). The oligonucleotides were synthesized and purified by Sangon Biotechnology Co. Ltd. (Shanghai, China). All the chemicals were used without additional purification. Apparatus. The morphological properties were obtained on JEM-1011 transmission electron microscopy (JEOL, Japan), JSM-7800F scanning electron microscopy (JEOL, Japan), and Dimension Icon scanning probe microscopy (Bruker, Germany). The UV-vis absorption spectra were recorded using a UV-vis spectrophotometer (UV-3600, Shimadzu, Japan). The zeta potential and dynamic light scattering (DLS) were acquired with a particle size analyser (90 Plus, Brookhaven, America). The cytometric analysis was performed on a FC500 Cytometer (Beckman Coulter, America). All the cell images were obtained with a Leica TCS SP8 STED 3X laser scanning confocal microscopy (Leica, Germany). The whole cell patch-clamp experiments were recorded with a MultiClamp 700B Microelectrode Amplifier and an Axon Digidata 1550 low-noise data acquisition system (Molecular Devices, America). Preparation of Gold Nanoparticles (AuNPs). AuNPs with diameter of approximate 15 nm were prepared according to the classical sodium citrate reduction method.29 Briefly, 100 mL of 0.01% HAuCl4 was heated to boiling and refluxing while 5



stirring, then 3.5 mL of 38.8 mM sodium citrate was quickly added. The color changed from pale yellow to deep red in a few minutes. After that, the system was refluxed with stirring for another 15 min, and then cooled down to room temperature. The sizes of obtained AuNPs were verified by TEM and DLS. Construction of the Primer@AuNP@Ab2 Conjugates. To prepare the SNA gold nanoparticle conjugates, AuNPs (~ 3.5 nM) were mixed with the thiolated primers (800 nM) and the thiolated spacers (80 nM), followed by shaking overnight. After 15 h, 2.0 M sodium chloride solution was added to the above solution gradually to achieve a final concentration of 0.2 M.30-33 Subsequently, the unreacted DNA sequences were removed by centrifugation (12000 rpm, 30 min) and the precipitate was washed twice with PBS. The pre-synthesized circle DNA templates (200 nM) were then annealed with the SNA-AuNP conjugates to achieve hybridization between primers and circle templates. Next, the secondary antibodies were conjugated to the oligonucleotides modified AuNPs via EDC and NHS chemistry.34-37 In this procedure, the SNA-AuNP conjugates were dispersed in PBS buffer (pH 7.0, 10 mM) containing 8 mM EDC and 2 mM Sulfo-NHS, and shaken for 30 min. Afterwards, the goat anti-rabbit IgG (10 µg/mL) was added to the above solution. The mixture was incubated at room temperature for 2 h under gentle stirring. After centrifugal ultrafiltration (12000 rpm, 15 min; Nanosep 300K, Pall), the obtained primer@AuNP@Ab2 conjugates were dispersed in PBS buffer (pH 7.4, 10 mM) for further use. The process was characterized by zeta potential and DLS. Besides, the products were deposited on conductive tapes, dried and coated with Pt, followed by observation on a scanning electron microscope. RCA Reaction in Vitro. In brief, the mixture of prepared circle DNA templates and primer sequences was annealed to ensure complete hybridization. After that, 2 µL phi29 DNA polymerase (10 U/µL), 5 µL dNTPs (10 mM), 2 µL BSA (10 mg/mL) and 10 µL of 10× reaction buffer (500 mM Tris-HCl, 10 mM MgCl2, 100 mM (NH4)2SO4, no DTT, pH 7.4) were added to the mixture (or SNA-AuNP conjugates) and incubated at 37 ºC for different times.38 The RCA products were characterized by polyacrylamide gel electrophoresis and AFM. Cell Culture and Imaging of hERG K+ Channels. MCF-7 cells were cultured in 6


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RPMI-1640, while HEK293 cells were cultured in DMEM. All media were supplemented with 10% fetal calf serum, penicillin (80 U/mL), streptomycin (0.08 mg/mL). The cells were cultured at 37 ºC in an atmosphere containing 5% CO2. Specially, HEK293 cells stably transfected with hERG K+ channel (hERG-HEK293) should be cultured with 0.4 mg/mL zeocin. For imaging of hERG channels in the cell membrane, 1 mL of different cells were seeded in confocal dishes respectively for 48 h. On the second day, the medium was changed, and the cells were cultured in succession with anti-hERG (4 µg/mL, rabbit, polyclonal) for 2 h and primer@AuNP@Ab2 conjugates (2 nM, 10 µL) for 1 h. Finally, RCA reaction was conducted with the assistance of phi29 DNA polymerase and Alexa Fluor 488 labeled dUTP, as described in the preceding section. All the confocal images were obtained under 488 nm excitation, while the emission was collected from 500 nm to 550 nm. Whole Cell Patch-Clamp Recordings. The experiments were performed according to the process reported in the literature.3,15,16,39 In this section, hERG-HEK293 cells were used. Borosilicate glass electrodes had tip resistances of 2-4 MΩ when filled with pipette solution (140 mM KCl, 2 mM MgCl2, 5 mM adenosine 5’-triphosphate disodium, 10 mM HEPES, 5 mM EGTA, pH 7.2-7.4). Junction potentials were zeroed before pipette contacted with the cell membrane in bath solution (137 mM NaCl, 4 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose, pH 7.4). In the

experimental

group,

cells

were

pre-incubated

with

anti-hERG

and

primer@AuNP@Ab2 conjugates. Electrophysiological experiments were conducted at room temperature.

RESULTS AND DISCUSSION Principle and Characterization of the Designed Dual-Amplification Strategy. First, polyacrylamide gel electrophoresis experiment was performed to track the synthesis of circle DNA template and test the rationality of the design (Figure 1a). According to change of the mobility, the nick in the template DNA was chemically closed, meanwhile, the ligation sequences and unreacted template sequences were 7



degraded by exonuclease III and exonuclease I (lane 6). Additionally, the extended ssDNA sequence of high molecular weight was observed (lane 8 and lane 9), indicating that the RCA reaction successfully occurred. The number of the bases contained in the long tandem repeated sequence was estimated to be much higher than 1500 base pairs (3000 bases) (Figure S3), which enabled its use for signal amplification. Gold nanoparticles (AuNPs) with a diameter of 15 nm (Figure 1c) were used for the carriers due to their excellent biocompatibility and their ability of easily being modified with oligonucleotides. After the AuNPs were functioned with primers and spacers, the maximum absorption in the UV-vis absorption spectrum was red-shifted, and the characteristic peak of DNA at 260 nm appeared (Figure 1b). The concentration of obtained SNA-AuNP conjugates could be quantified according to the Beer-Lambert Law. Next, based on mercaptoethanol-displacement fluorescence analysis and theoretical calculations (Figure S1), the number of oligonucleotides loaded on each AuNP was calculated to be about 71, consisting of 65 primers and 6 spacers. Combined with the results of gel electrophoresis, one primer@AuNP@Ab2 probe could carry more than 28470 reporter units after performing RCA reaction for 30 min. In other word, the ratio of signal to target could reach up to 104 within 30 min (details in SI). In addition, the synthetic process of primer@AuNP@Ab2 conjugates was verified by zeta potential analysis and dynamic light scattering (DLS) experiments (Figure S2). The results indicated that AuNPs were successfully assembled with the oligonucleotides and secondary antibodies. Then the SEM image of the obtained primer@AuNP@Ab2 conjugates showed an average diameter of 65 nm, which was larger than the actual size due to the presence of Pt on the surface (Figure 1c). To achieve the best RCA performance, the experimental conditions were optimized. In pre-experiment, SNA-AuNP conjugates underwent irreversible aggregation due to the presence of 4 mM DTT in reaction buffer, which was a condition recommended by the manual of phi29 DNA polymerase. Therefore, the stability of SNA gold nanoparticles in RCA reaction system should be first taken into consideration. As shown in Figure S5, no more than 200 µM DTT hardly had effect on the color and 8


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absorption spectra of SNA-AuNP conjugates, while RCA reaction was possible even without additional DTT. Considering all factors above, the 20 µM DTT residues from the prepared circle DNA template solution could be allowed, and no extra DTT would be added to the reaction system. Besides, the ratio of primers to spacers was optimized to be 10:1, meanwhile the reaction time was determined to be 30 min according to the electrophoresis results (Figure S4). The products obtained from RCA were characterized by AFM. Free primer-triggered

ssDNA sequences

did

not completely stretch

like

rigid

double-stranded DNA (dsDNA), but some of them stacked together into dendritic structures (Figure 2a). The height of DNA on the marked position A was determined to be 1-2 nm. By contrast, a radial conformation was observed after conducting RCA reaction on the DNA-modified AuNPs. Many long DNA strands grafted from the central AuNPs, objects with a height of approximate 15 nm in Figure 2b. Note that the plane sizes of DNA and AuNPs in AFM images were larger than theoretical sizes, which was a normal phenomenon in AFM characterization. Assessment of the Feasibility of the Primer@AuNP@Ab2 Probe in Cell Imaging. First, the optimum dosage of primer@AuNP@Ab2 for detection of hERG channels was determined by flow cytometry. When primary antibodies were enough, the fluorescence intensity gradually increased with the dosage until a plateau occurred at 100 µL (Figure 3b and Figure S6). Furthermore, a control cell line was used to estimate the intensity of background fluorescence in the signal amplification detection. The ordinary human embryonic kidney cells (HEK293) do not express detectable hERG K+ channels. When the cascade amplification method was conducted on the cell membranes of HEK293 cells, no distinguishable fluorescence was detected (bottom in Figure 3a). The results showed that there was almost no endocytosis within 30 min and the nonspecific adsorption on cell membrane could be avoided by washing with PBS. So the RCA reaction on SNA-AuNP conjugates could be used as a powerful method for relative zero-background and ultrasensitive imaging of hERG proteins. For in situ imaging hERG channels, HEK293 cells with stable expression of hERG (hERG-HEK293) were seeded in confocal dishes for 48 h. Then the cells were cultured 9



with 150 µL fresh media containing 4 µg/mL anti-hERG for 2 h, followed by incubation with primer@AuNP@Ab2 probe (10 µL, dispersed in 100 µL DMEM) for 1 h. After that, RCA reaction was conducted on cell membranes and the reaction time was 30 min. In the control group, cells were incubated with Alexa Fluor 488 labeled goat anti-rabbit IgG rather than the prepared probe, which could image hERG proteins on a one-to-one basis. Due to the steric hindrance and the complexity of extracellular environment, the signal amplification effect of the developed probe in cell imaging analysis was not as remarkable as RCA reaction performed in phi29 DNA polymerase buffer. Even so, there was still an obvious enhancement of fluorescence intensity in comparison to the results in the control group (Figure 3a). Additionally, counterstaining experiment and Z-stack analysis of hERG-HEK293 cells were performed to confirm the location of the primer@AuNP@Ab2 probes. On the one hand, there was no overlap between the probe and Hoechst 33342, a nuclei dye (see Figure S7). On the other hand, the change of fluorescence-emitting region during z-axis scan (Figure S8) and the cross-sectional views (Figure 3c) indicated that the signal was almost entirely from the membrane. The results further demonstrated that the reporting units barely entered the cells during 30-min starvation treatment, which ensured the low background noise in the cytoplasm. Electrophysiological analysis. Radically, the primary antibody bound to an extracellular epitope of hERG between S1 and S2 domains, a part of the voltage sensor domain (VSD). Upon being connected with the primer@AuNP@Ab2 probe, the normal movement of the VSD, driven by the change in the membrane voltage, may be hampered, thereby possibly influencing the gating characteristics of the channel. On the other hand, the reduction of current, in turn, could demonstrate the specificity of the fluorescence signal in cell imaging. Therefore, the whole-cell patch clamp technique was used to investigate the current curves (Figure 4c). The currents were evoked by a voltage protocol, during which cells were first depolarized to potentials between -60 and +40 mV and then repolarized to -40 mV (inset in Figure 4a). The currents showed obvious rectification characteristic, and the peak of tail currents could indicate the activation of hERG channels at the 10


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corresponding depolarizing voltage. Next, the peak values were plotted against the depolarizing voltages to obtain the steady activation curve, which was fitted to the Boltzmann

equation

A2+(A1-A2)/(1+exp((V-V1/2)/Vs)),

where

V1/2

was

the

half-maximal voltage and Vs was the slope factor. (Figure 4b). As shown in Figure 4a and 4b, after connection with the prepared probe, both the depolarizing current and tail current decreased at any depolarizing voltage. Then we performed a statistical analysis on the half-maximal voltage for activation (V1/2) and the peak of tail current after a depolarizing pulse of 40 mV (Ipeak). The half-maximal voltage only changed slightly (Figure 4d). In contrast, the peak current obviously decreased in the experimental group (Figure 4e). Imaging of hERG K+ Channels in MCF-7 Cells. Evaluating the expression of hERG channel proteins is of great importance for the study on tumor cell apoptosis and proliferation. Here, MCF-7 cells, in which hERG channels are rarely expressed, were selected as the model cells to verify the practicability of the developed primer@AuNP@Ab2 probe. Compared to the one-to-one imaging (top in Figure 5), much stronger fluorescence emission was observed in the experimental group under laser scanning confocal microscopy (bottom in Figure 5). These results demonstrated that the proposed dual-amplification strategy could light up relatively few hERG channel proteins which were invisible in traditional immunofluorescence assay. It is also worth mentioning that the signal amplification probe can image many kinds of membrane proteins when the anti-hERG is changed into other aptamer or antibody. So it may have extensive application in biological imaging.

CONCLUSION In conclusion, we have first developed a dual-amplification strategy based on spherical nucleic acids (SNA) gold nanoparticles and rolling circle amplification (RCA) for ultrasensitive imaging of hERG potassium channels in cell membranes. First, we synthesize the SNA-AuNP conjugates for the primary amplification by increasing the ratio of primer to spacer. Then the primer@AuNP@Ab2 probe triggers 11



the effective RCA reaction, during which thousands of tandem repeats are replicated. In vitro, the signal-to-target ratio can reach up to 104 within 30 min, making it possible to visualize hERG channels in cells with low-level expression. Though the complex extracellular environment affects the amplification effect, there is still remarkable enhancement of fluorescence intensity in cell imaging when compared to the phenomenon in control groups. Therefore, our prepared primer@AuNP@Ab2 probe could be used as for extreme sensitive imaging of hERG channels, and serve as a powerful tool for hERG-related biological study. More importantly, the cascade amplification method possesses excellent generality.

ASSOCIATED CONTENT Supporting Information Detailed description of the sequences, and some additional experimental results.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Phone/fax: +86-25-89687294 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by the National Natural Science Foundation of China (Grant No. 21327902 and 21535003). This work was also supported by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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Scheme 1. Schematic Illustration of the Dual-Amplification Strategy for Ultrasensitive Imaging of hERG K+ Channels in the Membrane a

a

The picture is not drawn in accordance with the actual proportion.

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Figure 1. Design and characterization. (a) Polyacrylamide gel electrophoresis analysis of DNA ladder (lane 1), template (lane 2), ligation (lane 3), primer (lane 4), L1 (lane 5), circle template (lane 6), R0 (lane 7), R40 (lane 8) and R60 (lane 9). Rn: n indicates the RCA reaction time (min). (b) UV-vis absorption spectra of AuNPs and SNA-AuNPs.

(c)

TEM

image

of

AuNPs

(left)

primer@AuNP@Ab2 conjugates (right).

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and

SEM

image

of


Figure 2. AFM images of the extended ssDNA (a) and long DNA-AuNP conjugates (b) obtained through RCA. At the bottom, curves were the height analyses on three marked positions.

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Figure 3. Imaging of hERG K+ channels in cell membranes. (a) Confocal images of hERG-HEK293 cells after incubation with Alexa Fluor 488-conjugated secondary antibody (control group, top) or the primer@AuNP@Ab2 conjugates (experimental group, medium) for 1 h. In experimental groups, RCA reaction was conducted to achieve the signal amplification. Bottom is the confocal image of a control cell line HEK293, which does not express hERG channels. (b) Flow cytometric analysis of hERG-HEK293 cells (1×106/mL, 1 mL) after incubation with 0, 25, 50, 75, 100, 125, 150 µL primer@AuNP@Ab2 conjugates (from Ctrl to Exp 6) at 37 ºC. (c) 3D merge image of a 20-step z-stack analysis (Figure S8) and the cross-sectional views. Obtained with Leica 3D viewer.

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Figure 4. Electrophysiological analysis. (a) Current curves of hERG-HEK293 cells after incubation with (down) or without (top) the prepared primer@AuNP@Ab2 conjugates. The insets show the scale bar and voltage protocol: 4-s depolarizing pulses from -60 to +40 mV and a 4-s test pulse at -40 mV to elicit tail current, holding potential was -80 mV. (b) The steady activation curves of hERG-HEK293 cells after incubation with (red) or without (black) the prepared primer@AuNP@Ab2 conjugates. (c) Bright-field image of the whole-cell patch clamp recording mode. (d) and (e), Statistical analysis of peak current density after 40 mV depolarizing pulse (d) and the half-maximal voltage for activation, V1/2 (e). Data from 33 control hERG-HEK293 cells and 28 experimental hERG-HEK293 cells.

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Figure 5. Imaging of hERG K+ channels in MCF-7 cells using Alexa Fluor 488 labeled secondary antibody (top) or the dual-amplification strategy (bottom).

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For Table of Contents Only

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In Situ Visualization of hERG Potassium Channel via Dual Signal

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