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Am J Physiol Cell Physiol 291: C266-C269, 2006. First published March 22, 2006; doi:10.1152/ajpcell.00633.2005
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METHODS IN CELL PHYSIOLOGY

Single plasma membrane K+ channel detection by using dual-color quantum dot labeling

Volodymyr Nechyporuk-Zloy, Christian Stock, Hermann Schillers, Hans Oberleithner, and Albrecht Schwab

Institute of Physiology II, University of Münster, Münster, Germany

Submitted 16 December 2005 ; accepted in final form 2 March 2006


   ABSTRACT
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 ABSTRACT
METHODS
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K+ channels are widely expressed in eukaryotic and prokaryotic cells, where one of their key functions is to set the membrane potential. Many K+ channels are tetramers that share common architectural properties. The crystal structure of bacterial and mammalian K+ channels has been resolved and provides the basis for modeling their three-dimensional structure in different functional states. This wealth of information on K+ channel structure contrasts with the difficulties to visualize single K+ channel proteins in their physiological environment. We describe a method to identify single Ca2+-activated K+ channel molecules in the plasma membrane of migrating cells. Our method is based on dual-color labeling with quantum dots. We show that >90% of the observed quantum dots correspond to single K+ channel proteins. We anticipate that our method can be adopted to label any other ion channel in the plasma membrane on the single molecule level.

Ca2+-activated K+ channel; migration

POTASSIUM CHANNELS are expressed ubiquituously in prokaryotic and eukaryotic cells, where one of their key functions is to set the cell membrane potential (9). Most K+ channels are tetrameric proteins, and they share a conserved sequence within the "pore loop" (2). Great progress has been made with respect to the resolution of their crystal structure. Both prokaryotic and eukaryotic K+ channels have been crystallized so that their structure could be determined with Å resolution (4, 7, 13, 15, 16). However, the vast heterogeneity of K+ channels still poses difficult questions. K+ channels not only form the largest family of ion channels. Depending on which genome is studied, 30–100 K+ channel genes are known. The diversity of K+ channels is further increased by the fact that they frequently form heteromers composed of subunits from different K+ channel family members (18). Thus it remains a great challenge to determine the exact molecular K+ channel composition of a given cell. These difficulties are in part due to the lack of easily accessible tools for single molecule detection.

In our view, quantum dots (QDs; 1, 17) allow the identification of K+ channel proteins on the single molecule level by means of fluorescence microscopy. QDs were introduced as inorganic dyes, which compared with organic dyes are more bright, stable against photobleaching (5), and have narrow, tunable, and symmetric emission spectra, whose maxima depend on the size of QDs (3). QDs used in our study are composed of a core of cadmium selenide that is surrounded by a shell of zinc sulfide. Water solubility and attachment of QDs to biological molecules such as antibodies, lectins, or nucleic acids is mediated by a coating with organic molecules covalently attached to the surface of the shell. QDs are versatile dyes that are used in applications that range from labeling tumor cells within an organism (24) to studying the signaling cascade of epidermal growth factor in living Chinese hamster ovary cells (14). We used a transformed renal epithelial Madin-Darby canine kidney cell line (MDCK-F), which serves as a model for studying the function and distribution of ion channels in migrating cells (21). The morphology of MDCK-F cells is ideal for resolving the distribution of Ca2+-activated K+ channels (10, 12; hIK1) at a single molecule level. They extend a large and flat lamellipodium into the direction of movement. It provides an almost two-dimensional surface for the detection of single hIK1 channel molecules within the plasma membrane.


   METHODS
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Materials. All reagents were of analytical grade. We used fetal bovine serum (FBS) from Biochrom (Berlin, Germany). QD565 and QD655 conjugates with goat F(ab')2 anti-mouse IgG were from Quantum Dot (Hayward, CA). Primary antibodies against hemagglutinin (HA) were from Roche Diagnostics (Mannheim, Germany).

Channel construct and transfected cell line. MDCK-F cells (19) were transfected as described previously with a modified hIK1 channel containing an HA tag in the extracellularly located S3-S4 linker (22) and maintained in CO2/HCO3–-buffered minimal essential medium (pH 7.4) supplemented with 10% FBS and 600 µg/ml geneticin at 37°C. Cells were seeded on poly-L-lysine-coated glass coverslips for the experiments.

QD labeling. We fixed MDCK-F cells with 0.5% glutaraldehyde in HEPES-buffered Ringer solution (pH 7.4) for 45 min directly in the incubator to preserve the intact architecture of the lamellipodium and the ruffled membrane at its leading edge. After fixation, the cells were washed five times in PBS (pH 7.4) at room temperature, and kept for 30 min in 100 mmol/l glycine/PBS solution. After being washed, the cells were blocked with FBS (10% in PBS) for 1 h at room temperature, and incubated with primary antibodies against the extracellular HA tag of hIK1 channels (1:600) for 1 h at room temperature. Antibody specificity was confirmed previously by Western blot analysis (22). The cells were then washed five times in PBS. For one-color labeling, we incubated the cells with goat F(ab')2 anti-mouse IgG conjugated with QDs (1:50) for 1 h at room temperature, and for multicolor labeling with a mixture of QD565 and QD655 goat F(ab')2 anti-mouse IgG conjugates (1:25 and 1:100, respectively), for 1 h at room temperature. Titers of secondary antibodies were chosen such that QD565 and QD655 labeled the cells at a similar density. Moreover, care was taken that the total density of QDs was low enough to clearly identify them individually. The cells were again washed five times in PBS and fixed by 0.5% glutaraldehyde in HEPES buffer (pH 7.4) for 45 min.

Microscopy and data acquisition. Immunofluorescence microscopy was performed with an inverted microscope (Axiovert 200, Zeiss, Oberkochen, Germany) equipped with a digital camera (Visitron, Puchheim, Germany) and a 100 x 1.45 oil-immersion objective. Data acquisition and analysis were performed with Metavue software (Visitron). We used the following filters: QD565, 420 nm excitation, 565 nm emission (XF302–1 filter; Omega Optical, Brattleboro, VT); QD655, 420 nm excitation, 655 nm emission (XF305–1 filter; Omega Optical). The number of QDs per cell was corrected for background level of QDs determined in cell-free areas. In case of double color labeling, only those cells which had the same number of green and red QDs were taken for further analysis. The pixel with the highest fluorescence intensity was taken as the optical center of a QD. Distance between QDs was then measured as the distance of their optical centers. Pixel shift due to filter change was determined by acquisition of two images of QD-labeled cells in one channel with a filter change (for example, red-green-red) in between. The two images were assigned to different colors, superimposed, and the distances of the optical centers were determined. The mean pixel shift amounted to 0.46 ± 0.05 pix (n = 100 QD pairs).

Statistics. Data are presented as means ± SE. N refers to the number of MDCK-F cells (see GoGoFig. 3).


Figure 1
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  		Fig. 1. Detection of Ca2+-activated K+ channel molecules (hIK1) channels on the surface of a renal Madin-Darby canine kidney (MDCK-F) cell by quantum dot (QD) labeling. The green spots correspond to hIK1 channel proteins labeled with QD565. The diffuse staining of the cell center is due to glutaraldehyde autofluorescence. Bar: 10 µm.

 

Figure 2
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  		Fig. 2. Multicolor QD labeling of hIK1 channel on the surface of a MDCK-F cell. A: leading edge and lamellipodium of the cell. Green and red spots correspond to single hIK1 channel proteins. Yellow spots indicate dual labeling with a pair of QD565 and QD655 with a distance of 1–2 pixels between their optical centers. B: a magnified view of the white rectangle. Bar, 3 µm. Also shown is the leading edge of the cell. The dashed white line marks four spots, whose intensity profiles are shown in C. Spots 1 and 3 are labeled only with red QD655, whereas spots 2 and 4 are dually labeled with green QD565 and red QD655 and therefore appear yellow. Bar, 0.5 µm. C: intensity profile of fluorescence signal. Vertical dashed lines mark the optical centers of fluorescence signal from single QDs, which are located closely to each other.

 

Figure 3
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  		Fig. 3. Relative frequency of dual-color QD binding to hIK1 channel proteins. The bars colored yellow give the frequencies of labeling hIK1 channel proteins with green QD565 and red QD655 simultaneously. The distances of the optical centers of the QDs are indicated. N = 14 MDCK-F cells.

 

   RESULTS AND DISCUSSION
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When the distribution of the hIK1 channel in the plasma membrane of MDCK-F cells is visualized with the use of QD labeling (see Fig. 1), the overall staining is similar to previous results obtained with conventional fluorescence labeling (22). The K+ channels are more concentrated at the leading edge of the cell than in the remaining membrane of the lamellipodium. The average density of QDs amounts to ~2 QDs/µm2, which is similar to the channel density calculated from earlier patch-clamp studies. To verify that a single QD corresponds to a single channel and not to a group of single channels, we applied dual-color labeling. We added a mixture of secondary antibodies conjugated either with "red" QDs that emit at 655 nm (QD655) or with "green" QDs that emit at 565 nm (QD565) (Fig. 2, A and B). The titers of the secondary antibodies were adjusted in such a way that they bind with the same efficacy to their target. Consequently, the numbers of QD655 and QD565 per cell are the same (Fig. 3). The merged cell surface image (Fig. 2, A and B) exhibits many green (QD565) and red (QD655) spots, and only a small fraction of "yellow" spots. Red and green spots could either represent single QDs that mark individual channel proteins or the binding of several red (QD655) or green (QD565) QDs to one channel protein. The appearance of yellow spots in the merged image indicates that red (QD655) and green (QD565) QDs are binding to the K+ channel simultaneously. The intensity profile (Fig. 2C) provides a quantitative analysis. Two pairs of red and green QDs are shown. Their optical centers are only 1–2 pixels or 60–120 nm apart from each other. It is only by the use of the dual-labeling technique that these QDs can be recognized individually. This would not be possible in case of single-color labeling. They would look like one bright single QD. However, most spots are clearly red or green and only a small fraction of spots (3.3%) is yellow with different distances between optical centers (Fig. 3). The fraction of yellow spots is hardly affected by doubling the antibody concentrations.

Each hIK1 channel protein is a tetramer, meaning that it has four binding sites for antibodies and QDs that can be completely or partially occupied. Assuming the equal binding of four QDs to the K+ channel the relative frequencies of red and green QDs should behave as predicted by the following binomial distribution: (a + b)4 = a4 + 4a3b + 6a2b2 + 4ab3 + b4. We derived a = 0.45 and b = 0.50 from our experiments (Fig. 3). a4 and b4 are the probabilities that four green or four red QDs bind to the channel, respectively. 4a3b + 6a2b2 + 4ab3 is the probability that different combinations of one, two, or three green and red QDs bind simultaneously. We can then calculate that almost 90% of the channels should be labeled by green and red QDs simultaneously and therefore appear yellow. This is in clear contrast to our experimental findings. We therefore modified our model calculation to the binding of only two QDs per channel protein: (a + b)2 = a2 + 2ab + b2. a2 and b2 are the probabilities that two green or two red QDs bind to the channel, and 2ab is the probability that one green and one red QD bind to the channel at the same time (yellow). We can then calculate that 45% of the channels should be labeled by one green and one red QD and therefore appear yellow which is also in contradiction to our experimental results. We therefore conclude that both models are not valid. In our view, the easiest explanation to account for this apparent discrepancy is to assume that most K+ channel molecules are labeled by only one QD.

If channels can be dually labeled by red and green QDs, they can also bind either two (or more) red or two (or more) green QDs, respectively. Our technique does not allow the distinction between binding of one or of multiple QDs of the same color. Thus, we must assume that in addition to 3.3% dually labeled yellow hIK1 channels, we also labeled 3.3% with more than one red or green QD, respectively. Nonetheless, 93.4% of the QDs observed are single and labeled single K+ channel molecules.

K+ channel proteins have a diameter of ~10 nm (11, 23). Antibodies have a size of 8 nm (8), and according to the manufacturer, the diameter of the QDs used in our study is 10 nm. Thus the maximal distance between the optical centers of two QDs bound to a single channel protein is in the order of 50 nm (Fig. 4). This corresponds to a distance of 1 pixel in the digitized image. Therefore, two QDs with a distance between optical centers of more than two pixels must be placed on two different K+ channel proteins. The geometric model (Fig. 4) in conjunction with the crystal structure of the voltage-gated K+ channel, Kv1.2 (15, 16) may also provide an explanation for the fact that most hIK1 channel proteins are labeled with only one QD. Voltage-gated K+ channels when viewed from the extracellular side resemble a four-leaf clover. S3-S4 linkers are in the "leaves" so that they are <10 nm apart from each other. Assuming a similar structure for Ca2+-activated hIK1 channels (they also have 6 transmembrane spanning domains) one can hypothesize that steric hindrance prevents the binding of more than one QD-labeled antibody to the HA tag in the S3-S4 linker.


Figure 4
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  		Fig. 4. Geometric model of QD binding to hIK1 channel proteins. The K+ channel structure, which is adopted from a voltage-gated K+ channel (4), somewhat resembles a four-leaf clover. The S3-S4 linker containing the hemagglutinin (HA) tag is situated in the "leaves." The maximal distance of the optical centers of two QDs amounts to ~50 nm. We used atomic coordinates of Kv1.2 (Protein Data Bank ID 2A79; Ref. 15) and atomic coordinates of the model of an entire human IgG1 molecule (20) for generation of the figure with PyMOL software (DeLano Scientific, San Carlos, CA). 1° Ab, primary antibody; 2° Ab, secondary antibody.

 
In summary, we have described a simple method for the detection of K+ channels on the single-molecule level. We anticipate that this method can be adopted for use with other ion channels as well. Moreover, by combining intra- and extracellular tags inserted into different subunits this method may also be used for identifying the subunit composition of heterooligomeric ion channels. Subsequently, separate binding of an extracellular domain of subunit A and an intracellular domain of subunit B after permeabilization with different-colored QDs should facilitate the stoichiometry analysis of the channel subunit composition. Total internal reflection fluorescence microscopy would provide the 10-nm resolution (6) needed for the spatial segregation of the two different dyes.


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This work was supported by Deutsche Forschungsgemeinschaft Grants RE 1284/2-1,2, SFB629 (A6), and SCHW 407/9-1,2.


   ACKNOWLEDGMENTS
 
We thank Dr. Dessy Nikova, Tetyana Nechyporuk, David Grünwald, and Dr. Ulrich Kubitscheck for fruitful discussions, as well as Hannelore Arnold and Sabine Mally for technical support.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Schwab, Institute of Physiology II, Univ. of Münster, Robert-Koch-Strasse 27b, D-48149 Münster, Germany (e-mail: aschwab{at}uni-muenster.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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