Probing Dynamic Heterogeneity in Aggregated Ion Channels in Live

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Probing Dynamic Heterogeneity in Aggregated Ion Channels in Live Cells Rajeev Yadav and H. Peter Lu* Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, United States S Supporting Information *

ABSTRACT: N-Methyl-D-aspartate (NMDA) receptor is a crucial ion channel requiring the concurrent binding of both agonists glycine and glutamate along with the membrane depolarization for its opening and mediates calcium influx, which triggers the signal transduction important for synaptic transmission and plasticity. The efficiency and regulations of such cellular physiological processes are crucially relying on inherent molecular spatial organization and interactions of these channels with ligands as well as trafficking in the confined local environment. To decipher their organization, we have combined two imaging microscopic approaches, photobleaching step counting and single particle tracking analysis, that allow detection of unique organized patterns of NMDA receptor ion channel in the cell membrane. A broad range of photobleaching steps is observed ranging from 1 to 16. Single particle tracking analysis is performed to see the surface mobility of NMDA receptors and measured diffusion coefficient for all the trajectories, which on correlating with photobleaching steps shows that the higher diffusion coefficient corresponds to the lower number of bleaching steps and vice versa. These studies clearly indicate that NMDA receptors are organized on the cell membrane in clusters with different stoichiometry. Our findings for the clustering effect of NMDA receptor ion channel on the live cell membrane represent an important step forward for understanding the functioning of NMDA receptor ion channels.

1. INTRODUCTION NMDA receptor ion channel is an important ion channel membrane protein among the glutamate receptor ion channels, which controls the synaptic transmission and plasticity during learning and memory functions.1−3 Moreover, it is a ligandgated and voltage gated ion channel composed of two copies for each GluN1 and GluN2 subunit and forms a heterotetramer.1−8 The structural characterization of NMDA receptor ion channel has been reported crystallographically with a resolution of 4 Å; the subunits are arranged in a dimer of heterodimer of GluN1-GluN2 with the 2-fold symmetry axis.7,9 Each subunit of NMDA receptor is composed with three subdomains, namely, amino terminal domain (ATD), ligand binding domain (LBD), and transmembrane domain (TMD). For activation, it requires binding of both agonists, glycine and glutamate, simultaneously bound at ligand binding domain (LBD) of GluN1 and GluN2 subunits, respectively, along with membrane depolarization to remove magnesium block from transmembrane domain (TMD).3−19 On opening, it mediates calcium influx and triggers the signal transduction, which is important for synaptic transmission and plasticity during learning and memory functions.1−3 The efficiency of such cellular physiological processes is crucially relying on inherent molecular spatial organization; and the regulations for these processes are mainly dependent on the interactions of these membrane proteins with ligands as well as trafficking in the confined local environment. The detailed mechanism is yet unclear in cell biology. In recent years, fluorescence © XXXX American Chemical Society

spectroscopic methods as well as model predictions based on theoritecal calculation have become powerful tools for understanding the interactions, compositions, heterogeneity, and conformational dynamic of proteins and ion channels.20−27 The advancements in the imaging techniques such as superresolution microscopy, fluorescence photobleaching analysis, molecular tracking, single molecule FRET, etc. can potentially reveal unique cellular organizational features, which are related to the ion channel interactions and dynamics, locations, and their compositions and stoichiometry within the live cells.25−50 For molecular understanding of membrane proteins within the live cells, we must decipher their molecular organization, structures, and compositions. In recent years, quantitative fluorescence microscopic methods such as stepwise photobleaching as well as ratio comparison to the standards have become very popular tools among the researchers to define the composition of complex biological systems within live cells.28−37 However, the bleaching step analysis has some limitations like missing the step as two dyes or fluorescent proteins can bleach at the same time; also it became impossible to detect the bleaching steps if the counting is high. The counting high number of bleaching steps is possible by using some mathematical aids; in this regard, there are a number of Special Issue: Prashant V. Kamat Festschrift Received: January 9, 2018 Revised: February 5, 2018 Published: February 5, 2018 A

DOI: 10.1021/acs.jpcc.8b00262 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C

were kept in incubator at 37 °C flowing with 5% CO2. Once the area of glass slide is covered by 50%, gene transfer is done for protein expression. For efficient gene transfer to expression of NMDA receptor, we followed a standard protocol mediated by transfection with Lipofectamine LTX and Plus reagent (Invitrogen, 11668-019). Cells are incubated with pDNAs for EYFP-GluN1a and GluN2B each 1 μg along with 3 μL of Plus reagent and 6 μL of LTX reagent in 500 μL of opti-MEM according to the protocol given by Invitrogen. During transfection, growth medium is replaced with the medium without antibiotics (Pen-strep) and phenol red. Before transfection, plasmid DNA is amplified using a standard method and used without any further purification for the expression of NMDA receptor in live HEK-293 cell. Plasmid DNA encoding with EYFP-GluN1a and EGFP-GluN2B are purchased from Addgene, whereas plasmid DNAs encoded with GluN1a and GluN2B are gift from Prof. David R. Lynch, University of Pennsylvania. 2.2. Single-Molecule Fluorescence Microscopy. The experimental setup is based on an inverted microscope (Olympus IX71) having differential interference contrast (DIC) imaging components and reported elsewhere.16 In brief, the sample is excited by a continuous wave laser source (λex = 473 nm, CrystaLaser) by sending the light through the total internal reflection fluorescence (TIRF) illumination combiner (model no IX2-RFAEVA-2), where the angle of laser beam is adjusted for TIRF mode. The beam is passed to the microscope through the back port, which is then focused onto sample with a high numerical-aperture objective (Olympous, UPlanSApo 1.2 NA, 60×) through dichroic beam splitter (Chroma Technology, 473rdc). The fluorescence is collected by the same objective and passed through a 500 nm long pass filter. Finally, fluorescence beam is focused to an electron multiplying charge coupled device (EMCCD) camera (Princeton Instruments, ProEM). The fluorescence signal is recorded in a video having 3000−5000 frames, and each frame has acquisition time of 10 ms. The readout time for EMCCD is 15.037 ms. We have used two different microscopic modes, TIRF and wide field, for the imaging as well as patch-clamp experiments, respectively. 2.3. Bleaching Step Analysis. EYFP fluorescence signal is used for the recording of imaging frames of the NMDA receptor ion channel as EYFP is genetically encoded to one of the subunits, GluN1a at N-terminal. However, GluN2B subunit remains unlabeled from any fluorescent tag. Thus, the two bleaching steps are expected for a single NMDA receptor. Here, we have used an automated program, PIF (Progressive Idealization and Filtering),35 for bleaching step analysis, which identifies the subunit stoichiometry of membrane proteins from TIRF imaging recordings. In this method, Laplacian of Gaussian (LoG) kernel filter is used to remove the background fluorescence, while Chung−Kennedy54 filter is used for intensity trajectory curves, generated for each fluorescent spot. 2.4. Single Molecule Tracking and Diffusion Analysis. Tracking analysis of NMDA receptor ion channel labeled with EYFP is performed for the movies recorded in TIRF microscopy using a plugin TrackMate of Fiji-ImageJ software. The center of the fluorescence spots was determined with a Gaussian fit using LoG (Laplacian of Gaussian) detector. The tracking analysis is done for first 200 frames only for each movie due to overcoming of the photobleaching effect of EYFP on continuous illumination for a long time. After getting the

reports that used computational based bleaching steps analysis. Recently, a fully automated single-molecule fluorescence counting method is developed as computational software for counting the subunits from photobleaching steps named as PIF (Progressive Idealization and Filtering), which can detect high copy of steps.35 Similarly, another computational procedure based on some mathematical algorithms is developed and applied to find out the number of cellulose synthase (CESA) in the cellulose synthesis complex and the stoichiometry, with the lower limit as 10 for copy number per particle.33 However, in the bleaching steps analysis, the photobleaching steps are observed from a diffraction limited spots, which is around 250− 300 nm and thus can have more than one group of biomolecules. Hence, if the counting of bleaching steps is high, it is not necessarily observed from a single moiety or a group but from more than one moiety colocalized within the diffraction limited area. For the current study, we have used NMDA receptor having two EYFP molecules, and we observed a range of bleaching steps from 1 to 16, which is not possible from a single NMDA receptor. Hence there might be a possibility of having more than one receptors within the diffraction limited area. For complete molecular understanding of biological processes and their organization within live cells, we must employ other methods along with bleaching steps analysis. Interestingly, some other optical microscopic methods like super-resolution imaging38−43 as well as single molecule tracking analysis44−53 can also give crucial information about the organization of these biosystems, with their related size and the possibility of colocalization if present. By use of a single particle tracking method, a broad range of diffusion coefficients are observed for glutamate receptors on live cell membrane, which has been explained in terms of molecular interactions.46,47 It has also been observed as the mobility of NMDA receptors having GluN2A subunits is slower than the receptor containing GluN2B subunits at the surface of cultured neurons.47 In this report, we have used PIF approach to detect the photobleaching steps for NMDA receptor ion channels in the live cell, where only GluN1a subunits are genetically tagged with EYFP, thus only two steps should be observed for a single NMDA receptor. However, we have observed a range of photobleaching steps from 1 to 16, clearly suggesting that NMDA receptors are colocalized or form a cluster within in the diffraction limited area. We have also performed single particle tracking analysis and observed a broad range of diffusion coefficients, which then correlate with the photobleaching step analysis, and observed that the fluorescent spots that have lower number of bleaching steps have higher diffusion coefficient and vice versa. These studies clearly indicate that NMDA receptors are located as heterogeneous clusters on the membrane.

2. MATERIALS AND EXPERIMENTAL METHODS Chemicals are purchased from Sigma-Aldrich, Invitrogen, Addgene, and ATCC for imaging and patch-clamp experiments. They are used for experiments without further purification. 2.1. Cell Culture and Protein Expression in HEK-293 Cell. A complete medium of DMEM (11995, Gibco/Life Technologies) containing 10% fetal bovine serum (SigmaAldrich) and 1% penicillin−streptomycin (Gibco/Life Technologies) is used to grow HEK-293 cells. After the cells are reached to a confluence of ∼75% on the surface of T-75 flask, cells are subcultured in T-75 flask and in 35 mm Petri dishes containing 25 mm glass slide for live cell imaging and the cells B

DOI: 10.1021/acs.jpcc.8b00262 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C fitted position of each spot in each frame, an algorithm for particle linking as LAP tracker (linear assignment problem) is used for getting the single molecule tracking trajectories, where the parameters for maximum linking distance, gap closing maximum distance, and maximum frame gap are used as 200 nm, 600 nm, and 10 frames, respectively. Diffusion coefficient, D, for all the trajectories are observed by fitting the first four points of mean squared displacement (MSD) curves versus time using eq 1, separately. MSD(Δt ) = rΔt 2 = 4DΔt

(1)

where square displacement (rΔt2) between the frames is calculated using position of X and Y coordinates from the single particle tracking trajectories as follows. rΔt 2 = (X t +Δt − X t )2 + (Yt +Δt − Yt )2

(2)

3. RESULTS AND DISCUSSION Our study is devoted to understanding the organization of NMDA receptor on the cell membrane, where we have used a correlated analysis for two single molecule imaging tools named as photobleaching steps counting as well as single molecule tracking analysis. We have used NMDA receptor composed of two types of subunits, GluN1a and GluN2B, where GluN1a is genetically encoded with a fluorescent protein EYFP at Nterminal, whereas GluN2B remains unlabeled from any fluorescent tag (Figure 1). Figure 1b shows a frame of a movie recorded for NMDA receptor ion channel in live HEK293 cell, and Figure 1c shows expected bleaching steps for single NMDA receptor. A typical movie recorded for NMDA receptors is shown in Supporting Information. PIF program is used for bleaching step analysis, where the fluorescence background is removed by convoluting a Laplacian of Gaussian (LoG) kernel filter in the image followed by filtering of each intensity trace with the Chung−Kennedy filter.35 The genetically encoded EYFP ensures that every NMDA receptor contains two tagged subunits, and thus a single NMDA receptor should have two photobleaching steps, if there is 100% maturation of EYFP, otherwise a low number of steps. However, we have observed a broad distribution range of photobleaching steps ranging from 1 to 15 as shown in Figure 2. These observed bleaching steps clearly indicate that NMDA receptor ion channels are distributed on cell membrane in a dissimilar fashion, where most of the NMDA receptors are singly dispersed as the occurrence is highest for the bleaching steps at around 2, whereas the remaining distribution of bleaching steps may correspond to two or more NMDA receptor ion channels within the diffraction limited spot area. Here it is to be noted that the photobleaching steps counting is from diffraction limited spot area of around ∼250−300 nm, whereas the lateral size of a single NMDA receptor is around ∼12−13 nm.7,9 Hence, there might be two possibilities for observing high number of bleaching steps, which are either NMDA receptors are colocalized within diffraction limited spot area or they make clusters on the membrane of live HEK cells. To resolve this issue, we have used single molecule tracking along with photobleaching steps counting method, which alone is not necessarily capable of giving any clear picture of organization of NMDA receptor. As we have observed from bleaching steps analysis, NMDA receptors are distributed over the cell membrane in a dissimilar fashion, where the density of receptors differs in different

Figure 1. (a1) Crystal structure of NMDA receptor (PDB code 4TLL) having four subunits, two GluN1 subunits and other two GluN2B in brown and green colors, respectively. It has three domains: ATD, LBD, and TMD. (a2) Schematic representation of heterotetrameric NMDA receptor ion channel (side view) in the membrane of the cell depicting the arrangement of four subunits, two GluN1a (orange color) and two GluN2B (blue color). GluN1a subunit is genetically encoded by fluorescent protein EYFP, as shown by yellow spots. (b) Fluorescence image of EYFP tagged with GluN1a subunit of NMDA receptor in live HEK-293 cell. (c) Expected changes in the fluorescence intensity show photobleaching steps for a single NMDA receptor, and out of four subunits only two are labeled. For bleaching steps analysis, we have used software Progressive Idealization and Filtering (PIF). Background fluorescence was removed by convoluting a Laplacian of Gaussian (LoG) kernel filter in the image followed by filtering with the Chung−Kennedy filter. Scale bar is 2 μm in panel b.

Figure 2. Broad distribution of photobleaching steps observed for NMDA receptor ion channels dispersed over cell membrane of live HEK-293 cells. Here, EYFP fluorescence signal is used for bleaching steps analysis of the NMDA receptor ion channel as EYFP is genetically encoded to one of the subunit, GluN1a at N-terminal, whereas GluN2B subunit remains unlabeled from any fluorescent tag. Thus, two bleaching steps are expected for a single NMDA receptor.

diffraction limited spots. Hence, we measured and compared the surface mobility using single-particle tracking analysis for C

DOI: 10.1021/acs.jpcc.8b00262 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C each diffraction limited spots observed for NMDA receptors (Figure 3) and the fluorescence signal for EYFP is used to study

the lateral surface motion of NMDA receptor ion channel in living HEK 293 cell. The tracking analysis is done from the same trajectories which are recorded for bleaching steps analysis; however only first 200 frames (∼3 s) are used for this analysis to avoid any bleaching effect. Parts b1, b2, and b3 of Figure 3 show the typical tracking trajectories for the spots having different bleaching steps, which are for two, four, and eight bleaching steps, respectively. These tracking trajectories clearly show that the step size of motion is becoming shorter on going from lower bleaching steps to higher bleaching steps. The difference in the step size can be clearly understand from the square displacement (rΔt2), which is calculated for the single step or first time lag (Δt = 10 ms). Figure 3c shows the cumulative distribution of single step square displacement observed from all three cases in Figure 3b as two, four, and eight bleaching steps. Here the slope of increment in the square displacement is becoming faster and faster, which also indicates that the higher is the number of receptors in the diffraction limited spot, the slower is the surface motion. For better understanding diffusion coefficient, D, is calculated by the linear fitting of the first four points of the mean square displacement (MSD) versus time as discussed in the experimental section, where the MSD is calculated for first (N = 200) frames for each trajectory of movies recorded in the TIRF mode and the diffusion coefficients for all the trajectories are observed in a long range from 19 × 10−4 μm2/s to 17 × 10−2 μm2/s (Figure 4a). Brownian diffusion causes the linearity in the MSD curve, which is generally observed in the initial time; however, with moving to the longer time MSD curves, it may deviate from the linear behavior caused by the anomalous behavior on the cell membrane.44,48,50,55,56 Thus, MSD curves are most commonly used to find the diffusion coefficient as well as anomalous behavior of a single particle tracking trajectory. However, the best estimate of the diffusion coefficient of a diffusion trajectory is obtained from the first few MSD measurements of small time steps because the slope of initial MDS points is assumed to have the smallest and insignificant contribution from anomalous behavior.36−40,46,47 Dahan et al. used the first five points to measure the lateral dynamics of glycine receptors in the neuronal membrane of living cells and characterized multiple diffusion domains as synaptic and extrasynaptic.44 Similarly, Pinaud et al. have used first two points to measure the instantaneous diffusion coefficient along with other methods and observed the diffusion behaviors of

Figure 3. Photobleaching steps for EYFP-NMDA receptor ion channels and their corresponding single particle tracking trajectories. (a1−a3) Photobleaching steps for three different diffraction limited spots observed for EYFP-NMDA receptor ion channel in TIRF mode show two, four, and eight bleaching steps, respectively. (b1−b3) Diffusion trajectories for the corresponding spots, b1−b3. (c) Cumulative distribution of single step (or first time lag, Δt = 10 ms) square displacement observed from all three cases in parts a1−a3 and b1−b3 as two, four, and eight bleaching steps and shown as black, blue, and red, respectively.

Figure 4. (a) Distribution histogram of diffusion coefficients observed for NMDA receptors in the live cell membrane. (b) Distribution of diffusion coefficients according to the photobleaching steps. The distribution of diffusion coefficients is broad, and the value is higher at low number of bleaching steps, while the distribution shrinks toward lower value on going toward higher photobleaching steps. D

DOI: 10.1021/acs.jpcc.8b00262 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry C GPI-anchored avidin protein in different membrane microdomains.55 Groc et al. have used the first four points to measure the diffusion coefficient of NMDA receptor with different combinations of subunits as GluN1-GluN2A and GluN1GluN2B and observed that the surface mobility of GluN2Acontaining NMDA receptors is much smaller than that of GluN2B containing ones.47 Quin et al. have provided a way to estimate the relative error in the diffusion coefficient observed from first few points (m) of MSD as [2m/3(N − m)]1/2, where N is the number of points in the trajectory or number of frames used for tracking analysis.56 For the current study we have used four points MSD and 200 frames; hence the estimated error is around 11%. Figure 4a shows the distribution of diffusion coefficients observed from all trajectories of around 15 movies ranging from 19 × 10−4 μm2/s to 17 × 10−2 μm2/s, which are very much consistent with reported values for the diffusion of membrane proteins.46,47,57,58 Hoze et al. have studied the trafficking of AMPA receptor on neuronal cells and observed the diffusion coefficient as 76 × 10−3 μm2/s and 51 × 10−3 μm2/s in two different neuron dendritic spines.46 Groc et al. have measured the diffusion coefficient for synaptic and extrasynaptic GluN2B-containing NMDA receptor and showed the results in the form of the interquartile range as 24 × 10−4 to 0.115 μm2/s with the median value of 250 × 10−4 μm2/s and 43 × 10−4 to 0.125 μm2/s with the median value of 500 × 10−4 μm2/s, respectively.47 However, we have observed the median value of diffusion coefficient of NMDA receptor as 183 × 10−4 ± 19 × 10−4 μm2/s in live HEK 293 cells, which is in the same range as observed previously. To further understand the molecular level NMDA receptor ion channel organization over the cell membrane, a correlation between two state of art imaging techniques, photobleaching steps counting and single particle tracking, has been established, where the observed diffusion coefficient from particle tracking analysis is correlatively compared with the number of bleaching steps observed from the same trajectory. Figure 4b shows the distribution of diffusion coefficients correlated with the bleaching steps, where on going from lower to higher bleaching steps, the distribution of diffusion coefficients become narrow and shrink toward lower value. For a quantitative comparison, we have made four random groups for the number of photobleaching steps as one-to-two, three-to-five, six-to-eight, and more than eight as per the bleaching steps distribution data from Figure 2. As it is known that the maturation efficiency for a fluorescent protein is around ∼80% in Xenopus oocytes,35,36 keeping this in mind, some of the consecutive higher bleaching step numbers like five, seven, and so on may correspond to the bleaching of two, three, and four NMDA receptors, respectively. Parts a1−a4 of Figure 5 show the distribution of diffusion coefficients for four predefined groups of bleaching steps. The diffusion coefficient for the first group having one and two bleaching steps (Figure 5a1) shows a broad distribution ranging from 110 × 10−4 μm2/s to 17 × 10−2 μm2/s with a median value of 420 × 10−4 ± 40 × 10−4 μm2/s, whereas the fourth group having nine and more bleaching steps (Figure 5a4) shows the distribution ranging from 19 × 10−4 μm2/s to 6 × 10−2 μm2/s with the median value as 70 × 10−4 ± 10 × 10−4 μm2/s. Moreover, the median values of all the groups have been observed as 420 × 10−4 μm2/s, 190 × 10−4 μm2/s, 100 × 10−4 μm2/s, and 70 × 10−4 μm2/s for first (Figure 5a1), second (Figure 5a2), third (Figure 5a3), and fourth (Figure 5a4) group, respectively. These values clearly suggest that on going from lower number NMDA receptor group (single

Figure 5. Analysis of diffusion coefficient for different groups of bleaching steps. The distribution histogram for the diffusion coefficients of group 1 which contains one and two bleaching steps (a1), group 2 which contains three to five bleaching steps (a2), group 3 which contains six to eight bleaching steps (a3), and group 4 which contains nine and more bleaching steps (a4). (b) Cumulative distribution of diffusion coefficient of all four groups shown by different colors as blue, pink, green, and red for first to fourth group, respectively. Black dotted line shows cumulative distribution of all diffusion coefficients as shown in Figure 4a.

NMDA receptor) to the higher number NMDA receptor group of photobleaching steps (more NMDA receptors), the diffusion coefficient shows a continuous shift from higher value to the lower values. This can only be possible if high number of NMDA receptors are tracking on the surface of cell membrane in a single confined assembly. Hence on going from lower NMDA receptor to higher number of NMDA receptors, the value of diffusion coefficient decreases due to the size of confined assembly increasing with the increase in the number of NMDA receptors within the assembly. The first group, which is expected for tracking of single NMDA receptor, has very similar values of IQR as already reported for single NMDA receptor.47 Figure 5b shows cumulative distribution of diffusion coefficient, where the increment for each point (bin size 7 × 10−3 μm2/s) is becoming faster on going from lower value to higher number of bleaching steps and the population in the first bin is around 0%, 10%, 30%, and 50% for first to fourth group, respectively, which is termed as immobile receptor population. This clearly indicates that the receptors that are in high number groups are immobile in nature. E

DOI: 10.1021/acs.jpcc.8b00262 J. Phys. Chem. C XXXX, XXX, XXX−XXX

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membrane represent an important step forward for understanding the functioning of NMDA receptor ion channels.

On the basis of the correlated analysis of two single molecule imaging methods, subunit counting by observing photobleaching steps, and surface mobility by single particle tracking, we have proposed that NMDA receptor ion channels are organized on the live cell membrane in clusters of different size (Figure 6). The lower diffusion coefficient for higher number of

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcc.8b00262. Movie recorded for NMDA receptors (AVI)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: 419-372-1840. ORCID

H. Peter Lu: 0000-0003-2027-428X Notes

The authors declare no competing financial interest.

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Figure 6. Proposed organization of NMDA receptor ion channels on the live cell membrane, where these are dispersed in random fashion, around 52% are singly dispersed, and others are in clusters with different number or in heterogeneous clusters. On the left side, a crystal structure of single NMDA receptor is shown.

ACKNOWLEDGMENTS We thank Prof. Anthony L. Auerbach, Department of Physiology and Biophysics, State University of New York, and Prof. David R. Lynch, University of Pennsylvania, for providing us with HEK-293 cell and plasmid DNA for NMDA receptor, respectively. We thank Dr. Rikard Blunck, Department of Physics, Université de Montréal, Canada, for providing PIF software. This work is supported by NIH NIGMS and the Ohio Eminent Scholar Endowment.

bleaching steps or the higher number of NMDA receptors is only possible when the NMDA receptors are making clusters. This is in support of our previous report, where we observed that the groups of NMDA receptors exist in the clusters of around 50 nm on the live cell membrane with different compositions.59 Very recently, it has been shown that within the synapses AMPA receptors are organized into a few clusters containing around ∼20 receptors in around ∼70 nm nanodomains, which are dynamic in their shape and position and can form and disappear within minutes.42 Hence, the clustering effect may be the one cause for the heterogeneity in the gating mechanism of NMDA receptor, which is still unclear to the scientific community; in this regard a lot of effort has been done until now to understand NMDA receptor ion channel gating mechanism.8,60−64 Popescu and co-workers have studied the gating mechanism of NMDA receptor composed of GluN1 and GluN2B subunits and showed that the long heterogeneous activations of NMDA receptor are due to the slow glutamate dissociation.8

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REFERENCES

(1) Mayer, M. L.; MacDermott, A. B.; Westbrook, G. L.; Smith, S. J.; Barker, J. L. Agonist- and Voltage-Gated Calcium Entry in Cultured Mouse Spinal Cord Neurons under Voltage Clamp Measured Using Arsenazo III. J. Neurosci. 1987, 7, 3230−3244. (2) Granger, A. J.; Nicoll, R. A. Expression Mechanisms Underlying Long-Term Potentiation: A Postsynaptic View, 10 Years on. Philos. Trans. R. Soc., B 2014, 369, 20130136. (3) Traynelis, S. F.; Wollmuth, L. P.; McBain, C. J.; Menniti, F. S.; Vance, K. M.; Ogden, K. K.; Hansen, K. B.; Yuan, H.; Myers, S. J.; Dingledine, R. Glutamate Receptor Ion Channels: Structure, Regulation, and Function. Pharmacol. Rev. 2010, 62, 405−496. (4) Mayer, M. L. Glutamate Receptors at Atomic Resolution. Nature 2006, 440, 456−462. (5) Furukawa, H.; Singh, S. K.; Mancusso, R.; Gouaux, E. Subunit Arrangement and Function in NMDA Receptors. Nature 2005, 438, 185−192. (6) VanDongen, A. M., Ed. Biology of the NMDA Receptor; CRC Press: Boca Raton, FL, 2009. (7) Lee, C.-H.; Lü, W.; Michel, J. C.; Goehring, A.; Du, J.; Song, X.; Gouaux, E. NMDA Receptor Structures Reveal Subunit Arrangement and Pore Architecture. Nature 2014, 511, 191−197. (8) Amico-Ruvio, S. A.; Popescu, G. K. Stationary Gating of GluN1/ GluN2B Receptors in Intact Membrane Patches. Biophys. J. 2010, 98, 1160−1169. (9) Karakas, E.; Furukawa, H. Crystal Structure of a Heterotetrameric NMDA Receptor Ion Channel. Science (Washington, DC, U. S.) 2014, 344, 992−997. (10) Zhang, W.; Howe, J. R.; Popescu, G. K. Distinct Gating Modes Determine the Biphasic Relaxation of NMDA Receptor Currents. Nat. Neurosci. 2008, 11, 1373−1375. (11) Mayer, M. L. Emerging Models of Glutamate Receptor Ion Channel Structure and Function. Structure 2011, 19, 1370−1380. (12) Lee, C.-H.; Gouaux, E. Amino Terminal Domains of the NMDA Receptor Are Organized as Local Heterodimers. PLoS One 2011, 6, e19180. (13) Jin, R.; Singh, S. K.; Gu, S.; Furukawa, H.; Sobolevsky, A. I.; Zhou, J.; Jin, Y.; Gouaux, E. Crystal Structure and Association

4. CONCLUSION In summary, combining two imaging microscopy approaches, photobleaching step counting method and single particle tracking analysis, allowed us to detect unique organized pattern of NMDA receptor ion channel in the cell membrane. First we have detected photobleaching steps for all the trajectories observed for NMDA receptor ion channels in the live cells; the observed counting of steps ranges from 1 to 16, indicating that NMDA receptors are colocalized or form a cluster within the diffraction limited area. We then performed single particle tracking analysis to see the surface mobility of NMDA receptors and measured diffusion coefficients for all the trajectories. The results show a broad range of the diffusion coefficient from 19 × 10−4 μm2/s to 17 × 10−2 μm2/s. Correlating with the photobleaching step analysis, we observed that the fluorescent spots that have lower number of bleaching steps have higher diffusion coefficient and vice versa. These studies clearly indicate that NMDA receptors are located as heterogeneous clusters on the membrane. Our findings for the clustering effect of NMDA receptor ion channel on the live cell F

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DOI: 10.1021/acs.jpcc.8b00262 J. Phys. Chem. C XXXX, XXX, XXX−XXX