Single Cell Titration-Type Assay for Plasma Membrane Cholesterol

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Single Cell Titration-Type Assay for Plasma Membrane Cholesterol Chemical Potential Meiling Zhang, Linyu Li, Danjun Fang, Thomas J. Kelley, and James D. Burgess Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00736 • Publication Date (Web): 16 Apr 2018 Downloaded from http://pubs.acs.org on April 16, 2018

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Analytical Chemistry

Single Cell Titration-Type Assay for Plasma Membrane Cholesterol Chemical Potential

Meiling Zhang1, Linyu Li1, Danjun Fang1*, Thomas J. Kelley3, James D. Burgess2*

1

School of Pharmacy and Collaborative Innovation Center for Cardiovascular Disease Translational

Medicine, Nanjing Medical University, Nanjing, Jiangsu, China, 211126； 2

Department of Medical Laboratory, Imaging, and Radiologic Sciences, College of Allied Health

Sciences, Augusta University and Augusta University Health System, Augusta, GA, USA 30912； 3

Departments of Pediatrics, Case Western Reserve University, Cleveland, OH, USA, 44106

Corresponding authors: Phone: 86-25-86868467 (D. F); 706-721-7626 (J. B) Email: [email protected] (D.F); [email protected] (J.B)

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ABSTRACT. In this paper, a titration-type assay is described that determines the minimum concentration of cholesterol in solution that is required to drive net influx of cholesterol to the plasma membrane and thus increase the cholesterol concentration. The increase in cholesterol in the plasma membrane is detected by cholesterol diffusion at the site of contact by a cholesterol oxidase-modified microelectrode. In the presented thermodynamic model, the minimum solution phase cholesterol concentration that drives influx to the plasma membrane is a close approximation of the true solutionphase equilibrium concentration of cholesterol produced from cellular cholesterol efflux and as such it is a quantitative measure of the chemical potential of cholesterol in the cellular plasma membrane. This assay provides a measure of cholesterol chemical potential in the living cellular plasma membrane through reference to a solution concentration which avoids invoking classic kinetic theory to relate a rate to a specific thermodynamic activity and which avoids uncertainty associated with mass transfer phenomena.


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Introduction. Historically it is well documented that cholesterol is a fundamental constituent of the mammalian cell plasma membrane (PM) where it influences physical properties such as membrane rigidity and permittivity.1 It is now also clear that the PM contains two states of cholesterol, an unbound state with high thermodynamic activity and a stabilized phospholipid associated state.2 Collectively, data from many groups indicate that the two PM cholesterol states are not in rapid dynamic equilibrium with one another despite proximity and the thermal energy of physiological temperature.3 The PM is in fact heterogeneous in its lateral composition as to accommodate the vast array of proteins and their functions, and it is certainly plausible that the two cholesterol states are segregated in lateral domains.2 The structural roles of cholesterol in the PM and as a physiological effector continue to be revealed. Relatively recently, for example, it has been established that the higher potential cholesterol state, termed active cholesterol, is directly involved in signaling cascades that regulate biosynthesis of cholesterol as well as its storage through ester-linkage to phospholipids.2 The Steck group clearly documents a threshold effect for PM cholesterol on trafficking machinery inside the cell. Their classic “J-curve” plots show multiple triggered consequences as the PM cholesterol concentration is increased above a certain level.4 Also, the McConnell group first proposes PM cholesterol as a thermodynamic switch in controlling cellular cholesterol homeostasis by invoking cholesterol-phospholipid binding stoichiometry as a mechanism producing a nonlinear increase in cholesterol activity with increasing cholesterol molar ratio in the PM.5 The ability to accurately measure active cholesterol in the PM is key to the study of diseases such as cystic fibrosis (CF).6 Active cholesterol concentration in the PM has been shown to be elevated approximately two-fold in both cultured CF cell models and in primary tissue from CF animal models.7,8 The increased PM active cholesterol is, at least in part, a consequence of an increased rate of de novo cholesterol synthesis for the CF disease state.9 This relationship is key because it shows that active cholesterol concentration correlates directly with intracellular signaling events that previous studies have shown to be related to CF inflammatory signaling.10-12 These relationships document PM active cholesterol concentration as a critically important biomarker for assessing new anti-inflammatory therapies and new therapies targeting CFTR, the mutated chloride ion channel that causes CF.13 Given this clear application in CF for the evaluation of PM active cholesterol and the importance of active cholesterol in overall understanding cellular cholesterol homeostasis for wildtype cells, measuring chemical potential of active cholesterol has merit in that it provides a quantitative value proportional to the active cholesterol concentration. Desorption of cholesterol from the PM and aqueous diffusion to acceptors in solution and/or bound to the PM is understood to be operant in the movement of cholesterol from cells as the initial step in reverse cholesterol transport.14

Work by the Rothblat group has firmly demonstrated that an

equilibrium condition exists between PM cholesterol and aqueous cholesterol by showing bi-



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directional flux of radio-labeled cholesterol between solution and the PM.3 Furthermore, these radiolabeled studies of cholesterol efflux from cells showed that a fraction of PM cholesterol was rapidly released to solution phase acceptors while the majority of PM cholesterol effluxed much more slowly.3 These fast- and slow-efflux pools of PM cholesterol are now referred to as active cholesterol and phospholipid associated cholesterol, respectively. There are a number of technical challenges in the evaluation of cell PM cholesterol. The main one that is of concern here is the characterization of cholesterol efflux from the cell PM at short time to ensure that the data are reflective of cholesterol originating from only in the PM, not internal cholesterol.

To achieve rapid efflux and thus capture data corresponding to PM cholesterol, the

efficient solution phase acceptor of cholesterol, 2-hydroxypropyl-cyclodextrin (CD), has been widely employed.3 The trade-off in probing initial efflux is that the active cholesterol pool is significantly depleted upon removal of enough cholesterol for analytical quantification. It is noted that work by the Rothblat group elegantly prevented depletion of the active cholesterol pool by having un-labeled cholesterol present in solution such that influx of un-labelled cholesterol occurred during efflux of the radiolabeled cholesterol. 3 Still, precise resolution of the average rate of efflux occurring in the initial seconds is not possible and pre-incubation of the cells with radiolabeled cholesterol presented uncertainty regarding preservation of the cell’s native state. In terms of structural identification of PM cholesterol, it is likely that transbilayer movement of cholesterol between the PM leaflets is rapid.15 Despite these limitations in analytical technology, the size of the active cholesterol pool has been characterized for various cells and under conditions known to affect PM chemistry and intracellular cholesterol trafficking.3,4 However, the size of the active cholesterol pool is not the same quantity as the thermodynamic activity (or chemical potential) which is an intensive property. Because active cholesterol is a cellular sensing trigger or switch for cholesterol homeostasis machinery inside the cell, the ability to characterize the chemical potential of the PM active cholesterol pool is perhaps more important from a mechanistic standpoint than is the amount of cholesterol making up this pool (i.e., pool size). As discussed below, this research group reports a method for quantifying the chemical potential exhibited by natively occurring active cholesterol in the PM of a single cell thus providing greater insight into the physical chemistry involved in the PM cholesterol switch/trigger.

The

chemical potential measurement reported here may be regarded as an analytical handle for the active cholesterol concentration in the cell PM.

A direct numerical estimate of active cholesterol

concentration would also require knowledge of the activity coefficients in the two states as well as the volume active cholesterol occupies in the PM. This research group has previously developed an electrochemical method for measuring the rate of active cholesterol diffusion in and from the PM at the point of contact between the microelectrode and the PM.16-18 Steady state microelectrode responses show that very little depletion of active cholesterol occurs on the time scale of the measurement (typically 5 s). The method provides a qualitative


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characterization of the active cholesterol concentration in the PM.

A number of experiments

comparing cell types and treatments vs. controls indicate altered diffusion rates that correlate with expected increases and decreases in PM active cholesterol concentration. While the microelectrode approach allows evaluation of the native cell state and avoids depletion of PM cholesterol, limitations are that kinetic theory is required to infer a thermodynamic activity from the diffusion rate and the measurements are thus not absolute in the strictest sense. An additional challenge is that routine comparisons of a particular cell or tissue type to a control group requires the use of the same electrode because of variance in performance between individual microelectrodes. The variation between different electrodes in response to a particular cholesterol flux is likely a consequence of dissimilarity in the platinum electrode surface roughness and actual surface area which is expected to affect the amount of enzyme covalently immobilized on the electrode surface. The ratio of the diffusional response at a particular microelectrode allows a quantitative comparison of the two samples where an increase or decrease in PM cholesterol activity is reported for a sample vs. a control.

Quality control and calibration for microelectrode production is

challenging which precludes the quantitative designation for individual microelectrode measurements of active cholesterol diffusion rate. In comparing data collected between different electrodes, results were reported for electrodes that exhibited a similar response at the control sample with the aim of showing the most repeatable increase or decrease at the sample being compared to the control. Heterogeneity in PM cholesterol activity between samples of the same type would cause an additional error in this reporting of a quantitative difference in PM cholesterol activity between control and target samples. Explicitly, to obtain a quantitative measure of the chemical potential of the native active cholesterol pool in the cell PM, the minimum solution cholesterol concentration needed to drive net influx of cholesterol to the cell PM is determined (Figure 1). Microelectrode active cholesterol diffusion analysis is used to qualitatively indicate the onset of the increase in active cholesterol diffusion rate that is associated with an increase in active cholesterol concentration in the PM. Thus, the experiment determines the minimum concentration of cholesterol outside the cell that results in the unidirection influx rate exceeding the unidirectional efflux rate and this concentration of cholesterol is taken as an estimate of the true equilibrium concentration of solution cholesterol.

Experimental Section. Preparation of saturated cholesterol solution. 1mg cholesterol was dissolved in 1 mL chloroform, dried under nitrogen flow, and mixed with 100 ml 100 ml saline (460 mM NaCl, 10 mM KCl, 22 mM MgCl2, 33 mM MgSO4, 10 mM CaCl2, 10 mM glucose, 10 mM 3-(N-Morpholino) propanesulfonic acid, pH 7.4–7.5). The mixture was stirred at room temperature for 3 hours and filtrated through the filter (pore size 0.22 µm) to get a clean solution saturated with free cholesterol.



Modification of cholesterol oxidase at microelectrode surface.

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100 µm diameter platinum

18

microelectrodes were prepared as described before . The electrodes were immersed into 5 mM hexane solution of 11-mercaptoundecanoic acid (Aldrich Chem. Co.) for 4 h to produce a submonolayer coverage of 11-mercaptoundecanoic acid on the electrode surface.

Then, the sub-

monolayer modified platinum surface was exposed to a 10 mM phosphate buffer saline (PBS, pH 7.4) containing 2 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC) (Sigma Chem. Co.) and 5mM N-hydroxysulfosuccinimide (NHS) (Fisher) for 30 min for conversion of the carboxylic acid end groups of the submonolayer to an NHS ester. Finally, this activated surface was immersed in a 1 mg/ml solution of recombinant cholesterol oxidase (Wako Chemicals USA, Inc., ca. 33.0 units/mg) for 12 h for covalent attachment of the cholesterol oxidase to the electrode surface. To investigate the relation of this active PM cholesterol chemical potential with the electrode performance, three microelectrodes are modified with cholesterol oxidase for 6, 9 and 12 h, respectively to load different amounts. Microelectrode determination of cholesterol in plasma membrane at single neuron. The microelectrode measurement of cholesterol diffusion was conducted using a two-electrode cell and a voltammeter-amperometer (Chem-Clamp, Dagan corp.). The three-pole Bessel filter of the ChemClamp was set to 100 Hz. The signal was further processed using a noise-rejecting voltmeter (model 7310 DSP, Signal Recovery Inc.) to digitally filter 60-Hz noise and to provide a dc voltage output with a time constant of 100 ms. An Ag/AgCl (1 M KCl) reference electrode was used for all experiments, and the applied potential was 820 mV versus NHE for all experiments. Prior to the electrochemical experiments, the electrode was positioned 500 µm from the surface of a single neuron in the buccal ganglion of Aplysia or oocyte (Fish egg) to acquire baseline data. The electrode was repositioned so that it was in physical contact with the cell PM surface for cholesterol diffusion analysis. The titration at the neurons was performed in 10 mM PBS (pH 7.4) with different concentration of cholesterol.

Results and discussions. Estimation of aqueous cholesterol concentration. Because the determined quantity in the titrationtype assay is a solution cholesterol concentration, it is important to note the low solubility of cholesterol in buffer containing no cholesterol acceptor and the concerns in the reproducibility in making the stock saturated solution. For the titration assay, the saturated cholesterol stock solution is judiciously prepared by agitation of cholesterol crystals in buffer for three hours and filtrated at a pore size of 0.2 µm. Analysis of the stock solution by repeated extraction into organic solvent, evaporative concentrating, and LC-MS analysis estimates the saturated cholesterol solution to be ca. 750 nM and cholesterol micelles are likely present.19 It is noted that the actual cholesterol monomer concentration may be much lower than 750 nM in the titration assay as the cholesterol micelles probably contribute to the LC-MS analysis.


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The low solubility of cholesterol in buffer may prevent application of this assay in traditional micro-well experiments where changes in cellular chemistry is the readout for increased PM cholesterol. In the micro-well experiments for study of consequences in cell biology triggered by cholesterol influx, the walls are coated with adherent cells so that the total amount of cell PM exposed to solution is large relative to the volume of solution containing the cholesterol to be delivered to the cells. Therefore, without the use of a cholesterol carrier/solubilizer such as CD, the capacity of the solution in the micro-well to deliver cholesterol may be too low to maintain a concentration that drives influx to the PM and thus trigger biochemical effects.

The premise of the titration assay is

demonstrated below without use of CD to deliver cholesterol to the cell PM as a means of showing the uncomplicatedness of the thermodynamic model. However, treatment of the cells with CD solution originally containing no cholesterol is used, subsequently, to partially deplete PM cholesterol as a means of demonstrating a dependence of the titration end-point on the initial active cholesterol chemical potential in the PM. Uptake of cholesterol into cellular membrane from saturated cholesterol solution. To demonstrate the uptake of cholesterol from saturated cholesterol solution (containing no CD, vide supra) and to show that the microelectrode method registers the influx by indicating an increased active cholesterol diffusion rate, a single neuron in the buccal ganglion of Aplysia is analyzed in 10 mM PBS to record the native PM active cholesterol diffusion rate. Aplysia neurons have proven to be an ideal sample for microelectrode cholesterol analysis in that tens of sequential measurements (contact and withdrawal cycles) can be conducted at the same electrode without any apparent loss of response. Also, simple PBS buffer is chosen to guarantee the performance and lifetime of the cholesterol sensor. Heterogeneity in the lateral distribution of cholesterol in the PM, as for the possible existence of cholesterol rich lipid rafts, is expected to have dimensions of nanometers.20 The 100 µm microelectrode would thus sample an average PM composition of raft and non-raft membrane. Subsequently, the neuron is exposed to saturated cholesterol solution for half an hour and the measured diffusion rate is compared to the rate for the native cell state. A larger diffusional response is observed compared to that measured prior to exposure of the cell to saturated cholesterol solution (Figure 2A). Figure 2B shows the ratio (iafter / ibefore) of the microelectrode responses before and after cholesterol uptake for seven single cell experiments. All cells studied show the increase (average ratio: 2.43 ± 0.49). We speculate that the difference in response ratios between different cells reflects heterogeneity of the cells and possibly differences in apparent Michaelis-Menton behavior for individual electrodes.16 It is noted that saturated cholesterol solution is too low (e.g., 750 nM) to produce a measurable current at the electrode (Figure 2A, trace c). Therefore, the possibility that the increased current response at treated cells is from direct reaction of solution cholesterol at the electrode is ruled out. These two experimental results confirm that the increase in the current response is from increased efflux resulting from elevated membrane cholesterol.



Titration at single neurons to determine chemical potential of membrane cholesterol. Figure 3 shows a series of microelectrode experiments for PM active cholesterol diffusion rates where the cell is incubated in buffer (see label: Cell) and buffer containing progressively increasing cholesterol concentration (50%, 75%, 87.5%, 90%, 97.5, and 99% of cholesterol saturated buffer). Almost the same background currents before each contact with the neuron suggest the negligible contribution of aqueous cholesterol on the electrode response. Upon contact of the electrode with the cellular membrane, steady-state current responses are collected. When the electrode is withdrawn from the cell, a noise spike is observed followed by decay of the signal to the background value. For the measurements below 87.5% saturated cholesterol, some cholesterol efflux has occurred and continues to occur from the PM. However, efflux is very slow to dissolve in buffer without a cholesterol acceptor and also the solubility of cholesterol in buffer is very low (estimated at ca. 750 nM) limiting the extent of efflux. The PM cholesterol lost during incubation is negligible and/or is replenished to the PM from internal cellular stores. It is noted that PM cholesterol is tightly regulated. After incubation in 90% and higher solution cholesterol, the cell exhibits an increased cholesterol diffusion rate at the microelectrode indicating increased active PM cholesterol thermodynamic activity (concentration). Taking the 90% saturated cholesterol solution as an estimate of the true equilibration state, which is obviously a slight over estimate, the PM active cholesterol chemical potential of the native cell can be assigned and quantitated, noting that the chemical potential of the two states is equal at equilibrium. Figure 4A shows the steady state current from four microelectrode experiments conducted at three different cells plotted vs. increasing cholesterol concentration. Different amounts of cholesterol oxidase were modified on three microelectrodes leading to the variation in the current response and the electrode sensitivity. While the absolute microelectrode currents are different for each experiment, the three cells exhibit the same active PM cholesterol chemical potential as all show the increase only after incubation with 90% cholesterol. Control experiments conducted for microelectrodes containing no immobilized cholesterol oxidase show no increase in signal with exposure of the cell to increasing cholesterol concentration (e.g., Figure 4A control). The slight decrease in Figure 4A might be caused by the partial blocking of electrode surface after multiple contacts with cellular membrane. The data show that 87.5% cholesterol does not drive net influx to the PM while 90% does drive net influx. Therefore, it is known that the true equilibrium solution concentration that would result from the native cell’s active cholesterol chemical potential is between 87.5% and 90% of the saturated concentration. Initial data for titration analysis of a different cell type, the fish egg oocyte (the other good cell line for cholesterol study), have been collected and the data suggest an end-point around 80% cholesterol solution. These data demonstrate proof of concept for quantitative analysis of the PM active cholesterol chemical potential where questions arising from kinetic theory, mass transfer,


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labeled cholesterol, use of cholesterol acceptors and the current variation between different electrodes are avoided. An alternate interpretation of the results that cannot be conclusively ruled out is that a, yet to be discovered, cholesterol receptor is activated for internalization of cholesterol at higher cholesterol concentrations. Further conjecture is that a particular size/shape of a cholesterol micelle that arises only above 87.5% of saturation activates a receptor for internalization of cholesterol. The notion that the current increase observed at 90% is a quantitative measure of PM cholesterol chemical potential and thus is a handle for active cholesterol concentration is supported by titration data on cells that have been partially depleted of active cholesterol (Figure 4B) by exposure to CD solution initially containing no cholesterol. Three independent experiments at three cells using three different microelectrodes show that 87.5% cholesterol solution is sufficient to drive influx of cholesterol to the PM of cholesterol depleted cells. Again, at least 90% cholesterol solution is required for delivery of cholesterol to native cells. These data show that the titration end-point is dependent on the concentration of active cholesterol initially present in the cell PM.

Conclusion. In this paper, this titration-type assay provides direct evidence for cellular recognition of cholesterol in aqueous solution and the data are consistent with a thermodynamic model for PM cholesterol uptake and release. Overall, the data support the notion that PM active cholesterol serves as the root sensory switch in cellular cholesterol homeostasis. Clearly, the titration assay offers quantitative information regarding the dependence of PM cholesterol on the concentration of cholesterol in surrounding solution.

Nevertheless, the mechanism of cholesterol recognition is

unknown. Despite the arguments from literature for direct influx by aqueous diffusion, selective cholesterol recognition by a receptor cannot be ruled out. In this case, the switch to increased active cholesterol in the PM could be coupled to intracellular trafficking of cholesterol and/or phospholipids with respect to the PM. No matter the mechanism of recognition, it is proposed that this titration-type assay provides a quantitative handle for the chemical potential of active cholesterol in the cell PM. Work is underway to evaluate the titration assay at cells that have biochemically altered cell PM active cholesterol.

Acknowledgments We thank Jiaqige Sheng for the estimation of aqueous cholesterol concentration and the National Natural Science Foundation of China (Nos. 2157050130) for support. The neuronal cells were a kind gift from Hillel J. Chiel.



REFERENCES 1. Simons, K.; Ikonen, E. Science 2000, 290, 1721-1726. 2. Lange, Y.; Steck, T.J. Chem Phys Lipids. 2016, 199, 74-93. 3. Haynes, M. P.; Phillips, M. C.; Rothblat, G. H. Biochemistry 2000, 39, 4508-4517. 4. Lange, Y.; Ye, J.; Steck, T. L. Proc. Natl. Acad. Sci. USA. 2004, 101, 11664-11667. 5. Radhakrishnan, A.; Anderson, T.G.; McConnell, H.M. Proc. Natl. Acad. Sci. USA. 2000, 97, 12422–12427. 6. Cianciola, N.L.; Carlin, C.R.; Kelley, T.J. Arch Biochem Biophys. 2011, 515, 54-63. 7. White, N. M.; Jiang, D.C.; Burgess, J. D.; Bederman, I. R.; Previs, S. F.; Kelley, T. J. Am J Physiol Lung Cell Mol Physiol 2007, 292, L476-486. 8. Jiang, D.C,; Fang, D.J.; Kelley, T. J.; Burgess, J. D. Anal.Chem. 2008, 80, 1235-1239. 9. Fang, D.J.; West, R. H.; Manson, M. E.; Ruddy, J.; Jiang, D.C.; Previs, S. F.; Sonawane, N. D.; Burgess, J. D.; Kelley, T. J. Respir res 2010,11, 61 10.Kreiselmeier, N. E.;Kraynack, N. C.; Corey, D. A.; Kelley, T. J. Am J Physiol Lung Cell Mol Physiol 2003, 285, L1286-1295. 11. Lee, J. Y., Elmer, H. L., Ross, K. R., and Kelley, T. J. Am J Physiol Lung Cell Mol Physiol 2004, 31, 234-240. 12. Rymut, S. M.; Kampman, C. M.; Corey, D. A.; Endres, T.; Cotton, C. U.; Kelley, T. J. Am J Physiol Lung Cell Mol Physiol 2016, 311, L317-327. 13. White, M.M.; Geraghty, P.; Hayes, E.; Cox, S.; Leitch, W.; Alfawaz, B.; Lavelle, G.M.; McElvaney, O.J.; Flannery, R.; Keenan, J.; Meleady, P.; Henry, M.; Clynes, M.; Gunaratnam, C.; McElvaney, N.G.; Reeves, E.P. EBioMedicine 2017, Sep;23,173-184. 14. Phillips, M. C. J. Biol. Chem, 2014, 289, 24020 -24029. 15. Steck, T. L.; Ye, J.; Lange, Y. Biophys J., 2002, 83, 2118-2125. 16. Li, Li.; Lu, B.Y.; Jiang, D.C.; Shin, M.; Kelley, T.J.; Burgess. J.D. Curr. Opin. Electrochem. 2017, 2, 82-87. 17. Devadoss, A.; Burgess, J. D. J. Am. Chem. Soc., 2004, 126, 10214–10215. 18. Jiang, D.C.; Devadoss, A.; Palencsar, M. S.; Fang, D.J.; White, N. M.; Kelley, T. J.;. Smith, J. D.; Burgess, J. D. J. Am. Chem. Soc. 2007, 129, 11352-11353. 19. Haberland, M. E.; Reynolds, J. A. Proc. Natl. Acad. Sci. USA, 1973, 70, 2313-2316. 20. Simons, K.; Ikonen, E. Nature. 1997, 387, 569–72.


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Figures and Captions. Figure 1. Scheme of single cell titration-type assay at the cells treated with saturated cholesterol solution. Figure 2. (A) Current responses observed at a single neuron cell before (trace a) and after (trace b) treatment of saturated cholesterol solution; trace c shows the current by the flow-injection of saturated cholesterol solution to oxidase modified microelectrode; the arrow indicates the time for contact between microelectrode and the cell surface or the exposure of microelectrode to saturated cholesterol; (B) the ratios of current responses (after/before) recorded at seven single cells treated with saturated cholesterol solution. Figure 3. The current responses observed at a single neuron cell for exposure to increasing cholesterol concentrations; up and down arrows indicate the time of contact and withdrawal, respectively, between the microelectrode and the cell. Increased microelectrode response occurs at 90% and greater cholesterol concentration. Figure 4. Titration end-points for (A) native neuron cells and (B) cells that have been partially depleted of PM cholesterol. The neurons titrated with increasing concentration of buffered cholesterol solution; three experiments at cholesterol oxidase modified electrodes (1-3) and one experiment at a platinum microelectrode containing no immobilized cholesterol oxidase (control).



Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



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Single Cell Titration-Type Assay for Plasma Membrane Cholesterol

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