Transmembrane potential measurement with carbocyanine dye diS-C3-(5): Fast fluorescence decay studies

Journal of Photochemistry

and Photobiology:

B: Biology, 4 (1990)

321 - 328

321

TRANSMEMBRANE POTENTIAL MEASUREMENT WITH CARBOCYANINE DYE diS-C,-(5): FAST FLUORESCENCE DECAY STUDIES M. fifPpt,P. HERMAN, J. PLAgEK and V. HROUDA Institute of Physics, Charles University, 121 16 Prague 2 (Czechoslovakia) (Received January 2, 1989; accepted April 17, 1989)

Keywords. Membrane potential, fluorescence decay, fluorescence lifetime, fluorescent probe, time-resolved fluorescence, carbocyanine dyes, diS-C,-( 5).

Summary The mechanism of carbocyanine dye diS-C,-( 5) fluorescence intensity variations with transmembrane potential changes has been studied using time-resolved fluorescence spectroscopy. Clear evidence is given of the transmembrane-potential-dependent partition of the dye among various sites with different fluorescence lifetimes. It was found that fluorescence decay profiles reflect the transmembrane potential changes.

1. Introduction The carbocyanine dye diS-C,-( 5) has been widely used to monitor transmembrane potential in various cells and organelles whose size precludes the use of microelectrodes [l] (see also refs. 2 - 6 for reviews). The fluorescence response of these dyes was demonstrated to be a result of the potentialdependent partition between the cells and the intracellular medium. With the membrane potential negative inside, the positively charged dye molecules are accumulated in the cell, the intracellular dye content being increased by hyperpolarization and vice versa. The observable changes in fluorescence result primarily from the dye uptake followed by the formation of nonfluorescent aggregates. Furthermore, a component of fluorescence from membrane-bound molecules was observed which has been red shifted by approximately 10 - 20 nm with respect to aqueous solutions of diS-C,-(5) [4,7,8]. This spectral shift is similar to changes of cyanine dyes fluorescence observed on going from water media to organic solvents of low polarity [l, 81.

+Author to whom correspondence should be addressed. loll-1344/90/$3.50

@ Elsevier Sequoia/Printed in The Netherlands

322

To be applicable for the quantitative determination of membrane potentials, the diS-Cs( 5) fluorescence response should be calibrated against a set of definite potentials. In many cells the diffusion potential of K+ ions is used for this purpose, which can be established by valinomycin (an ionophore specific for potassium) in the presence of various gradients of K+ ions. This calibration is often performed by the zero-point method where the concentration of K+ ions in the solution is adjusted so that on addition of valinomycin no fluorescence intensity change occurs [ 7,9 - 111. It has been well documented that in intact cells, particularly in those containing abundant mitochondria, the use of potential-sensing dyes is not free from problems. The most important problem concerns the toxicity of dyes and the contribution of mitochondrial potential to the apparent fluorescence response (see e.g. refs. 6, 11 and 12). Nevertheless, recent flowcytometric studies of the fluorescence of lymphocytes stained with various voltage-sensing dyes suggested again that the fluorescence techniques may be further considered as a perspective tool for the assessment of cell membrane potential [ 13,141. Apparently, flow-cytometry and other laser instrumentations with high sensitivity are likely to prevail in future applications. In this way, the toxic effect of the dyes can be minimized by decreasing the probe concentration in the sample. The detailed knowledge of fluorescence parameters of suitable dyes, in addition to understanding the origin of their changes in the systems under study, are obvious prerequisites for setting optimum experimental conditions. This paper presents an experiment designed to determine the effect of transmembrane potential on the fluorescence decay of stained cells. The fluorescence lifetime was treated as an emission parameter relevant to characterize different binding sites of probe molecules.

2. Materials and methods 2.1. Cells and chemicals Myeloid leukemia cells (ML-1 line) used in these experiments were kindly supplied by Dr. C. Haskovec, Institute of Hematology and Blood Transfusion, Prague. The cells were washed three times (120 g X 10 min) in a buffer containing 137 mM NaCl, 5.4 mM KCl, 1.43 mM CaCl,, 1 mM Na*HPO, , 0.8 mM MgS04, 10 mM N-(2-hydroxyethyl)piperazine-N’-2ethanesulfonic acid (HEPES) and 5 mM glucose (the pH was adjusted to 7.35). Washed cells were resuspended in this buffer and kept at 4 “C. Before measurements were recorded, all samples were adjusted to a density of (2 - 6) X lo6 cells ml-’ and shaken at 37 “C for 10 min. The cyanine dye 3,3’dipropylthiodicarbocyanine iodide (diS-C,-(5)) was supplied by Molecular Probes. A stock solution of the dye was prepared at 10e3 M in ethanol and stored in the dark at 4 “C. The cell suspensions were stained with the stock solution of diS-C,-( 5) to a final concentration of 2 I.~M and the system was left to equilibrate for 5 min at 37 “C.

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Absorption spectra of diS-C,-(5) solutions were measured Specord M40 spectrophotometer (Carl Zeiss, Jena, F.R.G.)

with

a

2.2. Fluorescence measurements Time-resolved fluorescence measurements were carried out with a timecorrelated single-photon-counting instrument, equipped with an RCA 8852 photomultiplier. The light source was a synchronously pumped cavity dumped dye laser system from Spectra Physics (model 171-19 Ar+ laser and the time model 375 dye laser) tuned to X,,, = 632 nm. After deconvolution, resolution of the system is better than 100 ps. The fluorescence was collected at right angles to the excitation beam direction. A dichroic polarizer was placed at the magic angle into the emission to avoid the effect of molecular reorientation on the fluorescence time decay profiles. The emission wavelength was selected by a monochromator (Model H20, Jobin-Yvon, France) at 645 nm with a spectral slit width of 8 nm. The stained cell suspension was gently stirred during the experiment to prevent cell sedimentation. Data analysis was performed by means of the Marquardt non-linear least-squares deconvolution procedure which permits us to fix any lifetime rf in the model fluorescence decay function f( t ):

f(t) = f

ai w(---t/Ti)

forN=

l-3

i=l

The quality of fit was judged their autocorrelation function.

by the x2 test, distribution

of residuals

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2.3. Calibration of the dye fluorescence response against the diffusion potential of K+ ions For the fluorescence vs. membrane potential calibration the method according to Rink et al. [lo] was adopted. The value of the diffusion potential of K+ ions, which was established in cells after valinomycin clamping, was calculated using the equation E, = (RTIF) ~n([K+lo~t/[K+lin), where R, T and F have their conventional meanings and [K+lout and [ K+]in are the concentrations of extracellular and intracellular K+ ions, respectively. The intracellular potassium concentration in ML-1 cells was approximated by the value of 145 mM, which was found earlier in a very similar leukemic cell line HL-60 [ 151.

3. Results and discussion 3.1. Effect of dye binding to cells The diS-C,-(5) fluorescence decay was studied in samples of various cell-to-dye concentrations. The cell concentration ranged from the pure buffer to 12 X lo6 cells ml-‘. The total dye concentration in all suspensions was 2 PM.

324

=P(--V~~)

(2)

Since certain parts of the dye remain free in the extracellular solution [l], we may obviously fix one component of this decay law to the value r, = 0.9 ns, which is typical of the diS-C,-(5) fluorescence in the buffer. Then the double-exponential analysis of our experimental data yielded r2 = 2.00 + 0.18 ns. Differences between lifetimes in samples containing various cell concentrations did not exceed the limit of experimental error (< 0.18 ns). The lifetime of the cell-associated fluorescence r2 is higher than the fluorescence lifetime in aqueous solutions. This effect obviously reflects a

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Fig. 1. Dependence of diS-Ca-(5) fluorescence decay profiles on the ML-1 cell concentration: curve P, diS-CJ-(5) fluorescence in pure buffer; curves 1, 3, 6 and 12, cell suspensions containing 1, 3, 6 and 12 x lo6 cells per 1 ml, respectively. Measured at the dye concentration of 2 PM.

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binding of the dye molecules in a less polar environment (presumably any type of cellular membrane) where the fluorescence quenching is less efficient. Considering the general rule relating lifetimes to fluorescence quantum yield (see for example ref. 16) we conclude that the quantum yield of diS-C3-( 5) fluorescence in the membrane environment is enhanced by about twofold for the dye solution in buffer. This interpretation is also in good agreement with the increase in the diS-C3-( 5) fluorescence lifetime which was observed on going from aqueous solution to less polar ethanol. In ethanol the fluorescence decay is monoexponential with a lifetime of 1.5 + 0.1 ns. In contrast to the stable lifetime TV,fluorescence increased considerably with increasing cell-todye concentration. It was revealed by changes in the ratio of pre-exponential factors a2 (longer component related to the cellassociated fluorescence) to a, (shorter component related to the fluorescence from the buffer) (Fig. 2). Hence, the analysis of diS-C,-(5) fluorescence decay is capable of quantitative estimation of relative amounts of fluorescing monomeric dye in different environments, i.e. bound to cell structures and free in aqueous solution. 3.2. Dependence of diS-C3-(5) fluorescence decay on transmembmne potential To search for a relationship between membrane potential and the decay of the diS-C,-(5) fluorescence we have investigated its response to variations of the diffusion potential of K+ ions in ML-1 cells. In this experiment the KC1 concentration in the cell medium was varied from 5.4 to 105.4 mM, i.e. the diffusion potential of K+ ions established after valinomycin clamping varied from -87 to -8.5 mV. For this range of membrane potentials the best fit of the fluorescence decay was again the sum of two

0

2

4

6 CELL

0

10

CONCENTRATION

Fig. 2. Dependence of the preexponential Measured at the dye concentration of 2 /JM.

factor

ratio

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az/al

1

on the cell concentration.

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exponentials (eqn. (2)) with r1 = 0.9 ns and 72 = 2.0 ns. The component lifetimes were independent of the membrane potential value. The observed differences were lower than the experimental error. Changes in the membrane potential are reflected by the ratio of preexponential factors ~*/a,, which estimates the proportion of cell-associated fluorescence intensity relative to the fluorescence intensity in cell-free medium. Moreover, a common loss of total fluorescence intensity was found in hyperpolarized samples. As shown in Fig. 3, the a,/a, ratio increased with the absolute value of the diffusion potential of K+ ions, e.g. on the condition stimulating dye uptake by cells. A good correlation is observed. The decay of cell-associated fluorescence alone cannot be used for direct measurements of membrane potential. The fact that r2, the lifetime of cell-associated fluorescence, remained ,constant during the valinomycin calibration shows clearly that changes in membrane potential do not considerably influence the environment of fluorescing cell-bound molecules. Furthermore, the nanosecond decay measurements provided additional evidence of the main mechanism of fluorescence intensity changes which follow the uptake of diS-C,-(5) by cells. To summarize: (i) not more than two distinct components contribute to the decay of diS-C,-(5) fluorescence in cell suspensions, one of them being the fluorescence from diS-C,-(5) solution in extracellular medium, and (ii) the quantum yield of the cellassociated fluorescence is increased by about twofold relative to that in the extracellular medium. Since the quantum yield of the detectable cellassociated fluorescence is higher than the fluorescence quantum yield in K+ -67.5 v

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Fig. 3. Dependence of the pre-exponential factor ratio az/al on the extracellular KC1 concentration in the presence of valinomycin. The arrow shows the ~/a, value for cells in the standard buffer ([KC11 = 5.4 mM) before the addition of valinomycin. Measured with 3.5 X 10” cells ml-’ at the dye concentration of 2 /AM.

321

extracellular medium, we must attribute the loss of total fluorescence intensity in hyperpolarized samples to the formation of non-fluorescent dye aggregates within cells, as suggested already by Sims et al. [11. 4. Conclusions Using the method of time-resolved fluorescence it was established that diS-C3-( 5) emission in cell suspensions comprises two distinct components: fluorescence of the dye solution in extracellular medium and cell-associated fluorescence. The analysis of the fluorescence decay in cell suspensions confirmed that the major changes in the diS-C,-(5) fluorescence intensity are due to the formation of non-fluorescent aggregates in cells. Marked dependence of diS-C,-(5) fluorescence decay on the diffusion potential of K+ ions was found, which is promising from the point of view of a direct application of the time-resolved technique to measure the transmembrane potential. However, further investigations should be performed to determine the limitations of this approach. Acknowledgment The authors wish to thank Dr. C. Haskovec from the Institute of Hematology and Blood Transfusion, Prague, for supplying the ML-1 cell samples. References 1 P. J. Sims, A. S. Waggoner, C. H. Wang and J. F. Hoffman, Studies on the mechanism by which cyanine dyes measure membrane potential in red blood cells and phosphatidylcholine vesicles, Biochemistry, 13 (1974) 3315 - 3330. 2 A. S. Waggoner, Optical probes of membrane potential, J. Membr. Biol., 27 (1976) 317 - 334. 3 A. S. Waggoner, Dye indicators of membrane potential, Ann. Rev. Biophys. Bioeng., 8 (1979) 47 - 68. 4 C. L. Bashford and J. C. Smith, The use of optical probes to monitor membrane potential, Methods Enzymol., 55 (1979) 569 - 566. 5 C. L. Bashford, The measurement of membrane potential using optical indicators, Biosci. Rep., I (1981) 183 - 196. 6 T. M. Chused, H. A. Wilson and R. Y. Tsien, Probes for use in the study of leukocyte physiology by flow cytometry. In D. Lansing Taylor, A. S. Waggoner, F. Lanni, R. F. Murphy and R. S. Birge (eds.), Application of Fluorescence in Biomedical Sciences, Alan R. Liss, New York, 1986, pp. 531 - 544. 7 S. B. Hladky and T. J. Rink, Potential difference and the distribution of ions across the human red blood cell membrane. A study of the mechanism by which the fluorescent cation diS-Ca-(5) reports membrane potential, J. Physiol., 263 (1976) 287 - 319. 8 R. J. Tsien and S. B. Hladky, A qualitative resolution of the spectra of a membrane potential indicator diSC!s-(5) bound to cell components and to red blood cells, J. Membr. Biol., 38 (1978) 73 - 97.

328 9 J. F. Hoffman and P. C. Larris, Determination of membrane potentials in human and amphiuma red blood cells by means of a fluorescent probe, J. Physiol., 239 (1974) 519 - 552. 10 T. J. Rink, C. Montecucco, T. R. Hesketh and R. J. Tsien, Lymphocyte membrane potential asssessed with fluorescent probes, Biochim. Biophys. Acta, 595 (1980) 15 - 30. 11 R. M. Johnstone, P. C. Larris and A. A. Eddy, The use of fluorescent dyes to measure membrane potential: A critique, J. Cell. Physiol., 112 (1982) 298 - 301. 12 T. C. Smith, The use of fluorescent dyes to measure membrane potentials: A response, J. Cell. Physiol., 112 (1982) 302 - 305. 13 H. A. Wilson and T. M. Chused, Lymphocyte membrane potential and Ca2+ sensitive potassium channels described by oxonol dye fluorescence measurements, J. Cell. Physiol., 125 (1985) 72 - 81. 14 H. A. Wilson, B. E. Selingmann and T. M. Chused, Voltage sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components, J. Cell. Physiol., 125 (1985) 61 - 71. 15 J. J. Gargus, E. A. Adelberg and C. W. Slayman, Rapid changes in bidirectional K+ fluxes preceding DMSO-induced granulocytic differentiation of HL-60 human leukemic cells, J. Cell. Physiol., 120 (1984) 83 - 90. 16 C. Parker, Photoluminescenbe ofSolutions, Elsevier, Amsterdam, 1968.