Eur. J. Biochem. 267, 5879±5890 (2000) q FEBS 2000

On the kinetics of voltage formation in purple membranes ofHalobacterium salinarium

Richard W. Hendler, Lel A. Drachev, Salil Bose and Manoj K. Joshi*

Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA

The kinetics of the bacteriorhodopsin photocycle, measured by voltage changes in a closed membrane system

using the direct electrometrical method (DEM) of Drachev, L.A., Jasaitus, A.A., Kaulen, A.D., Kondrashin, A.A.,

Liberman, E.A., Nemecek, I.B., Ostroumov, S.A., Semenov, Yu, A. & Skulachev, V.P. (1974) Nature 249, 321±324

are sixfold slower than the kinetics obtained in optical studies with suspensions of purple membrane patches. In

this study, we have investigated the reasons for this discrepancy. In the presence of the uncouplers carbonyl

cyanide m-chlorophenylhydrazone or valinomycin, the rates in the DEM system are similar to the rates in

suspensions of purple membrane. Two alternative explanations for the effects of uncouplers were evaluated:

(a) the `back-pressure' of the DmÄ H1 slows the kinetic steps leading to its formation, and (b) the apparent

difference between the two systems is due to slow major electrogenic events that produce little or no change in

optical absorbance. In the latter case, the uncouplers would decrease the RC time constant for membrane

capacitance leading to a quicker discharge of voltage and concomitant decrease in photocycle turnover time. The

experimental results show that the primary cause for the slower kinetics of voltage changes in the DEM system is

thermodynamic back-pressure as described by Westerhoff, H.V. & Dancshazy, Z. (1984) Trends Biochem. Sci.

9, 112±117.

Keywords: photocurrents; electrogenicity; energy-transduction; back-pressure; electrometric measurements.

The phenomenon of regulation of the kinetics of energy-transducing pathways in closed vesicular systems, by thebuildup of an electrochemical potential across the limitingmembrane, known as `back-pressure' [1], has been firmlyestablished in a variety of systems. One of the earliest examplesis that of respiratory control [2], but many other examples existas for example photocycle kinetics for bacteriorhodopsin inliposomes [3], cell envelopes [4], and whole cells [5].

Previous studies [6], using the same current clamp methodsas we use here, have shown similar kinetics for voltagetransitions as found by optical transitions in the closed vesiclescited above, and as we report here using the direct electricalmeasurement (DEM) system.

In the current paper, which utilizes the uncouplers carbonylcyanide m-chlorophenylhydrazone (CCCP) and valinomycin,we demonstrate that the kinetics of voltage generation in purplemembranes (PM) from Halobacterium salinarium are stronglyaffected by DmÄ H1. An earlier observation that indicated thepossible influence of DmÄ H1 on voltage formation and dissipa-tion was that of Kleinschmidt and Hess [7]. However, theconclusion, based on experiments utilizing uncouplers, that

DmÄ H1 can directly influence the kinetics of voltage transitionsof photocycle intermediates through back-pressure effects is notreadily deducible because of a possible alternative explanationbased on the RC time constant of the system. In this paper, wedescribe how effects resulting from by changes to RC timeconstants can be separated from those due to back-pressure. We findthat back-pressure is the predominant factor in the slow kinetics ofvoltage generation and dissipation that we have measured.

E X P E R I M E N T A L P R O C E D U R E S

Direct electrical measurements

The apparatus used for the DEM [8] and the use of anonbiological membrane as a support for the biologicalmembrane under study [9] have been previously described. Inour work, we tried both a collodion film (0.2 m thick) and aporous Teflon film, impregnated with phospholipids dissolvedin decane as the supporting membrane. The Teflon film wasprepared from plumber's polytetrafluoroethylene thread sealTeflon tape [10]. Our best results were obtained with tapemanufactured in Malaysia, which is readily available inhardware stores. The tape was heated to 90±100 8C, thenheld by clamps and stretched to about 20 times its width. Wefound no significant difference in the results using the twodifferent supports and therefore most experiments reported herewere performed with the Teflon tape, which was easier to workwith. There were no significant differences in the observedkinetic responses using different phospholipids (cardiolipin,plant lecithin, asolectin, native PM lipids) or different hydro-carbon solvents (hexane, decane, hexadecane). Maximumamplitudes of the electrical response were obtained with plantlecithin (Avanti Polar Lipids, USA) in decane. We haveestablished that decane, at a similar concentration to that used

Correspondence to R. Hendler, Laboratory of Cell Biology, National Heart,

Lung and Blood Institute, National Institutes of Health, Bethesda,

MD 20892, USA.

Abbreviations: DmÄ H1, DC, electrochemical potential for protons,

membrane potential; CCCP, carbonyl cyanide m-chlorophenylhydrazone;

FCCP, p-(trifluoromethoxy)-phenylhydrazone; DEM, direct electrical

measurement; PM, purple membrane; BLM, black lipid membrane;

Mf, the fast M intermediate of the bacteriorhodopsin photocycle; Ms,

slow M intermediate of the bacteriorhodopsin photocycle; Mss, slower M

intermediate than Ms.

*Present address: Unilever Research Center, 64 Main Road, Whitefield,

Bangolore 560 066, India.

(Received 25 April 2000, revised 3 July 2000, accepted 4 July 2000)

in the DEM experiments, does not slow the bacteriorhodopsinphotocycle in PM suspensions, measured optically (notshown). Varying the concentration of this solution from 5 to70 mg´mL21 in the DEM system did not influence the kineticsof the electrical responses. However, the most stable andlong-lived membranes were obtained at the highest lipidconcentrations. In most of the preparations, we used aconcentration of 25 mg´mL21. Our highest amplitude responseswere obtained with a 2-week-old solution stored at 15 8C. Thepreparation formed a colorless gel, which was liquified beforeuse by heating to 70 8C.

The resistance of the membrane support was in the range of0.5±1.5 GV, which is 0.6±1.8 � 108 V´cm22, assuming thatthe area of the membrane is 0.12 cm2. The capacitance ofthe membrane measured during the discharge phase was0.7±1.0 � 1028 F or about 6±8 � 1028 F´cm22. If theresistance of the membrane was changed after addition ofuncouplers or aging, the capacitance remained constant. PMwas added to one compartment of the apparatus at aconcentration of 100 mg bacteriorhodopsin per mL, in100 mm NaCl containing 20 mm Mes at pH 6.0. Flash-inducedelectrical responses were registered with an A/D converter aftermagnification using an operational amplifier with a timeresponse of 10 ms (AD 549, Analog Devices, USA). The datawere analyzed in matlab for a sum of exponentials model,using our own nonlinear least squares regression programsbased on Marquardt±Levenberg procedures. The native pre-paration (i.e. absence of uncoupler) was usually fitted witheight exponentials consisting of five electrogenic and threedecay phases.

Because the supporting Teflon membrane consists of twoareas, one covered by the attached PM, and one free, thevoltage generated by the photocycle is divided across the tworesistances that are present. The measured voltage signal isactually the difference between that generated by the bacterio-rhodopsin and the voltage drop across the covered portions ofthe Teflon membrane. The magnitude of generated potential isrelated to the magnitude of the measured voltage by a factorwhich is the sum of the resistances of both the free and coveredportions of the Teflon divided by the resistance of only theuncovered portion. It is clear that this ratio must be greater than1. Although the exact value is not known, in one study whereDEM system was used in conjunction with the electrochromicshift of the carotenoid absorption spectrum, a value of < 2.4was reported for this ratio factor [11]. Therefore, the actualmembrane potential would be approximately 2±2.5 times themeasured voltage.

Correction for influence of RC constant on the amplitudes ofthe electrical responses

As shown in the equation presented below, the influence of RCon the amplitude of the transition for component i of thephotocycle is expressed in the term, RC/(RC 2 ti). To removeany possible influence of RC on the amplitude response of aphotocycle intermediate, the fitted amplitude of the experi-mental data is multiplied by the reciprocal of the term [i.e.(RC 2 ti)/RC]. In the case of experimental data, the largest andquantitatively most significant value of RC constant was used.

Other details

To follow the kinetics of spectral responses, a bacteriorhodopsin/liposomal suspension was prepared by sonication according toprocedures of Quintanilha [12]. An asolectin suspension(100 mg´mL21) in buffer (20 mm Mes, 100 mm NaCl, pH 6)

was sonicated for 15±20 min until a clear preparation wasobtained. PM was added to the sonicated suspension to aconcentration of 1 mg´mL21 and then the suspension wassonicated for an additional 10 min

Optical measurements with whole cells of H. salinariumwere performed as recently described [5].

R E S U LT S

Time profiles for voltage generation and dissipation

Figure 1A shows the development and dissipation of voltagesduring single turnovers of bacteriorhodopsin in the presence ofdifferent concentrations of CCCP. CCCP appears to acceleratethe kinetics of the bacteriorhodopsin photocycle while decreas-ing the magnitude of the total signal and altering the shape ofthe voltage/time profiles. Figure 1C shows simulated databased on an electric circuit analogy of the PM system(described below), in which the RC time constant of membranecapacitance has been progressively decreased. The family ofcurves obtained in these simulations also shows an apparentacceleration of the photocycle and a progressive decrease in themagnitude of the total signal.

Simulation of data based on an electric model of the purplemembrane

An electric circuit analogy of voltage development in PMcontains three components; a current generator (the bacterio-rhodopsin photocycle), and both an electrical resistance andcapacitance (supplied by the membrane). When the RC con-stant is high (i.e. relative to the time constants of the photo-cycle), as in the absence of uncoupler, the photocycle currentinitially charges the capacitor, leading to a build up ofmembrane potential. Subsequently, the capacitor dischargesthrough the resistor leading to a dissipation of the potential.Uncoupler lowers membrane resistance and therefore the RCtime constant, leading to a more rapid dissipation of thevoltage. This will both lower the magnitude of the maximumsignal and accelerate the time for a single photocycle turnover.The equation which describes the influence of RC on thevoltage/time profile in a single turnover is,

V i�t� � I0ti

C

RC

ti 2 RC

� ��e2t/ti 2 e2t/RC�

where ti is the time constant for an isolated step in thephotocycle, I0 is the amount of current produced at zero time, Ris membrane resistance, and C is membrane capacitance. Asmentioned above, there are two interesting limits indicated bythis equation. When RC q ti, Vi � (Iti /C)(1 2 e2t/ti), volt-age increases with time constant ti. On the other hand, whenRC p ti, Vi � IR*(e2t/ti), voltage decreases with time con-stant t. This is the case, especially for the slow components,when a high concentration of uncoupler is present.

To simulate the five electrogenic steps that were isolated bycurve-fitting to the experimental data, five of the above basicequations, using each of the experimentally determined tvalues, were summed into a single expression. Each initialI0ti /C term was then adjusted to reflect the relative amount ofcurrent provided by each exponential process. These valueswere obtained by averaging the relative amplitudes provided byeach exponential in a number of control (i.e. no uncoupler)experiments. The combined five-exponential expression wassolved using different values for the RC constant. The startingvalue for the RC time constant was taken from computerfittings to the decay phase of actual experimental data obtained

5880 R. W Hendler et al. (Eur. J. Biochem. 267) q FEBS 2000

in the absence of uncoupler. In order to express the various RCvalues in terms of equivalent CCCP concentrations, theapproach described below was used.

The peak voltage attained in all cases is a balance betweenthe charge and discharge processes. If decreases in RC constantare responsible for the changes observed in the experimentaldata, we can use the times of peak signal occurrence as ameasure for equivalent CCCP concentrations. Figure 2 shows

the times of peak voltage signal as a function of CCCPconcentration observed under experimental conditions. Equi-valent CCCP concentrations for different RC values wereobtained using the relationship of peak times and CCCPconcentrations shown in the figure.

Isolation of electrogenic and decay processes

Curve-fittings to a sum of exponentials model were performed,as recently described [13], using asymptotic error analysis toinsure against errors of ill-conditioning and over-determination.

Fig. 1. Effects of CCCP and changes in the RC time constant on kinetics and magnitude of voltage formation during a single turnover of the

bacteriorhodopsin photocycle. The ordinate shows measured voltages across the combined Teflon/PM interface. Membrane potentials across the biological

membrane are from 2 to 2.5 times the value of the measured voltages as explained in the text. (A) Raw data including both the rise and fall of the voltage

signals measured at different CCCP concentrations. The curves, in the order of decreasing maxima, represent CCCP concentrations of 0, 1, 5, 10, 20, and

40 mm, respectively. (B) Isolated electrogenic portions of total curves shown in (A). The curves shown in (A) were fitted to sums of exponentials. The fitted

exponentials and amplitudes for the rising (i.e. electrogenic) components, normalized to the same maximum value, are shown proceeding leftwards from the

arrow in the order of 0, 1, 5, 10, 20, and 40 mm CCCP concentrations, respectively. (C) Simulated data based on the electric circuit analogy to PM discussed

in the text. The curves in the order of decreasing maxima were obtained using RC values of 500, 300, 100, 50, 10, and 1 ms, respectively. These values

correspond to CCCP concentrations of 0.37, 0.73, 2, 4.3, 15.4, and 42 mm, respectively, determined as described in the text. (D) Isolated electrogenic portions

of curves shown in (C), determined by curve-fitting, as described for (B) above. The order of curves proceeding leftwards from the arrow represent CCCP

concentrations of 4.3, 2, , 1, 15.4, and 42 mm, respectively.

Fig. 2. Plot of the times for maximum voltage signal as a function of

CCCP concentration. These data were taken from the curves shown in

Fig. 1A. The experimental data are shown as points. The solid line is an

empirical fit to the data. The numbers on the right-hand y-axis denote total

signal in mV.

Fig. 3. Sum of squares obtained by fitting raw data for voltage vs, time

to six to nine exponentials.

q FEBS 2000 Voltage formation in H. salinarium (Eur. J. Biochem. 267) 5881

Eight exponentials were required to fit the data obtained fromexperiments with PM in the absence of uncoupler (Fig. 3).Figure 4A shows raw experimental data as points and the fittedcurve as a solid line. The residuals of the fit are shown in thebottom of the figure. Table 1 shows the fitted time constants forboth the charge and discharge processes in the presence ofvarying concentrations of CCCP. The five electrogenic timeconstants obtained from the control case (no uncoupler) wereused for the simulations based on the electric circuit analogy.For the starting value of the RC time constant in the simulateddata, a single average value of 500 ms was taken. Using thesame time base as was used in the control experimental caseplus the five time constants just described, data points weregenerated from the combined electrical equations describedabove. These data points are shown in Fig. 4B plus the solidline fitted to the simulated data, as well as the residuals of the

fit. Table 2 shows the time constants that were obtained in thefitting procedures. It is immediately clear that the five charge-building time constants used in the simulation (i.e. 0.045, 0.7,3.3, 11, and 33 ms) are always recovered, regardless of thevalue of RC time constant tested. However, when the value ofthe RC time constant becomes lower than that of a charge-building time constant, there is a reversal of amplitudes, so thatthe amplitude of the components with time constants largerthan that of the RC time constant become negative and theamplitude of the RC time constant becomes positive. This isillustrated in Table 2 by the use of bold type. For example,when the RC time constant is reduced from 50 to 10 ms, theelectrogenic components with time constants of 11 and 33 ms,which had positive amplitudes when they were lower than the50 ms RC time constant, exhibited negative amplitudes whenthe RC time constant was reduced to 10 ms. The italic type for

Fig. 4. Raw and fitted voltage data. (A) Points show experimental data obtained in the absence of uncoupler. The line through the points is the result of an

eight-exponential fit. The lower line, at 0 mV, shows the residuals of the fit. (B) Points show data obtained by simulation using the five electrogenic kinetic

constants obtained from the experimental data shown in (A), an average for the decay constants obtained from the data shown in (A), and an RC value of

500 ms.

Table 1. Influence of CCCP on kinetics of voltage changes in bacteriorhodopsin photocycle. Fitted time constants are shown in ms. The relative

amplitude of each electrogenic component, expressed in percent, is shown in Fig. 9.

Concentration of CCCP (m)

0 1 5 10 20 40

Electrogenic transitions

1 0.045 0.023 0.023 0023 0.020 0.022

2 0.700 0.185 0.110 ± ± ±

3 3.3 1.5 0.820 0.190 0.154 0.146

4 11 7.2 3.5 1.7 1.2 1.2

5 33 22 14 8.6 4.6 3.6

Decay transitions

6 330 191 55 30 9.1 18

7 950 389 100 90 19

8 2500 676

5882 R. W Hendler et al. (Eur. J. Biochem. 267) q FEBS 2000

the 10 ms component shows where the amplitude of the RCtime constant changed from negative to positive. These samekinds of changes occurred when the RC time constant (i.e. at1 ms) became lower than the charge time constant of 3.3 ms.

Comparison of the effects of uncoupler concentration andchanges in RC time constant on the rising phase of voltagechange

The position of the peak signal as well as its magnitude couldbe influenced by changes in either the charge or dischargeterms. The apparent acceleration seen in these composite curves

shown in Fig. 1A,C could be caused by changes in any or all ofthese processes. More specific information on the effects ofuncoupler and changes in the RC constant can be obtained byisolating the fitted charge terms from the discharge terms. Thisis shown in Fig. 1B for the experimental data and 1D for thesimulated data. In the experimental case (Fig. 1B) there was asteady acceleration of the electrogenic process accompanied byan accentuation of the relative importance of the earliest kineticsteps. In other words, CCCP was more effective in uncouplingthe voltage contributions of the slower components than thoseof the faster. The corresponding curves obtained from theelectric simulations do not show these trends (Fig. 1D). There

Fig. 5. Effect of CCCP on individual time constants and magnitude of voltage generation during a single turnover of the bacteriorhodopsin

photocycle. The t values for the three slowest electrogenic events occurring in PM are shown as functions of CCCP concentration. III, II, and I, refer,

respectively, to the slowest, second slowest, and third slowest events as determined by curve-fitting procedures (see text). The magnitude of the total signal in

mV is indicated by the right-hand y-axis and the top curve in the figure. The medium is described under Materials and methods.

Table 2. Influence of RC constant on simulated kinetics of voltage changes in bacteriorhodopsin photocycle. Fitted time constants are shown in ms. The

bold type represent cases where electrogenic components become discharging components when they their time constants become larger than the tested RC

time constant as explained in the text. Similarly, the amplitude of the RC transition changes from negative to positive when its time constant becomes faster

than that of a previously electrogenic component (italic type at RC � 10 ms). The relative amplitude of each electrogenic component, expressed in percent,

are shown in Fig. 11.

Concentration of CCCP (m) [RC (ms)]

0.37 [500] 0.73 [300] 2 [100] 4.3 [50] 15.4 [10] 42 [1]

Electrogenic Transitions

1 0�.045 0�.045 0�.045 0�.045 0�.045 0�.045

2 0�.700 0�.700 0�.700 0�.700 0�.700 0�.700

3 3�.3 3�.3 3�.3 3�.3 3�.3 ±

4 11 11 11 11 10 ±

5 33 33 33 33 ± ±

Decay Transitions

6 500 300 100 50 11

33

1

3.3

11

33

q FEBS 2000 Voltage formation in H. salinarium (Eur. J. Biochem. 267) 5883

is no steady progression of the curves in the direction of fasterdevelopment of electric potential or as clear a case of theorderly increase in relative quantitative importance of theearliest events (cf. Fig. 1B).

The modulation of the kinetics of voltage generation by thephenomenon of back-pressure is expected to be a continuousprocess of decrease in the magnitudes of forward rate constantsas the membrane potential is lowered. Figure 5 shows the timeconstants of the three slowest electrogenic processes (i.e.components 3, 4, and 5 in Table 1) as a function of increasingCCCP concentration. There is an immediate and continuouslowering of all of these time constants from 1 to 40 m CCCPconcentration. Another expected consequence of CCCP addition,the progressive diminution of the total voltage signal is also seen.

The results obtained by decreasing the value of the RC timeconstant were quite different (Fig. 6). The t values of the three

slowest components remained constant until the RC constantfell below that of the slowest electrogenic process (i.e.RC � 10). There were then abrupt displacements in the curvesto make up for the loss of the previously slowest componentsand the change in sign of the RC constant from minus to plus.

The experimental results obtained with the uncouplervalinomycin resembled those obtained with CCCP and differedfrom those obtained in the simulation based on the decrease inRC constant (cf. Figs 7 and 1, Fig. 8 vs. Figs 5 and 6, andTable 2 vs. Tables 1 and 3). In the most uncoupled case (6 mvalinomycin, Table 3), the 1.3 ms component may be the RCconstant, and the dissipative processes at 5 and 20 ms may berelated to the 9.7 and 28 ms components seen in the fullycoupled case, as discussed above.

The changes in shape of the voltage/time profiles caused byCCCP and valinomycin in the raw experimental data tracesshown in panels B of Figs 1 and 7 show greater losses ofelectrogenicity in the slowest electrogenic steps of the photo-cycle. That is, as the uncoupler concentration was raised, therise in voltage generation in the earlier part of the curvesbecame steeper. More detailed information on the effects of theuncouplers on the remaining electrogenicities of the individualsteps of the photocycle is presented in Fig. 9 (CCCP) and 10(valinomycin). These figures each show two panels, A and B. InFig. 9A, the amplitudes of fitted components may be modifiedby the RC time constant. It is possible to remove RC-relatedeffects on the amplitudes using the RC correction proceduredescribed under Experimental procedures. The corrected data,which represents the intrinsic time constants of the photocycleevents uninfluenced by the electrical response time of themembrane, are shown in panels B of the figures. In all of thepanels, the reduced magnitudes of the signals in the presence ofuncoupler were normalized to 100% for each concentration ofuncoupler. The figures then show the influence of uncoupler onthe percent of total signal generated by each isolated step of thephotocycle. In Fig. 9, the five curves shown with solid points inthe concentration range of 0±5 mm use the data shown inTable 1. The heavy numbers, 1±5, identify the transitions fromthe fastest to the slowest transition in sequence. Because, above

Fig. 6. Effect of changes in RC time constant (expressed as equivalent

CCCP concentration in mm) on time constants of the three slowest

photocycle events and total signal in the simulations based on the

electric circuit analogy to PM. The designations for the curves are the

same as described in the legend to Fig. 5.

Fig. 7. Effect of valinomycin on kinetics of voltage formation and total signal during a single turnover of the bacteriorhodopsin photocycle. See

legend to Fig. 1A,B for additional information.

5884 R. W Hendler et al. (Eur. J. Biochem. 267) q FEBS 2000

Fig. 8. Effect of valinomycin on individual time constants and magnitude of voltage generation during a single turnover of the bacteriorhodopsin

photocycle. See legend to Fig. 5 for additional details.

Fig. 9. Effect of CCCP on relative

electrogenicities of individual time-resolved

transitions in bacteriorhodopsin photocycle.

The percentage Total Signal was computed from

the total signal at each CCCP concentration. The

heavy numbers in the left part of the figure refer

to components with increasing time constants

(from the fastest to the slowest) as shown in

Table 1. The lines with solid points required five

electrogenic components in the fittings. The

dotted lines and open points were fit with four

electrogenic components. The lighter, italicized

numbers from 1 to 4 refer to the four component

fits and are listed in order of increasing time

constants. (A) Results of direct fitting of

experimental data. The time constants obtained

here could be influenced by the electrical

response time of the system as expressed by the

RC value. (B) The same data as shown in (A),

but corrected for delays due to the RC constant

as described in the text. These data reflect the

values of the intrinsic time constants of the

components of the photocycle. See text for

further details.

q FEBS 2000 Voltage formation in H. salinarium (Eur. J. Biochem. 267) 5885

Fig. 10. Effect of valinomycin on electrogenicities of individual time-resolved transitions in bacteriorhodopsin photocycle. Refer to legend to Fig. 9 for

further details.

Table 3. Influence of valinomycin on kinetics of voltage changes in bacteriorhodopsin photocycle. Fitted time constants are shown in ms. The relative

amplitude of each electrogenic component, expressed in percent, is shown in Fig. 9.

Concentration of valinomycin (m)

0 0.2 0.4 2 4 6

Electrogenic Transitions

1 0�.029 0�.033 0�.032 0�.025 0�.025 0�.023

2 0�.274 0�.171 0�.171 0�.086 0�.084 ±

3 2�.1 1�.4 1�.3 0�.508 0�.445 0�.057

4 9�.7 6�.6 4�.7 2�.3 2�.0 0�.235

5 28� 20 15 9�.2 6�.7 1�.3

Decay Transitions

6 233 105 53 37 17 5

7 733 394� 207� 149� 40 20

8 2424� 170 49

295

5886 R. W Hendler et al. (Eur. J. Biochem. 267) q FEBS 2000

5 mm only four electrogenic steps could be resolved, a decisionmust be made as to how to continue to trace the electrogenicityof each step at the higher CCCP concentrations. Using theprinciple that increasing concentrations of CCCP will alwayslower a time constant, we can consider the slowest time con-stant at 10 mm CCCP (8.6 ms) as a continuation of the curve forthe slowest time constant at 5 mm CCCP (14 ms). Similarly, thetime constant of 1.7 ms at 10 mm must be a continuation of thecurve for the second slowest time constant. Therefore, the timeconstant at 0.19 ms must be associated with the curve for thethird slowest time constant. This leads to the conclusion that thesecond fastest time constant seen at 5 mm CCCP (0.11 ms) waseither lost or blended with the fastest time constant at 0.02 ms.The same logic was used in the case of valinomycin (Fig. 10).With this approach, we can now compare the uncoupling effectsof CCCP and valinomycin on the electrogenicity of eachseparate transition. First, we will concentrate on panels A,which include possible effects of the RC constant.

In the absence of added uncoupler, the two slowestcomponents are the most electrogenic, followed by the fastestcomponent (Figs 9A and 10A). Components 2 and 3 are theleast electrogenic. As uncoupler is added, the slowest com-ponent becomes relatively more electrogenic at the expense ofthe next slowest component. The relative electrogenicities ofcomponents 1, 2, and 3 do not change by much. A peak in relativeelectrogenicity of component 5 is reached at about 5 mm CCCPand about 2 mm valinomycin. As the uncoupler concentration israised, the relative electrogenicity of component 1 dramaticallyrises. In the case of valinomycin, which appears to be the strongeruncoupler, the curves for the slowest and fastest componentsintersect at about 4.5 mm so that the fastest component becomesthe most electrogenic component. With CCCP the same trendslead to a crossover of the slowest and fastest electrogeniccomponents at about 23 mm concentration.

Additional information is obtained by comparing the datain panels A and B of each figure. If the changes in relative

Fig. 11. Effect of RC time constant (expressed as equivalent CCCP concentration in mm) on electrogenicities of individual time-resolved transitions

in bacteriorhodopsin photocycle as modeled in the electric circuit shown to PM. Refer to the legend of Fig. 9 for additional details.

q FEBS 2000 Voltage formation in H. salinarium (Eur. J. Biochem. 267) 5887

importance of the individual transitions seen as a function ofuncoupler concentration in panel A are due entirely to RCeffects and not to back-pressure, then these changes will beremoved in the RC-corrected data. If, on the other hand, thechanges are primarily due to effects of back-pressure, they willstill be seen in panel B after making the RC correction. As canbe seen in both Figs 9 and 10, the basic pictures in panels A andB are very much the same, indicating that the changes seen inthe early parts of the curves are primarily due to back-pressureand not RC.

The dependability of using this comparison approach forassessing the importance of back-pressure as an influence onthe collected data is illustrated in applying the method to datasynthesized entirely by adjusting RC values, which are free ofback-pressure effects. These data are shown in Fig. 11. First, letus consider the range of data where the RC value was greaterthan the slowest time constant. Referring to Table 2, thisincludes the columns for RC values of 500, 300, 100, and 50,which correspond to CCCP concentrations up to 4.3 mm. In theuncorrected curves (panel A), the two slowest components

(4 and 5) appear to change dramatically in their contribu-tions to the overall electrogenicity. When the influence of RCon the fitted amplitudes is removed by applying the RCcorrection, the true picture of the time constants for the actualcomponents is seen (panel B). There was no change in therelative contribution of each. This is consistent with Table 2,where it is seen that the intrinsic time constants are independentof the RC value. The apparent changes in relative electro-genicities seen in Fig. 11B at higher equivalent CCCPconcentrations, are due to the fact that the total amount ofvoltage on which the percentage contribution is calculated isaltered as the slowest time constants are no longer electrogenicwhen they are higher than the RC constant (Table 2). Thisexercise with synthetic data shows that the true picture for theelectrogenic contributions of the individual components,independent of influences of RC on the responses, is obtainedby applying the RC correction procedure. Therefore, the quitedifferent results seen with the experimental data are consistentwith the interpretation of the major influence of back-pressureon the relative electrogenicities of the individual transitions as afunction of membrane potential.

Experiments with whole cells

The slowness of the photocycle in the DEM system, and theability of uncouplers to accelerate the process are quite similar toeffects seen in optical studies with intact cells [5]. It wastherefore of interest to perform parallel titrations withuncoupler, using intact cells, monitored by optical means, incomparison to the results obtained using the DEM system. Inthe absence of added uncoupler, the single cycle turnover timewas about 80 ms in both systems. The ability of CCCP toaccelerate the photocycle in the intact cell system is shown inFig. 12. The effects of CCCP on the time constants of theisolated transitions are shown in Fig. 13, where a dramaticdecrease in tav is seen. It is also shown that similarly to theeffects observed in the DEM system, the slower M species ismost affected by uncoupler. The effects of CCCP on the relative

Fig. 12. Effect of CCCP on turnover time of bacteriorhodopsin in whole

cells, studied optically at 569 nm. The curves begin from the second time

point at 50 s. The curves from right to left were obtained at 0, 0.5, 2.5, 4.5,

6.5, 8.8, 10.5, 15, 20, 30 mm CCCP, respectively. The values at 0 time are

not shown (i.e. log � ±1).

Fig. 13. Effects of CCCP on tav, and the t values of Ms, Mf, and L

decays as measured optically in whole cells of H. salinarium. tav is the

sum of the mol fraction of Mf times tf and Ms times ts.

Fig. 14. Effects of CCCP on amounts of Ms, Mf, and total M turnover

measured optically in whole cells of H. salinarium. A series of optical

spectra was taken at each of 500 time points for each concentration of

CCCP tested. All data were analyzed by least squares-based singular value

decomposition and isolated difference spectra obtained for each transition

in the photocycle. A polynomial was fitted across the peak centered at

412 nm and the amplitude of this peak is presented in units of 103 A412 in

the figure.

5888 R. W Hendler et al. (Eur. J. Biochem. 267) q FEBS 2000

amounts of both the slow (Ms) and fast (Mf) M intermediatesare shown in Fig. 14. This figure dramatically demonstratesthat the ratio of these two forms of the M intermediate isregulated by DmÄ H1 in addition to the well-known regulationby the intensity of incident light [13]. Higher levels ofDmÄ H1 strongly decrease the mol fraction of Mf.

Direct test of the existence of electrogenic transitions thatare optically silent

In the summary, possibility (b) was based on the idea thatelectrical events far away from the retinal may have occurredwithout affecting the optical properties of the chromophore. Tocheck this possibility, we prepared a simple closed liposomalvesical suspension. These liposomes should be susceptible tothe same back-pressure effects that operate in the closedvesicular system used for the DEM studies. It was found thatboth the DEM and liposomal preparations had the sametransitions (data not shown). In other words, there were noelectrical events observed in the DEM system that were absentin the optically observed system.

D I S C U S S I O N

Although the ability of an electrochemical potential gradient toslow the kinetics of enzymatic reactions leading to its formationis well-established in a variety of closed vesicular systems (seeintroduction), it has not been established that this phenomenonis responsible for the relatively slow kinetics of voltage forma-tion, measured directly in isolated membranes and liposomes.The most common demonstration of the phenomenon, knownas `back-pressure', is the ability of uncouplers, which lowerelectrochemical potential, to accelerate the kinetics of theenzymatic pathways. In the case of the DEM and relatedsystems for measuring voltages and currents, however, thissimple demonstration is insufficient because uncouplers willalso lower the RC time constant and therefore the electricaltime response of the system. An early indication that back-pressure may be operative in the formation and dissipation ofvoltage in PM was that of Kleinschmidt and Hess [7]. Theystudied current flow in PM fixed to a black lipid membrane(BLM). They resolved three kinetic steps and found that theslowest of these (< 10 ms) was accelerated threefold byaddition of < 0.8 mm carbonyl cyanide p-(trifluoromethoxy)-phenylhydrazone (FCCP), at low light intensity. At 2 and 4 mmFCCP, however, the transition became slower. No effects ofuncoupler on the faster transitions at < 500 and 20 s were seen,and the possible influence of uncoupler on the RC constant wasnot considered.

Another system for measuring the kinetics of voltageformation in PM uses oriented, open PM membranes embeddedin polyacrylamide gel contained in a cuvette that allowssimultaneous voltage and optical measurements on the samesample [15,16]. A comparison of the turnover times for thephotocycle in that system [16] with those we report here usingthe DEM system shows that although the photocycle in theirsystem was more uncoupled than that in the DEM system, itwas still partially coupled. For example, the time for maximumvoltage formation in the uncoupled DEM system (i.e. in thepresence of 10 mm CCCP) was 8 ms compared to 82 ms in theabsence of uncoupler. The data of Ludmann et al. [16] showedthat maximum voltage formation required < 40 ms. This corre-sponds to the time for attainment of maximum voltage in thepresence of < 1 mm CCCP in the DEM system. Therefore,although the system using open PM fragments attained the

maximum signal in less time than that in the DEM system, itwas still considerably longer than the time for a single turnoverin an unoriented suspension of open PM fragments. There aresome distinct advantages in the use of these systems, originallydescribed by Keszthelyi and Ormos [17]. Principally, becauseboth the voltage and optical measurements are obtained on thesame sample, one can assign electrogenicity to particular stepsin the photocycle. MuÈller et al. [18], using this system,published simultaneous values for obtained from both opticaland photocurrent measurements on the same sample of orientedPM (their Table 1). In these data, the t values based on opticalmeasurements were the same as normally obtained withcompletely uncoupled suspensions of PM (i.e. 2.5 ms for Mf,5.0 for Ms, and 19 ms for Mss). The t values based on photo-current measurements were not the same (Mf was not seen, Ms

was 4.5 ms, and Mss was 13 ms). Clearly, this preparation wascompletely uncoupled. These data show a marked difference inthe extent of coupling compared to that seen in the DEM approach.

We have demonstrated here that the kinetics of electro-genicity determined with an efficient voltage-forming systemsuch as that of Drachev et al. [8] reflect control exerted by`back-pressure' [1]. Our findings on the time constants in thefully coupled system correspond well with the time constantssummarized by Holz et al. [6] from data published by fourlaboratories (their Table 1). For the slowest of four kineticconstants reported from eight studies from four laboratories.These values vary from 4.5 to 33 ms. From the studies wereport here, back-pressure may provide an explanation for thisrange of values. Although Holz et al. used a differentsupporting membrane (BLM with octocilimine), they used asonicated PM preparation as we have done. From their Fig. 4, itis seen that the time constants obtained in a six exponential fitto their data correspond closely to the constants we find in thestudies reported here. The amplitudes of their two slowestcomponents (7±10 ms and 20±33 ms) were 27 and 33%,respectively, corresponding to the < 30% value we find for ourtwo slowest components (Figs 9 and 10). Also, in agreementwith our findings, Holz et al. did not see an influence of flashintensity on the kinetic parameters of the response.

As demonstrated in the current work, the release of back-pressure both accelerates the response of the slowest com-ponents and increases the relative electrogenicity of the slowestcomponent by as much as 50%. It is reasonable to try toassign the particular steps isolated in the current work toknown steps in proton transfer. Although, a generally acceptedphotocycle sequence has not been established, we can base ourspeculations on one recent model [19]. Three stages of protontransfer in the ms range are envisioned: (a) D96! Schiff base,(b) cytoplasm !D96, and (c) D85 !E204, where the geo-metric distances for these three stages are approximately equal.Possibly, that slowest component of voltage formation isidentified with the O ! bacteriorhodopsin transition (i.e. D85!E24) as discussed in [20]. Stages (a) and (b) correspond toMf and Ms spectral transitions. In PM suspensions (pH 7,23 8C), using optical methods, the t values of these steps are 2and 5 ms, respectively. In whole cells, the optically monitoredtime constants for Mf and Ms are 3 and 30 ms, respectively [5]and in cell envelope vesicles, they are 3 and 23 ms, respectively[4]. Our singular value decomposition-based optical studieswith bacteriorhodopsin liposomes show two major M transi-tions with t values of 25 ms (60% total M) and 7 ms (30% totalM) and two minor transitions with t values of 3 ms and 160 ms(each < 4% of total). In the presence of 50 mm CCCP, onlythree transitions were seen with t values of 3, 8, and 18 ms andrelative amounts of 24, 34, and 42% total M, respectively.

q FEBS 2000 Voltage formation in H. salinarium (Eur. J. Biochem. 267) 5889

As has been demonstrated with other proton pumps, forexample cytochrome oxidase liposomes, any given preparationmay exhibit a different degree of tightness of coupling asmeasured by the respiratory control coefficient. In the currentcontext, this means that the kinetics of electrogenicity in anygiven case may vary along the lines indicated in Figs 9 and10 of this paper. When optical kinetics are determined on aseparate preparation from the one used for voltage measure-ments, the degree of coupling can be quite different in the twocases. This means that the same time-constants determined inparallel experiments for voltage and optical changes cannot beassumed to be reporting identical events. This realization iscrucial in trying to assign electrogenicity to individual tran-sitions determined in parallel optical and voltage-measurementstudies.

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