Eur. J . Biochem. 89, 169-179 (1978)

Potassium Uniport and ATP Synthesis in Halobacterium halobium Gottfricd WAGNER, Rainer HARTMANN, and Dieter OESTERHELT lnstitut fur Botanik und Phannazeutische Biologie der Universitat Erlangen, and Institut fur Biochemie der Universitat Wiirzburg (Received February 7/March 28, 1978)

Light-driven potassium ion uptake in Hul(hzcterium halohiurn is mediated by bacteriorhodopsin. This uptake is charge-balanced by sodium ions and not by proton release. Light-induced shifts in concentrations of divalent cations were found to be negligible. The transient changes in extracellular pH (alkaline overshoot) can be understood by the concomitant processes of ATP synthesis, proton/ sodium exchange and potassium uptake. The driving force of potassium ion uptake is the membrane potential, no ATP-dependent potassium transport process is found. Fluorescence measurements indicate a high permeability of the membrane to potassium ions compared to sodium ions. Therefore the potassium ion diffusion potential contributes to the membrane potential (about 30 mV/decade) and thereby influences the ATP level. Sudden enhancement of the diffusion potential by the potassium ionophore monactin leads to the expected transient increase in cellular ATP level. Due to the large size (up to 100-fold) of the potassium ion gradient and its high capacity (intracellular concentration up to 3 M) the potassium ion gradient can well serve the cell as a long term storage form of energy. The potential difference across biological membranes (A$,) was initially regarded as a diffusion potential ( A $ d ) carried by the ionic gradients of K' and to a lesser extent by other ions like Na' and Cl-. Goldman [l] and Hodgkin and Katz [2] deduced the relationship between A $d and the ionic concentrations from the Nernst equation: A$d

R.T F

= -___

of electrogenic pumps are expressed by the following equation [4] :

where Zp is the current density I generated by the electrogenic pump and g , is the membrane conductivity. The total membrane potential A$,,, then is the sum of the diffusion potential and the electrogenic potential. A$,

Ul0 and bli are concentrations of ion j in the external solution and the cytoplasm of a cell, respectively, and Pj is the permeability of the membrane to ion j. Nevertheless, many experiments with animal and plant cells clearly demonstrated large values of A$,,, which were difficult to explain in terms of ion diffusion processes alone [3]. In addition, in energy-coupling membranes of mitochondria, chloroplasts and bacteria, it was shown that large changes of A$,,, are induced by charge translocation (protons) during electron flow through the respective redox chains. Such membrane potentials ( A $ e ) created by the action

+A$e.

(3) As a consequence, bioenergetic processes which can be driven by membrane potential, such as ATP synthesis, will tend to equilibrate with the electric potential difference carried by ionic gradients and electrogenic pumps. In addition ionic gradients will be influenced by the action of electrogenic pumps. Halobacterium halobium provides a good example of these bioenergetic relationships. Conversion of light energy into chemical energy in this organism is based on the action of the electrogenic proton pump bacteriorhodopsin [5 - 71. The proton ejection from the cell creates an increase in membrane potential and an increase of pH difference across the cell mem=A$d

170

Potassium Uniport and ATP Synthesis in Halobacterium hulobium

brane [8 - 111. Both components can be combined to describe the change in the electrochemical potential of the proton (LIP"+) also called proton motive force ( d p )in the chemiosmotic theory of Peter Mitchell [12]. Besides ATP-synthesis which is induced by an increase in total proton motive force (i.e. A $ plus dpH), a change in the membrane potential component of dp also induces ionic shifts of Na' and K' . Since Halobacteria live in concentrated salt solution these ionic shifts can reach the molar range [13-161. In this paper we report on the light-induced uptake of potassium, its relation to photophosphorylation and sodium release and the possible function of the high intracellular potassium pool as a long term storage form of light energy absorbed by bacteriorhodopsin.

silicone oil. Pellets from two tubes were resuspended together in 25 ml of double distilled water which resulted in cell lysis and homogeneous dissolution of the pellets. The signals from the atomic absorption spectrophotometer were calibrated by internal standardization and by using suitable reference solutions and blanks [21,22]. The intracellular concentration of ions (L3i; mmol/ kg cell water) was corrected for the extracellular carry over (blO; mmol/l) by the following equation [23]: Li,, - ljlpellet - (fraction extracellular space x u],,) . (4) 1 - fraction extracellular space

MATERIALS AND METHODS

Ion-Selective Electrode Measurements ( H + and K+)

Strain and Culture Conditions

Bacterial suspensions (about 10 ml, absorbance at 578 nm between 3.0 and 4.0) were magnetically stirred and continuously flushed with nitrogen in a sealed and thermostated glass vessel (t = 25 "C); for irradiation light from a 250-W quartz iodine lamp filtered through an OG 515 cut off filter (Schott, Mainz) was used. Concentration changes of protons and potassium ions in the suspending medium were detected by an Ingold combination glass electrode and a Philips liquid membrane potassium electrode IS 560-K. A calomel electrode (Knick, Berlin) placed in a cell filled with basal salt and connected by an agar bridge to the measuring cell served as a reference for the potassium electrode. The electrodes were shielded against light and signals were amplified by two electrometers (pH-Meter Knick, Berlin, and PHM 64 Radiometer Kopenhagen) connected to a two-channel chart recorder (Servogor Metrawatt, Numberg). Concentration changes were calibrated with standard solutions of HCl and KCl. Concentration changes of potassium less than 0.1 mmol/l in the presence of 4290 mmol/l sodium could well be detected due to the high Na" rejection of theK' electrode used (selectivityconstant 2.6 x and the low potassium concentration in the suspension medium (5 mmol/l); a freshly assembled electrode produced the half-maximal signal after 10 to 15 s. Net ionic fluxes (mol x m-2 x s-') were calculated from the known membrane area of the cell suspensions and the concentration changes per time.

Halobacterium halobium mutant RIMI deficient in gas vacuoles and bacterioruberine was cultured and harvested as described before [15,17,18]. Cells were resuspended in basal salt of varying composition (see legends of the figures) and were used immediately or starved for 24-48 h in the dark at room temperature. The cell density was adjusted to an absorbance of 4.0 (578 nm, d = 1 cm; Eppendorf model 1101M), corresponding to a protein concentration of 2 mg/ml or a cell volume of 5.45 pl/ml [8,15]. Since the average halobacterial cell dimensions are that of a cylinder 5 pm long and 0.5 pm wide [19], 1 ml of the suspension contains 5.55 x lo9 cells with a total membrane area of 457 cm'; out of the 5.45 pl internal cell volume 3.2 yl are water [20]. Atomic Absorption Spectroscopy Concentrations of Na', K+, Ca2' and Mg2" were determined employing an atomic absorption spectrophotometer (Beckman 1248). Cell suspensions (350 pl, absorbance of 4.0 at 578 nm) were layered on 50 pl of silicone oil (CR 500, Wacker, Miinchen) containing 20 % bromobenzene (v/v) in transparent polypropylene tubes (Greiner, Niirtingen). The tubes were placed in a Beckman Microfuge 152 with a transparent plastic cover and irradiated with light from a 250-W quartz iodine lamp or a 1200-W HMI lamp (Spectra 1200, Pasel KG) filtered through a water-cooled OG 515 filter (Schott) ; a heat-transparent mirror reflected the light onto the samples. At the end of the irradiation period the cells were spun down; after 5 min more than 99.5% of the cells had passed the oil layer (50% after 15 s) [8]. The tubes were cut across the silicon oil layer and the parts containing the cell pellet shaken in 0.5 ml of diethyl ether for removal of

1-

The amount of medium dragged through the silicon layer during centrifugation was found to equal to the internal water content of the cells (for determination see [20]).

Determination of A T P , ADP and Inorganic Phosphate Aliquots of the cell suspensions (100 pl) were rapidly injected into 5 ml of ice-cooled phosphate buffer (10 mmol/l, pH 7.4) containing 0.1 mmol/l

G. Wagner, R. Hartmann, and D. Oesterhelt

171 0 c

EDTA where the cells lysed immediately. An aliquot was used for determination of ATP by the bioluminescence method as described earlier [15]. Another aliquot was used for ADP determination. ADP was converted to ATP by reaction with phosphoenolpyruvate and pyruvate kinase (both from Boehringer, Mannheim) and ATP determined as before. ADP was then taken as the difference between the two measurements. Inorganic phosphate was determined according to Baginsky et al. [24].

-al Fluorescence Measurements

1

Y

All fluorescence measurements were carried out on a Perkin Elmer MPF 3 spectrofluorimeter using a thermostated fluorimetric cuvette (cross section 5 x 10 mm). The perspex lid of the cuvette had inlets for nitrogen and for addition of ionic standard solutions. An electrically driven stirrer guaranteed complete mixing within a few seconds. The cyanine dye used in these experiments was 3,3'-dipropylthiodicarbocyanine iodide, a generous gift from Dr Alan Waggoner of Amherst College (Massachussetts). The excitation wavelength was set at 622 nm (band width 4 nm), and the fluorescence emission was measured at 670 nm (band width 26 nm). Diluted basal salt had to be used as suspension medium for reasons explained under 'Results'. After addition of the dye (0.5 pl of a 1 mM ethanolic solution) and valinomycin (2.5 pl of a 1 mM ethanolic solution) or equivalent amounts of ethanol instead of valinomycin, the bacterial suspension (1 ml) in the fluorimetric cuvette was flushed with nitrogen for 15 min in the dark. During this time a constant fluorescence emission signal developed. The increase in fluorescence upon addition of known amounts of KCl (or other ions) was recorded and expressed as a percentage change in fluorescence. A F

= 100

[(FafterlFbefore) -

11.

(5)

In control samples containing no cells, a fluorescence decrease instead of an increase occurred upon changing the KCl concentration. This decrease was maximally 1- 2 % when going from 2 mmol/l to 100 mmol/l. Therefore no corrections were made. RESULTS Changes in Proton, Sodium and Potassium Ion Concentrations When cells of H . halobium are exposed to light, a net uptake of K'. by the cells is observed. This can be measured either by atomic absorption spectroscopy (Fig. 1) or by a K+-selective electrode (Fig. 2). The action spectrum of this potassium uptake closely follows the absorption spectrum of bacteriorhodopsin documenting that halobacterial potassium transport 1

B toooO

10

20

30

40

50

60

Time (rnin)

Fig. 1. Halobuclerial potassium uptake and sodium release in light us meusured by atomic absorption spectroscopy. Cells were resuspended in basal salt of 4270 mmol NaC1/1, 27 mmol KCI/l, 81 mmol MgS04 . 7 HzO/l and 20 mmol Tris-maleate/l pH 7.0. Samples of 350 ~1 were prepared and treated as described under Materials and Methods. The irradiance at the sample surface was 250 W/m2. The vertical bars indicate the standard error of the mean (S.E.M.)

in light is coupled to the action of this light-driven proton pump (Fig.3). Starvation of the cells in the dark leads to a leakage of K' which lasts for more than 24 h [25]. Due to this loss the net uptake of K i in light following a dark period depends on the amount of K' released during starvation and ranges between 600 mmol/kg cell water after a 2-h period of starvation (Fig. 1) and more than 2000 mmol/kg cell water after a 48-h period of starvation (data not shown). The maximal cellular concentration of K' in light was found to be about 3000 mmol/kg cell water (Fig. 3 ) . The potassium uptake is charge balanced mostly by a release of N a + and not by a release of H +(Table 1). The observed shifts in H', Mg", Ca2+and anions are small [26]. Therefore the overall process consists of a n Na " /Kf exchange rather than a net uptake of a potassium salt. The time course of the changes in K + , Na' and H' concentrations are, however, more difficult to interpret. In spite of the fact that bacteriorhodopsin electrogenically pumps protons into the medium, there is a transient net uptake of H + observed after illumination (Fig. 2, Table 2) [25]. At 50 W/m2, this 'alkaline overshoot' starts after a 45-s delay and lasts for several minutes due to the low irradiance applied (Fig. 2, onset of illumination). Upon illumination K + uptake also starts after a delay but then increases progressively with increasing irradiance (Fig. 2 and 4). Since these ion fluxes are by far too large to be accounted for by an electrogenic process, both the transient net uptake of H' and the net uptake of K4., require a charge balancing process which is very likely N a + extrusion (see Fig. 1 and Table 2 last column). Net fluxes of Na' are not as easily detected as are H i or K + fluxes: the high Nai concentration in the

Potassium Uniport and ATP Synthesis in Halobucterium halobium

172

I

Dark

50

100

250

500

I

1000

Dark

Irradiance ( W/rn2) Fig. 2. Relation betcwn thr cellular concentration of A T P , the uptake of K' and the extrusion of H' as a.function of irradiance. Cells were resuspended in basal salt of 4290 mmol NaCI/I, 5 mmol KC1/1, 81 mmol MgSOL 7 H20/1 and 10 mmol Tris-maleate/l pH 7.0; the temperature was 25 'C Potassium and proton transport were measured simultaneously by ion-selective electrodes, ATP was determined from 10O-pl samples collected during the experiment. Light (250 W) was filtered through a cut-off filter (OG 515 Schott); irradiance WBS adjusted by neutral density filters (Schotl, Mainz). The sample volume at the time of electrode calibration was 10 ml at a cell density giving an absorbance at 578 nm of 3.25; this is equivalent to a bacterial membrane area of 0.37 mz (see Materials and Methods). From this value and the data shown in the graph, net ionic fluxes per membrane area and time can be calculated

__

Table 2. Cationic netJluxes at the onset ofillurnination (50 W / m 2 ) Concentration changes of K' and H + were measured by electrodes, changes in Na' concentration by atomic absorption spectroscopy. + and - indicate uptake and release of ions, respectively

TVl 2 . 0 1

I

/Pi

Time interval

K+

H'

Na

+

~

Fig. 3. Action spectrum of potassium uptake. The potassium uptake

min

nmol x m-2 x s-'

0- 1 1- 2 2- 3 3- 4 4- 5 5- 8 8-10

+ 1.25 + 2.75 2.75 + 2.75 + 2.75

+

+ 0.88 + 6.5 + 6.5 + 3.25 + 1.5 -

- 0.5 - 6.15 - 0.8 - 1.3

+ 2.7 - 2.1

- 4.45

(0) was determined by a K'-selective electrode and is given as potassium flux per membrane area and time (nmol x m-' x s - l ) at

photon flows of 100 nmol/s over the wavelength range tested. Monochromatic light was selected by interference filters (Anders, Diendorf) with a band width of 5 nm. For comparison, the absorption spectrum (0) of bacteriorhodopsin is also given, taken from [15]

Table 1. Changes in intracellular eutionic concentrations during illumination for 60 min (250 Wlm') For experimental details, see legend to F1g.l and Materials and Methods. + and - indicate increase and decrease, respectively, of ionic concentration. The extrusion of H + was measured with a glass electrode. Mean values of at least five independent determinations are shown f S.E.M.

K'

H+

Na'

CaZ

+

Mg"

mmol/kg cell water

+ 637 k 7

- 28.9

3.6

- 534 &

5

+ 23.7 & 7.1 + 18.3 rt 5.8

salt medium prevents measurable concentration and hence, the successful use of an changes of "a'],, ion selective electrode. Therefore, net fluxes of Na' were calculated from determination of intracellular Na'. by atomic absorption spectroscopy (see Material and Methods). The results are compiled in Table 2 together with the net fluxes of H' and K'. The Na+ data are scattered, but nevertheless show that Na' release accompanies the transient proton uptake, whereas K '-uptake lags behind. Driving Force for Potassium Ion Uptake

There are several possible sources of energy that might drive K' uptake. Light supplies the halobacterial cell with an increased membrane potential and, in time, with an increased pH-gradient and an elevated

G. Wagner, R. Hartmann, and D. Oesterhelt

-101;

'

200

173

400

1000

600 800 Irradiance ( W l r n ' )

Fig. 4. Light-dependent potassium uptake as a junction of externul p H . For experimental details, see Fig. 2 and 3. The vertical bars indicate S.E.M. Inset: redrawing of potassium uptake at low light intensities on an extended scale

ATP level. All/,,, could drive K + uptake provided an appreciable permeability of the cell membrane for potassium ions exists or ionophores specific for K" are present in the membrane. Alternatively, K' uptake could be driven by ATP through an ATP-hydrolysing transport system. If ATP hydrolysis were the driving force for K' uptake in H . halobium, an ATP-hydrolysing enzyme activated by Naf and K f would be present (Naf/KfATPase). This type of transport enzyme is found in animal cells, and is specifically inhibited by cardiac glycosides like ouabain [27]. Therefore, we tested ouabain inhibition of K uptake, but no significant effect was found using ouabain concentrations up to 1.4 mmol/l (data not shown). Similar results have been obtained with other microorganisms indicating a lack of a eukaryotic type of Na'/K+-ATPase [28, 291. Furthermore, the different time lags of the Na' and K' transport, shown in Table 2, argue against a strictly coupled transport of these ions. However, this does not exclude other ATP-fuelled K + uptake systems in H . halobium ; therefore experimental proof was sought based on the following consideration. An ATPhydrolysing transport enzyme of K " is expected to catalyse K' uptake as a function of the cellular concentration of ATP. Hence, a comparison of the lightdependent increase in [ATP]i with the light-dependent increase in the rate of K' uptake should indicate whether or not K uptake is driven by ATP hydrolysis (see Discussion). The data in Fig.4 show that K' uptake at the highest irradiance used (1000 Wjm') is still far from light saturation. In contrast, [ATPIi reaches a constant value at 50 W/m2 (Fig. 2), and the initial rate ofATP synthesis saturates at 250 W/m2 [15]. Hence there is no correlation between [ATP], or rate of ATP-synthesis and the rate of K' transport. Thus,

1200

-> E

-

..+

l i

L

Y)

N

'E

0

-,E 2 0 2

15-

5

10-

._ E,

5-

m

+

I

. 5.5

6.0

6.5

7.0

7.5

8.0

External pH

Fig. 5 . Proton gradient ( A p H ) (x). membrane potential (A+,,,) (01, proton motive force ( A p ) (0)and potassium uptake ( 0 ) as u functiun c ~ f external pH. Fot experimental details, see Fig. 2. Irradiance for potassium uptake was 250 W/m2. Data of A pH. A$,,, and Ap are taken from [20]. The experimental procedure is describcd in detail in [8]

an ATP-hydrolysing enzyme system for the uptake of K' seems unlikely in H . halobium. Therefore, the direct energy source for potassium ion uptake is most likely to be the electrochemical proton gradient (proton motive force d ~ ) . In Fig. 5 , the pH dependence of the rate of lightinduced K" uptake is compared with that of lightinduced changes in dpH, and dp. It is evident that d p H and A p decrease steadily within the pHrange from 5.75 to 8.0, whereas A$,,, increases. Similarly, the rate of K" uptake increases with pH and

174

Potassium Uniport and ATP Synthesis in Halobacterium halobium

Cell concentration ( m g protein I m l )

Basal salt concentration (Oh,

Fig. 6. Potassium-induced fluorescence changes (AF) of the carbocyanine dye as a function of? ( A ) dye concentration; ( B ) bacterial cell concentration; ( C ) basal salt concentration. In each of the experiments only one parameter was varied and the others kept constant at the concentrations indicated by the arrow: dye concentration = 5 x lo-' mol/l; bacterial concentration = 0.35 mg protein/ml; basal salt concentration = 1760 mmol/l (= 40%) consisting of 1726 mmol NaC1/1, 2 mmol KC1/1, 32 mmol MgS04 . 7 HzO/l and 10 mmol Trismaleate/l pH 7. Stock suspensions of cells in basal salt of 4290 mmol NaCI/l, 2 mmol KCI/l, 81 mmol MgS04 . 7 HzO/l and 10 mmol Trismaleate/l pH 7 were diluted with buffered basal salt of reduced ionic strength; the final salt concentration after dilution is given in % of basal salt. Fluorescence changes were induced by a rapid shift in the external potassium concentration from 2 mmol/l to 20 mmol/l in presence of 2.5 pmol valinomycin/l through addition of KCl. The cell suspensions were stirred continuously and thermostated at 25 "C

therefore correlates with A$, rather than with ApH or Ap. Furthermore, K' uptake was found to increase slightly when ApH is decreased in presence of the membrane permeable buffer compound 5,5-dimethyloxazolidin-2,4-dione (50 mmol/l) at pH, = 7.0 (data not shown). Thus, a A $,-driven uptake mechanism of K + seems likely in H. halobium. However, for such a uniport mechanism to work, a membrane permeability highly selective to K" is essential and was tested as described in the following section. Permeability of the Halobacterial Cell Membrane to Potassium Ions

If the membrane permeability to K'. (PK)greatly exceeds the permeabilities to all other ions present, a simple Nernst-relation should exist between the concentration gradient of K + across the cell membrane and the diffusion potential (A$,, ; see Introduction). This can be tested provided charge transport across the membrane due to electrogenic components [see Eqn (3)] is switched off completely. To this purpose, cell suspensions were flushed in the dark with NZfor at least 15 min; this treatment relaxes light and oxygen induced enhancements of Arc/,, A pH and [ATPIi [15] (H. Michel, personal communication). Membrane potentials in small organisms like bacteria cannot be measured by microelectrodes. An indirect but reliable technique is the use of charged membrane-permeable fluorescent dyes of the cyanine type which change their fluorescence intensity upon

changes of A$,. Several dyes were checked by comparison with microelectrode measurements and gave satisfactory results [30,31]. We generally used the carbocyanine dye 3,3'-dipropylthiodicarbocyanine iodide (see Material and Methods) albeit the merocyanine dye 3,3'-dipentyloxadicarbocyanine iodide [30],gave comparable results. Fluorescence changes due to a rapid increase in the external K" concentration are shown in Fig.6A-C. A plateau in signal height was found at varying dye and bacterial concentration (Fig. 6A and B, respectively). The concentrations used for routine measurements are indicated by arrows showing that the ratio of dye to bacterial concentration was kept as low as possible. Fig. 6C shows that fluorescence measurements had to be performed in diluted basal salt. The highly concentrated salt solution (usually 4380 mmol/l, see legends of Fig.1 and 2) did not allow detection of any fluorescence change. Only after dilution to a salt strength of 2630 mmol/l (= 60 %) potassiuminduced fluorescence changes became measurable and increased up to the point of cell lysis near to 880 mmol/l (= 20 %) [32]; beyond this point, fluorescence emission changes immediately dropped. For routine measurements, a salt strength of 1760 mmol/l (= 40%) was used and carefully set during all fluorescence measurements (see arrow in Fig. 6C). This lowered salt concentration had no measurable effect on ATP synthesis or on potassium uptake (Table 3). Changes in fluorescence upon rapid changes in external potassium ion concentration are shown in

G. Wagner, R. Hartmann, and D. Oesterhelt

175

Fig. 7. In the presence of valinomycin the potassiuminduced increase of fluorescence is about twice as high as in absence of valinomycin. Comparable amounts of other ions such as Na' had no effect on fluorescence, neither in presence nor in absence of valinomycin. In 40 basal salt suspensions (1760 mmol/l) the cellular potassium concentration averaged at about 1029 95 mmol/kg cell water. From this value and from the applied shifts in the external potassium concentration the respective changes in potassium gradient across the cell membrane can be calculated. These changes are plotted logarithmically against the corresponding changes in fluorescence in Fig. 8. The slopes of the two lines in the presence and in the absence of valinomycin, respectively, differ by a factor of two. For a quantitative consideration of these results a calibration of fluorescence signals in terms of voltage (mV) can be obtained. Often valinomycin is used for calibration [l 11 assuming that under saturating concentrations of this K'--ionophore, A $ d is

dominated by A + K [see Eqn (l)], i.e. 59 mV result from a gradient in K' concentration of a decade, Thus, taking total A $ d equal to A$K in the presence of valinomycin the data in Fig. 8 show that under physiological conditions (i.e. absence of valinomycin) the potassium gradient in Halobacterium is able to deliver about half of its maximal electrochemical potential, i.e. approximately 30 mV per decade of K' concentration difference. Assuming that the other half is wasted due to leakiness to Na', a ratio of the Na+PNJPK permeability ( P N ~to) the K '--permeability (PK) = a = 0.05 can be calculated using Eqn (1) and the following measured data : [K+]i = 1000 mmol/l, [K'l0 = 10 mmol/l, [Na+]i = 800 mmol/l, [Na'], = 1720 mmol/l, A$d = - 60 mV, t = 25°C.

Table 3 . Rates of photophosphorylation and of potassium uptake in diluted basal salt For experimental details, see Fig. 2 Basal salt concentration

ATP synthesis

Potassium uptake

pmol x mg protein-' x s-'

nmol x m-2 x s-l

40

16

100

16

22.3 11.4

I\

I

33.3 21.9

\

W valinornyci ith

valinomyci

44.5

0'

52.9

-0.55

-0.43

-0.30

-0.18

0

Change in potassium gradient [ A I g ( [ K ' ] ; / [ K ' l . l ] ~

W/m' Irradiance

50

250

500

Fig. 8. Change in fluorescence ( A F) as a function of' the change in cellular potassium gradient in presence and in absence of valinomycin. The bars indicate S.E.M.

1000

I I I

I

I I

,I

-----+----_. I

W + J O

20

I

I

I I

With I' valinomycin

I

1I

I#--

I

I I

I

I

!

!

40

60

80

I

Fluorescence increase 15%

I

External potassium concentration ( r n m o l l l )

Fig. I . Potassium-induced fluorescence changes ( A F) in halobacterial cell suspensions measured in presence and in absence of valinomycin. The external potassium concentration was increased stepwise within 2 to 3 s by injection of KCI solution. For further details, see Fig. 6 and the text

Potassium Uniport and ATP Synthesis in Halobacterium halobium

176 Light

-

Dark

L

(Y

c

-;5.0 a, 0

f -

2.0t

n im57E . . . . ' -m\ 1.0

.... U

1

0.1 0

20

40

60

[K'],=

100

80

2700mrnolI I

120

140

Time ( m i n )

.

Fig. 9. The cellular concentration of ATP as a function of' external potassium concentration. Parallel samples of cells were resuspended in: (a) basal salt of 4270 mmol NaCl/I, 27 mmol KCI/l and 81 mmol MgS04 . 7 HzO/I containing 10 mmol Tris-maleate/l at pH 7; (b) in basal salt of 1570 mmol NaCl/l, 2700 mmol KCI/I and 81 mmol MgS04 . 7 HzO/l containing 10 mmol Tris-maleate/l pH 7. The intracellular potassium concentrations were insignificantly different and averaged at 2466 72 mmol/kg cell water. The cell suspensions (absorbance at 578 nm = 4.0) were kept under nitrogen atmosphere and illuminated for 10 min (irradiance 500 W/m2, OG 515), f = 25°C

a :

Light

Dark

L

c

+

Monactin

1

Time (rnin)

Fig. 10. The cellular concentration of' ATP as influenced hy the K' ionophorc rnonactin at low and ur high external potassium concentrations. For experimental details, see Fig. 9. Monactin was added anaerobically (final concentration = 5 pmol/l)

Similar values of CI are found in other K+-selective sea water organisms like Grijjithia pulvinata [33]. This result is only a quantitative estimate but clearly demonstrates a high potassium selectivity of the cell membrane in H . halobium. Thus, the coupling mechanism for the uptake of K' in light has been demonstrated to be uniport driven by A+m. This result is also relevant to the bioenergetics of Halobacteria. In the chemiosmotic theory Peter Mitchell postulates that the free energy change in ATP hydrolysis ( A G A ~ Pin) bioenergetic systems like chloroplasts, mitochondria and bacteria is in equilibrium with A p , which is the sum of 59 x A pH and A$,,, [12]. When Halobacterium is in starving conditions and in darkness A$,,, is mainly determined by the potassium gradient. Therefore under these conditions the ATP level should be a function of the K'. gradient, thus allowing the light-induced accumulation

of molar concentrations of potassium ions to serve as chemical energy pool for the cell.

Potassium Ion Gradient and ATP Synthesis The cellular level of ATP is at its maximum in light and decreases in the dark. This decrease is highly dependent on the external potassium concentration (Fig. 9): a potassium concentration of 27 mmol/l in the basal salt mixture induces a drop of [ATP]i from its initial level to about 1.5 mmol/kg cell water and a further decay with a half-life of 75 min, whereas in basal salt of a potassium concentration of 2700 mmol/l, [ATPIi drops to about 0.82 mmol/kg cell water and further decays with a half-life of about 35 min. These differences apparently are due to the hundred-fold difference in the potassium gradient, since [K'.li in both cell samples was 2466 k 72 mmol/kg cell water.

G . Wagner, R. Hartmann, and D. Oesterhelt

177

H+-ATPase

EacteriorhodQpsin H+

H'INa' antiport

K+ uniport

Potassium gradient [ l g ( [ K t I i / [K'],)]

Fig. 1 1 , Correlation between the phosphorylation potential A T P i l A D P x phosphate) und the cellular potassium grudietit in presence and in uhsence of monactin. Parallel samples were resuspended in basal salt of different potassium concentrations varied at the expense of the sodium concentration from 3 mmol/l to 2700 mmolil (see Fig. 9). The intracellular potassium concentrations were insignificantly different and averaged at 2323 70 mmol/kg cell water. After 10 to 30 min in darkness under nitrogen atmosphere, 100 to 350 111 samples were collected for ATP, ADP, phosphate and potassium determination. Monactin (final concentration = 5 pmol/l) or equivalent amounts of ethanol were added 5 min before start of sampling. Phosphorylation potential is given in (mol-')

Fig. 12. Scheme of the main cutionic transport components in H. halobium. The dominant transport component bacteriorhodopsin translocates protons from inside to outside using light energy. From this electrogenic proton translocation, an increase in the proton motive force ( A p ) results due to the increasing membrane potential ( A $m inside more negative) and the increasing proton gradient ( A pH; inside more alkaline). Protons running downhill (dp) will drive ATP synthesis and sodium extrusion through the H '-ATPase and the H '/Na' antiporter, respectively. K uniport is driven by A $,, and serves to keep the proton circuits closed

Our assumption that the potassium ion gradient influences the cellular ATP level via its diffusion potential is further substantiated by the experiment shown in Fig. 10 using a K' ionophore (see also Fig. 8). In contrast to valinomycin which has no effect in undiluted basal salt on halobacterial potassium permeability [9,34], monactin is active [36] and was used in this experiment (Fig. 10). A sudden and considerable increase in cellular concentration of ATP is seen upon addition of monactin at low (27 mM) external K' concentration whereas a slight decrease in ATP occurs upon addition of monactin at high (2700 mM) external potassium concentration. The correlation of the phosphorylation potential and the potassium gradient before and after addition of monactin is shown in Fig. 11. From these data the change in Gibbs free energy of ATP-hydrolysis ( ~ G A T Pas) a function of the potassium gradient can be calculated : monactin was found to enhance the slope of the linear correlation between dGATPand the potassium gradient by a factor of two ; this two-fold enhancement is in close agreement with the findings in Fig.8, using valinomycin in cell suspension of reduced salt strength.

unclear. An omnipermeable membrane, as suggested by Ginzburg [37] is certainly unrealistic (see also [9, 10,23,38]). In contrast, the existence of K'-selective membrane channels or of an intrinsic K + ionophore seems reasonable, but remains unproven. Analysis of K' uptake in light as a function of time reveals a delay in the start of K' uptake whereas Na'. is immediately released in a burst and is chargebalanced by transient proton uptake. In an elegant study using halobacterial cell vesicles Lanyi and MacDonald [39] showed that the bursting H i /Na' movements can be explained by an electrogenic H + / Na'-antiporter working with a stoichiometry greater than 1. However, why does K' uptake lag when the antiporter is already working efficiently? The following consideration attemps to answer this question. Fig. 12 shows that in light at least four ion transport systems are at work in the halobacterial cell membrane: the dominant proton pump bacteriorhodopsin, the dp'consuming' proton translocating ATPase (see [25]), the Ap-'consuming' H'/Na" antiporter and the A$,,,'consuming' K+-uptake. It is evident from the initial alkaline overshoot that although bacteriorhodopsin is the dominant pump and provides the driving force that maintains the membrane potential, it does not immediately balance the proton influx. This only happens after a certain sequence of reactions: as soon as bacteriorhodopsin starts to pump protons outwards, the two components near equilibrium, i.e. the proton translocating ATPase and the H+/Na'-antiporter,

DISCUSSION It is now clear that H. halohiurn is capable of rapid net potassium uptake in light, making use of a uniport mechanism of transport [12], (see also [35]). However, the way K + permeates the cell membrane is still

178

start to translocate protons back into the cell for the sake of ATP-synthesis and sodium release, respectively. However, K + drifts outwards as long as A$m is lower than i.e. as long as both, ATPase and H'/Na'-antiporter, 'consume' H'. Within minutes, the rate of ATP-synthesis slows down due to exhaustion of ADP and proton influx is reduced. Provided proton influx by the H'/Na+-antiporter is also limited, e.g. due to its turn over rate, acidification of the suspension medium and a further increase in A I),,, due to charge translocation result [see Eqn (2)]. As soon as All/m is greater than the value of d$K, potassium uptake starts. The model also explains the extended delay of potassium uptake at pH 8.0, and the transient release of K' at pH 5.5 (see Fig.4). Earlier findings showed that at pH, 8.0 bacteriorhodopsin pumps protons at a rate which is one half of that observed at pH, 7.0 [10,40]. Hence, the light-dependent increase in membrane potential and the A$m driven K' uptake will also be slower at pH, 8.0 in particular under low irradiance. Furthermore, evidence has been presented that membrane conductivity to K' is decreased in the alkaline pH range, at least in whole cells [41]. In contrast, at pH, 5.5 when light is switched on membrane conductivity seems to be increased and causes a transient drop in membrane potential. Such lightdependent changes in membrane conductivity accompanied by transient drops in membrane potential have been observed in a large number of plant cells [42]. In Halobacteria the transient release of K+ in light may be "explained by similar phenomena (see [44]). Furthermore, it is intriguing to ask whether under these conditions the N a + / H '-antiporter (and the proton-translocating ATPase) could run backwards resulting in a burst of H' as is observed at pH values more acidic than 5.5 [10,40,43]. The conclusion most relevant to the bioenergetics of H . halobium is that under non-respiratory conditions in darkness the cell uses the potassium gradient established in light to maintain a certain cellular concentration of ATP. Hence, the potassium gradient serves as the ultimate source of energy when oxidative and photophosphorylation fail. This energy pool decays with a half-life in the order of hours which is long enough to overcome dark periods e.g. the night. In terms of the chemiosmotic theory, the halobacterial cell transforms light energy into a chemical gradient, rather than into an electrical gradient. This way, the cell increases its effective electrical capacity up to 1 kF/m2 as compared to the electrical membrane capacity of about 10 mF/m2 that is usually found in biological membranes [12,33]. We would like to thank Dr E. Grell for the gift of a sample of monactin. We are grateful to Dr L. Randall for help in improving the style of the English. This work was supported by the Drutsche Forsc~iungsgc.mrinschaft.

Potassium Uniport And ATP Synthesis in Halohuctei ium halohzum

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G. Wagner*, Institut fur Botanik und Pharmazeutische Biologie der Friedrich-Alexander-Universitltzu Erlangen-Nurnberg, SchloBgarten 4, D-8520 Erlangen, Federal Republic of Germany R. Hartmann and D. Oesterhelt, Instilut fur Biochemie der Julius-Maximilians-Universitlt Wurzburg, Rontgenring 11, D-8700 Wurzburg, Federal Republic of Germany -~

* To whom correspondence should be addressed.