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Electrogenicity of Potassium Transport in Chlorella
Institut fUr Physikalische Chemie der Universitat Wien, WahringerstraBe 42, A-I090 Wien, Austria
Electrogenicity of Potassium Transport in Chlorella H. W.
TROMBALLA
With 6 figures Received June 22,1979 . Accepted August 9,1979
Summary The nature of coupling between K and proton fluxes across the plasmalemma of Chlorella /usca has been investigated by measuring K accumulation and membrane potential in parallel. The membrane potential was determined through the uptake of the lipid soluble cation tetra phenyl-phosphonium. In media of pH 6.5 at K concentrations between 0.1 and 0.2mM the membrane potentials were -115 ± 10mV, and always by 75-90 mV more positive than the Nernst potential of K. The membrane potential was light independent and remained constant up to 10-3 M external K, but at higher concentrations a depolarisation took place. The strong variation of the membrane potential with external pH from 0 mV at pH 4 up to -200 mV at pH 10 had almost no effect on K accumulation. Net K uptake induced by propionate and K release induced by methylammonium were not accompanied by changes of the membrane potential. At concentrations around 3 ,uM in the light and 1 ,uM in the dark the uncoupler CCCP caused net K uptake and at higher concentrations K release, but the membrane potential was not affected in either case. These results rule out secondary active uptake of K by passive uniport, driven by a membrane potential due to an electrogenic proton pump. Primary active transport by an electroneutral or only slightly electrogenic K/H-exchanging ATP-ase is postulated. Key words: Chlorella, membrane potential, potassium uptake, proton transport.
Introduction In Chiarella Fusca net K fluxes across the plasmalemma are coupled to proton movements in the opposite direction (TROMBALLA, 1978). This K/H-exchange serves as a pH-stat compensating changes of the pH of the cytoplasm induced by exogenous agents - as permeant acids and bases - as well as by production of acidic or basic metabolites within the cell. The purpose of the present work has been to elucidate the nature of coupling between Hand K fluxes across the plasmalemma. According to the chemiosmotic hypothesis accumulation of cations is driven by a membrane potential generated by an electrogenic proton pump (MITCHELL, 1970; Abbreviations: CCCP - carbonyl cyanide m-chlorophenyl-hydrazone; DA = dark/air; LA = light/air; PIPES = piperazine-N-N-bis-ethane sulfonic acid; TPP = tetraphenylphosphonium.
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HAROLD, 1977). In eukal'yotic cells this pump is a cell membrane-bound ATPase transporting protons from the cytoplasm into the external medium. Specific carrier molecules facilitate the distribution of cations (especially K) across the membrane. Evidence for this mechanism - called «electrically driven diffusion» or «passive uniport» (cf. BENTRUP, 1978) - was mainly obtained with prokaryotic cells (HAROLD and PAPINEAU, 1972) and fungi (SLAYMAN and SLAYMAN, 1974; PENA, 1975). In the case of electrically driven diffusion the membrane potential must be more negative or at least equal to the Nernst potential of K (EK)' In plants this condition is frequently fullfilled (cf. FINDLAY and HOPE, 1976) but sometimes it is not (COCUCCI et aI., 1976; LIN and HANSON, 1976). So chemiosmotic cation transport in plants, although likely in some cases, is not conclusively proven. On the other hand, chemiosmotic mechanisms for sugar uptake seem to be common (KOMOR and TANNER, 1976; KOMOR, 1977). Frequently a closer link between K and proton fluxes is suggested for plants, namely an ATP-driven nH/K exchange with n> 1, also called «electrogenic antiport» (cf. BENTRUP, 1978; POOLE, 1978). In this case the membrane potential must not necessarily be more negative than EK but changes of active K fluxes should be accompanied by changes of the membrane potential. The main alternative to electrogenic mechanisms is a electroneutral 1 : 1 exchange of univalent cations brought about by an ATP driven pump exchanging K from the outside against protons and probably also Na from the inside. In this case the correlation between the membrane potential and K movements would not need to be as close as in the case of electrogenic mechanisms. Also a variable stoichiometry of the pump is possible, as postulated for red beet (POOLE, 1973) and maize roots (LIN and HANSON, 1976; COCUCCI, 1976). In order to decide between electrogenic and nonelectrogenic K transport mechanisms, knowledge of the membrane potentials under various conditions of K uptake is necessary. Measurements of the membrane potential of Chlorella Fusca by means of microelectrodes (BARBER, 1968 a; LANGMULLER and SPRINGER-LEDERER, 1974) resulted in surprisingly low values around -50 mV. As the smallness of the Chlorella cell leads to serious technical problems, an alternative method for the determination of the membrane potential was desirable. So a method has been adopted that was applied before to Chlorella vulgaris (KOMOR and TANNER, 1976): measurement of the uptake of the lipid soluble cation tetraphenylphosphonium (TPP).
Materials and Methods Chiarella Jusca (211-8 b Gottingen) was cultivated and harvested as described earlier (TROMBALLA, 1978). The algae were suspended in a K free medium containing 10-4 M CaCl 2 and a buffer consisting of 5 mM Tris, 3 mM HCl and PIPES to give a final pH of 6.5. If necessary, more acidic or alcaline pH values were obtained by adding HCl or tetramethylammonium hydroxide, respectively. Cell density was 1.25 Ofo (volume Ofo packed cells deterZ. PJlanzenphysial. Bd. 96. S. 123-133. 1980.
Electrogenicity of K transport in Chiarella
125
mined by hematokrit), corresponding to 2.5 g dry weight/I. The suspensions were aerated at 30°C and illuminated (LA conditions) or kept in the dark (DA conditions). Before the start of the experiments the algae were preconditioned 2 h under LA, and for DA experiments an additional hour under DA conditions. After this time K release from the algae had ceased and the K concentration in the supernatant was between 4· 10-5 and 1.4 . 10-4 M, and changed only slightly (TROMBALLA, 1978). K accumulation and TPP uptake were determined in parallel in aliquots taken from the same algal suspension. In one vessel after addition of 10-5 M unlabeled TPP chloride K was determined by atomic absorption spectrometry of samples of the supernatant obtained by 1 min centrifugation at 3000 g. At the end of the experiment 1 ml suspension was boiled shortly with 10 ml 0.2 M HCL. Analysis of the supernatant of this solution gave the K and Na content of the algae. In the second vessel 10-5 M 3H-labeled TPP chloride (specific activity 160 MBq/mol) were added to the algal suspension (time zero). In most cases TPP uptake was followed by taking aliquots of the supernatant obtained by 1 min centrifugation at 3000 g. The 0.5 ml samples were mixed with 0.5 ml 1 M HCl and 10 ml of a scintillator cocktail containing 5 g PPO and 0.5 g POPOP in 1 1 of a 1 : 1 mixture of toluene and Triton X 100. The radioactivity of the samples was referred to the radioactivity of the first sample taken after 20 seconds (100 0/0). As the penetration of TPP through the cell membrane is rather slow, at this time only the small amount of TPP adsorbed to the surface of the cells had disappeared from the supernatant (only 1-2 Ofo of the total TPP). So it was not necessary to account by calculation for surface binding. For sufficiently accurate measurements of the initial velocity of TPP uptake it was necessary to determine the radioactivity of the algae. A 1 ml aliquot of the experimental suspension was added to 2 ml buffer in a hematocrit tube and centrifuged 1 min at 3000 g. The supernatant was quantitatively removed by rinsing with water and wiping dry the upper part of the tube and by sucking up the rest of the supernatant from the capillary of the tube by means of a syringe. The algal pellets were extracted 1 h with 1 ml 1 M HCI. After centrifugation 0.5 ml of the supernatant were mixed with 0.5 ml water and 10 ml scintillator cocktail as described above. It had been found that in Chiarella Fusca 66 Ofo of the hematokrit volume represent internal cell space (TROMBALLA and BRODA, 1972). Thus 11 of the experimental suspension of 1.25 Ofo density contains 8 ml cell water. The internal concentrations of K or TPP were computed under the assumption that these substances are uniformly distributed over this volume. From the accumulation factors Cin/Cout the Nernst potentials EK and ETPP were calculated. ETPP was assumed to represent the membrane potential.
Results
The internal K concentration of the Chiarella cell is around 1.7· 10-1 M and almost independent of the K concentration in the medium (SHIEH and BARBER, 1971). Therefore the measurements were carried out mainly in media of low K content as only under such conditions the full capacity of the algae to accumulate and hold internal K can be seen. Moreover for a freshwater alga external K concentrations of about 10-4 M are surely nearer to natural conditions than the 10-2 M present in the nutrient medium. In all experiments, K released at first into the K free medium during the preconditioning phase was partly taken up again (compare Fig. 1). This shows that the high K accumulations observed were due to active uptake and not a consequence of the low K permeability of the plasmalemma.
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TROMBALLA
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Fig. 1: TPP uptake and K distribution. pH 6.5; TPP uptake: 0 LA, • supernatant: 0 LA, • DA; total K content was 1.5 . 10-3 Molll suspension.
5
h
DA; K in the
TPP uptake by Chlarella was rather slow. Stationary accumulation was reached after 3-4 h only. No significant differences in TPP uptake were found between LA and DA conditions. At pH 6.5 K accumulation always exceeded TPP accumulation by a factor of about 20. In the experiment shown in Fig. 1 TPP and K accumulation after 4 hours were 84 and 2250 fold, respectively, in LA, and 78 and 1400 fold in DA. This corresponds to Nernst potentials for ETPP and EK of -116 and -201 mV in LA, and of -114 and -188 mV in DA, respectively. The mean results of 6 experiments of this kind are listed in Tab. 1. The dependence of the membrane potential on the external K concentration at two pH values is shown in Fig. 2. Up to 10- 3 M K, the membrane potential was constant. At higher concentrations a depolarisation took place which was less than predicted by the Nernst equation, i. e. at higher external K concentrations the membrane potential became finally more negative than EK. External Na influenced the Z. PJlanzenphysiol. Ed. 96. S. 123-133. 1980.
Electrogenicity of K transport in Chiorella
127
Table 1: TPP and K accumulation in Chiorella at pH 6.5 (mean of 6 experiments). The average difference of the Nernst potentials EK-E TPP was 87 ± mV in LA and 76 ± 6 mV in DA. TPP
K
LA
accumulation Nernst potential
99±30 -116± 9 mV
2880±1100 -206±10mV
DA
accumulation Nernst potential
83±25 -114± 7 mV
1600±650 -190±12mV
-200~----------------------------~
mV
- 150
-100
- 50
-5
-4
-3
-2
-1
log c
Fig. 2: Dependence of the membrane potential on the external concentrations c [M] of K and Na. K: • buffer pH 6.5, + buffer pH 6.5 containing CI04- instead of CI-, X nutrient medium pH 6.5, 0 buffer pH 8.2. Na: .. buffer pH 6.5. The internal K concentration of the algae was 1.6-1.8 . 10-1 M and the internal Na concentration was 1-1.5 . 10-3 M. The straight line gives EK •
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membrane potential to a lesser extent than K. In order to test the influence of anions on the membrane potential the chloride of the buffer was replaced by perchlorate, an ion penetrating into the Chlarella cell only very slowly (TROMBALLA and BRODA, 1971). No significant alteration was found. In nutrient medium of pH 6.5 the membrane potential was equal to that in buffer of pH 6.5, also containing the K concentra:tion of the nutrient medium (10- 2 M). Thus the other ions present in the culture medium seem to have no significant influence on the membrane potential. The dependence of the membrane potential on external pH is shown in Fig. 3. The strong variation from 0 mV at pH 4 up to -200 mV at pH 10 is surely due to the pH changes only, for at all pH values tested the external K concentrations were below 1.5· 10-4M, i. e. in a range where K does not affect the membrane potential (Fig. 2). As previously found (TROMBALLA, 1978), salts of membrane permeant acids cause net K uptake, and salts of permeant bases K release. Na propionate and
mV
- 200
- 150
-100
- 50
"
5
6
7
8
9
10 pH
Fig. 3: Dependence of the membrane potential and EK on external pH. LA conditions; 0 membrane potential, • E K• The straight line gives the slope of the Nernst equation. At all pH values external K concentrations were below 1.5 . 10-4 M, the internal K concentrations between 1.5 and 1.8 . 10-1 M.
z. Pf/anzenphysiol. Rd. 96. S. 123-133. 1980.
Electrogenicity of K transport in Chlorella
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methyl ammonium chloride, chosen as typical examples, clearly had opposite effects on K movements (Fig. 4 a), but had no influence on accumulation and initial uptake velocity of TPP (Fig. 4 b, Fig. 5). The uncoupler CCCP, up to a concentration of 5.10-5 M, had no effect on TPP accumulation and initial uptake velocity either (not shown). Nevertheless a surprising effect on net K movements was found: At intermediate concentrations of uncoupler a persisting net K uptake was observed after a short K release (Fig. 6 a and b). Maximum K uptake was obtained with 3 ,uM
a)
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Fig. 4 a and b: Effect of Na propionate and methylammonium chloride on net K movements (a) and on TPP uptake (b). LA conditions, pH 6.5 . • control, 0 5 . 10-4 M Na propionate, X 5 . 10-4 M methylammonium chloride added at time zero.
z. Pflanzenphysiol. Bd. 96. S. 123-133. 1980.
130
H. W.
TROMBALLA
lOr-
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5
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Fig. 5: Effect of Na propionate and methylammonium chloride on initial velocity of TPP uptake. LA conditions, pH 6.5 . • control, 0 5 . 10-4 M Na propionate, X 5 . 10-4 M methylammonium chloride added at time zero.
CCCP in LA and 1 ,aM CCCP in DA. At high CCCP concentrations K release was found, as expected.
Discussion In plant cells membrane potentials more negative than EK are common (FINDLAY and HOPE, 1976; RAVEN, 1976). The build-up of these potentials is attributed to the action of electrogenic pumps at the plasmalemma. This is suggested by the strong dependence of the potentials on light and on metabolic inhibitors. The observations made with Chlorella do not fit this scheme. A membrane potential equal to or more negative than EK is only observed at K concentrations and/or pH values unphysiologically high for a freshwater alga (Fig. 2, Fig. 3). It can therefore be ooncluded that in natural conditions - neutral pH and low external K - the membrane potential is not negative enough to lead to the K accumulations observed. This holds especially for pH 4 where despite a membrane potential of 0 m V K is accumulated about 1000-fold. The hyperpolarization of the membrane potential with increasing pH may be attributed to increasing electrogenicity of a proton pump. Z. P/lanzenphysiol. Bd. 96. S. 123-133 .. 1980.
Electrogenicity of K transport in ChIarella
t
t fA M CCCP added ::.:: ~
,
f'-M CCCP added
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Fig. 6 a and b: Effect of CCCP on net K movements in LA (a) and DA (b). Before addition of CCCP the curves are identical, with K concentrations 0.85 . 10-4 M in LA and 1.35 . 10-4 M in DA. For better comparison, the curves are drawn separately.
However, this has no effect on K transport as seen by the constancy of EK over the wide pH range (Fig. 3). Little information exists about the internal compartmentation of the ChIarella cell. Hence the absolute values of the membrane potential calculated from TPP distribution may be doubted. Nevertheless, a ra:tio of 20 between the accumulations of K and of TPP appears too high to be attributed to preferential binding of K in parts of the cell. Moreover, if such compartments existed the effect would also be found at higher external K concentrations. However, the opposite - i. e. a stronger TPP accumulation - is the case (Fig. 2). If K were taken up by electrogenic mechanisms the marked effects of propionate and methyl ammonium on net K uptake should be accompanied by changes in the membrane potentials. The lack of such effects (Fig. 4 band 5), just as the independence of the membrane potential on changes in illumination (Fig. 1), are further arguments against electrogenic K uptake. As the penetration of TPP is rather slow, short transients of the membrane potential, after addition of the salts or after change in illumination, may escape detection even when the initial velocity of TPP uptake is followed. But short transients surely cannot be responsible for nhe persisting effects on K uptake (Fig. 1, Fig. 4 a). It is accepted that uncouplers abolish membrane potentials my making membranes permeable to protons. In eukaryotic cells a double action of CCCP on transport Z. PjIanzenphysiaI. Bd. 96. S. 123-133. 1980.
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H. W. TROMBALLA
phenomena occurs (PENA, 1975): a) breakdown of the membrane potential at the cell membrane due to proton permeability, b) inhibition of the ATP-driven pumps at the plasmalemma by a block in the ATP production of the cell. In any case K following the membrane potential via passive uniport should be released upon addition of CCCP. Actually no action of CCCP on the membrane potential was found and at middle concentrations of uncoupler initial K release was followed by persisting net K uptake (Fig. 6 a and b). These unexpected effects can only be explained by assuming primary active K/H exchange. In an intermediate concentration range the uncoupler should effect the plasmalemma only while the ATP production of the cell should remain unimpaired. This was found by FELLE and BENTRUP (1977) with Riccia. So the protons entering at first through the leaky plasmalemma against K release can be extruded by an increased action of the ATP-driven K/H exchange system which tends to hold the internal cell pH constant. If K is taken up electroneutrally the pump itself does not contribute to the membrane potential. In this case, the potential should depend on the distribution and the permeabilities of all ions present at both sides of the membrane. From Fig. 2 and 3 it can be seen that the membrane potential is mainly influenced by protons and to a lesser extent by K. Unfortunately the data do not fit the equations of Goldman or Hodgkin-Horowicz (JAFFE, 1974), probably because the permeability of the membrane and the electrogenicity of the pump change with the external concentrations of K and H. So at present it cannot yet be explained why in ChIarella the membrane potential can be more positive than EK. But at least secondary active K uptake via electrically driven diffusion can be ruled out. K transport in ChIarella is rather primary active and proceeds in exchange against protons and Na (TROMBALLA, in preparation). As K efflux from ChIarella is energy dependent it probably also occurs via the same system (BARBER, 1968 b). The stoichiometry of this univalent ion exchange should be 1 : 1 or near to it, i. e. it is electroneutral or only slightly electrogenic. Similar conclusions have been reached for maize roots by COCUCCI et al. (1976). The author thanks Professor E. BRODA for his interest. Financial support from the «Fonds zur Forderung der wissenschaftlichen Forschung» of the Republic of Austria and of the «HochschuljubiHiumsstiftung der Stadt Wien» is gratefully acknowledged. Tritiated TPP was a generous gift of Dr. P. GECK (Frankfurt/Main).
References BARBER, J.: Measurement of the membrane potential and evidence for active transport of ions in Chiarella pyrenaidasa. Biochim. Biophys. Acta 150, 618-625 (1968 a). - The efflux of potassium from Chiarella pyrenaidasa. Biochim. Biophys. Acta 163, 531-538 (1968 b). BENTRUP, F. W.: Cell physiology. Cell electrophysiology and membrane transport. Progr. Botany 40, 84-98 (1978). Cocuccr, M., E. MARRE, A. BALLARIN DENTI, and A. SCAccr: Characteristics of fusicoccininduced changes of transmembrane potential and ion uptake in maize root segments. Plant Sci. Letters 6, 143-156 (1976). Z. Pflanzenphysial. Bd. 96. S. 123-133. 1980.
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FELLE, H. and F. W. BENTRUP: A study of the primary effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on membrane potential and conductance in Riccia fiuitans. Biochim. Biophys. Acta 464,179-187 (1977). FINDLAY, G. P. and A. B. HOPE: Electrical properties of plant cells: Methods and findings. In: LUTTGE, U. and M. G. PITMAN (Eds.): Transport in plants II, Part A Cells, 51-92. Springer, Berlin, Heidelberg, New York, 1976. HAROLD, F. M.: Ion currents and physiological functions in microorganisms. Ann. Rev. Microbiol. 31, 181-203 (1977). HAROLD, F. M. and D. PAPINEAU: Cation transport and electrogenesis by Streptococcus faecaiis. J. Membrane BioI. 8, 27-62 (1972). JAFFE, L. F.: The interpretation of voltage-concentration relations. J. theor. BioI. 48, 11-18 (1974). !(,OMOR, E.: Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics, mechanism, and regulation. Planta 137, 119-131 (1977). KOMOR, E. and W. TANNER: The determination of the membrane potential of Chiorella vulgaris. Evidence for electrogenic sugar transport. Eur. J. Biochem. 70, 197-204 (1976). LANGMULLER, G. und SPRINGER-LEDERER: Membranpotential von Chlorella Fusca in Abhangigkeit von pH-Wert, Temperatur und Belichtung. Planta 120, 189-196 (1974). LIN, W. and J. B. HANSON: Cell potentials, cell resistance and proton fluxes in corn root tissue. Effect of dithioerythrito!' Plant Physio!. 58, 276-282 (1976). MITCHELL, P.: Membranes of cells and organelles: Morphology, transport and metabolism. In: Symposia of the Society for General Microbiology No. XX, Prokaryotic and Eukaryotic Cells, 121-165 (1970). PENA, A.: Studies on the mechanism of K+ transport in yeast. Arch. Biochem. Biophys. 167, 397-409 (1975). POOLE, R. J.: The H+ pump in red beet. In: ANDERSON, W. P. (Ed.): Ion transport in plants, 129-134. Academic Press, London, New York, 1973. - Energy coupling for membrane transport. Ann. Rev. Plant Physio!. 29, 437-460 (1978). RAVEN, J. A.: Transport in algal cells. In: LUTTGE, U. and M. G. PITMAN (Eds.): Transport in plants II, Part A Cells, 129-188. Springer, Berlin, Heidelberg, New York, 1976. SHIEH, Y. J. and J. BARBER: Intracellular sodium and potassium concentrations and net cation movements in Chiorella pyrenoidosa. Biochim. Biophys. Acta 233, 594-603 (1971). SLAYMAN, C. L. and C. W. SLAYMAN: Net uptake of potassium in Neurospora. Exchange for sodium and hydrogen ions. J. Gen. Physio!. 52, 424-443 (1968). TROMBALLA, H. W.: Influence of permeant acids and bases on net potassium uptake by Chlorella. P!anta 138, 243-248 (1978). TROMBALLA, H. W. und E. BRODA: Das Verhalten von Chiorella Fusca gegenuber Perchlorat und Chlorat. Arch. Mikrobiol. 78,214-223 (1971). - - Der freie Raum synchroner Chlorella. Arch. Mikrobio!' 86, 281-290 (1972). Dr. H. W. TROMBALLA, Institut fur Physikalische Chemie, Universitat, WahringerstraBe 42, A-I090 Wien, Osterreich.
Z. P/lanzenphysioi. Bd. 96. S. 123-133. 1980.
Electrogenicity of Potassium Transport in Chlorella H. W.
TROMBALLA
With 6 figures Received June 22,1979 . Accepted August 9,1979
Summary The nature of coupling between K and proton fluxes across the plasmalemma of Chlorella /usca has been investigated by measuring K accumulation and membrane potential in parallel. The membrane potential was determined through the uptake of the lipid soluble cation tetra phenyl-phosphonium. In media of pH 6.5 at K concentrations between 0.1 and 0.2mM the membrane potentials were -115 ± 10mV, and always by 75-90 mV more positive than the Nernst potential of K. The membrane potential was light independent and remained constant up to 10-3 M external K, but at higher concentrations a depolarisation took place. The strong variation of the membrane potential with external pH from 0 mV at pH 4 up to -200 mV at pH 10 had almost no effect on K accumulation. Net K uptake induced by propionate and K release induced by methylammonium were not accompanied by changes of the membrane potential. At concentrations around 3 ,uM in the light and 1 ,uM in the dark the uncoupler CCCP caused net K uptake and at higher concentrations K release, but the membrane potential was not affected in either case. These results rule out secondary active uptake of K by passive uniport, driven by a membrane potential due to an electrogenic proton pump. Primary active transport by an electroneutral or only slightly electrogenic K/H-exchanging ATP-ase is postulated. Key words: Chlorella, membrane potential, potassium uptake, proton transport.
Introduction In Chiarella Fusca net K fluxes across the plasmalemma are coupled to proton movements in the opposite direction (TROMBALLA, 1978). This K/H-exchange serves as a pH-stat compensating changes of the pH of the cytoplasm induced by exogenous agents - as permeant acids and bases - as well as by production of acidic or basic metabolites within the cell. The purpose of the present work has been to elucidate the nature of coupling between Hand K fluxes across the plasmalemma. According to the chemiosmotic hypothesis accumulation of cations is driven by a membrane potential generated by an electrogenic proton pump (MITCHELL, 1970; Abbreviations: CCCP - carbonyl cyanide m-chlorophenyl-hydrazone; DA = dark/air; LA = light/air; PIPES = piperazine-N-N-bis-ethane sulfonic acid; TPP = tetraphenylphosphonium.
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HAROLD, 1977). In eukal'yotic cells this pump is a cell membrane-bound ATPase transporting protons from the cytoplasm into the external medium. Specific carrier molecules facilitate the distribution of cations (especially K) across the membrane. Evidence for this mechanism - called «electrically driven diffusion» or «passive uniport» (cf. BENTRUP, 1978) - was mainly obtained with prokaryotic cells (HAROLD and PAPINEAU, 1972) and fungi (SLAYMAN and SLAYMAN, 1974; PENA, 1975). In the case of electrically driven diffusion the membrane potential must be more negative or at least equal to the Nernst potential of K (EK)' In plants this condition is frequently fullfilled (cf. FINDLAY and HOPE, 1976) but sometimes it is not (COCUCCI et aI., 1976; LIN and HANSON, 1976). So chemiosmotic cation transport in plants, although likely in some cases, is not conclusively proven. On the other hand, chemiosmotic mechanisms for sugar uptake seem to be common (KOMOR and TANNER, 1976; KOMOR, 1977). Frequently a closer link between K and proton fluxes is suggested for plants, namely an ATP-driven nH/K exchange with n> 1, also called «electrogenic antiport» (cf. BENTRUP, 1978; POOLE, 1978). In this case the membrane potential must not necessarily be more negative than EK but changes of active K fluxes should be accompanied by changes of the membrane potential. The main alternative to electrogenic mechanisms is a electroneutral 1 : 1 exchange of univalent cations brought about by an ATP driven pump exchanging K from the outside against protons and probably also Na from the inside. In this case the correlation between the membrane potential and K movements would not need to be as close as in the case of electrogenic mechanisms. Also a variable stoichiometry of the pump is possible, as postulated for red beet (POOLE, 1973) and maize roots (LIN and HANSON, 1976; COCUCCI, 1976). In order to decide between electrogenic and nonelectrogenic K transport mechanisms, knowledge of the membrane potentials under various conditions of K uptake is necessary. Measurements of the membrane potential of Chlorella Fusca by means of microelectrodes (BARBER, 1968 a; LANGMULLER and SPRINGER-LEDERER, 1974) resulted in surprisingly low values around -50 mV. As the smallness of the Chlorella cell leads to serious technical problems, an alternative method for the determination of the membrane potential was desirable. So a method has been adopted that was applied before to Chlorella vulgaris (KOMOR and TANNER, 1976): measurement of the uptake of the lipid soluble cation tetraphenylphosphonium (TPP).
Materials and Methods Chiarella Jusca (211-8 b Gottingen) was cultivated and harvested as described earlier (TROMBALLA, 1978). The algae were suspended in a K free medium containing 10-4 M CaCl 2 and a buffer consisting of 5 mM Tris, 3 mM HCl and PIPES to give a final pH of 6.5. If necessary, more acidic or alcaline pH values were obtained by adding HCl or tetramethylammonium hydroxide, respectively. Cell density was 1.25 Ofo (volume Ofo packed cells deterZ. PJlanzenphysial. Bd. 96. S. 123-133. 1980.
Electrogenicity of K transport in Chiarella
125
mined by hematokrit), corresponding to 2.5 g dry weight/I. The suspensions were aerated at 30°C and illuminated (LA conditions) or kept in the dark (DA conditions). Before the start of the experiments the algae were preconditioned 2 h under LA, and for DA experiments an additional hour under DA conditions. After this time K release from the algae had ceased and the K concentration in the supernatant was between 4· 10-5 and 1.4 . 10-4 M, and changed only slightly (TROMBALLA, 1978). K accumulation and TPP uptake were determined in parallel in aliquots taken from the same algal suspension. In one vessel after addition of 10-5 M unlabeled TPP chloride K was determined by atomic absorption spectrometry of samples of the supernatant obtained by 1 min centrifugation at 3000 g. At the end of the experiment 1 ml suspension was boiled shortly with 10 ml 0.2 M HCL. Analysis of the supernatant of this solution gave the K and Na content of the algae. In the second vessel 10-5 M 3H-labeled TPP chloride (specific activity 160 MBq/mol) were added to the algal suspension (time zero). In most cases TPP uptake was followed by taking aliquots of the supernatant obtained by 1 min centrifugation at 3000 g. The 0.5 ml samples were mixed with 0.5 ml 1 M HCl and 10 ml of a scintillator cocktail containing 5 g PPO and 0.5 g POPOP in 1 1 of a 1 : 1 mixture of toluene and Triton X 100. The radioactivity of the samples was referred to the radioactivity of the first sample taken after 20 seconds (100 0/0). As the penetration of TPP through the cell membrane is rather slow, at this time only the small amount of TPP adsorbed to the surface of the cells had disappeared from the supernatant (only 1-2 Ofo of the total TPP). So it was not necessary to account by calculation for surface binding. For sufficiently accurate measurements of the initial velocity of TPP uptake it was necessary to determine the radioactivity of the algae. A 1 ml aliquot of the experimental suspension was added to 2 ml buffer in a hematocrit tube and centrifuged 1 min at 3000 g. The supernatant was quantitatively removed by rinsing with water and wiping dry the upper part of the tube and by sucking up the rest of the supernatant from the capillary of the tube by means of a syringe. The algal pellets were extracted 1 h with 1 ml 1 M HCI. After centrifugation 0.5 ml of the supernatant were mixed with 0.5 ml water and 10 ml scintillator cocktail as described above. It had been found that in Chiarella Fusca 66 Ofo of the hematokrit volume represent internal cell space (TROMBALLA and BRODA, 1972). Thus 11 of the experimental suspension of 1.25 Ofo density contains 8 ml cell water. The internal concentrations of K or TPP were computed under the assumption that these substances are uniformly distributed over this volume. From the accumulation factors Cin/Cout the Nernst potentials EK and ETPP were calculated. ETPP was assumed to represent the membrane potential.
Results
The internal K concentration of the Chiarella cell is around 1.7· 10-1 M and almost independent of the K concentration in the medium (SHIEH and BARBER, 1971). Therefore the measurements were carried out mainly in media of low K content as only under such conditions the full capacity of the algae to accumulate and hold internal K can be seen. Moreover for a freshwater alga external K concentrations of about 10-4 M are surely nearer to natural conditions than the 10-2 M present in the nutrient medium. In all experiments, K released at first into the K free medium during the preconditioning phase was partly taken up again (compare Fig. 1). This shows that the high K accumulations observed were due to active uptake and not a consequence of the low K permeability of the plasmalemma.
z. P/lanzenphysial. Bd. 96. S. 123-133. 1980.
126
H. W.
TROMBALLA
~----------------------------------------------~
e---------e ____________
1,5
e +J
C
+J
c
eo eo
100
C
L0)
D-
--0--0 __ 0 ~, ----0____0_ CI
::J
V)
'-i!:::::::-
~r!=~==
0)
.c. c 0.... 0....
1,0
eo eo
+J
c
L0)
Cl. ::J
V)
-0
• 0
O)
.c. c
~
~
50
0,5
:2:
-..x I
0
~
~
2
3
4
Fig. 1: TPP uptake and K distribution. pH 6.5; TPP uptake: 0 LA, • supernatant: 0 LA, • DA; total K content was 1.5 . 10-3 Molll suspension.
5
h
DA; K in the
TPP uptake by Chlarella was rather slow. Stationary accumulation was reached after 3-4 h only. No significant differences in TPP uptake were found between LA and DA conditions. At pH 6.5 K accumulation always exceeded TPP accumulation by a factor of about 20. In the experiment shown in Fig. 1 TPP and K accumulation after 4 hours were 84 and 2250 fold, respectively, in LA, and 78 and 1400 fold in DA. This corresponds to Nernst potentials for ETPP and EK of -116 and -201 mV in LA, and of -114 and -188 mV in DA, respectively. The mean results of 6 experiments of this kind are listed in Tab. 1. The dependence of the membrane potential on the external K concentration at two pH values is shown in Fig. 2. Up to 10- 3 M K, the membrane potential was constant. At higher concentrations a depolarisation took place which was less than predicted by the Nernst equation, i. e. at higher external K concentrations the membrane potential became finally more negative than EK. External Na influenced the Z. PJlanzenphysiol. Ed. 96. S. 123-133. 1980.
Electrogenicity of K transport in Chiorella
127
Table 1: TPP and K accumulation in Chiorella at pH 6.5 (mean of 6 experiments). The average difference of the Nernst potentials EK-E TPP was 87 ± mV in LA and 76 ± 6 mV in DA. TPP
K
LA
accumulation Nernst potential
99±30 -116± 9 mV
2880±1100 -206±10mV
DA
accumulation Nernst potential
83±25 -114± 7 mV
1600±650 -190±12mV
-200~----------------------------~
mV
- 150
-100
- 50
-5
-4
-3
-2
-1
log c
Fig. 2: Dependence of the membrane potential on the external concentrations c [M] of K and Na. K: • buffer pH 6.5, + buffer pH 6.5 containing CI04- instead of CI-, X nutrient medium pH 6.5, 0 buffer pH 8.2. Na: .. buffer pH 6.5. The internal K concentration of the algae was 1.6-1.8 . 10-1 M and the internal Na concentration was 1-1.5 . 10-3 M. The straight line gives EK •
z. Pjianzenphysioi. Bd. 96. S. 123-133. 1980.
128
H. W. TRoMBALLA
membrane potential to a lesser extent than K. In order to test the influence of anions on the membrane potential the chloride of the buffer was replaced by perchlorate, an ion penetrating into the Chlarella cell only very slowly (TROMBALLA and BRODA, 1971). No significant alteration was found. In nutrient medium of pH 6.5 the membrane potential was equal to that in buffer of pH 6.5, also containing the K concentra:tion of the nutrient medium (10- 2 M). Thus the other ions present in the culture medium seem to have no significant influence on the membrane potential. The dependence of the membrane potential on external pH is shown in Fig. 3. The strong variation from 0 mV at pH 4 up to -200 mV at pH 10 is surely due to the pH changes only, for at all pH values tested the external K concentrations were below 1.5· 10-4M, i. e. in a range where K does not affect the membrane potential (Fig. 2). As previously found (TROMBALLA, 1978), salts of membrane permeant acids cause net K uptake, and salts of permeant bases K release. Na propionate and
mV
- 200
- 150
-100
- 50
"
5
6
7
8
9
10 pH
Fig. 3: Dependence of the membrane potential and EK on external pH. LA conditions; 0 membrane potential, • E K• The straight line gives the slope of the Nernst equation. At all pH values external K concentrations were below 1.5 . 10-4 M, the internal K concentrations between 1.5 and 1.8 . 10-1 M.
z. Pf/anzenphysiol. Rd. 96. S. 123-133. 1980.
Electrogenicity of K transport in Chlorella
129
methyl ammonium chloride, chosen as typical examples, clearly had opposite effects on K movements (Fig. 4 a), but had no influence on accumulation and initial uptake velocity of TPP (Fig. 4 b, Fig. 5). The uncoupler CCCP, up to a concentration of 5.10-5 M, had no effect on TPP accumulation and initial uptake velocity either (not shown). Nevertheless a surprising effect on net K movements was found: At intermediate concentrations of uncoupler a persisting net K uptake was observed after a short K release (Fig. 6 a and b). Maximum K uptake was obtained with 3 ,uM
a)
5
~-x
..... c
X-X-
X
X
•
•
•
L.
C1l ..... C1l C
L.
a.
3
::J (/)
..c.
c
2 it;:-e_.
~
......... 0
~ '-J"
----0 - - 0_ _ 0
I
0
0
a ..... 100 c
.....C1l C1l C
L.
a. ::J
(/)
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b)
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c
Cl. Cl.
~f
"--~-3-*
50 -
I-
;-.:
•
...........0
I
1
2
3
L.
5
h
Fig. 4 a and b: Effect of Na propionate and methylammonium chloride on net K movements (a) and on TPP uptake (b). LA conditions, pH 6.5 . • control, 0 5 . 10-4 M Na propionate, X 5 . 10-4 M methylammonium chloride added at time zero.
z. Pflanzenphysiol. Bd. 96. S. 123-133. 1980.
130
H. W.
TROMBALLA
lOr-
~/
Q)
C1J
O'l
nl Q)
..c
c 0.. 0..
5
/
~
,I
~
~
~
I
10
20
mrn
Fig. 5: Effect of Na propionate and methylammonium chloride on initial velocity of TPP uptake. LA conditions, pH 6.5 . • control, 0 5 . 10-4 M Na propionate, X 5 . 10-4 M methylammonium chloride added at time zero.
CCCP in LA and 1 ,aM CCCP in DA. At high CCCP concentrations K release was found, as expected.
Discussion In plant cells membrane potentials more negative than EK are common (FINDLAY and HOPE, 1976; RAVEN, 1976). The build-up of these potentials is attributed to the action of electrogenic pumps at the plasmalemma. This is suggested by the strong dependence of the potentials on light and on metabolic inhibitors. The observations made with Chlorella do not fit this scheme. A membrane potential equal to or more negative than EK is only observed at K concentrations and/or pH values unphysiologically high for a freshwater alga (Fig. 2, Fig. 3). It can therefore be ooncluded that in natural conditions - neutral pH and low external K - the membrane potential is not negative enough to lead to the K accumulations observed. This holds especially for pH 4 where despite a membrane potential of 0 m V K is accumulated about 1000-fold. The hyperpolarization of the membrane potential with increasing pH may be attributed to increasing electrogenicity of a proton pump. Z. P/lanzenphysiol. Bd. 96. S. 123-133 .. 1980.
Electrogenicity of K transport in ChIarella
t
t fA M CCCP added ::.:: ~
,
f'-M CCCP added
o
o .-.-.----....1.'----.-.----..
o--o-cr-o----o--__ o______ o 1 0--0-0.-.. _
0,3 .-.-.-.-.-.---....
0
LI)
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0
~
c
C1l
131
LI)
~
C1l C
LCll
c..
::l If)
Cll
-£ c
::.::
a)
o
I
I
2 h
o
I
I
2 h
Fig. 6 a and b: Effect of CCCP on net K movements in LA (a) and DA (b). Before addition of CCCP the curves are identical, with K concentrations 0.85 . 10-4 M in LA and 1.35 . 10-4 M in DA. For better comparison, the curves are drawn separately.
However, this has no effect on K transport as seen by the constancy of EK over the wide pH range (Fig. 3). Little information exists about the internal compartmentation of the ChIarella cell. Hence the absolute values of the membrane potential calculated from TPP distribution may be doubted. Nevertheless, a ra:tio of 20 between the accumulations of K and of TPP appears too high to be attributed to preferential binding of K in parts of the cell. Moreover, if such compartments existed the effect would also be found at higher external K concentrations. However, the opposite - i. e. a stronger TPP accumulation - is the case (Fig. 2). If K were taken up by electrogenic mechanisms the marked effects of propionate and methyl ammonium on net K uptake should be accompanied by changes in the membrane potentials. The lack of such effects (Fig. 4 band 5), just as the independence of the membrane potential on changes in illumination (Fig. 1), are further arguments against electrogenic K uptake. As the penetration of TPP is rather slow, short transients of the membrane potential, after addition of the salts or after change in illumination, may escape detection even when the initial velocity of TPP uptake is followed. But short transients surely cannot be responsible for nhe persisting effects on K uptake (Fig. 1, Fig. 4 a). It is accepted that uncouplers abolish membrane potentials my making membranes permeable to protons. In eukaryotic cells a double action of CCCP on transport Z. PjIanzenphysiaI. Bd. 96. S. 123-133. 1980.
132
H. W. TROMBALLA
phenomena occurs (PENA, 1975): a) breakdown of the membrane potential at the cell membrane due to proton permeability, b) inhibition of the ATP-driven pumps at the plasmalemma by a block in the ATP production of the cell. In any case K following the membrane potential via passive uniport should be released upon addition of CCCP. Actually no action of CCCP on the membrane potential was found and at middle concentrations of uncoupler initial K release was followed by persisting net K uptake (Fig. 6 a and b). These unexpected effects can only be explained by assuming primary active K/H exchange. In an intermediate concentration range the uncoupler should effect the plasmalemma only while the ATP production of the cell should remain unimpaired. This was found by FELLE and BENTRUP (1977) with Riccia. So the protons entering at first through the leaky plasmalemma against K release can be extruded by an increased action of the ATP-driven K/H exchange system which tends to hold the internal cell pH constant. If K is taken up electroneutrally the pump itself does not contribute to the membrane potential. In this case, the potential should depend on the distribution and the permeabilities of all ions present at both sides of the membrane. From Fig. 2 and 3 it can be seen that the membrane potential is mainly influenced by protons and to a lesser extent by K. Unfortunately the data do not fit the equations of Goldman or Hodgkin-Horowicz (JAFFE, 1974), probably because the permeability of the membrane and the electrogenicity of the pump change with the external concentrations of K and H. So at present it cannot yet be explained why in ChIarella the membrane potential can be more positive than EK. But at least secondary active K uptake via electrically driven diffusion can be ruled out. K transport in ChIarella is rather primary active and proceeds in exchange against protons and Na (TROMBALLA, in preparation). As K efflux from ChIarella is energy dependent it probably also occurs via the same system (BARBER, 1968 b). The stoichiometry of this univalent ion exchange should be 1 : 1 or near to it, i. e. it is electroneutral or only slightly electrogenic. Similar conclusions have been reached for maize roots by COCUCCI et al. (1976). The author thanks Professor E. BRODA for his interest. Financial support from the «Fonds zur Forderung der wissenschaftlichen Forschung» of the Republic of Austria and of the «HochschuljubiHiumsstiftung der Stadt Wien» is gratefully acknowledged. Tritiated TPP was a generous gift of Dr. P. GECK (Frankfurt/Main).
References BARBER, J.: Measurement of the membrane potential and evidence for active transport of ions in Chiarella pyrenaidasa. Biochim. Biophys. Acta 150, 618-625 (1968 a). - The efflux of potassium from Chiarella pyrenaidasa. Biochim. Biophys. Acta 163, 531-538 (1968 b). BENTRUP, F. W.: Cell physiology. Cell electrophysiology and membrane transport. Progr. Botany 40, 84-98 (1978). Cocuccr, M., E. MARRE, A. BALLARIN DENTI, and A. SCAccr: Characteristics of fusicoccininduced changes of transmembrane potential and ion uptake in maize root segments. Plant Sci. Letters 6, 143-156 (1976). Z. Pflanzenphysial. Bd. 96. S. 123-133. 1980.
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FELLE, H. and F. W. BENTRUP: A study of the primary effect of the uncoupler carbonyl cyanide m-chlorophenylhydrazone on membrane potential and conductance in Riccia fiuitans. Biochim. Biophys. Acta 464,179-187 (1977). FINDLAY, G. P. and A. B. HOPE: Electrical properties of plant cells: Methods and findings. In: LUTTGE, U. and M. G. PITMAN (Eds.): Transport in plants II, Part A Cells, 51-92. Springer, Berlin, Heidelberg, New York, 1976. HAROLD, F. M.: Ion currents and physiological functions in microorganisms. Ann. Rev. Microbiol. 31, 181-203 (1977). HAROLD, F. M. and D. PAPINEAU: Cation transport and electrogenesis by Streptococcus faecaiis. J. Membrane BioI. 8, 27-62 (1972). JAFFE, L. F.: The interpretation of voltage-concentration relations. J. theor. BioI. 48, 11-18 (1974). !(,OMOR, E.: Sucrose uptake by cotyledons of Ricinus communis L.: Characteristics, mechanism, and regulation. Planta 137, 119-131 (1977). KOMOR, E. and W. TANNER: The determination of the membrane potential of Chiorella vulgaris. Evidence for electrogenic sugar transport. Eur. J. Biochem. 70, 197-204 (1976). LANGMULLER, G. und SPRINGER-LEDERER: Membranpotential von Chlorella Fusca in Abhangigkeit von pH-Wert, Temperatur und Belichtung. Planta 120, 189-196 (1974). LIN, W. and J. B. HANSON: Cell potentials, cell resistance and proton fluxes in corn root tissue. Effect of dithioerythrito!' Plant Physio!. 58, 276-282 (1976). MITCHELL, P.: Membranes of cells and organelles: Morphology, transport and metabolism. In: Symposia of the Society for General Microbiology No. XX, Prokaryotic and Eukaryotic Cells, 121-165 (1970). PENA, A.: Studies on the mechanism of K+ transport in yeast. Arch. Biochem. Biophys. 167, 397-409 (1975). POOLE, R. J.: The H+ pump in red beet. In: ANDERSON, W. P. (Ed.): Ion transport in plants, 129-134. Academic Press, London, New York, 1973. - Energy coupling for membrane transport. Ann. Rev. Plant Physio!. 29, 437-460 (1978). RAVEN, J. A.: Transport in algal cells. In: LUTTGE, U. and M. G. PITMAN (Eds.): Transport in plants II, Part A Cells, 129-188. Springer, Berlin, Heidelberg, New York, 1976. SHIEH, Y. J. and J. BARBER: Intracellular sodium and potassium concentrations and net cation movements in Chiorella pyrenoidosa. Biochim. Biophys. Acta 233, 594-603 (1971). SLAYMAN, C. L. and C. W. SLAYMAN: Net uptake of potassium in Neurospora. Exchange for sodium and hydrogen ions. J. Gen. Physio!. 52, 424-443 (1968). TROMBALLA, H. W.: Influence of permeant acids and bases on net potassium uptake by Chlorella. P!anta 138, 243-248 (1978). TROMBALLA, H. W. und E. BRODA: Das Verhalten von Chiorella Fusca gegenuber Perchlorat und Chlorat. Arch. Mikrobiol. 78,214-223 (1971). - - Der freie Raum synchroner Chlorella. Arch. Mikrobio!' 86, 281-290 (1972). Dr. H. W. TROMBALLA, Institut fur Physikalische Chemie, Universitat, WahringerstraBe 42, A-I090 Wien, Osterreich.
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