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A M . ZOOLOGIST 10:377-392 (1970) Movement of Ions and Electrogenesis in Microorganisms CLIFFORD L. SLAYMAN' Department of Physiology, Yale School of Medicine, New Haven, Connecticut 06510 SYNOPSIS. The relationship between movement o£ ions and the electrical properties of microorganisms (algae, fungi, and bacteria) are reviewed, with particular emphasis on the giant alga, Nitella, and the fungus, Neurospora. The hypothesis is presented that there are two basically different components to the membrane potential o£ both organisms: (1) one arising from the diffusion of sodium, potassium, and hydrogen ions down their chemical gradients, and (2) one associated with the utilization of energy and the active efflux of hydrogen ions, and attributed to an "electrogenic H+ pump." Numerous discrepancies between the measured electrical properties of the algae or fungi and the predictions of ordinary ion-diffusion theory can be accounted for by such an H+ pump, and its existence is further supported by a few indirect experiments on the bacteria. The three groups of organisms which I would like to discuss—algae, fungi, and bacteria—have in common one critical feature which has strongly influenced their evolution: a rigid constraining cell wall, usually composed o£ polysaccharide, which encloses the plasma membrane and cytoplasm. The cell wall permits them to live in a much broader range of environments than is generally possible for animal cells, since they can counter severe osmotic forces with hydrostatic pressure. In animal cells, which appear to be in osmotic equilibrium with the environment, the process of extruding sodium ions is addressed mainly to the task of maintaining osmotic equilibrium by making the plasma membrane effectively impermeable to sodium; any sodium which diffuses into the cells is actively "pumped" out. However, microorganisms with cell walls are like higher plants in having little need for osmotic regulation, and it seems likely that the properties of their major ion transport systems have been determined in evolution almost entirely by the need to maintain cytoplasmic concentrations of The author would like to thank Drs. E. J. Williams, F. M. Harold, and H. Kitasato for permission to use data and modified figures from previous publications. The author has been supported by a U.S. Public Health Service Career Development Award (GM 20164) and by a Research Grant (GM 15858) from the National Institute of Ceneral Medical Sciences. ions which are optimal for enzymatic activity. The process of sodium extrusion, for example, has been de-emphasized, and the primary function of the so-called sodium pump has shifted to that of accumulating intracellular potassium. Of course the sodium pump also plays this role in animal cells, but from a teleologic point of view it appears that once the osmotic constraint is removed, the pump mechanism is altered and can extrude practically any available cation in exchange for potassium: sodium, hydrogen ions (Conway and O'Malley, 1946; Rothstein and Enns, 1946; Zarlengo and Schultz, 1966), ammonium ions (Conway and O'Malley, 1946), and perhaps also other amino cations (Slayman and Slayman, 1968). A second characteristic of ion-transport processes in the microorganisms, which has probably co-evolved with the cell wall, is the small size of diffusion fluxes, or leaks. In nerve and muscle cells passive diffusion can account for 75% or more of potassium influx (Hodgkin and Keynes, 1955); in the fresh-water algae the figure is somewhat smaller, 30-70% (MacRobbie, 1962); but among the fungi and bacteria that have been examined, it is 5% or less (Rothstein, 1956; Epstein and Schultz, 1966; Slayman and Slayman, 1968). Indirect evidence suggests that for most other ions diffusion fluxes make up still smaller fractions of the 377 378 CLIFFORD L. SLAYMAN total. The major fluxes, then, are metabolically dependent and presumably occur through distinct active transport systems, or pumps. Numerous ion pumps have been identified among the microorganisms: for both basic and acidic amino acids (Grenson, et al., 1966; Frank and Hopkins, 1969; Pall, 1969); for monovalent anions such as chloride and bicarbonate (MacRobbie, 1964, 1965; Hope, 1965; Raven, 1968); for sulfate and phosphate (Dreyfuss and Pardee, 1966; Borst-Pauwels, et al., 1965; Rothstein, 1963; Weiden, et al., 1967); for divalent cations (Fuhrmann and Rothstein, 1968; and, of course, for the monovalent cations potassium, sodium, and hydrogen ions. Several authors have pointed out previously {e.g., Rothstein, 1964) that specific accumulative pumps together with small diffusion fluxes are necessary for an organism to exist economically in very dilute media, where concentration ratios (C,/Co) of 103 to 105 must be maintained. Of the many different ion-transport systems that have been examined in microorganisms, I have chosen only one to review in the present discussion, one whose role is currently the subject of much debate and speculation: the hydrogen ion efflux system. Most plants and microorganisms produce excess organic acids (Schultz, et al., 1963; Zarlengo and Abrams, 1963; Ranson, 1965) either for storage or for secretion, and in some cases acids released into the medium can reach 0.02-0.2 M (Conway and O'Malley, 1946; Kempner, 1966), the same range as observed for acid secretion by the gastric mucosa. It is not clear, in a teleologic sense, why many organisms do this, but it is clear that they expend considerable metabolic energy in overcoming both chemical and electrical gradients. In some cases there is also evidence that die secretion of H+ is directly involved in producing the electrical gradients. The acidsecreting system is then said to be "electrogenie," meaning that it is capable of extruding hydrogen ions without chemical coupling to anions or to the counter-movement of other cations. In what follows, I shall try to indicate how the electrogenic pumping of hydrogen ions by the algae, fungi, and bacteria could account for a number of otherwise perplexing and incongruous phenomena. CLASSICAL DESCRIPTION OF ALGAL MEMBRANE POTENTIALS A full description of ion transport in any system requires many different kinds of information for each ionic species concerned: measurements of both the electrical and chemical gradients; measurements of net fluxes and of separate unidirectional fluxes; and attention as well to the movements of any other substances, particularly water, to which ion fluxes may be coupled. The small size of most microorganisms makes some of this information, particularly that on the electrical gradients, very difficult to obtain. Hence, it is only for a family of "giant" algae known as the Characeae that anything resembling a full description is available. The Characeae are represented most familiarly in the laboratory by Nitella, a fresh-water form which grows in stalks, with branch points or "nodes" at intervals of several centimeters. Ordinarily, the entire distance between nodes is occupied by a single cell which is, therefore, several centimeters long and may be as much as a millimeter in diameter. It is this internodal cell which is the subject for most of the studies on ion transport in algae. In many ways it is an ideal cell. Fluid-filled microcapillary electrodes can readily be inserted to measure the difference in voltage between the cell's interior and a macroelectrode in the culture medium. The large size of the cells also permits uptake or loss of radioisotopes to be measured from single cells—a feat which is not yet possible with the bacteria or fungi. And finally, the chemical composition of a single cell can be determined on droplets blown or squeezed from the cut end of the cell (MacRobbie, 1966). There are, naturally, some complications. One minor one is that Characean internodal cells are excitable; when electrically depolarized or when damaged they show reversible changes in membrane permeability and voltage which resemble the action potential of nerves (though on a much slower time scale). Careful handling of the cells, therefore, is necessary in any study of the resting electrical characteristics. A second and more serious complication arises because of the cell wall, which acts both as an unstirred layer and as a 379 MOVEMENT OF IONS AND ELECTROGENESIS TABLE 1. Distributions of Ions tn Nilella Internal concentrations Ions K Na Cl Em Equilibrium potentials Cytoplasm (mM) Vacuole (mM) cones. (mM) Plasma memb. (mV) 119 i t 3 14-t-2 65 ± 3 75-1-2 65-1-2 160-1- 3 0.1 1.0 1.3 —178 — 66 + 99 —138-1-2 Tonoplast (mV) + 12 —39 +23 + 18-1- 1 Results summarised from Spanswick and Williams, 1964. Concentrations and voltages given as Mean •+- 1 S.E. sodium (EXa), and —— j 99 mV for chloride (E,;.,).1 From these figures we must conclude that sodium is excluded by the cells, and both potassium and chloride are accumulated, the latter very strongly. The results can be taken as presumptive, though not airtight, evidence that sodium is actively transported outward and that potassium and chloride are actively transported inward across the plasma membrane. Whether there is also active transport across the tonoplast is an unsettled quesThe resting potential difference (ECJ.t) tion. As can be seen from Table 1 (column between the cytoplasm of Characeae and 6), the equilibrium diffusion potentials the external medium, and that (Evat.) be- for both potassium and chloride are within tween the vacuole and the medium vary a few millivolts of the observed potential somewhat with species (Stolarek, 1968) difference across the tonoplast. It is posand with conditions (Findlay, et ah, sible, because of uncertainties in the mea1969). But for the common species, N. surements of both the ionic concentrations Iranslucens, in an artificial pond water and the difference in voltage across the containing 1.0 mM NaCl + O.I mR RC1 + tonoplast, that potassium and chloride are 0.1 mM CaCL, Spanswick and Williams in fact at equilibrium. Sodium ions appear (1964) have given mean values of —138 to be concentrated in the vacuole, but the mV for Ecyt and —120 mV for Evac. The distribution of sodium between the cytolatter value is really the sum of two poten- plasm and the vacuolar fluid is still sometial differences, the larger one (—138 mV) what unsettled. It is clear in any case that across the plasma membrane, and a second the major gradients are developed across (_|_]8 mV) across the tonoplast. Ecyt is of the plasma membrane, not the tonoplast, such size that none of the three major and this presumably means that the major inorganic ions is close to its diffusion conversion of metabolic energy into the equilibrium across the plasma membrane. work of ionic transport takes place across Cytoplasmic concentrations of potassium, 1 principle, the chemical activity, rather than sodium, and chloride are given in Table 1 the Inconcentration, of all ions should be used to (column 2), from which calculated concen- compute equilibrium potentials. I have chosen not tration ratios (Ccyt/C0) are 1190, 14, and to use activities for three reasons: (1) they have 50, respectively. The Nernst equation then not generally been determined in cell cytoplasm; (2) most resulting corrections would be small and, gives equilibrium diffusion potentials of consequently, (3) would not simplify interpretation — 178 mV for potassium (EK), —66 mV for of any phenomena to be presented. Donnan regime having fixed negative charges. The wall can distort apparent fluxes and transmembrane voltages, particularly during transients, and its presence needs to be considered in the interpretation of most flux and electrical measurements. A third complication is that the volume of the cell is largely taken up by a fluid vacuole, which is surrounded by only a very thin (2-5^) layer of cytoplasm. Each surface of the cytoplasm is delimited by a membrane: the (inner) "tonoplast" enclosing the vacuole, and the (outer) plasma membrane. Difficulties frequently arise in attributing given phenomena to the tonoplast or to the plasma membrane, but—as we shall show below—the plasma membrane is generally much more important than the tonoplast. 380 CLIFFORD L. SLAYMAN 0.05 -too Externol K + conc. (mM) 0.10 0.20 0.50 1 I I I 1.0 1 ' 6 -120 o -140 -160 FIG. 1. Effect of the external concentration o£ potassium on E c r t in Nitella translucens. Solutions contained KC1 -)- NaCl; potassium and sodium varied at constant ionic strength, with CK -f- C s , = 1.1 mM. The curve is drawn from Equation (1) with'C Kcj , t = 93 mM and C Nacyt = 37 mM; the least-squares estimate (Marquardt, 1963) of PN,/PK is 0.27. The straight line represents the equilibrium diffusion potential for potassium. Data averaged for 27 cells; standard error at each point, less than 2 mV. Modified from Spanswick, Stolareck, and Williams (1967). the plasma membrane. An important method for distinguishing those ionic gradients which contribute to membrane potentials from those which do not has been to examine the way in which voltage varies when the extracellular concentration of an ion is changed, under conditions where the internal concentration is stable. Such studies on Nitella and on another pond alga, Chara corallina (Hope and Walker, 1961), have indicated that diffusion of potassium makes the major contribution—as is to be expected from the value of EK already mentioned. Figure 1 shows the experimental relationship between the steady-state Eeyt and the external potassium concentration for internodal cells of Nitella bathed in zero-calcium solutions. Small changes in the concentration of potassium near 1 mM shift the potential in the manner predicted by the Nernst equation, i.e., 58 mV per log unit change in CK 0 . However, at lower concentrations, near 0.1 mM, the slope is much less steep. The data can be fitted quite adequately by supposing the membrane to have a finite permeability to sodium, according to the equation (Goldman, 1943; Hodgkin and Katz, 1949): ;= —In - -•• l_i_, (1) in which R, T, and F have their usual meaning, and PK, PNa represent the membrane permeability to potassium and sodium. The procedure for fitting Equation (1) to the data in Figure 1 yields an estimate of the permeability ratio: PN O /PK = 0.27. [Because Ecyt in Nitella and Chara is relatively insensitive to the anions of the external medium (Hope and Walker, 1961), PCI is generally assumed to be negligible (PCI/PK = 0.01).] Thus, in respect to the dependence of their membrane potentials on the extracellular levels of potassium and sodium ions, the algae behave very much like animal cells, though perhaps algal membranes discriminate less strongly between different cations and more strongly between cations and anions than is true for nerve and muscle. SOME ANOMALIES It has been known for many years (Hill and Osterhout, 1938) that the behavior shown in Figure 1 depends upon careful rinsing of the cells and on maintaining them in a zero-calcium solution. Small amounts of calcium added to the medium desensitize the membrane to potassium, so that variations in CKQ between 0.1 and 1 mM have practically no effect on the measured Ecyt or Evac. A recent demonstration of the calcium effect was given by Kitasato (1968), whose results are replotted in Figure 2; it can be seen that even at potassium concentrations as high as 100 mM the 381 MOVEMENT OF IONS AND ELECTROGENESIS External K+conc. (mM) 1.0 10 —i | 1 O.I "" (Goldman, 1943; Hodgkin and Katz, 1949): 100 r— 1— • -80 Jcyt^O ,, (2a) T Na / ° - (2b) / T ci o ro Pci = O iternal potential, A / / > 1-100 u 1 ,_ ci / / / -o 1 Jcyi (2c) c o • -140 / FIG. 2. Effect of potassium on Ev,c of Nitella clavata in the presence o£ calcium. Solutions contained KsSO, + 2 mM NaCl + 1 mM CaCl. + 1 mM MgSO4 -f- 0.2 mM Tris buffer, pH 5.3. The smooth curve is drawn through the averages for 2-3 determinations. The straight line gives the potassium equilibrium potential, with C Kcrt — 93 mM. Redrawn from Kitasato (1968). slope of voltage versus log (CK0) reaches only 35-40 mV, very significantly less than the Nernst slope. Another difficulty arises when (luxes of sodium, potassium, and chloride are measured and used to compute the ionic permeabilities and the overall membrane resistance. There is general agreement that steady-state unidirectional fluxes across the plasma membrane of Nitella are approximately as follows, at 25°C: K+, 1 pmole/sec; Na+, 0.5 pmole/sec; Cl~, 2 pmolcs/sec (all values are computed on the basis of one cm2 of membrane area; 1 pmole = 10~32 moles). Under steady-state conditions, influx and efflux must be equal, but—as has already been mentioned —active transport is involved in influx of potassium and chloride and in efflux of sodium. Hence, the diffusion equations can be applied only to efflux of potassium and chloride and to influx of sodium. By assuming the electrical field through the membrane to be constant, we can obtain the following three relationships for the membrane permeability to specific ions in which the J's are the influxes (0—»cyl) and effluxes (cyt—>0), and <j> == FEL.yt/RT. Using Ecvt = —138 mV, with the concentrations from Table 1 (converted to moles/ cm3) and the fluxes quoted just above (pmoles/cm2.sec), we arrive at the following values of permeability: P K = 3.7 X 10~7 cm/sec PNa = 0.91 X 10- 7 P01 = 0.056 X 10- 7 . The resulting permeability ratios are P Na /P K = 0.25 and P C i/P K = 0-015, nearly the same as those discussed in connection with Figure 1. [The agreement is probably fortuitous, since measurements of flux are routinely made on cells bathed in calciumcontaining solutions and should properly be compared with the results of Figure 2.] Membrane resistance, r, can now be computed by a third equation derived from the same theory. RT (3) ]-•- where Co = P K C K o + P x B o ^ and C lTt = P K C K J + Px.C x ." Tt + P C iC C y + Co and Coyt can be evaluated from the calculated P's, and r is found equal to 2.2 X 10"- ohm. cm2, or 220 kohm. cm2. Direct estimates of membrane resistance in the Characeae are made most simply by driving a known current (I) through the cell—e.g., between a micro-electrode placed in the vacuole and an external reference electrode (see e.g., Walker, 1960)-and re- CLIFFORD L. SLAYMAN 382 cording the resulting displacement of Evac (AE). Surface resistance is then calculated from Ohm's Law, modified to allow for some non-uniformity of the membrane current, which arises because of the cylindrical shape of the cells (Taylor, 1963). Values actually obtained with this procedure on Nitella vary considerably, depending on conditions, but overall averages from the literature lie near 15 kohms.cm2 for cells in zero-calcium solutions, and 45 kohms. cm2 for cells in the normal artificial pond water, containing 0.1-1.4 mM calcium. While calcium clearly produces an increase in surface resistance, even the higher figure is five-fold smaller than the 220 kohms.cm2 predicted from the ionic fluxes. Any correction which must be applied to the measurement of resistance because of the cell wall or tonoplast would increase, rather than decrease, the discrepancy. One likely interpretation of this result is that the flux measurements are somehow in error. Either potassium, sodium, and chloride are not the major current carrying ions, as has been suggested by Kitasaio (1968); or the apparent fluxes of these ions, measured with radioisotopcs, are badly underestimated because of backfluxes between the cell-wall envelope and the cell interior, as argued by Walker and Hope (1909). The latter interpretation remains essentially qualitative. It is surely correct in part, but that it could account for the entire discrepancy between measurements of flux and electrical measurements seems improbable, particularly since it does not deal with a number of metabolic effects that we shall discuss later. A ROLE FOR H + IONS The possibility that ions other than K+, Na+, or Cl~ might carry substantial current across algal membranes forces a search for the unidentified ions. The one additional ion whose extracellular concentration most strongly affects Ecyt or ETac in Nitella is H+ (Fig. 3). In the range of pH 6 to pH 4 the average slope of voltage vs. pH is 53 mV/pH unit for a medium con- -200 L FIG. 3. Influence of external pl-l on Evoc of Nilclla clavala. Solutions contained 0.05 mM KsSO, -|- 2 mM NaCl -f 1 mM CaCL + 1 mM MgSO4 + 0.2 mM Tris buffer. pH was adjusted by adding H0SO4. Solid line: equilibrium potential for H+, assuming pH cyt = 5.5. Dashed curve: calculated from Equation (1) wilh terms for H+ added (see text). CK^ Cs-.ic t> and I'SH/PR the same as in Figure 1: leastsquares estimates (Marquardt, 1963) of P H /PK. 1.72 X 10'. Solid curve: same as dashed curve, but shifted by —67 mV. Each point represents the average for 2-9 determinations; standard errors for pH 5.3 and below, 2-9 mV; for pH 6 and above, 20-27 mV. Data from Kitasato (1968). taining 1 mM calcium, so that the membrane appears considerably more sensitive to hydrogen ions than to potassium ions (compare Fig. 2). An argument (Walker and Hope, 1969) that the effect of pH on voltage is indirect, operating through altered permeabilities to potassium, sodium, or chloride, is unlikely, since the apparent efflux of potassium is independent of external pH as long as the membrane potential is held constant. This has been shown by a "voltage clamp" experiment, in which a micro-electrode is used to measure Evac and also to control a current generator; the current generator is in turn arranged to drive just enough current through the Nitella membrane to hold Evac at a predetermined value. Efflux of potassium is followed simultaneously with 42K. Results from one such experiment are given in Figure 4, with the upper portion showing 383 MOVEMENT OF IONS AND ELECTROGENESIS be approximately equal to— and e* = 0. Then, 1 FIG. 4. Influence o£ external pH on efflux of potassium and membrane current at constant voltage; Nitella clavata. Solutions as in Figure 3. Cell preloaded with *°K; Evac damped at —110 mV. Current supplied by the voltage clamp is expressed in pmoles/cm=.sec, for direct comparison with the K fluxes; it may be converted to ^amps/cm2 by the multiplying factor 0.0965. Inward current, negative; outward current, positive. Modified from Kitasato (1968). potassium efflux plotted against time, and the lower portion showing the clamp current required to maintain Evac at —110 mV. The measured K-efflux evidently does not change between pH 8 and pH 4, although the current changes by a factor of 10 or more. [Figure 4 further emphasizes the fact that membrane current needed to hold Evac at any specified value is very much larger (10-100 fold) than the potassium flux.] In a different kind of experiment, the apparent chloride efflux was found to vary only by a factor of 2 over the whole range of pH's and voltages plotted in Figure 3, so that from Equation (2c) there could be little change in PCI. Kitasato has proposed, therefore, that hydrogen ions themselves carry substantial current through algal membranes. If this interpretation is correct, then membranes of Nitella maintained in calciumcontaining media should have an H+ resistance (rn) of 57 kohms.cm2, which is electrically in parallel with 220 kohms.cm2 for the other ions combined (giving a total membrane resistance of 45 kohms.cm2; see above). P n can be calculated from an equation similar to (2b), but for H+ ions; as long as Ecyt is very negative, passive efflux of hydrogen ions will be small, r n will RT F2 > cyt> (4) For an external pH of 5.5, at which Ecvt = — 138 mV (compare Fig. 3 and Table 1). P H = 1.46 X 1°~ 3 cm/sec. This figure is more than three orders of magnitude larger than P K . [No physical explanation for such a large H+ permeability has yet been offered.] Even by taking into account the large permeability to hydrogen ions, however, it does not appear possible to describe the resting membrane potential of Nitella in terms of ionic diffusion. Computation from Equation (1), with terms PHCH O and PHCHtTt added, gives a curve which is identical in shape to that drawn through the data of Figure 3, but which is displaced 67 mV upward (positive), and is represented by the dotted line in Figure 3. The discrepancy is in part a reflection of the fact that the equilibrium potential for H+ (straight line, EH, in Fig. 3) is positive to the actual membrane potential at all pHo's tested. If hydrogen ions were to reach diffusion equilibrium, the internal pH would vary from 5 (at pH 0 = 8) to about 3 (at pH0's below 6). Hence, a powerful, active, transport process must operate to keep the pH within the cells high, or the H+ concentration low. Independent evidence of such a process has been presented by Spear, Barr, and Barr (1969), who estimated the net efflux of acid from Nitella to be 5-20 pmoles/ cm2.sec. Their technique, which employed the pH indicator, phenol red, identified discrete bands (0.5-0.8 mm wide) along the length of internodal cells which release acid, and alternating bands which do not. A parallel study of the depolarization or hyperpolarization resulting from pH shifts indicated that the entire surface, including both pumping and non-pumping bands, is sensitive to external pH. 384 CLIFFORD L. SLAYMAN At this point it becomes expedient to postulate that the hydrogen ion pump is in fact electrogenic, that it can produce a stream of hydrogen ions and thereby generate a potential difference across the cell membrane. The real membrane potential would then be expected to lie somewhere between the intrinsic EMF of the electrogenic pump and the diffusion potential generated by existing ionic gradients. As long as the resultant value of Ecyt is larger (more negative) than the diffusion potential for a particular cation (Ex), there will be a net driving force, Ecyt—Ex, moving the cation inward toward diffusion equilibrium. Thus, regardless of whether other ion-specific pumps exist, an electrogenic hydrogen-ion pump should act to concentrate cations and (by a similar argument) to extrude anions. Those ions which are most permanent will, of course, tend to reach equilibrium most rapidly. Under the conditions used to obtain the data in Figure 3, EK is approximately —178 mV (see Table I), which—allowing for a slight difference between the measured Evac and Ecyt—is very close to the probable limiting value of Ecvt at high extracellular pH's. METABOLIC EXPERIMENTS: THE FUNGUS Neurospora Satisfactory measurements of membrane potentials have been reported for only one microorganism other than the algae: the pink bread mold, Neurospora. And when the experiments are carried out under conditions comparable to those used with the pond algae, almost exactly parallel results are obtained. Although this organism does not approach the size of the Characeae, its filaments (hyphae) frequently reach 20 ^ in diameter, large enough for microelectrode studies. In certain other respects, Neurospora is simpler to study than the algae. It does not possess a large central vacuole, so that both microelectrode measurements and flux measurements deal with differences across the plasma membrane, without contributions from a tonoplast. The cell wall in this microorganism apparently does not have a high density of fixed negative charges, at least when the external pH is kept near or below G (Slayman and Slayman, 1970), which should remove much of the difficulty encountered in making accurate flux measurements on Nitella or Cham. Finally, there appears to be only one significant way for Neurospora to obtain metabolic energy, i.e., from mitochoiiclrial electron transfer coupled to oxidative phosphorylation. Like all fungi, Neurospora lacks chloroplasts and therefore the photophosphorylative pathways, and it cannot obtain sufficient energy from glycolysis to grow in the absence of oxygen (Denny, 1933). Table 2 shows the distributions of potassium and sodium, as well as the resting membrane potential, for hyphae bathed in zero-calcium medium containing 0.1 mM KC1 + 5 mM NaCl -f- 2% sucrose, pH 5.9. E(.yt is not far from EK and the membrane potential is also sensitive to variations of extracellular potassium, having a slope of 45 mV/log unit change of concentration in media containing only KC1 and sucrose (Fig. 5). Calcium greatly desensitizes the membrane to potassium, and addition of 0.1-1 mM calcium to the medium reduces the slope to 17 mV/log unit. The steady-state unidirectional potassium fluxes are approximately 1.2 pmoles/cm2. sec (Slayman and Tatum, 1965) in media containing 0.1 mM KC1. Calculations of the type discussed above lead to estimates of several parameters: PK = 1.6 X 10~6 cm/sec (Equation 2a), a permeability ratio P Na /P K = 0.2 (Slayman, 1965a) and a membrane resistance of 48 kohms.cm2 (Equation 3). The measured surface resistance of Neurospora is less than 10 kohms.cm2 (Slayman, 1965&). But another property of the resting membrane potential is particularly conspicuous in Neurospora: an extreme sensitivity to metabolic inhibitors. Figure 6 illustrates the effect of addition (up arrows) and washout (down arrows) of sodium TABLE 2. Distributions of Ions in Neurospora Ions K Na Internal External Equilibrium concentrations concentrations potentials (mM) (mM) (mV) 180 ± 3 0.13 5 —183 _ 26 —193 ± 4 Results summarized from Slayman and Tatum (1964) and Slayman (I965n). Results given as Mean •+• 1 S . E . 385 MOVEMENT OF IONS AND ELECTROGENESIS 01 -too 0.3 1.0 3 10 30 1 1 1 1 1 -120 - -140 KCI + s u c r o s e / ' -160 -180 200 - T _ - / ^ T.—-~ K C I + l m M C o C l z + sucrose -220 -240 -9cn FIG. 5. Effect o£ external potassium on E cyt of Neurospora, with and without calcium. Solutions contained KCI + 2% sucrose or KCI + 1 mM CaCl2 -f- 2% sucrose. Each point represents the average potential measured in 10-30 cells; standard error at each point, less than 2.5 mV. Slopes: without calcium, 45 mV/log unit; with calcium (right hand segment.) 17 inV/log unit. Data from Slayman (1965a). 10 0 i azide. Within one minute the inhibitor produces a shift of Ecyt from —227 mV to —44 mV (0.1 mM NaN3) or to —19 mV (1 mM NaN3). Washout produces a similarly rapid recovery, but is normally accompanied by a small oscillation of voltage before the steady level is re-established. Cycles of this kind can be repeated as long as the micro-electrode is held in a cell. Under conditions of rapid flow, the decay of voltage proved to be exponential with time, having a maximal rate-constant of 0.18 sec- 1 (Slayman, et al., 1970). This rapid voltage response to metabolic inhibitors—being unaccompanied by either a significant shift of membrane resistance (Slayman, 1965&) or a measurable decrease in the ionic content of the cells (Slayman and Tatum, 1965)—would not be expected for a membrane potential arising primarily from ionic diffusion; and it is not ordinarily observed in nerve and muscle (cf. Hodgkin and Keynes, 1955). Time (min) 15 I 20 25 I 30 -200 t 4 t Azide I.OmM Azide wash 0.1 mM FIG. 6. Metabolic dependence spora; voltage record from a tions contained 10 mM KCI -\sucrose -(- Cv saturated at 1 of E r r t in Neurosingle hypha. Solu1 mM CaCU -|- 2% aim: sodium azide added at up arrows (f), and washout begun at down arrows (\). The effect of 0.1 mM azide is slightly siibmaximal. 386 CLIFFORD L. SLAYMAN electrogenic ionic pump, fueled by ATP or a closely related substrate would account very well for these metabolic data on Neu- 3.L w 2 \ rospora. O 1 \ T 05 & •5?_, \ \ 2 • \ \ \ \ 1S1 0.2 V, V \ 01 \ i 0 i i 10 5 Time (sec) \ 15 \ No "— 10 20 30 Time (sec) FIG. 7. Rapid decay o£ intracellular ATP at the onset of inhibition by cyanide; liquid-cultured cells of Neurospora. Control medium contained 20 mM dimethylgluiaric acid (an inert buffer), pH 5.8 + 25 mM K (OH) + 1% glucose. The suspension was filtered rapidly, and buffer containing KCN was sucked through the cell mat. Mats were then frozen at intervals in liquid nitrogen, lyophilized, extracted in perchloric acid, and analyzed with rue-fly hiciferase. Data: O, 1 mM KCN; • . 10 mM KCN. Dashed curve redrawn from the straight line in the semilogarithmic plot (inset). Rate conslant for ATP decay: 0.18 sec"1; half-time: 3.7 ziz 0.2 sec. Redrawn from Slayman, Lu, and Shane (1970). That the effect of metabolic inhibitors (cyanide, carbon monoxide, dinitrophenol, anoxia, and low temperature, as well as azide) is not spurious or unrelated to the effects on metabolism is indicated by a very close relationship between measured levels of ATP in Neurospora and the value of Ecyt. Experiments carried out in parallel with the azide-voltage experiments, but on liquid cultures of Neurospora, revealed a normal ATP level of 2.5-3.0 mM, which falls rapidly to 0.3 mM following addition of azide or cyanide (Fig. 7). Here, too, the most rapid decay is exponential with time, having a rate constant of 0.18 sec"1, as computed from the semilogarithmic plot in the inset of Figure 7. Thus, the voltage/time curve (Fig. 6) is superimposable on the ATP/time curve; the rate-constant for both processes corresponds to a half-time of 3.7 seconds. An ]f the potential does indeed arise from an electrogenic ionic pump, the sign (cell interior negative) would demand the active extrusion of a cation or the active uptake of an anion. Identification of the process in Neurospora with hydrogen-ion ejection rests on two kinds of evidence. First, the membrane potential seems nearly independent of the influxes of anions or the effluxes of cations other than hydrogen. Anionic influxes in Neurospora appear to be small, generally about 10% of potassium flux at equivalent extracellular concentrations (Slayman and Slayman, 1968); furthermore, as is shown in Table 3, the resting membrane potential is indifferent to the kind of anion in the external medium, whether chloride, nitrate, sulfate, phosphate, bicarbonate, or dimethylglutarate (chosen as an inert buffer). It is not likely, then, that an electrogenic uptake system for any of these anions is important. While external cationic concentrations do influence the resting membrane potential, the rapid active extrusion of sodium ions which accompanies addition of potassium to low-K, sodium-loaded cells is not associated with any significant increase of membrane potential. Thus, (Table 3) E cyt in cells containing 42 mM K+ and 138 mM Na+ averages —165 mV under steady state conditions in 10 mM NaCl -f- 1 mM CaCIo, and —170 mV within one minute after switching to 10 mM KC1 -f- 1 mM CaCl2. Sodium extrusion at this time can exceed 10 pmoles/cm2.sec. Furthermore, internal Na+ can be reduced to less than 2% of its normal concentration without any effect on Ecyt. Evidently, no electrogenic sodium transport system—of the kind postulated for some rapidly pumping nerve and muscle tissues (Thomas, 1969; Rang and Ritchie, 1968; Adrian and Slayman, 1966)—can play a major role in Neurospora. The second kind of evidence is that net 387 MOVEMENT OF IONS AND ELECTROGENESIS TABLE 3. Lack of effect of anionic substitutions and NajK pumping on membrane in Neurospora Composition of cells Ion tested Control cond itions Medium E c ,, (mM) (mV) Normal Normal Normal NO3" SOr H.POr 25 NaCl + 37 KC1 10KC1 25 NaCl + 37 KC1 Normal Normal DMG HCO 3 - Normal Low-Na Lou'-K Na* 10 KC1 10 K + 6.7 phosphate + 1 CaCI.,, pH 6.9 10 KC1+ 1 CaCI, 10 KC1 4- 1 CaCI, 10 NaCl 4- 1 CaCla Testing conditions Eerl Medium (mM) (mV) —126 ± 4 25 NaNOj 4- 37 KNO3 —145 ± 9 SK^SO, —126 ± 4 25 Na 4- 37 K 4- phosphate at pH5.9 —141 ± 6 10 K 4- 8DMG, pH5.8 —217 ± 6 10 KHCO3 4- 1 CaCl2 45% CCX, pH 7.0 —181 ± 4 —174 i t 3 — 165 ± 7 potential —125 i t 3 —149 ± 6 — 128 ± 7 —131 ± 3 —212 it 6 10 NaCl 4- 1 CaCI, —209 ± 4 10 KC1 4- 1 CaCU —170 ± 7 All media contained, in addition to the ingredients listed, 2% sucrose or 1% glucose. Sodium in the lou-Na cells was estimated at 0.2 mM; potassium in the low-K cells, at 42 mM. Voltages are given as Mean ± S.E. efllux of hydrogen ions has indeed been found to vary in parallel with the membrane potential under a variety of conditions. Efllux of hydrogen ions has been estimated from pH changes produced by suspensions of liquid-cultured cells in a standard, calibrated buffer solution (Slayman and Slayman, 1970). The technique is similar to that used for study of bacteria and mitochondria (Pressman, 1967), and has yielded normal steady-state fluxes of 5-30 pmoles/cm2.sec, the same range as observed by Spear, Barr, and Barr (1969) on Nitella. More importantly, partial inhibition produced by submaximal concentration of cyanide or azide or by lowered temperature gives roughly proportional changes in membrane potential and net efllux of H+ (Slayman, unpublished). Conspicuous transients of membrane potential, such as the overshoot associated with relief from azide inhibition (see Fig. G) or anoxia are also mimicked by H + efllux (Fig- 8) . Both the peak voltage (—240 mV) and the peak H+ efflux (15.5 pmoles/cm2.sec) are reached 20 seconds following readmission of oxygen to previously anoxic cells. Thereafter, both voltage and H+ efflux show a dip and gradual rise toward the final steady level. A precise match between the time courses for the slower events has not been obtained, perhaps partly because the measurements can be made only in parallel experiments and partly because the resistance of the membrane changes significantly during the recovery phase. UNCOUPLING AGENTS: EFFECTS ON IONIC TRANSPORT 7N BACTERIA I have already mentioned the fact that bacteria, as well as the algae and fungi, can release substantial amounts of acid into the medium. The questions of whether this release of acid is active—in the sense of requiring ATP utilization—and of whether it is also electrogenic have not yet been approached directly. But a certain amount of indirect evidence favoring an electrogenic H+ pump has appeared, mainly from studies with agents which are traditionally known to uncouple oxiclative phosphorylation. The organism which has been studied most extensively in these experiments is the Gram-positive species, Streptococcus faecalis. The particular feature of S. faecalis which makes it useful in the present context is that it lacks the cytochrome system for respiration (Smith, 1961), and obtains nearly all of its energy from glycolysis (Smith and Sherman, 1942). In the presence of glucose it maintains an internal concentration of ATP of ,V7 mM (Forrest, 1965; Harold and Baarda, 1968), with quantitative conversion of glucose to lactic acid (Smith and Sherman, 1942). Lactate and hydrogen ions are released into the medium at a rate of 50-70 mmoles/kg cell water. 388 CLIFFORD L. SLAYMAN 16 14 o d) 12 V) CM E o 10 Anoxic o 8 3 X 3 0 -2 2 -100 _ 4 Time 6 8 10 (min) -120 E 1 -140 u UJ _- -160 ^o -180 | -200 - -220 -240 -260 FIG. 8. Comparison o£ the time courses for membrane voltage and net H+ efflux following a period of anoxia; Neurospora. Solution: 4 mM dimethylglutaric acid, pH 5.8 + mM K(OH) -f 20 mM KC1 + mM CaCl2 + 1% glucose. pH recorded con- tinuously from stirring suspension of liquid-cultured cells in a closed chamber: H* efflux calculated from & pH. Record of ECJ.t obtained from agarcultured hypha, MOVEMENT OF IONS AND ELECTROGENESIS min (Zarlengo and Abrams, 1963; Zarlengo and Schulu, 1966), or 20-30 pmoles/cm^sec (1.6M spheres; Luria, 1960), again in the same range as observed with Nitella and Neurospora. In addition to this steady-state release o£ H*, low-K cells show a large increment of H* release upon being returned to K-containing media. In this process the ratio, K.+ influx: H* efflux: ATP utilized, appears to be 1:1:1 (Zarlengo and Schultz, 1966). or the steady-state levels of ATP (Harold and Baarda, 1968). Yet all five block or greatly reduce movements of ions into and out of the organism. Figure 9 shows the effect of 6 X 1°"° M TCS on potassium uptake by low-K cells. Both the initial rate of entry of potassium and the apparent steady level, CKcyt, are reduced approximately sixOn the basis of the fact that 5. faecalis fold. Exit of sodium (from sodium-loaded generates ATP only by glycolysis, one cells), entry of rubidium, entry of phoswould expect uncouplers of oxidative phate, and even uptake of amino acids phosphorylation to have no effect on ener- (alanine) are similarly blocked by uncougy metabolism, either the synthesis of ATP pling agents (Harold and Baarda, 1968; or its utilization for ion transport. A vari- Harold, et al., 1970). ety of uncoupling agents has been tested: Many, though certainly not all, of these dicumarol, pentachlorophenol, tetrametheffects might be accounted for—as sugyldipicrylamine, carbonylcyanide m-chlorogested by Harold and his coworkers—by phenylhydrazone (CCCP), and 3,5,3',4'supposing that (1) an electrogenic cationic tetrachlorosalicylanilide (TCS). As expectpump, capable of extruding either hydroed, these inhibitors do not substantially gen ions or sodium ions (in Na-loaded affect either synthesis of ATP in S. faecalis cells), normally keeps Ecvt at a large negative value; and (2) uncoupling agents produce depolarization by making the mem400 brane highly permeable to hydrogen ions. Control This mode of action, originally postulated to explain the effects of dinitrophenol on mitochondria (Mitchell and Moyle, 1967), is supported by independent evidence on 300 artificial phospholipid membranes, whose electrical resistance has been shown to diminish 10-1000 fold in the presence of low concentrations of 2,4-dinitrophenol or 200 CCCP (Hopfer, et al., 1968). At the same time the membranes become selective for hydrogen ions, displaying membrane potentials as high as 54 mV for each 10-fold difference in H+ concentration imposed t 100 across the membranes. TCS 10 20 30 Time (min) FIG. 9. Effect of an uncoupling agent (3,5,3', 4'-tetrachlorosalicylanilide) on K+ uptake by Kdepleted, Na-loaded Streptococcus faecalis. Solutions contained 10 mM NasSO, -f- glucose; pH stat at 7.5; 2 mM K+ added at 0-min. Potassium uptake was 90% balanced by sodium loss. Upper curve: control; lower curve: 6 x 10~° M TCS. Redrawn from Harold, Baarda, and Pavlasova (1970). In keeping with the results on artificial membranes, Harold, Pavlasova, and Baarda (1970) have found that S. faecalis treated with TCS loses its ability to maintain a pH gradient between the cytoplasm (normally alkaline) and the external medium. Presumably this occurs because the leak to hydrogen ions is increased sufficiently so that most of the electrogenic H+ efflux is carried passively back into the cells, along the electrochemical gradient. The results in Figure 9, then, could be 390 CLIFFORD L. SLAYMAN interpreted to mean that 5/6 of the normal inward potassium current is replaced by H+ in the presence of TCS. CONCLUSIONS Jt should be evident, from the results I have presented above, that the electrogenic ion pump is an emerging hypothesis rather than a proven fact. This is true for ionic transport systems in other kinds of organisms, just as it is for hydrogen ion transport in microorganisms, indeed, only within the past six or eight years has a need to modify the classical ion-diffusion concept of biological membrane potentials been generally accepted. Evidence has been accumulating for a much longer period of time, but the great success of the ionic hypothesis in describing both resting membrane potentials and action potentials in nerve and muscle tended to override the few disparate observations. This was particularly true because the signs of electrogenic ion pumping are present in nerve and muscle (Thomas, 1969; Rang and Ritchie, 1968; Adrian and Slayman, 1966) only transiently, following periods of extended ionic depletion, so that they could in principle be accounted for by transient changes in permeability, unstirred layer effects, and coupling of ion flows to movements of other substances. However, the electrical behavior of the microorganisms, as well as of certain kinds of animal secretory epithelia [especially the gastric mucosa (Rehin, 1966) ], differs from that of nerve and muscle in showing large, metabolically dependent potential differences under apparent steady-state conditions. The same systems also carry out more conspicuous steady-state active transport of ions than do nerve or muscle, so that it is natural to ask whether the electrical behavior is directly related to any of the active transport processes. The probable existence of an electrogenic hydrogen-ion pump among the microorganisms raises the teleological question of what its purpose might be. From the point of view of ridding a cell of excess H+ alone, the pump would be very inefficient; the membrane's large passive permeability would allow the difference in voltage to drive extruded hydrogen ions back into the cell. [In nerve and muscle only relatively impermeant ions such as sodium are presently thought to be electrogenically pumped.] Of course, diffusion of hydrogen ions does not account for all of the passive movements of ions through algal or fungal membranes, which have finite (if smaller) permeabilities to potassium, sodium, and chloride ions, and perhaps to organic anions as well. A possible role of the H+ pump in maintaining CK,.,.* near its normal steady-state value has already been mentioned. In a similar manner, the potential produced by the pump should serve to exclude inorganic anions or to extrude metabolic, organic acid anions. No direct measurements of permeability of the membrane to glycolytic anions or anions of the Krebs cycle have yet been made, but it is clear that under many circumstances the net efflux of H+ is balanced by the net efflux of these anions (Zarlengo and Schlutz, 1966; Conway and Downey, 1950). In the realm of more frank speculation, it is conceivable that a high resting membrane potential is essential to the functional integrity of the plasma membrane: either by controlling the selective permeability of the membrane, or perhaps by influencing orientations or conformations of protein within the membrane and thereby controlling enzymatic activity. We now know at least 25 different iontransport systems in which electrogenic pumping has been postulated. Some of these may well prove spurious, but it is becoming increasingly plausible that separation of charge (i.e., electrogenesis) may be an inherent, fundamental property of active ion-transport mechanisms. If this is indeed the case, then the electrogenic hydrogen-ion pumps among the microorganisms should be of special interest, serving as model systems to which the powerful techniques of biochemical and genetic analysis can be applied much more effectively MOVEMENT OF IONS AND ELECTROGENESIS 391 Harold, F. M., E. 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