Download Movement of Ions and Electrogenesis in Microorganisms

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. Pavlasova, and J. R. Baarda.
1970. A transmembrane pH gradient in Streptococcus faecalis: origin, and dissipation by proton
conductors and N,N'-dic)clohexylcarbodiimide.
Biochim. Biophys. Acta 196:235-244.
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