Electrophysiology of plasma membrane vesicles

WRIGHT, ERNEST M. Electrophysiology of plasma membrane vesicles. Am.

J. Physiol. 246 (Renal Fluid Electrolyte Physiol. 15): F363-F372, 1984.-
In both renal and gastrointestinal physiology, it has become popular to
study epithelial transport phenomena using vesicles isolated from the apical
and basolateral cell membranes. Transport in vesicle preparations is usually
monitored with radioactive tracers, but more recently attention has been
directed to electrophysiological methods. As it is impossible to measure the
electrical properties of membranes in small vesicles (~500 nm diam) with
classical electrophysiological techniques, indirect methods have to be em-
ployed. In this review I focus on the application of voltage-sensitive optical
probes to measure membrane potentials in brush border membrane vesicles.
Optical signals are calibrated with diffusion potentials generated with
known ion gradients in the presence of ionophores, e.g., EKs with K
gradients in the presence of valinomycin. Membrane potential measure-
ments can be used 1) to illustrate the specificity and kinetics of sugar-,
amino acid-, and carboxylic acid-Na cotransport systems in brush border
membranes, and 2) to determine the ion permeability of brush border
membranes. All organic solutes known to be transported by Na cotransport
across brush border membranes depolarize the membrane in a Na-depend-
ent, saturable manner. The results agree, both qualitatively and quantita-
tively, with electrophysiological data obtained in the intact renal tubule
and with tracer uptake in vesicles. Bi-ionic potential measurements dem-
onstrate that brush border membranes are permselective to anions and
cations, but there are indications that the permeabilities are somewhat
dependent on the method of vesicle preparation and the experimental
conditions. However, electrical potential measurements provide insight into
the mechanisms of ion transport in vesicle preparations, and the application
of patch-clamp techniques should provide further gains in the future.
brush border membrane vesicles; Na cotransport; sugars and amino acids;
carboxylates; ion permea .bility; membrane potentia 1s; voltage-sensitive dyes
PLASMA MEMBRANE VESICLES have hadamajorimpact
on the study of epithelial transport since they were first
introduced by Hopfer over a decade ago (see Refs. 16 and
25). Vesicles have been employed to great advantage in
the study of gastric acid secretion and the absorption of
amino acids, sugars, metabolic intermediates, sodium,
phosphate, and calcium in the intestine and the proximal
renal tubule. Although the vast majority of studies re-
ported so .far have employed radioactive tracers to ex-
amine the kinetics of solute transport, it has become
obvious that it is essential to measure and/or control the
electrical potential difference (PD) across vesicular
membranes. Membrane potential is an important driving
force in the transport of ions, amino acids, metabolic
intermediates, and even in the transport of neutral mol-
ecules by electrogenic processes. Classical electrophys-
iological techniques cannot be used to monitor mem-
brane potentials in vesicles, and it is necessary to employ
indirect techniques. In this Editorial Review I discuss
the application of voltage-sensitive dyes to recording
membrane potentials in vesicles. Two topics are empha-
sized: electrical properties of Na cotransport systems and
ion permeability.
Voltage-Sensitive Dyes
Optical techniques for recording membrane potentials
have been developed over the last decade. In the early
1970s Cohen and his collaborators initiated a search for
extrinsic probes of membrane potential (see Refs. 1, 5,
and 7). The probes developed are divided into two classes:
the fast-response and the slow-response dyes. The fast-
response dyes respond to changes in membrane potential
in less than milliseconds, and their application has been
directed at recording action potentials in excitable tis-
sues. The slow dyes respond in seconds, and their major
advantage is that they show large absorbance (Ab) and
fluorescence (F) changes with changes in membrane
potential (0.5l%/mV). Hoffman and Laris (10) first
used cyanine days to record the membrane potential of
0363-6127/84 $1.50 Copyright 0 1984 the American Physiological Society F363
F364
3oc
A
DEPOLARIZATION
HYPERPOLARIZATION
2s
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ii IS-
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ii 0 /
Ir. lo-
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-2o- 0
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1 1 1 1 4 b 1 I 1 1 4
0 0.2 0.4 0.6 0.8 I.0 0 -0.2 -0.4 -0.6 -0.8 -1.0
Log [K+lo/IKli Log [K+lJ Wli
E. M. WRIGHT
FIG. 1. Effect of valinomycin-induced K+ diffusion
potentials on fluorescence of diS-C&5). A: effect of
depolarizing conditions on dye fluorescence. Brush
border vesicles equilibrated in 15 mM KCl, 135 mM
choline chloride, 1 mM Tris-HEPES, pH 7.5, were
added to dye solutions containing 15-100 mM KC1
(isosmolarity maintained at 150 mM with choline
chloride), 1 mM Tris-HEPES, pH 7.5, 2 PM diS-Ca-
(5), and 2 PM valinomycin. Final membrane protein
concentration was 0.19 mg/ml. Resulting fluorescence
of membrane-dye suspension was monitored for 1 min,
and zero-time fluorescence of suspension was esti-
mated by extrapolation of fluorescence record to time
zero. Zero fluorescence was arbitrarily chosen as the
fluorescence determined when intra- and extrave-
sicular K+ concentrations were equal. Ten arbitrary
units represent a change in total fluorescence of 10%.
B: effect of hyperpolarizing conditions on fluores-
cence. Conditions as in A except that vesicles were
preequilibrated in 100 mM KC1 and 50 mM choline
chloride and were placed in solutions with KC1 con-
centrations ranging from 10 to 100 mM. Final mem-
brane protein concentration was 0.17 mg/ml. From
Wright et al. (31).
red blood cells, and these slow dyes were subsequently
applied to such subcellular organelles as synaptosomes
and renal brush border membranes. Among important
reviews of the characterizations of optical signals, signal
size, mechanism, difficulties, methods, and applications
are Refs. 5 and 7. One practical starting point for the use
of dyes is the review by Bashford and Smith (1) that
provides advice about the choice of dyes, their source,
storage, fluorescence and absorbance instrumentation,
choice of wavelengths, calibration of signals, artifacts,
and the pharmacological effects of the dyes.
The cyanine dyes have been the most popular in stud-
ies with membrane vesicles mainly because of their large
signals (in the range 0.3-0.6% AF/mV) and their earlier
success in red blood cell studies. Useful dyes include 3,3-
dipropylthiadicarbocyanine iodide [diS-Ca-(5)], 3,3-di-
ethylthiadicarbocyanine iodide [diS-C&)], and 3,3-di-
ethyloxadicarbocyanine iodide [diO-Cz-(5)]. These and
other probes can now be obtained commercially. Spec-
trophotometric measurements of cyanine dye signals are
made in either the absorbance or fluorescence mode in
commercially available instruments. We have recorded
fluorescence signals of diS-Cs-(5) from intestinal and
renal brush border membranes in an Aminco spectro-
photofluorometer (model SPF 500) with the excitation
wavelength set at 620 nm and the emission wavelength
at 669 nm (23, 31). The dye concentration was in the
range 1.5-3 PM, the protein 0.2-0.3 mg/ml, and the ionic
strength of the solutions 0.11 M. Absorption of the dye
to the cuvette is minimized by the use of polystyrene
cuvettes. More recently we monitored the absorbance
response of this dye in a dual-wavelength spectropho-
tometer (Aminco DW2 UV/VIS) as described by Salama
et al. (18) for chromaffin granules (see also Ref. 12)?
Stieger et al. (26) report that the fluorescence sensitivity of diS-
C&(5) is too low for the measurement of membrane potential changes
in rat small intestinal membrane vesicles in the absence of La3+. In our
laboratory, diS-C3-(5) is satisfactory with rabbit intestinal brush border
vesicles.
Calibration of Optical Signals
In large cells (e.g., squid axons) and bilayers it is a
simple procedure to obtain direct calibration of the op-
tical responses of dyes using straightforward electro-
physiological techniques. In vesicles, which are smaller
than the tip of a microelectrode (diam 100-400 nm), this
is impossible, and therefore it is necessary to rely on
indirect methods. The most popular method is to cali-
brate dyes with K diffusion potentials. The approach is
to preload the vesicles with solution containing a known
K concentration and then record the level of the fluores-
cence or absorbance signal when the vesicles are added
to cuvettes containing a wide range of K concentrations.
Valinomycin, the K ionophore, is added to ensure the
development of K equilibrium potentials, i.e., E, = Ek
= RT/F In K,/Ki, where E, is the membrane potential
(intravesicular space relative to the outside solution), Ek
is the K equilibrium potential, K, is the external K
concentration (activity), and Ki is the intravesicular K
concentration. Since the dye signals may vary with ionic
strength and pH, the ionic strength of the internal and
external solutions is maintained constant with imper-
meant ions and the solutions are well buffered.
A calibration curve for DiS-C&-(5) in brush borders
using K plus valinomycin diffusion potentials is shown
in Fig. 1. In both the hyperpolarizing and depolarizing
directions there is a linear relationship between fluores-
cence and Ek: ~0.3% AF/mV in the hyperpolarizing
direction and -0.5% AF/mV in the depolarizing direc-
tion. Similar curves have been reported for DiS-C&-(5) in
renal brush border membranes (Fig. 1 of Ref. 2), and for
DiO-C&-(5) renal brush border and basolateral mem-
branes (Fig. 3 of Ref. 12). A problem, indicated in the
legend to Fig. 1, is the fact that the K diffusion potentials
are unstable in KC1 solutions. In these experiments it
was necessary to extrapolate the fluorescence record to
time zero. This is because the chloride permeability
(PcJ # 0, and it can be avoided by replacing Cl with the
impermeant gluconate or isethionate (see Fig. 6 of Ref.
EDITORIAL REVIEW F365
18 and Fig. 1 of Ref. 9).
Alternative protocols for the calibration of dyes in-
clude the use of H diffusion potentials (Figs. 5 and 15 of
Ref. 18 and Fig. 4 of Ref. 32) and Na diffusion potentials
(Fig. 2 of Ref. 9). In intestinal, renal, and choroid plexus
brush borders we have found that the calibration of the
DiS-Q-(5) signals with diffusion potentials was inde-
pendent of both the ion and the ionophore used to
generate the potential.
Na-Cotransport Systems
It is well known that sugars and amino acids are
actively transported across the epithelial cells of the
small intestine and proximal renal tubule. The first stage
of absorption is the uphill transport of the solutes into
the epithelium across the brush border1 membrane via
Na cotransport systems, and the second is the downhill
diffusion of the -solutes from the epithelium into the
blood across the basolateral membranes. Transport
across both the brush border and basolateral membranes
has been extensively examined using membrane vesicles
(see Refs. 16 and 25 for reviews). Sugar and amino acid
transport generate electrical potentials across the epithe-
lia; these originate with the Na cotransport across the
brush border-membrane (see Schell et at (23) for refer-
ences). In brush border vesicles, sugar and amino acid
uptake are sensitive to the membrane potential, and it is
therefore concluded that the transporters are electro-
genie.
rr
* L-PHE
D-PHE
NO
+
L-PHE
K
+
I I
1
i
1
30 set
20
FIG. 2. Effect of L- and D-phenylalanine on fluorescence of diSCa-
(5) in renal brush border membranes. In each experiment 0.6 mg protein
was added to 3 ml buffer containing 100 mM NaCl (or 100 mM KCl),
100 mM mannitol, 10 mM Tris-HEPES, pH 7.5, and 1.5 PM DiS-&-
(5). Fluorescence signal was obtained by activating at 620 nm and
recording the emission at 669 nm at a bandwidth of 2 nm. A: addition
of L- andD-phenylalanine (7 mM) 1 min after start of experiment: B:
effect of L-phenylalanine when uptake buffer contained either 100 mM
NaCl or 100 mM KCl. Arrow marks the time amino acid was added to
membrane suspension. From Schell et al. (23).
T
0.002A
FIG. 3. Depolarization of renal brush border membrane vesicles by
L-proline. Absorbance signal of DiS-C&-(5) was recorded at 655 vs. 685
nm in a dual-wavelength spectrophotometer. Base line was recorded
for 1 min in presence of 100 mM NaCl, LiCl, or KCl, and L-proline
(5.4 mM) was added to cuvette by a mixing device in less than 0.2 s.
Dye concentration was 2 PM and membrane protein 200 pg/ml. Note
that in the presence of both NaCl and LiCl proline produced a transient
depolarization of membrane potential of 8-10 mV, which reached a
peak in 5 s. (Schell and Wright, unpublished data.)
Amino acids. Figure
brane voltage (diS-Cs-
2 illustrates the changes in mem-
(5) fluorescence) associated with
Na-coupled amino acid transport in renal brush borders.
In both experiments shown, the base-line fluorescence
was recorded in the absence of the amino acid for 1 min
in either 100 mM NaCl or 100 mM KCl, and then either
L- or D-phenylalanine (7 mM) was added to the cuvettes.
In the presence of a N
produced an immediate
aC1 gradien
increase in
.t, L-phenylalanine
fluorescence (-10
units), which corresponds to a membrane depolarization
of -20 mV. No depolarization was observed with D-
phenylalanine in either NaCl or KC1 (not shown). The
Na-dependent depolarization produced by L-phenylala-
nine was transient, presumably due to the dissipation of
the Na and phenylalanine gradients across the mem-
brane. The initial phase of the E, changes is not seen in
Fig. 2 owing to the time required to add the substrate to
the cuvette (-15 s). However, the initial time course of
the depolarization can be readily observed using a rapid-
mixing device (Fig. 3). In the presence of an initial 100
mM NaCl gradient, 5.4 mM r.,-proline produced a maxi-
mum depolarization in 5 s with a half time of -1 s. This
slow depolarization is not due to instrumentation or
dye problems, since control experiments with K plus
valinomycin diffusion potentials indicated that the dye
responds fully with the response of the recorder (0.25 s
for full-scale response). In the intact rat proximal tubule,
Fromter (8) reported that sugars and amino acids depo-
larized the cell with rise times (lo-90%) of 90-260 ms.
Accordingly, the response in vesicles at 22C is slow
relative to that in the intact epithelium at 37C. The
reason for this discrepancy is unclear.
Although no depolarization was produced by proline
in KCl, the signal in LiCl was comparable, but smaller,
than in NaCl. This was the case with all amino acids
tested (L-proline, L-phenylalanine, and L-alanine).
F366
In rabbit brush border
the e ffect 0 f 17 amino aci
membranes we have examined
.ds and determined the concen-
4
tration dependence of the potential change. In all cases
the concentration dependence can be fitted to an expres-
sion of the type AF = (AF,,, x [S])/(K, + S) where
AFm,, is the maximum Na-dependent AF change, [S] is
the substrate concentration, and K, is the concentration
of S producing 0.5 AF,,,. For L-phenylalanine the J&
was 0.76 mM, and the range for aliphatic, hydroxyamino,
dicarboxylic, basic, imino, aromatic and P-amino acids
.was in the range 0.37-8.5 mM. Kragh-Hansen et al. (12)
found a Na-dependent depolarization of rabbit brush
borders with L-phenylalanine but not with D-phenylala-
nine. Murer and his collaborators (3,27,28) also reported
that certain amino acids (glutamic acid, L-lysine, and L-
cysteine) exhibit Na-dependent potentials in rat renal
brush border membranes.
In general, all amino acids that are transported by Na
cotransport have been observed to depolarize the brush
border membrane in vesicle preparations, and the &,
values agree closely with those obtained from radioactive
tracer experiments in vesicles and microelectrode exper-
iments in the intact renal tubule (Table 1). A similar
correlation was reported for intestinal brush borders
(Table 5 of Ref. 23).
There are wide variations in the maximum membrane
depolarizations produced by various solutes in both ves-
icles and the intact tubule: AF,,, in rabbit vesicles ranges
from 25 (valine) to 200% (serine) of that obtained for L-
TABLE 1. Apparent K, values for renal
brush border membranes
Km, mM
PD, Vesicles,
Rabbit
Tracers, Vesicles,
Rabbit
PD, Cells,
Rat
Amino acids
Neutral
L-Phenylalanine
L-Methionine
L-Alanine
Glycine
L-Proline
L-Hydroxyproline
MeAIB
L-Glutamine
L-Cysteine
Basic
L-Lysine
Acidic
L-Glutamate
D-Glucose
Carboxylic acids
Succinate
Citrate?
L-Lactate
Pyruvate
0.76
0.96
2.6
2.35
0.38
0.69
2.6
2.2
1.8
0.33
0.18 0.70"
0.68 1.0*
0.19 0.48*
0.25 0.12*
1
1
0.5
1.5
-1
-0.5
0.86*
1.8
0.65
6.6
0.31
0.30
0.32
5.5
6.9
0.8
0.09
1.1
Comparison of K, values obtained for renal brush border membranes
by PD measurements in rabbit vesicles, tracer measurements in rabbit
vesicles, and PD measurements in intact rat proximal tubules. Tracer
uptakes were carried out under open-current conditions except where
indicated by an asterisk, in which the membrane PD was short-circuited
with K plus valinomycin. Data are from Refs. 19-21, 23, 27, and
28. t Unpublished.
E. M. WRIGHT
phenylalanine (Table 1 of Ref. 23), and the APD,, in
tubules ranges from 3-32 mV (19-21). Although these
variations reflect, at least in part, different Na-depend-
ent transport systems with different Vmax values, other
effects cannot be ruled out, e.g., variations in Na/amino
coupling coefficients.
Glucose. D-Glucose also depolarizes renal brush borders
in a stereospecific, Na-dependent fashion (2, 12, 23, 31).
In the presence of a Na gradient the depolarization is
saturable and the K, for vesicles and the intact epithe-
lium is in the range 0.3-l mM (Table 1).
Metabolic intermediates. Mono- and dicarboxylic acids
are also transported across renal brush border mem-
branes by Na cotransport systems. The Krebs cycle
intermediates are transported by a dicarboxylic acid car-
rier, which is highly specific for &carbon, terminal di-
carboxylic acids in the trans configuration (30, 31). The
divalent form of the tricarboxylic acid, citrate, is handled
by this system. Using diS-C&j), we demonstrated that
the substrates for this transporter depolarized rabbit
renal brush borders (31). Figure 4 shows two recent
recordings obtained with citrate and succinate (1.9 mM)
using a dual-wavelength spectrophotometer. These acids
depolarized the membranes in the presence of NaCl but
not in the presence of LiCl or KCl. The maximum
depolarization (-6 mV) and the time course (t112 - 2 s)
was very similar for both substrates. It is odd that it
takes so long for the membrane PD to depolarize in view
of the observation that [4C]succinate uptake into vesi-
cles is only linear for l-2 s (30). The concentration
dependence of the Na-dependent depolarizations fol-
lowed saturation kinetics, and the K, values (0.18 and
0.68 mM) are close to those obtained using radioactive
tracers (Table 1). The citrate K, values are much lower
(-0.01 mM) if the concentration of the divalent species
is used rather than the total chemical concentrations.
Our results have been confirmed by Kragh-Hansen and
colleagues (11-13).
A separate transport system exists in renal brush
borders for short-chain monocarboxylic acids (17). L-
Lactate and pyruvate depolarize the brush border and
the Km values are again close to those obtained with
radioactive tracers (Table 1). Although our results with
mono- and dicarboxylic acids agree closely with From-
ters microelectrode experiments (8) on the rat proximal
tubule, there is one discrepancy that is not yet explained.
Namely, that citrate did not depolarize the cell potential
in the rat tubule. This is not simply due to species
differences in citrate transport as we have observed Na-
dependent citrate uptake in rat renal brush border mem-
branes.
More limited studies with basolateral membranes (11,
12) showed that phenylalanine, glucose, and citrate did
not depolarize the vesicles. This is not unexpected in
view of Fromters microelectrode experiments and the
characteristics of basolateral transport systems for these
solutes.
Cotransport mechanisms. Although the above studies
have provided a valuable link between vesicle experi-
ments and the intact renal tubule, a question remains
about what can be learned about the Na-cotransport
EDITORIAL REVIEW F367
A
Succinate
B
Citrate
I
0.002A
li K
Li
FIG. 4. Effect of succinate (A) and citrate (B) (1.9 mM) on optical added by a rapid-mixing device. Note that both citrate and succinate
response of diSC&) in renal brush border vesicles. Absorbance of
dye was recorded at 655 vs. 685 nm in a dual-wavelength spectropho-
produced a transient increase in the signal, which corresponded to a
depolarization of membrane potential (6-10 mV) only in NaCl. Exper-
tometer. Base-line signals were recorded for 1 min in presence of 100
mM NaCl, LiCl, or KC1 gradients (out + in), and substrates were
imental conditions were identical to those in Figs. 2 and3 (Schell and
Wright, unpublished data.)
75
7 50
25
30 60 90 120
ml
FIG. 5. Effect of cis Na on the kinetics of succinate transport in function of AF/S [from Schell et al. (23)]. Note that as the cis Na
renal brush borders. A: initial rate of succinate uptake, J, is plotted as concentration was varied from 10 to 100 mM there was no change in
a function of J/S, where S is the cis succinate concentration [from J,, or AF,, but there was an increase in the succinate affinity
Wright et al. (30)]. B: Na-dependent AF of diS-Ca-(5) plotted as a (- slope).
mechanisms from dye experiments. It is clear that the
dye experiments provide a rapid and convenient method
for screening the specificity of electrogenic Na cotrans-
porters and provide an alternative method to radioactive
tracers. This is particularly useful when radioactive
tracers are either costly or unavailable commercially.
One example is in the study of the decarboxylate trans-
porter in which we had to rely on competition experi-
ments to evaluate substrate specificity (31). In this case
the potential studies strongly suggested that substrates
which inhibit succinate transport are actually trans-
ported.
With a judicious choice of controls it is also possible
-------L-- I-------- .1_ --- -1 ---.-. _.--J
I 2 3 4 5
AF/ [ Succinate]
to obtain information about the kinetics of the coupled
transporters. For example, the kinetics of succinate
transport has been examined with tracers and dyes (23,
30, 31). Varying the cis Na concentration (Fig. 5) shows
that Na behaves as a competitive activator of succinate
transport, i.e., the Km values decrease with increasing
[Na] in the absence of changes in J,, or AF,,. On the
other hand, increasing the trans Na concentration re-
duces both uptake and the membrane potential, and this
trans inhibition is due mainly to a decrease in J,,,.2
2 A general observation with dyes and tracers is that tram Na reduces
the optical signal and uptake with sugars, amino acids, and metabolic
F368 E. M. WRIGHT
Likewise, we have confirmed the specificity and kinetics
of the action of Li on succinate transport (30), i.e., cis Li
is a potent competitive inhibitor and trans Li is a non-
competitive inhibitor of Na-dependent succinate uptake
(Schell and Wright, unpublished observations). Similar
experiments can be carried out with other drugs on these
electrogenic transport processes, e.g., phlorizin on glu-
cose transport (2) and H$+ on succinate transport
(Schell and Wright, unpublished observations). We find
that Hg+ behaves as a noncompetitive inhibitor of suc-
cinate transport with a Ki of 8 PM.
40
1
Caution has to be exercised with these types of studies
and artifacts have to be rigorously excluded. First, a
number of drugs and reagents are known to react. with
cyanine dyes to produce nonfluorescent complexes,
e*g*,
perchlorate, thiocyanate, phloretin, and dinitrophenol(1,
5). These effects can be detected by spectral analysis of
the dye solutions. Second, potential-independent optical
changes can occur, e.g., variations in ionic strength and
pH may influence the state of aggregation of the dye and
the extent of dye binding to plasma membranes and/or
brush border core material. These changes can be mini-
mized by working at constant ionic strength and pH, and
the voltage dependence of the signal can be examin .ed by
manipulations designed . to short-circuit membrane volt-
ages, e.g., with permeable ions and ionophores. We have
been able to short-circuit succinate-dependent voltage
changes with K plus valinomycin (30). Third, it is im-
. portant to confirm that the dyes themselves do not alter
the transport properties of the membranes. This can be
tested with radioactive tracer experiments in which the
kinetics of transport are measured in the presence and
absence of dye. So far, it has been established that diS-
C&5) does not alter the initial rate or time course of
glucose, succinate, and phenylalanine transport (2, 23,
31)
Finally, care must be exercised in the interpretation
of the kineti
ments . It is
.c parameters deduced from voltage measure-
widely recognized that cotransport kinetics
are voltage dependent. For example, negative voltages
increase the initial rate of s luccinate uptake by lowering
the &-, with no change in J,,, (30). Therefore the kinetics
obtained under open-circuit conditions may be distorted
by 1) the magnitude of the resting potential; 2) the
depolarization produced by the substrate; and 3) the
actual voltage dependence of K, and J,,,. The problem
can be illustrated by one example. It has been reported
that changes in anion composition alter the potentials
produced by glucose, phenylalanine, malate, and citrate
in renal brush borders (U-13). The potentials, as re-
corded by DiO-C&5) absorbance changes, decrease in
magnitude in the sequence Cl > SO* > gluconate. Kragh-
Hansen and colleagues (ll-13) suggested that this effect
was simply a reflection of the anion permeabilities (Pcl
> ho, > Pgluconate ) the implication being that the more 9
permeable the anion the more negative the interior of
the vesicle and the higher the rate of cotransport. Their
intermediates. However, the kinetics of the tram inhibition may be
different from substrate to substrate, e.g., in the case of succinate there
is a decrease in K,, but with glucose there may be a large increase in
K, (compare Refs. 23 and 2).
0
30
\
0
AAb
\
a
0
I I 1 1 I
16 32 48 64 80
AAbhuccinatel
FIG. 6. Effect of anions on succinate-dependent PD changes in renal
brush border membranes. Change in absorbance of diS-C&-(5) (685 vs.
655 nm) due to succinate (see Fig. 4) was measured in 100 mM Na
gluconate, NaCl, and NaBr, and plotted against AAb/[ succinate]. Note
that maximum depolarization decreased as permeability of anion in-
creased (P,l, < Pcl < P &; see Table 2), while there was no apparent
change in the succinate affinity (- slope). (Schell and Wright, unpub-
lished data.)
argument is flawed, as we would expect that the sub-
strate-induced depolarization would be short-circuited
by the more permeable anions. In fact, this is what we
observed with diS-C&j) signals. As shown in Fig. 6 for
succinate, we found that the more permeable the anion
( PBr > Pcl > Pgluconate, see below and Table 2), the lower
the substrate-induced depolarization. To confirm that
the anions only affect the membrane potential, we mea-
sured the rate of substrate uptake as a function of anion
composition. When the membrane potential was short-
circuited with K plus valinomycin, we found, as expected,
that the initial rate of D-glucose, L-phenylalanine, and
L-lactate uptake was independent of the anion present
(15). This indicates that for these substrates, variations
in membrane potential and rate of uptake measured
under
tions
open-circuit conditions are simply
in anion permeability. In the case
due to varia-
of succinate,
however, a more complex anion effect was observed. The
initial rate of uptake varied by an order of magnitude
with different Na salts even when the membrane voltage
was short-circuited: the
fluorine and gluconate
highest
and the
rates were observed
lowest with iodine.
witYh
The
anion sequence corresponded to the inverse of the anion
permeability sequence, and further kinetic analysis re-
vealed that the permeable anions behaved as weak com-
petitive inhibitors of succinate transport. This suggests
that variations in both voltage and. uptake with anions
under open-ci
permeability,
rcuit conditions are due to
membrane potential, and
changes
interacti
in anion
.ons with
the succinate binding site on the transporter. Accord-
ingly, from optical measurements alone it is often diffi-
cult to interpret changes in potential without additional
information.
Ion Permeability
Over the last two years we have exploited the voltage-
sensitive dyes to measure the ion permeability of mem-
EDITORIAL REVIEW F369
brane vesicles. The principle is to measure bi-ionic dif-
fusion potentials produced by known ion concentration
gradients across the membrane of the vesicles and then
use the constant-field equation to solve for the relative
ion permeability coefficients. This is an accepted bio-
physical method for characterizing the ion permeability
of model and biological membranes, including epithelia.
We have measured the permeability of brush border
membranes prepared from rabbit jejunum, rabbit renal
cortex, and bovine choroid plexus by a standard Ca
differential centrifugation procedure (9, 32). The exper-
imental protocol was to 1) calibrate the voltage-depend-
ent optical signal of diS-C&) with K and/or Na diffu-
sion potentials (Fig. 1) using K gluconate plus valino-
mycin or Na gluconate plus N,N,N,N-tetrabenzyl-3,6-
dioxaoctan-diamide (ETH 1097) (one of Professor W.
Simons Na ionophores); 2) generate ion gradients across
the membrane in the absence of the ionophores and
record the re tsulting bi- ionic membrane potentials; and
3) insert the intra- and extracellular ion concentrations
and the value of the diffusion potential into the constant-
field equation and solve for the ion permeabilities. Since
the optical signal of diS-Ca-(5) varies with ionic strength
and pH, all experiments were carried out in well-buffered
solutions of constant ionic strength (maintained con-
stant with impermeant ions such as choline, gluconate,
and isethionate).
One experiment on renal brush borders is shown in
Fig. 7, in which the vesicles were preloaded with 10 mM
K gluconate plus 90 mM choline gluconate. The figure
shows the signals when vesicles were added to cuvettes
containing 1) 10 mM K gluconate + 90 mM choline
gluconate, 2) 100 mM K gluconate, 3) 1 mM K gluconate
+ 99 mM choline gluconate, and 4) 100 mM KBr. The
in - out
60
100 mM
50
Val
KGlu
N
IOOmM K Glu
IOOmM K Br
F 40
IOmM KGlu ,+, wfi IOmMKGlu
30
IOOmM KBr &
I mM KGlu ncly
+M ImMKGlu
I i
FIG. 7. Observed fluorescence of diS-C&-(5) in presence of rabbit
renal brush borders with various transmembrane ion gradients. Vesicles
were preloaded with 10 mM K gluconate, 90 mM choline gluconate,
100 mM D-mannitol, and 10 mM Tris/HEPES (pH 7.5). They were
added to cuvettes containing I) 100 mM K gluconate, 2) 10 mM K
gluconate + 90 mM choline gluconate, 3) 100 mM KBr, and 4) 1 mM
K gluconate + 99 mM choline gluconate, and JO0 mM mannitol and
10 mM Tris/HEPES (pH 7.5). Concentration of diS-C&-(5) was 1.5 PM
and protein 0.3 mgjml. At arrow valinomycin (4 pg/ml) was added to
each cuvette. Fluorescence, F, was recorded at 669 nm while exciting
at 620 nm.
signal recorded in the absence of ion gradients represents
the base line, i.e., E, = 0 mV. Note that addition of
valinomycin to this cuvette produced less than 1 unit
increase in F. In the presence of 100 mM K gluconate F
increased by 22 units in the absence and presence of
valinomycin. In the presence of valinomycin, E, = Ek =
58.5 log Ko,t/Kin = +58.5 mV (concentrations may be
used since the ionic strength of all solutions is constant).
Therefore, in the absence of valinomycin, the AF for the
100-10 K gradient corresponds to +58.5 mV, and this
implies that in the native membrane Pk >> Pgluconate,
P
choline*
When the vesicles were added to the 1 mM K
gluconate solution, AF decreased by 11.5 units in the
absence of valinomycin and 13 units in the presence of
valinomycin. Thus, in the absence of the ionophore, the
fluorescence reduction corresponded to -51 mV. Regard-
less of which assumption is made about the permeability
of gluconate and choline, i.e., Pgluconab/Pk = Pchoiine/Pk,
or P
choline
>> P
gluconate,
or P
gluconate
29 P
choline 9
solution of
the constant-field equation necessitates Pgluconab/Pk,
P choline/PK < 0*005-
When 100 mM K gluconate in the cuvette was replaced
with 100 mM KBr the fluorescence decreased below the
base line by 6 units. This corresponds to a bi-ionic
potential of -26 mV. In this case the constant-field
equation is
E, = 58.5 log
PK [Klo + Pgluconate [gluconateli
PK [K]i + PB~ [Br]o + Pcholine [choline]i
Assuming P&oline/PK = 0.005 and Pg~u~ona~/Pk = 0.005,
then
E, 100 + 0.005 [loo] = -26 mV = 58.5 log
10 + PBr/PK [loo], + 0.005 [go]
which on rearranging and solving yields PBr/Pk = 2.7.
Eight similar experiments made on two different mem-
brane preparations yielded an average PBr/Pk ratio of
4.9 t 0.1 (SE). Note that on addition of valinomycin to
the KBr solution, the membrane potential jumped to &,
i.e., PK (valinomycin) >> P &, and as KBr diffused into
the vesicle, E, returned slowly to 0 mV.
The net influx of KBr into the vesicles at any time t
after addition of valinomycin (Jr& can be estimated
from the change in Ki and the volume of the vesicles (V
- 1 pl/mg protein), assuming that the volume is constant,
i.e., JkBr = V(Kr - Ki)/(t mol/(mg protein X S) where Ki
= 10 mM and Kf is obtained from Ef, (&) using the
Nernst equation. In Fig. 7 the initial rate of fall of Ek in
KBr is -0.5 mV/s, and this yields an influx of -150
pmol/(mg x s).~ The value of PBr can then be obtained
from the flux equation
P
Br =
J
Br
RT (1 - expZFEmRT)
~~Gk.l pro1
where JBr = JkBr, and solving gives PBr - 1 nl/(mg X s).
This compares with P No3 reported for intestinal mem-
branes by this technique-(g). These P values are in the
3 In the first few seconds after the addition of valinomycin the KBr
influx only increased the internal osmotic pressure by less than 2%,
and so volume changes may be neglected.
F370 E. M. WRIGHT
range of the permeabilities [ 1 .-lo nl/(mg x s)] measured
by tracers (9). It is, however, difficult to express these P
values in terms of vesicle area owing to the lack of
information about the number of vesicles per milligram
of membrane protein. Nevertheless, the P values must
be low relative to other vesicle preparations, e.g., toad
bladder apical membrane vesicles, because the half time
for equilibration of renal and intestinal brush borders
with isotopes is in the range of minutes rather than
milliseconds (9). Apical membrane vesicles from toad
urinary bladder, which contain amiloride-sensitive Na
channels, exhibit half times for Na uptake of -200 ms
(4), and it can be calculated that vesicles with a radius
of 200 nm and one channel with a conductance of 10 pS
per vesicle should equilibrate with external ions in milli-
seconds. Another rough estimate of the permeabilities
can be obtained from the radius of the vesicles (100 X
lo-' m) and the water space (2 hl/mg protein). This
implies that there are 5 x 1011 vesicles/mg protein with
an area of 600 cm2/mg protein. Ion permeabilities in the
range of l-10 nl/(mg X s) are then equivalent to 0.2-2 X
lo-' cm/s of membrane area. Thus it would appear that
brush border vesicles from leaky epithelia are rather tight
to ions.
Bi-ionic potential measurements have been made on
renal and intestinal vesicles preloaded with K, Na, and
Cl as the permeant ions (see Figs. 1 and 2 of Ref. 9 and
Fig. 3 of Ref. 32), and the P ratios are independent of
the ion pairs used to generate the gradients. This indi-
cates that the optic al response of diSC&) is not spe-
cifica lly dependent on the ions present but only on the
magnitude of the membrane diffusion potenti .al. The
relative P values ob tained for some common ions at 22OC
are summarized in Table 2.
Owing to the lack of firm data about the ionic perme-
ability of brush borders in intact epithelia, due mainly
to the high paracellular shunts, it is difficult to judge the
physiological significance of the vesicle results. One clue
comes from estimates of the brush border potential using
physiological extra- and intracellular ion concentrations
and the P values from Table 2. The predicted membrane
potential is -15 to -20 mV, which is substantially lower
than anticipated for the brush border electromotive force.
Potential problems with the vesicle data may arise be-
cause the permeability properties of vesicles at 22C may
not reflect the permeability of brush borders in the intact
epithelium at 38OC. For example, the vesicle isolation
procedure may damage the membrane, ion channels
may be under the control of factors missing in the
vesicles, there may be large temperature effects between
22 and 3 8"C, a .nd the dye may alter the i .on permeability
of the vesicles.
There are a number of experimental observations bear-
ing on these points. First, we have repeated the renal
brush border experiments at 38C and attempted to alter
ion permeabilities by ion channel blockers. Increasing
the temperature from 22 to 38C decreased the Pcl/Pk
ratio from 1.4 t 0.2 to 0.3 t 0.2, and this effect was
blocked (60-70%) with tetraethylammonium, Ba2+, and
Cs+ but not with tetramethylammonium, suggesting the
presence of a temperature-sensitive K channel in these
TABLE 2. Ion permeability of brush border vesicles
Kidney Intestine
Li 1.7 1.6
Na 0.4 0.4
K 1.0 1.0
NH, 2.1 5.0
Cl 1.3 0.8
Br 4.9 1.1
HC03 1.2 0.35
Relative ion permeabilities (Pi/Pk) were measured as illustrated in
Fig. 7 and described in the text. All experiments were conducted at
22C, and the permeability values are means determined in quadrupli-
cate in two to six preparations. The standard errors were less than 10%
of the mean. Pncos was estimated using 60 mM KHC03 solutions
equilibrated to pH 7.5 with COZ. Data taken from Refs. 9, 29, and 32.
membranes (29). Second, we have compared the proper-
ties of rabbi .t intestinal bru sh borders prepared two dif-
ferent ways from the same starting material, i.e
l ?
by the
standard Ca2+ procedure and by a divalent ion-free pro-
cedure (Mandel, Harms, Stevens, Schell, and Wright,
unpublished observations). Results suggest that although
there is little difference in the kinetics of sugar and
amino acid transport, Ca-free membranes are more se-
lective to K over Cl than the standard preparation. Both
sets of results suggest that the ion permeabilities of
membrane vesicle preparations are dependent on both
the isolation methods and the experimental conditions.
Third, there are reports that d&C&) is a potent inhib-
itor of Ca-activated K channels in human erythrocytes
(24). Although we know that diS-C&5) does not inhibit
cotransport systems (sugars, amino acids, and metabolic
intermediates) in brush borders, we have no information
yet about the dye effects on ions in our systems. This
may be tested by the measurement of the effects of dyes
on radi .oactive tracer fluxes and the use of dyes,
e*g*,
oxonols, not known to inhibit Ca-activated K channels.
Finally,t thiadicarbocyanine dyes are known to alter the
membrane permeability properties (14), particularly at
low ionic strength where they act as channels or as anion
carriers depending on the membrane and the dye. These
perturbations can be minimized by working at high (0.1
M) ionic strengths.
Despite these potential problems pertaining to the
physiological significance of the ion permeability of
vesicle preparations, the bi-ionic potentials may be used
to explore transport mechanisms. One example is the use
of pharmacological and other agents, e.g., tetraethylam-
monium, Cs, and Ba, to examine ion permeation, and
another example is probing the interactions of Na with
cotransport systems. In the latter case we have examined
the effect of sugars, amino acids, and metabolic inter-
mediates on the sodium permeability of renal brush
borders. The approach was to measure bi-ionic potentials
in the presence and absence of organic substrates. Not
unexpectedly, we found that the cotransported solutes
specifically increased the effective sodium permeability
in a concentration-dependent fashion. The maximum
increase in P$ ranged from twofold for succinate to
lo-fold for L-phenylalanine at a NaCl concentration of
EDITORIAL REVIEW
F371
100 mM. This is consistent with an increase in Na
conductance of the brush border membrane (8).
Conclusions
The primary objectives of this review have been to
demonstrate that it is possible to make quantitative
determinations of membrane potentials in epithelial
membrane vesicle preparations by using voltage-sensi-
tive dyes and to show how PD measurements in vesicles
can provide useful information about the transport prop-
erties of epithelial transport phenomena. With brush
border Na cotransporters for organic substrates, it is
clear that all are electrogenic and that they are associated
with an increase in membrane sodium conductance.
There is a remarkable correlation between the kinetic
parameters obtained in vesicles and intact epithelial cells
by electrophysiological methods. Problems remain, how-
ever, regarding the molecular basis of the Na conduct-
ance changes and the time course of the membrane
depolarizations. In the case of ions, bi-ionic potentials
have been used in vesicles to measure permselectivity of
brush borders in the absence of paracellular shunts, and
the results provide information about ion permeation
processes. A major difficulty yet to be resolved concerns
the physiological relevance of the vesicle data. One prob-
lem is that ion permeation may change due to the isola-
tion procedure and dye-membrane interactions. Prelim-
inary evidence suggests that presence of temperature-
sensitive K channels in renal brush border vesicles. How-
ever, the major limitation encountered so far with elec-
trophysiological studies of epithelial membrane vesicles
is that methods have not been developed for the meas-
urement of membrane currents. Perhaps it is possible to
extend the patch-clamp pipette technique (6) to epithe-
lial vesicles to record the kinetics of ion channels and
Na cotransporters.
I am grateful to Robert Gunther, Ian Kippen, Sally Krasne, Richard
Schell, Bruce Stevens, and Stephen Wright for their many contribu-
tions to our collaborative studies on the use of diS-C&-(5) in membrane
vesicles.
This work was supported in part by Public Health Service Grants
NS-09666 and AM-19567.
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Ernest M. Wright
Department of Physiology,
School of Medicine, University of California, Los Angeles, California 90024