Transport of water and urea in red blood cells

MACEY, ROBERT I. Transport of water and urea in red blood cells. Am. J.
Physiol. 246 (Cell Physiol. 15): Cl95C203, 1984.--Evidence for water
channels in red blood cells is reviewed. In an entropically driven reaction,
organic mercurials decrease water permeability, elevate the activation en-
ergy, and reduce the ratio of osmotic to diffusional water permeabilities to
unity so that water transport properties of red blood cells are hardly
distinguishable from lipid bilayers. It is concluded that mercurials close the
water channels. A variety of kinetic, pharmacological, and comparative
evidence converges on the conclusion that urea and other solutes are
excluded from water channels. Urea apparently permeates the red cell
membrane via a facilitated diffusion system, which plays an important role
when red blood cells traverse the renal medulla; rapid urea transport helps
preserve the osmotic stability and deformability of the cell, and it helps
prevent dissipation of extracellular osmotic gradients. Water apparently
traverses the channel via a single-file mechanism; the very low channel
permeability of H+ is explained if the channel contains fixed charge, or
alternatively, if the mobile water molecules within the channel do not form
a continuum. An alternative unitary pore hypothesis for simultaneous
transport of water, ions, and small solutes is also discussed.

permeability; erythrocyte; renal medulla; channels; mercurials; p-chloro-

mercuribenzene sulfonate; kidney

A MOSAIC MODEL of the plasma membrane consisting of Evidence for Water Channels
patches of protein perforating a background of lipid
bilayer has been a paradigm for transport studies for Functional evidence for the existence of water pores
over 40 yr. It emphasizes the existence of at least two in the red cell membrane comes from comparisons with
paral .lel permeation pathways and provides a physical lipid bilayers.
basis for postulating the presence of polar channels or 1) In the first place, many transport properties of
pores that span the membrane. Membrane pores are a water through the membrane can be predicted from the
useful construct in so far as they offer a simple expla- bulk properties of water. Thus the activation energy for
nation of 1) the rapid pen.etration of small polar solutes both self diffusion of water and viscous transport is about
and water, 2) the permeation selectivity based on molec- 4.6 kcal/mol (22), which is similar to the 4-6 kcal/mol
ular size of the permeant, 3 ) the coupled transport of measured for the diffusional and osmotic permeabilities
solutes and water, and 4) the differences between dsmotic of water in red blood cells (57). Further the effects of
and diffusional permeabilities of water. In the simplest both deuterium oxide (27) and hydrostatic pressure (D.
cases, pores are modeled as equivalent right circular 1111.Karan and R. I. Macey, unpublished observations)
cylinders, with the proviso that the actual shape and on osmotic water flow can be predicted from correspond-
tortuosity may be quite different from the model (54). In ing effects on water viscosity. The results imply that as
more complicated cases where very specific selectivity a water molecule traverses the membrane, it encounters
and/or an electrical gating is introduced, it has become polar groups similar to the polar environment it finds in
customary to refer to the transport device as a channel the bulk solutions external to the membrane. In contrast,
rather than a pore. activation energies for permeation through lipid bilayers
This article-examines the case for pores or channels are much higher, ranging from 11-14 kcal/mol (16).
in red blood cells. More specifically, we compare the Although these observations do not establish the exist-
transport properties of water and urea with respect to ence of water channels or pores, they do underscore the
the criteria listed above. We argue that current evidence differences between normal water transport in red blood
supports the notion of a highly specific water channel cells and simple diffusion through the lipid bilayer.
where transport takes place in single file and that urea 2) The water permeability of red blood cells is much
is excluded from the water channels but is transported higher than corresponding permeabilities of lipid bilay-
by a facilitated diffusion system, which serves a physio- ers. Precise comparisons are compromised by the high
logical function in the renal circulation. degree of variability for bilayers of different composi-
0363-6143/84 $1.50Copyright0 1984the AmericanPhysiological’Society Cl95
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Cl96
tions. Nevertheless, water permeability of red blood cells
is higher in almost all instances; in the case of artificial
lecithin-cholesterol bilayers, the osmotic permeability of
red blood cells is approximately 10 times higher (20).
3) The reflection coefficients for a significant number
of small polar nonelectrolytes have been reported to be
less than unity even when corrections for the volume
contribution of the solute has been accounted for (19,
44). These results imply a coupling between water and
solute movement, which is most easily interpreted in
terms of water channels or pores. In artificial lipid layers
that are not treated with ionophores, reflection coeffi-
cients are essentially unity.
4) In red blood cells, the osmotic or filtration perme-
ability (PJ is several times higher than the diffusional
permeability (Pd) (45); in artificial bilayers they are equal
(7). Although it has been repeatedly emphasized that this
type of result can arise from unstirred layer artifacts (11,
4l), this is probably not so for red blood cells (34). Both
theoretical (49) and experimental (3) estimates of extra-
cellular unstirred layer thickness indicate that it is below
3 pm and could not perturb the measurements to any
extent. The same conclusion is true for intracellular
unstirred layers where distances are less than 2 pm and
where the diffusion coefficient for water does not differ
substantially from that in bulk dilute solution (43, 47).
The finding that Pf > Pd implies that osmotic flow is
nonrandom; presumably it occurs by bulk transfer of
water where water molecules are transported in groups
or clusters rather than individually. This would be ex-
pected in a system of channels or pores. The ratio Pflpd
reflects some geometric property of the channel: in mac-
roscopic pores, PflPd is directly proportional to the
square of the pore radius (54), whereas in tightly con-
strained channels like gramicidin where water permeates
by single-file diffusion, Pflpd is equal to the number of
water molecules within the channel (12, 31, 50).
Although none of the four criteria listed above provides
an unequivocal proof, they are all readily explained by
the existence of channels or pores.
Closing Water Channels
Studies of water permeability require techniques for
manipulating the water channels. Although water perme-
ability is insensitive to most transport reagents, it is
dramatically and reversibly inhibited by mercurial
sulfhydryl reagents such as p-CMBS (p-chloromercuri-
benzene sulfonate), chlormerodrin, and mersalyl (36).
When saturating doses (22 mM) of p-CMBS are applied
to red blood cells, most of the evidence for channels
disappears (34, 37). In particular, p-CMBS decreases Pf
by an order of magnitude, it increases the activation
energy from 4.8 to 11.5 kcal/mol, and it decreases the
discrepancy between Pf and Pd so that they become equal.
The water transport properties of p-CMBS-treated red
blood cells become virtually indistinguishable from those
of lipid bilayers. A simple interpretation is that mercu-
rials react with sulfhydryl groups in proteins associated
with water channels and result in closure of the channels.
This view is substantiated by comparisons with chicken
red blood cells (5,14), which apparently have no channeis
(i.e., they have a low Pf, Pf = Pd, and activation energy
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Copyright © 1984 American Physiological Society. All rights reserved.
R. I. MACEY
= Il.4 kcal/mol); water transport in chicken red blood
cells is not influenced by p-CMBS. This also suggests
that p-CMBS has no influence on primitive lipid bilayers.
Assuming then that p-CMBS acts only on the chan-
nels, it can be used operationally to decompose the gross
water permeability into its component parts 1) p, the
permeability through pores or channels, and 2) 4, the
permeability through- the lipid background matrix. Be-
cause these pathways are in parallel, the measured
permeability is given by
P(c) = P(C) + 4 (1)
where c denotes the concentration of mercurial reagent,
and P is the permeability (either Pf or Pd). When c is
sufficiently large (p sz:0), so that q can be estimated by
measuring P(c) with saturating doses of mercurial (i.e.,
c -> 2 mM for p-CMBS), then
4 = pw (2)
P(C) = P(c) - pw (3)
where 00 denotes a “saturating dose.” For osmotic flow,
P&) = O.lPf(O) (36); it follows that in the normal cell
approximately 90% of the osmotic flow takes place
through channels and 10% through lipid bilayer.
By measuring the concentration dependence of both
Pf and Pd as a function of c, we (Moura, Chien, and
Macey, manuscript in preparation) have provided evi-
dence that the ratio of osmotic to diffusional permeabil-
ities referred to the channel, i.e., Lpf(c)]/[Pd(c)] = gP
remains constant over a substantial range of c. Because
gP probably reflects the geometrical or water packing
properties of the channels (12, 31, 50, 54), the result is
most easily interpreted if channel closure is all or none;
the reagent acts by reducing the number of channels, but
it does not affect the ones that remain open. If there are
only two channel states, open or closed, then the reaction
that closes channels can be represented as
K
nC + M(open) + C,M(closed) (4)
where C is the reagent, M(open) represents a membrane
receptor and associated open channel, C,M represents
the reagent-receptor complex associated with a closed
channel, n is the reaction stoichiometry, and K is the
equilibrium constant given by K = (C,M)/(C)“(M). Not-
ing that (&M)/(M) is the ratio of the number of closed
to open channels; we have
pm - P(c) P
GM) _
- K(c)n (5)
P(c) -P(m) = (M)
-P(c) = 1 + [w)I[P(O)IKw (6)
P(O) I + K(C)n
By studying the c dependence of Eq. 6, we (Moura, Chien,
and Macey, manuscript in preparation) showed that n =
1 and K = (0.2 mM)-’ at T = 37°C. Further, by studying
the temperature dependence of Eq. 6 at three different
concentrations, we estimated that the channel closure
reaction (4) is characterized by a AH” = 25 kcal/mol and
a AS" = 100 cal .deg-’ mol? Thus channel closure is
l
energetically unfeasible but is driven by a huge increase
in entropy. These figures are not characteristic of simple
BRIEF REVIEW
mercaptide formation, which generally has a negative
AH” and smaller entropy change (59). However, the
parameters cited above refer not only to a single reaction
with the receptor site but also to all subsequent steps
involved in closing the channel.
Positive enthalpies and large positive entropies, such
as those found here, are characteris ‘tic of som.e macroas-
l
sembly processes that are probably drl .ven by hydropho-
bic interactions (1, 13, 24). Hydrophobic reacti .ons are
entropy driven presumably because the degrees of free-
dom of water molecules near hydrophobic surfaces are
more limited. Thus when a hydrophobic surface disap-
pears from the water phase, the entropy of the surround-
ing water increases. Even though the AH” may be posi-
tive 1,the 1.arge positive AS” favors removal of the hydro-
phobic surface. Kauzmann (28) estimates an entropy
increase of the order of 20 cala deg-l mol-” for each
l artifact, and using a curve-fitting routine to estimate 0
nonpolar aliphatic side chain that leaves an aqueous
environment to enter a nonpolar region.
If the large entropy change in channel closure is due
to hydrophobic interactions, we can expect the reaction
to include the disarray of water molecules. This is con-
trary to what would be anticipated if channel closure
were due to immobilization or “freezing” of the putative
water molecules that traverse the channel. Although it
may be tempting to identify the inferred changes in water
structure with water lying within the channel (as shown
in Fig. 4), there is no evidence that this must be so.
Independence of Water and
Solute Transport
The conclusion that p-CMBS blocks water channels
can be exploited to test the solute-tran sportin g capaci.tY
of the channels; blockin g th .e channels should abolish or
at least inhibit the transport of any solute that usesthem
as a primary means for transport. Studies using concen-
trations of p-CMBS sufficient to block the majority of
water channels show that cation leakage is increased by
at least an order of magnitude (56), whereas both anion
exchange and net electrogenic anion flux (29,46) appear
unaffected. These results make it unlikely that ions are
transported via the water channels. (However, the pos-
sibility remains that any inhibitorv effect ofp-CMBS on
cation transport through water channels was masked by
leakage induced in parallel pathways.) A similar conclu-
sion applies to many small, polar nonelectrolytes (36)
which had been used extensively in the past to probe the
size of the channel (19). Of a large number of solutes
tested, only urea, methyl urea (37), and certain small
amides (52) are affected by p-CMBS. However, other
evidence strongly indicates that this inhibition by p-
CMBS is fortuitous, that these solutes traverse special
facilitated diffusion pathways, and that they are not
transported by water channels to any significant extent.
The case for urea is more detailed; it is particularly
interesting because the exclusion of urea (radius 2 A)
from water channels has strong implications for the
structure of the channel. Early attempts to measure
reflection coefficients in red blood cells appeared to
establish a strong coupling between urea and water trans-
port (19). The reflection coefficient (a) was estimated at
about 0.6. This value is the lowest reported for any solute
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Copyright © 1984 American Physiological Society. All rights reserved.
Cl97
tested and is well below the near unity value that would
be expected in the absence of urea-water coupling. How-
ever, the method used to estimate 0 employed light
scattering to measure cell volume ch .nges and required
an extrapolation to estimate the volume flow at time t =
0. Doubts about the validity of the extrapolation were
raised in Levitt’s (31) numerical investigation of the
Kedem-Katchalsky equations, and more refined meas-
urements by Owen and Eyring (44) estimated that 0 =
0.79 for urea. Finally, in a recent paper, Levitt and
Mlekoday (33) have pointed out that previous investi-
gations of urea and water transport by light-scattering
techniques may be inaccurate because of failure to ap-
preciate refractive index corrections, which need to be
taken into account as large concentrations of urea equil-
ibrate between cell and surroundings. Correcting for this
from the cell volume kinetics following a step change in
urea concentration, Levitt and Mlekoday (33) find a best
fit for c = 0.95. (In the absence of coupling, the theoret-
ical value for u is also 0.95.) Thus the original evidence
that u rea and water transport is tightly coupled is no
longer viable. Because urea has the smallest g that has
been reported, the same conclusion may hold for the
other nonelectrolytes. The u for ethylene glycol, for
example, was originally reported to be 0.63 (19). This
was later estimated at 0.86 by Owen et al. (44) and more
recently found to be c = 1.0 by Levitt and Mlekoday
(33)
In’dependence of water and urea transport can also be
demonstrated with a number of inhibitors (e.g., phlore-
tin, nitrophenols, and amide and urea derivatives). These
reagents reduce urea permeability by huge factors (e.g.,
~50) without appreciably changing water permeability
(26, 34, 36, 39, 62). Further evidence for independence
comes from comparati .ve studies (60). Duck red blood
cells h.ave a high water permeability but low lipid bilayer
type urea permeability; in contrast, amphiuma red blood
cells have a high urea permeability but a low lipid bilayer
type water permeability. Finally, the fact that p-CMBS
inhibits both urea and water transport appears to be
fortuitous. This is shown in Fig. 1, A and B, where both
the kinetics and concentration dependence of inhibition
of water an.d urea transport are compared. Clearly urea
1s inhibited much faster and at a. much lower conce ntra-
tion ofp-CMBS than water. Given the ubiquitous nature
of sulfhydryl groups in the red cell membrane (51) and
the high reactivity of organic mercurials, it is not sur-
prising that the reagent inhibits more than one process.
Facilitated Transport of Urea
If urea is excluded from water channels, how does it
permeate? The lipid solubility of urea is low, and its
permeability through lipid bilayers (4 X 10D6cm/s; Ref.
18) is correspondingly low-much lower than its perme-
ability through red cell membranes (1.2 x 10B3cm/s; Ref.
39), so it appears as though urea transport in red blood
cells does not take place by simple diffusion either
through channels or bilayers. Further, the fact that it is
markedly inhibited by the known transport inhibitors p-
CMBS and phloretin stiggests facilitated diffusion for
urea transport. More compelling evidence comes from
Cl98 R. I. MACEY

60

FIG. 1. Osmotic water permeability (open circles) and urea permea- dependence on time following introduction of 1 mM p-CMBS at t = 0.
bility (closed circles) measured by osmotic perturbation method (8, 32). B: permeability of cells (Pf) that have been equilibrated with indicated
Permeabilities are normalized by control values. A: permeability (pf) concentration of p-CMBS.

port into and out of the cell, respectively, and the maxi-
A
I .o
mal permeability (i.e., at 0 concn) was 1.2 X 10m3 cm/s.
Further, Mayrand and Levitt (39) have measured the
0.9 inhibition constants for over 40 competitive inhibitors
of urea transport; several of them are very potent, having
0.8 a inhibition constant in the range of 30 PM.

0.7
Fast Urea Transport Stabilizes
Y
Red Blood Cells in Renal Medulla
0.6
A special transport system for urea in the mammalian
red blood cell does not appear to fulfill any significant
physiological function in most parts of the body; how-
ever, rapid transport with high Michaelis constant is
probably essential in the renal circulation. As blood
reaches the deeper portions of the renal medulla, it is
1 I f i I I I exposed to high concentrations of both NaCl and urea,
0 20 40 60 80 100 which can reach a combined osmolarity as high as 1,400
Time (msec) mosM. In entering these regions, a red blood cell with
poor urea transport may be in danger of severe shrinkage
FIG. 2. Equilibrium exchange time for urea (data from Ref. 58).
Efflux of 14C urea is measured by a continuous flow filtration method and possibly even hypertonic hemolysis (40). The transit
(45) after equilibrating cells with various concentrations of urea. Open time through capillaries of the medullary vasa recta is
circles, 145 mM urea + isotonic saline; filled circks, 290 mM urea + unusually long, about 24 s (30), so that even with a slow
isotonic saline; open triangh, 1,450 mM urea + isotonic saline. Ordi- transport, the cell may have time to equilibrate and upon
nate y represents log[ s( m) - s(t)], where s(t) is the extracellular specific
activity of 14C urea at time E. Values of y have been normalized to
leaving the medulla, it will not only dissipate the urea
facilitate comparisons. gradient but it will also swell to dangerous volumes. The
extent of these osmotic disturbances are examined in a
simulation that compares a normal urea transporting red
kinetic studies, which show saturation (4, 23, 38, 39, 58) blood cell with one whose urea transport has been inhib-
and competition (4, 26, 39, 62). Data illustrated in Fig. 2 ited so that its permeability is reduced to that of a lipid
show an example of saturation kinetics found with 14C bilayer.
urea equilibrium exchange studies. The rate of exchange The results illustrated in Fig. 3 are based on numerical
is clearly decreased as the concentration of urea is in- integration of the Kedem-Katchalsky equations. In both
creased, finally becoming below the limits of resolution cases, it was assumed that the transit time through the
of the apparatus at 1,450 mM. It would be difficult to vasa recta was 24 s (30) and that in passing from the
ascribe this to a generalized membrane deterioration at cortex to the tip of the medulla, osmolarity varied from
high concentrations of urea, because an independent 300 to 1,200 mosM. Further it was assumed that urea
study (9) has shown that red cell water exchange remains contributed approximately 50% of the solute load in the
invariant at concentrations of urea ranging from 0 to 1.5 medulla and that the concentration profiles were essen-
M. A detailed study of isotope exchange (39) and net tially parabolic as illustrated in Fig. 3.
transport (33) indicates that the urea transport system Note that both the cell with intact urea transport and
follows asymmetric carrier kinetics. The dissociation the “lipid bilayer” cell both shrink considerably, but as
constants were estimated at 508 and 117 mM for trans- expected the lipid bilayer cell shrinks the most. On

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l3RIEF REVIEW Cl99

Single-File Water Transport

If urea is excluded from the water channel by steric
factors, it follows that there must be at least one patch
within the channel where the radial dimension lies be-
tween 1.5 (radius of water molecule) and 2 A (radius of
. urea molecule). Within this patch, water must be con-
strained to move by single-file diffusion. Although the
extent of the patch is unknown, it is reasonable to assume
that it presents the rate-limiting barrier for water trans-
port so that measured permeability coefficients reflect
single-file transport. It follows that the constricted patch
of the channel contains from five to nine water molecules
as estimated from the ratio of osmotic to diffusional
permeability (see Refs. 2, 12, 31, 35, 50 and Moura,
Chien, and Macey, manuscript in preparation).
Comparison of Red Cell and
---
0 24 40 Gramicidin Channels
rime Ised Human red cell water channels and gramicidin share
a number of relevant similarities. Like red cell channels,
300 300 mow the gramicidin channel is readily permeable to water, yet
it is sufficiently narrow (radius -2 A) to exclude urea
and other nonelectrolytes. Further, similar to the red cell
channel, the gramicidin channel has a PflPd between five
and six; presumably it contains five to six water mole-
cules (50).
Striking as these similarities seem, significant differ-
ences appear when ion transport is considered. Red cell
water channels virtually exclude Na+, K+, and H’,
whereas gramicidin channels are freely permeable to
these ions. Although quantitative comparisons of chan-
nel properties from gross permeability measurements are
FIG. 3. Simulation of red cell volume as cell enters renal medulla. compromised by lack of knowledge of the number of
Upper curves show volume (normalized to isotonic volume) as a func- channels in each case, the problem can be circumvented
tion of time after entering the hypertonic medulla at t = 0 and returning
to isotonic cortex at t = 24 s. Solid curve represents volume changes by normalizing solute permeability data with Pd, the
calculated by numerical integration of Kedem-Katchalsky equations diffusion permeability of water. Thus the statement that
modified to incorporate a facilitated diffusion model with parameters gramicidin excludes urea (relative to water) is reflected
measured by Mayrand and Levitt (39). Dotted and dashed traces are in the fact that P,,,& < 0.001 (17); similarly for red
simulations using unmodified Kedem-Katchalsky equations with urea
permeabilities (lo-” and 4 x lo-” cm/s) characteristic of lipid bilayers.
blood cells treated with phloretin, P,,,/Pd < 0.005 (58).
Bottom curve shows assumed concentration (osmolarity) dependence The case for K+ permeation in gramicidin channels is
of extracellular fluid as cell traverses medulla. indicated by the ratio Pk+/Pd = 0.7, whereas the im-
permeability of red blood cells is expressed by Pk+/Pd =
10-7.
Channel transport of H+ is particularly interesting;
exiting from the medulla, the difference between the two the gramicidin channel is very permeable to H+ with
cells becomes much more dramatic; swelling of the intact Pn+Ipd > 120 (tzl), yet its transport does not appear to
cell is hardly perceptible, whereas the lipid bilayer type be coupled with water (32). This result has been inter-
cell not only swells to nearly 1.5 times its normal size, it preted in terms of a Grotthus conduction mechanism
remains engorged for sometime afterwards. Thus even if where protons pass from one water molecule to the next
it does withstand hemolysis, it has lost considerable along a continuous row of water molecules that span the
deformability and can be expected to encounter difficul- membrane without any translation of water. Interpreta-
ties in passing through the microcirculation. Finally, tion of H+ (or OH-) transport in red blood cells is
calculations show that the urea concentration in cells complicated because it involves at least three mecha-
entering the medulla is close to zero, while those that nisms (61): 1) HCOg-Cl- exchange, 2) H+-Cl- cotrans-
lack rapid urea transport exit containing approximately port, which requires an intact anion exchanger (25), 3)
150 mM/l, which is still trapped within the cell and a residual OH- or H+ permeability which remains after
destined to be unloaded in extracellular spaces through- inhibition of anion exchange by 4,4’-diisothiocyano-Z,Z’-
out the body; the contribution of urea to the hyperton- stilbene disulfonate (DIDS) treatment. Because DIDS
icity of the medulla would be compromised by these cells. has no effect on water transport, it is apparently this last
In sum, a rapid transport of urea in the red blood cell residual component that contains the water channel
appears to have a physiological role; it helps preserve the contribution to Hf transport. As emphasized by Wieth
osmotic stability and deformability of the cell and it et al. (62), this residual transport is very slow; with an
helps stabilize osmotic gradients in the renal medulla. extracellular pH = 3.5 (intracellular pH = 7.34), they

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Copyright © 1984 American Physiological Society. All rights reserved.
c200
measured an H+ (or OH-) flux of only 1 pmol cm-’ s-l. l l
Assuming zero membrane potential, this corresponds to
a permeability of about 3 x 10D6 cm/s, which could
represent H+, OH-, or some combination of the two.
Further, by altering the membrane potential with vali-
nomycin, they showed that most of this flux is electrically
neutral. The electrogenic H’ (or OH-) permeability ap-
pears to be an order of magnitude lower; possibly the
DIDS-insensitive monocarboxylate system described by
Deuticke (10) accounts for the remaining nonelectro-
genie H+ flux. Hence, it seems safe to assume that for
water channels, PH+ < 3 x lo? Accordingly, PH+Ipd <<
10B3, and we can conclude that H+ is virtually excluded
from the red cell water channel; apparently a Grotthus
conduction is not operative here. These results for H+
would follow if the channel were positively charged, but
an additional negative channel charge would be required
to explain the low electrogenic OH- and other anion
permeability. An alternative model (35), Fig. 4 considers
a hypothetical water channel where some of the water
molecules are isolated from one another by a patch of
relatively low dielectric so that Grotthus conduction
would be prevented. The channel could be formed en-
tirely by proteins, or as suggested by Brahm (Z), it could
be formed at lipid protein interfaces. Isolation of water
molecules has been advanced by Farrington (15) to ex-
FIG. 4. Hypothetical water channel. IWed circles represent mobile
water molecules; circles with a halo represent relatively immobile water.
Stippled region represents an area of high dielectric where there is
minimal contact between adjacent water molecules. Lower channel is
“open”; upper channel has been closed and is associated with decrease
in immobile water suggested by the large positive entropy change that
accompanies channel closure.
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Copyright © 1984 American Physiological Society. All rights reserved.
R. I. MACEY
plain the seven to nine orders of magnitude difference in
proton conduction in two very similar crystals (p-alu-
mina and p”-alumina). A low dielectric barrier might also
be responsible for the poor transport of other ions. The
hypothetical “immobile” water molecules shown lining
the channel are considered part of the channel structure.
This provides a polar environment for the LCmobile”
water, and release of immobile water during channel
closure could account for the large positive entropy
change.
Location of Water Channels
Although the location of the water channel is not
known with certainty, the observations by Solomon and
his co-workers (55) tend to implicate band 3 proteins.
They estimate that approximately 3 x lo5 channels are
required to account for the magnitude of Pd and, assum-
ing only one channel per protein, they point out that
only three integral membrane proteins (glycophorin,
band 3, and band 4.5) are present in sufficient quantity
to supply this number. Glycophorin is dismissed because
it lacks sulfhydryl groups; band 3 is selected because it
has a “cryptic” sulfhydryl group which, similar to the
receptor for inhibition of water transport, does not react
with n-ethylmaleimide but appears to react with p-
CMBS with an apparent dissociation constant of 0.2
mM. Arguments based on numbers of water channels are
suspect at this stage, because there is no unequivocal
way to estimate them. The estimation by Solomon et al.
(55) assumes, among other things, a value for %teric
hindrance,” which is obtained by curve fitting the perme-
ability data of a number of nonelectrolytes to the Renkin
equations. Application of these equations seemsinappro-
priate, because most probing solutes show saturation
kinetics (39). Urea for example has a permeability that
varies by orders of magnitude with concentration. Which
permeability should be used in the Renkin equations?
In my view, the discovery of a cryptic sulfhydryl group
in band 3, which appears to bind p-CMBS Faith a disso-
ciation constant of 0.2 M, is the principal if not the only
evidence linking band 3 with water channels. Earlier
evidence by Brown et al. (6) attempted to implicate band
3 by labeling the channels with radioactive 5,5,‘-di-
thiobis-2-nitrobenzoic acid (DTNB). Identification of
DTNB labeling with water channels is based on the
assumption that DTNB is an effective inhibitor of water
transport-a result that has not been clearly demon-
strated. The original observation of inhibition of Pf by
DTNB (42) was based on prolongation of hemolysis
times, which was simply assumed to be due a slower
water permeation. No account was taken of possible
changes in membrane fragility or of initial volume
changes, which generally follow electrolyte leakage
caused by sulfhydryl reagents. In stopped-flow experi-
ments where special attention was directed at initial
volume artifacts, we (Chien and Macey, unpublished
observations) found that DTNB had no effect on Pf,
whereas Levitt and Mlekoday (33) found a small 30%
inhibition. Further, studies of diffusional permeability
by Brahm (2) show no effect of DTNB on Pd. Later
studies (53) using radioactive p-CMBS as a channel
marker are compromised by its loose binding to the
BRIEF REVIEW CZOl

transport site; p-CMBS inhibition can be reversed by can induce a cation leakage-therefore, passive cation
saline washes. Thus, although band 3 remains a plausible fluxes are linked to the same channel. 4) Thiourea inhib-
site for water channels, evidence from radioactive tagging its the passive flux of urea and amides; it also prevents
with inhibitors is equivocal at best. p-CMBS from inhibiting water transport, and it inhibits
the p-CMBS inhibition of binding of DBDS, an inhibitor
Large Pores in the Membrane? of anion transport. Therefore, nonelectrolyte permeabil-
ity is linked to the same channel with water and anions.
The view expressed in this paper is that water channels The notion of a single transport pathway for permea-
are fairly specific. They appear to transport water and tion of a large number of permeants not only has the
very little else and may not even form a continuum in basic appeal of simplicity, it is also in accord with the
the conventional sense. Solutes also appear to have spe- fact that there is only a small number of integral proteins
cific transport pathways. It should be pointed out that in the red cell membrane. However, as indicated below,
this view is not universally accepted. The diametric it may also give rise to more problems than it answers.
opposite, a universal pore that transports m.ost solutes, In particular, linking the water channels with ion trans-
has been recently proposed by Solomon and co- workers port must account for the following.
(55). In brief, these authors postulate the existence of an 1) Both anion exchange and conductance are dramat-
aqueous pore 9 A in diameter that passes between the ically inhibited by DIDS, which has no effect on urea
two monomers of band 3 protein. This pore serves as a permeability or on water permeability. This is explained
common pathway for essentially all solutes that are by assuming that once DIDS enters the channel and
sufficiently small, including water, an ions, cations, and reacts with the protein site, conformational changes oc-
nonelectrolytes. cur which “internalize” the DIDS so that it no longer
Effects of p-CMBS on permeability are ascribed to “blocks” the channel. Not only is DIDS removed from
an assumed p-CMBS-induced conformational change, the channel, but it also implies that the channel pertur-
which distorts the pore, causing it to become na rrow and bations resulting from these conformational changes
more tortuous. In these terms, reduction of the ratio PIf have no effect on either urea or water permeability.
pd (to unity) and elevation of the activation energy for 2) p-CMBS dramatically inhibits water permeability,
water transport (to levels ch aracteristic of lipi .d bilayers) but it has no effect on anion exchange or conductance
are ascribed to increased frictional interactions as the (29). According to the hypothesis, water channels are not
pore narrows. closed by action of p-CMBS but they become so narrow
Problems with this interpretation arise because it has that Pf and Pd become equal. Further, according to
been shown both experimentally and theoretically that Solomon et al. (55), the large hydrated size of Cl- (radius
as the diameter of a channel narrows to approach single- of first shell E3.9 W) makes it highly unlikely that Cl
file diffusion, the ratio Pf/Pd does not become unity but ions can pass each other in the channel. Under these
rather becomes equal to the number of water molecules circumstances, it is hard to visualize how the dramatic
in the channel (31,50). Further, the fluorescence changes change in channel dimensions required to account for
observed by Solomon et al. (55), which are presumed to changes in water permeability has no effect on anion
reflect the conformational changes resulting from action permeability. The demonstration that p-CMBS causes
of p-CMBS, occur “with a time course of 1-5 min”; this an increase in cation leakage in vesicles containi .ng band
is several times shorter than the time required for inhi- 3 is provocative but it does not identify the water channel
bition of water transport (see Fig. 1A). Finally, the fact as a major pathway for cation leakage. The salient feature
that both pf/pd and the activation energies do approach is that water permeability decreases dramatically,
predicted values for pore closure seemshard .to overlook. whereas cation permeability increases. Assuming the two
To explain saturation kinetics for urea transport, the processesoccur on the same pathway requires additional
single-pore hypothesis assumes that the entrance to the ad hoc assumptions, e.g., there is removal of positive
pore is lined with H+ bonding sites; p-CMBS-induced charge from the channel, and somehow cations snake
conformational change inhibits by perturbing the posi- their way through the narrowed channel.
tion of these sites. To explain the low conductivity to Further, linking water and nonelectrolyte transport
cations and anions, it is assumed that the channel con- must account for the following.
tains both positive and negative charges; the positive 1) Both the concentration dependence and the time
charges are presumed to be rendered ineffective by p- dependence of p-CMBS action on urea transport do not
CMBS. Finally, confinement of all small polar per- compare with corresponding results on water transport.
meants, including water, anions, cations, and nonelectro- Examination of Fig. lB, for example, shows that at 0.11
lytes to the same transport pathway is based On the mM p-CMBS, urea permeability is reduced by>a factor
following observations. 1) The permeability data from a of 10, whereas water permeability is hardly inhibited.
number of small nonelectrolytes can be reconciled by 2) Permeation of small polar nonelectrolytes is as-
“curve fitting” them to the Renkin equations (48), which sumed to be highly dependent on H+ bonding at the
are based solely on steric hindrance in a single pore. 2) channel entrance. It is not clear why a unitary action of
Binding of the anion exchange inhibitor 4,4’-dibenza- p-CMBS has a profound effect on the H’ bonding for
mido-2,2’-stilbene disulfonic acid (DBDS) is inhibited some solutes, e.g., urea and no effect on others, e.g.,
by p-CMBS in N-ethylmaleimide-treated cells-there- ethylene glycol.
fore anion and water transport are linked. 3) Interaction 3) Although thiourea inhibits urea permeability and
of p-CMBS with the cryptic sulfhydryl group of band 3 interferes with the action of p-CMBS on water permea-

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Copyright © 1984 American Physiological Society. All rights reserved.
c202 R. I. MACEY

bility, this does not imply that urea and water share the Conclusion: Independent Pathways
same pathway; it is more easily explained by the fact
that p-CMBS inhibition of water permeability is readily The unitary pore hypothesis is a bold attempt to unify
reversible and that thiourea complexes with p-CMBS at a bewildering array of transport pathways into one single
low concentrations (Orme and Macey, unpublished ob- pore, and it is unfortunate, but perhaps not surprising,
servations). Thus thiourea inhibition may be similar to that the pore must be endowed with a large number of
the action of cysteine; it merely removes the lightly ad hoc properties to account for an equally large number
bound p-CMBS by compleigation. of diverse observations. For acceptability, not only must
4) Successful curve fitting of permeability data to the each of these properties be plausible when examined by
Renkin equation cannot be regarded as evidence for a itself, but they also must be consistent with each other
common permeation pathway, if as indicated above, the and with observation. As indicated above, it is not clear
probing solutes show concentration-dependent permea- how this consistency can be obtained without the intro-
bility. duction of even more ad hoc assumptions. In other words,
Finally the observations of Wieth and Brahm (60) the unitary pore hypothesis has lost its basic appeal-
show that a fully functional band 3 anion transport simplicity.
system can exist independently of the other solute or The bulk of the evidence supports the functionaE in-
water transport systems. Comparing water, urea, and dependence of transport pathways. This does not imply
anion transport in human, chicken, duck, and amphiuma that different loci in the same membrane macromolecule
red blood cells, they find that human cells have a high cannot be involved in the operation of more than one
water and high urea permeability; chicken cells have low “independent” transport system. Water transport in par-
water and low urea permeability; duck cells have high ticular appears to take place through exclusive narrow
water and low urea permeability; amphiuma cells have channels but there is no necessity to assume that water
low water and high urea permeability. In other words, forms a continuum through these channels; in fact, the
any combination can be found in some species, yet the low H ion permeability in these channels is easily ex-
properties and the capacity of the anion exchange system plained if there is no continuum. There remains the
is similar in all four types. Thus it is possible to have a possibility of incidental water transport through other
fully functional anion exchange and urea tran sport with ion channels; however, its magnitude appears to be
little or no evidence for aqueous channels (e. g ., am- dwarfed when compared to permeation through water
phiuma cells) or to have a fully functional anion ex- channel or lipid bilayer routes.
change, high water permeability, and very poor urea
transport (e.g., duck cells or human cells with 0.1 mM p-
CMBS). It is also possible to have a functional anion
exchange with no evidence for water channels and very This work was supported by National Institutes of Health Grant
poor urea transport (e.g., chicken cells or human cells GM-18819.
This review was based on the Jacobs-Parpart-Ponder Memorial
with 2 mM p-CMBS). Finally, using DIDS in human Lecture presented to the Red Cell Club at the Annual Meeting of the
cells, it is possible to knock out anion exchange and leave Federation of American Societies for Experimental Biology, Atlanta,
water and urea permeability intact. GA (April 1981).

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Robert I. Macey
Department of Physiology-Anatomy
University of California
Berkeley, California 94720

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