(Received for publication, August 29, 1995; and in revised form, October 26, 1995)
From the
The aquaporin-1 (AQP1) water transport protein contains a
polymorphism corresponding to the Colton red blood cell antigens. To
define the fraction of membrane water permeability mediated by AQP1,
red cells were obtained from human kindreds with the rare Colton-null
phenotype. Homozygosity or heterozygosity for deletion of exon I in AQP1 correlated with total or partial deficiency of AQP1
protein. Homozygote red cell morphology appeared normal, but clinical
laboratory studies revealed slightly reduced red cell life span in
vivo; deformability studies revealed a slight reduction in
membrane surface area. Diffusional water permeability (P) was measured under isotonic
conditions by pulsed field gradient NMR. Osmotic water permeability (P
) was measured by change in light
scattering after rapid exposure of red cells to increased extracellular
osmolality. AQP1 contributes
64% (P
= 1.5
10
cm/s) of the
total diffusional water permeability pathway, and lipid permeation
apparently comprises
23%. In contrast, AQP1 contributes >85% (P
= 19
10
cm/s) of the total osmotic water permeability pathway, and lipid
permeation apparently comprises only
10%. The ratio of
AQP1-mediated P
to P
predicts the length of the aqueous pore to be 36 Å.
It has long been argued whether the fundamental process of
membrane water permeability results from diffusion of water through the
lipid bilayer, transit of water through protein pores, or the sum of
both processes (reviewed by Finkelstein(1987)). Diffusional water
permeability (P) (
)represents
transmembrane flow of water in the absence of an osmotic gradient;
human red cells exhibit P
3
10
cm/s at 25 °C (Brahm, 1982). Osmotic water
permeability (P
) represents transmembrane
flow of water driven by an osmotic gradient; human red cells exhibit P
20
10
cm/s at 25 °C (Moura et al., 1984). Recognition that
red cell water permeability is inhibited by mercurials was taken as
evidence that protein water pores must exist (Macey and Farmer, 1970),
but it remains uncertain how much membrane water permeability is due to
lipid and how much is due to protein pores.
Discovery of the water transporter AQP1 (CHIP28) in red cells and renal tubules (Denker et al., 1988; Smith and Agre, 1991; Preston and Agre, 1991) led to the identification of the aquaporin family of water transporters (reviewed by Knepper(1994); Chrispeels and Agre, 1994). Analysis of AQP1 cDNA (Preston et al., 1992) and highly purified AQP1 protein (Zeidel et al., 1992b, 1994) permitted molecular characterization of water transport. The structure of aquaporins has been resolved by site-directed mutagenesis (Preston et al., 1993, 1994a; Shi et al., 1994; Jung et al., 1994) and by two-dimensional (Walz et al., 1994a, 1995; Mitra et al., 1994, 1995; Jap and Li, 1995) and three-dimensional electron crystallography (Walz et al., 1994b).
The physiological
importance of the collecting duct aquaporin homolog became apparent
when mutations in the AQP2 gene were found in some patients
with nephrogenic diabetes insipidus (Deen et al., 1994). The
Colton blood group antigens (Co and Co
)
represent a surface polymorphism in the AQP1 molecule (Smith et
al., 1994), and extremely rare individuals (Colton-null) became
sensitized to Co
and Co
fetal red cells during
pregnancy (Lacey et al., 1987). The physiological importance
of AQP1 was thrown into question when all three unrelated individuals
with the Colton-null phenotype were found to be homozygous for
disruptions in the AQP1 gene, yet none suffered an obvious
clinical defect (Preston et al., 1994b).
Detailed
biochemical and biophysical studies of red cells from homozygous
Colton-null individuals and their heterozygous relatives have not
previously been performed. Thus, it is not certain if they are entirely
normal clinically or how they may compensate for absence of AQP1, the
major red cell water transporter. Existence of red cells with a
specific deficiency of AQP1 protein should provide the purest system
for determining the fractions of diffusional (P) and osmotic (P
) water permeabilities mediated by AQP1
and will permit refined calculation of the length of the aqueous
pathway.
S and S are water signal
intensities in the presence and absence of gradient, k
and k
are forward and back rate constants,
and p
and p
are the mole
fraction of spins in each compartment; in K =
g
,
is the nuclear gyromagnetic ratio, and g and
denote gradient strength and length;
is the
separation between two gradients.
Experimentally, the gradient
strength g was changed from 3-55 G/cm, and the signal
decay (S) recorded with at least two values of (50 and
100 ms) and simultaneously fitted to to obtain four unknowns (D
, D
, k
, and k
). Fitting was by
Powell function (Press et al., 1992). Permeability in cm/s was
obtained by multiplying the forward rate constant k
by volume to surface area ratio (4.57
10
cm). Diffusion was measured in a General Electric Omega 400 NMR
Spectrometer equipped with a triple axis gradient unit (up to 130
G/cm); 5-mm sample tubes were analyzed at 20 °C.
where V(t) is relative red cell volume as a
function of time, P is in cm/s, SAV is
the vesicle surface area to volume ratio, MVW is the molar
volume of water (18 ml/mol), and C
and C
are initial concentrations of total
intracellular and extracellular solute. Red cell radii were calculated
from cell volume.
Figure 1:
Genetic and morphologic analysis of
AQP1-deficient, Colton-null red cell phenotype. A, geneology
of Colton deficiency in three generations of kindred 1; open
figures, normal Colton antigens; half-closed figures,
reduced Colton antigens; closed figures, absent Colton
antigens. B, Wright's stain of peripheral blood from
Colton-null subject IIa. C, Southern blot of 10 µg of
genomic DNA obtained from leukocytes of selected members of kindred 1
after digestion with PstI and electrophoresis through 1%
agarose, probed with P-labeled AQP1 cDNA.
Figure 2: Biochemical analyses of red cell membrane proteins prepared from an unrelated control individual (+/+), subject IIa who is homozygous for Colton-deficiency (-/-), and subject IIIa who is heterozygous for Colton deficiency (+/-). Top, Coomassie Blue-stained SDS-PAGE slab of red cell membranes. Packed membranes were serially diluted, and 10 µl were applied to the gel. Bottom, immunoblots of a duplicate SDS-PAGE slab probed with affinity-purified antibodies to spectrin, anion exchanger (AE1), glucose transporter (GLUT1), urea transporter (UT), and aquaporin-1 (AQP1). Note, the AQP1 blot reveals glycosylated subunits (broad bands with slower mobility) and nonglycosylated subunits (sharp bands with faster mobility).
Figure 3:
Osmotic deformability profiles of red
cells. Peripheral red cells from an unrelated control individual
(+/+, dashed line) and an individual homozygous for
AQP1 deficiency (subject IIa, -/-, solid line)
were analyzed by osmotic gradient ektacytometry, a technique that
determines whole cell deformability while the osmolality of the
suspending medium is continuously being changed. Consistent with a
small reduction in membrane surface area and a small decrease in
surface-to-volume ratio, the Colton-null red cells exhibit a minor
reduction in maximum deformability (at 290 mosM/kg) and a
small shift in the osmolality value at which red cells exhibit minimum
deformability in hypotonic medium (
140
mosM/kg).
Figure 4:
Diffusional water permeability (P) of red cells measured by pulsed field
gradient NMR spectroscopy. A, Water signal intensity versus K
values for a series of red blood
cell suspensions. B, coefficient of diffusional permeability
values computed from measurements in absence (black bars) and
presence of 1 mM PCMBS (stippled bars). Shown are
mean values ± S.D. (n = 8 measurements on
different control individuals and subject IIa; n = 4
measurements of subjects Ia and IIIa).
Figure 5:
Osmotic water permeability (P) of red blood cells. A,
representative tracings of red cells abruptly exposed to twice the
external osmolality with time course of water efflux monitored in a
stopped flow spectrophotometer. B, coefficients of osmotic
water permeability in the absence (black bars) and presence of
1 mM PCMBS (stippled bars). Shown are mean values
± S.D. (n = 6 measurements of different control
individuals; n = 4 measurements of subject IIa; n = 2 measurements of subjects Ia and IIIa). Note that
tracings in panel A are exponential
fits.
These studies have further defined the biophysical behavior
of AQP1-mediated water permeability in red cells. Several years before
identification of the aquaporins, indirect studies using mercurial
inhibitors suggested that 90% of the red cell osmotic water
permeability and
50% of diffusional permeability results from
transit of water through hypothetical pores in the membrane, while the
remainder is due to passage of water through the lipid bilayer
(reviewed by Finkelstein(1987)). Red cells totally devoid of AQP1 but
lacking other defects provided the membranes needed for direct
measurement of non-AQP1-mediated parameters. Our determinations are
close to the predicted values, and their significance is underscored by
determination of intermediate values from red cells from heterozygotes,
although it is not clear why heterozygote values were less than half of
the normal values.
Low molecular weight solutes do not permeate
through AQP1 (Zeidel et al. 1992a, 1992b, 1994), and urea is
now known to be transported across red cell membranes by a protein
unrelated to the aquaporins (Olives et al., 1995). Therefore,
it is reasonable to conclude that water goes through AQP1 by single
file diffusion (reviewed by Finkelstein(1987)). Since the diameter of
water is 2.72 Å, the diameter of the aqueous pore should be
slightly larger (3-4 Å). It has been theorized (Levitt,
1974) that for a water pore to be sufficiently narrow so that
individual water molecules cannot pass each other, the ratio of P/P
= N,
where N is the number of water molecules in single file within
the pore. Water permeability measurements in cells totally deficient in
AQP1 permitted correction of P
and P
to that specifically mediated by AQP1. The ratio P
/P
= 13.2 predicts
the length of the aqueous pathway of AQP1 to be 13.2
2.72
= 36 Å, somewhat shorter than the estimated
50-Å width of the lipid bilayer (Fig. 6). The
structure of AQP1 is being investigated by membrane crystallography of
functionally active molecules (Walz et al., 1994a), and their
tetrameric organization has been resolved to <7 Å (Walz et
al., 1995; Mitra et al., 1995; Jap and Li, 1995). Studies
of site-directed mutant molecules indicate that the tetramer contains
four functionally independent pores, although it remains uncertain
where the pores reside within the protein (Jung et al., 1994;
Preston et al., 1993). Further refinements in the membrane
crystallization process may make it possible to visualize aqueous
pores, which are predicted to be 4 Å in diameter and 36 Å
in length.
Figure 6:
Schematic of single file diffusion of
water through a pore. The equivalent length of the pore (L) is
36 Å (N, the number of water molecules 2.72
Å, the molecular diameter of water). The assumed width of the
bilayer is 50 Å.
These studies also provide further insight into the
physiological consequences of red cell membrane water permeability. The
surprising observation (Preston et al., 1994b) that total
deficiency of AQP1 does not produce obvious clinical manifestations has
been confirmed; however, subtle evidence for shortened red cell life
span and reduced membrane surface area was observed (Table 1, Fig. 3). Red cells from the individuals homozygous for
disruptions in the AQP1 gene do not appear to contain other
aquaporins, but measurements of the P of these
cells revealed a smaller but still significant amount of diffusional
water permeability (
36% of the P
of control
red cells). This non-AQP1-mediated diffusional water permeability may
partially explain the lack of hematologic manifestations of the
AQP1-deficient state. In contrast, the osmotic permeability
measurements of red cells totally deficient in AQP1 was more
dramatically reduced (<15% of the P
of control
red cells). The physiological importance of P
is
thought to involve the need for rapid osmotic water movements when red
cells permeate the hypertonic renal medulla, but it remains unknown if
AQP1deficient individuals retain full renal concentration mechanisms.
If osmotic water permeation occurred exclusively through AQP1, P/P
= 1 in red cells
totally deficient in AQP1. Our studies provided a ratio of 3.4,
suggesting that pathways other than AQP1 and diffusion through the
lipid bilayer must exist in red cell membranes. This paradigm is
supported by analysis of the P
in red cells
totally deficient in AQP1 (Fig. 4), which appears to be
comprised of a small PCMBS-inhibitable pathway in addition to the
PCMBS-resistant pathway, and their composite actions exhibit a high
activation energy indicating that they are not aqueous pores. While the
identities of these two minor water permeation pathways are unknown, it
may be speculated that the PCMBS-inhibitable pathway is a protein, and
the glucose transporter has been shown to transport water at a very low
rate (Fischbarg et al., 1990; Zeidel et al., 1992a).
The PCMBS-resistant pathway may represent simple diffusion through the
lipid bilayer. Thus, the movement of water across the red cell membrane
may reflect the complex behaviors of multiple membrane components in
addition to AQP1.