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INTRODUCTION |
The shape and deformability of the human red blood cell
(RBC)1 membrane are thought
to be maintained by a combination of protein-protein and protein-lipid
interactions. Prominent among the former are associations within the
spectrin-actin skeleton (often termed horizontal interactions), and
bridges between the membrane skeleton and various membrane-spanning
proteins (frequently called vertical interactions) (1, 2). The
horizontal interactions are considered important, since they maintain
the architecture and stability of the protein network underlying the
lipid bilayer (3-5). The vertical interactions are thought to be
essential, since they permit stress sharing between the lipid bilayer
and protein skeleton (6), and because they prevent aggregation of
membrane-spanning proteins and the consequent vesiculation of
protein free lipid domains (7, 8).
Although several minor integral membrane proteins are
believed to attach to the membrane skeleton (9-11), the band
3-ankyrin-spectrin bridge and the glycophorin C-protein
4.1-spectrin/actin bridge are thought to constitute the major tethers
between the membrane skeleton and the lipid bilayer (12). Evidence that
the band 3-ankyrin-spectrin bridge is critical to membrane mechanical
properties derives primarily from analyses of cells with genetic
defects in one or more members of this bridge. Thus, mutations leading to reduced expression or abnormal folding of band 3, ankyrin, or
spectrin generally lead to membrane vesiculation and hereditary spherocytosis (3, 4, 13-16). Other studies aimed at manually disrupting the band 3-ankyrin-spectrin bridge also document a causal
relationship between rupture of the bridge and loss of normal cell
morphology and stability (6).
Evidence for the importance of the glycophorin C-protein
4.1-spectrin/actin bridge to membrane shape and flexibility also stems
from investigations of natural defects in the bridging components. Thus, deficiencies in either protein 4.1 or glycophorin C commonly result in elliptocytic cells with compromised mechanical properties. However, although such observations might initially be construed to
suggest that the protein 4.1-mediated bridge to the membrane skeleton
is critical to membrane stability, observations by Narla and colleagues
(17-20) raise questions regarding the validity of such an
hypothesis. Thus, quantitative investigations of the above elliptocytic
cells have revealed that a natural deficiency in glycophorin C is
invariably accompanied by a decrease in protein 4.1 (17, 18) and that
reconstitution of only the spectrin-actin binding domain of protein 4.1 into protein 4.1-deficient cells fully restores the membrane's
mechanical properties (19, 20). Because the above reconstitution
procedure does not re-establish protein 4.1's attachment to
glycophorin C or the lipid bilayer, these data suggest that the protein
4.1-mediated bridge has little impact on membrane stability.
In order to examine the contribution of the glycophorin
C-protein 4.1-spectrin/actin tether to red cell membrane structure in
greater detail, we have undertaken to manually disrupt the bridge
without compromising protein 4.1's established role in stabilizing the
spectrin-actin junctional complex. For this purpose, we have explored
methods to break the protein 4.1-mediated tether in intact cells and
have examined the impact of this rupture on membrane mechanical
properties. We report here four novel and one previously published
protocol for weakening/severing the protein 4.1 linkage to glycophorin
C. Although not all of the protocols allow for an unambiguous
evaluation of the role of the glycophorin C linkage in establishing
membrane mechanical properties, the collective results support the
contention that the glycophorin C-protein 4.1-spectrin/actin bridge
does not contribute prominently to membrane stability.
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EXPERIMENTAL PROCEDURES |
Materials--
Phorbol 12-myristate 13-acetate
(PMA), 4
-phorbol 12,13-didecanoate (4
-PDD), staurosporine, and
calyculin A were purchased from Calbiochem. Phosphoenolpyruvate (PEP),
anti-actin antibody, phenylmethanesulfonyl fluoride, pepstatin A,
leupeptin, and dimethyl sulfoxide (Me2SO) were purchased
from Sigma. Human blood was drawn by venipuncture into acid citrate
dextrose anticoagulant following informed consent and used on the same
day as withdrawal.
Protocols for Rupturing the Glycophorin C-Protein 4.1 Bridge in Intact RBCs--
RBCs were sedimented at 800 × g for 5 min, washed twice in phosphate-buffered saline (PBS,
pH 7.4) and added to an
-cellulose-Sigma Cell column to remove white
cells and platelets. Collected RBCs were then washed with HEPES buffer
(125 mM NaCl, 3 mM KCl, 16 mM
HEPES, 10 mM glucose, 2 mM CaCl2, 1 mM MgCl2, pH 7.4) and resuspended in the same
buffer prior to subjection to one of the following protocols for
severing glycophorin C-protein 4.1 interactions. In the first
methodology, washed RBCs were incubated at 40% hematocrit for 1 h
at 37 °C with increasing concentrations of phosphoenolpyruvate. Phosphoenolpyruvate is rapidly transported into erythrocytes and converted to 2,3-bisphosphoglycerate (21), which is reported to inhibit
protein 4.1 binding to the red cell membrane in vitro (22).
In a second protocol, RBCs at 5% hematocrit were loaded with
Ca2+ by treatment with 1 µM A23187 and
various calcium-EGTA buffers (23) for 30 min at 37 °C. The influx of
Ca2+ would be expected to down-regulate protein
4.1-membrane interactions based on its ability to prevent 4.1 binding
to band 3 and glycophorin C in vitro (24, 25). Third, RBCs
at 5% hematocrit were incubated for 30 min at 37 °C with 0.1 µM phorbol ester (PMA dissolved in Me2SO) in
order to activate protein kinase C (26). As controls, the inactive
analog of PMA, 4
-PDD, or an equal volume of Me2SO was
added to analogous cell suspensions. Whenever protein kinase or
phosphatase inhibitors were also employed, they were added to the red
cell suspensions 30 min prior to PMA treatment. Fourth, RBCs suspended
at 5% hematocrit in deoxygenated HEPES buffer were bubbled with
N2 gas for ~5-30 min (depending on the volume of the
suspension) at room temperature to deoxygenate the cells. To evaluate
the reversibility of this treatment, reoxygenation was carried out by
mixing the deoxygenated RBCs in oxygenated HEPES buffer. Finally, RBCs
at 5% hematocrit were equilibrated for 3 h in MES buffers (27) of
pHs ranging from pH 5.5 to 7.4.
Preparation of Membrane Skeletons--
After the desired
incubations, packed RBCs were collected and mixed with an equal volume
of PBS. Membrane skeletons were then isolated by incubating the
suspensions in two volumes of 1% C12E8 (Nikko
Chemical Co.) in PBS supplemented with 0.5 mM
dithiothreitol, 20 µg/ml leupeptin, 20 µg/ml pepstatin A, 1 mM EDTA, and 80 µg/ml phenylmethanesulfonyl fluoride. The
mixture was loaded onto a 35% sucrose cushion and centrifuged at
85,500 × g for 90 min to separate the solubilized
membrane components from the pelleted membrane skeletons. The pellet
was solubilized in SDS buffer, and its protein concentration was
measured using the bicinchoninic acid assay.
Electrophoretic and Immunoblot Analysis of Membranes and Membrane
Skeleton Components--
Samples dissolved in SDS sample buffer were
separated on 10% Laemmli gels, as described elsewhere (28). After
transfer to nitrocellulose membranes, nonspecific protein binding was
blocked by incubating the nitrocellulose (Bio-Rad) for 1 h in 5%
milk-TBST (50 mM Tris, 200 mM NaCl, 0.05%
Tween 20, pH 7.5). The nitrocellulose membranes were then incubated
with a mouse monoclonal anti-glycophorin C antibody (BRIC 10, a kind
gift from D. J. Anstee, International Blood Group Reference
Library, Bristol, United Kingdom), anti-actin, anti-adducin (a
kind gift from V. Bennett, Duke University, Durham, NC), anti-band 3, anti-protein 4.1, or anti-glycophorin A in 1% milk-TBST for 1 h.
The resulting nitrocellulose membranes were washed three times with
0.1% milk-TBST, incubated with horseradish peroxidase-conjugated
secondary antibody in 1% milk-TBST for 30 min, and washed three times
with 0.1% milk-TBST. Finally, the membranes were incubated with ECL
chemiluminescent reagent (Amersham Pharmacia Biotech) and exposed to
x-ray film. When reprobing the same nitrocellulose sheet was
necessary, the nitrocellulose membrane was incubated with stripping
buffer (100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) for 45 min at 70 °C, washed three
times with TBST, and then relabeled with a different antibody and
developed, as described above. Quantitation of the glycophorin C
content in the membrane skeletons by densitometry was invariably performed by normalizing its staining intensity to that of actin in the
same gel lane. Using this method, the glycophorin C content of control
membranes at pH 7.4 was arbitrarily set at 100%.
Analysis of the Phosphorylation of Membrane Proteins--
RBCs
suspended at 50% hematocrit in HEPES buffer were incubated with 0.5 mCi of 32PO4/ml of packed cells for 2 h at
37 °C. The cells were washed twice with HEPES buffer before PMA
treatment and SDS-polyacrylamide gel electrophoresis, as described
above. Identification of phosphorylated membrane polypeptides was made
by analyzing the resulting polyacrylamide gel on a phosphorimager
(Cyclone, Packard Instrument Co.).
Measurement of 2,3-BPG Content in RBC--
The concentration of
2,3-BPG in deproteinized extracts of phosphoenolpyruvate-treated
erythrocytes was measured using a Sigma diagnostic kit. Briefly, red
cells were suspended at 50% hematocrit in PBS, mixed with three
volumes of trichloroacetic acid, and pelleted at 8000 × g. The supernatant was neutralized with NaOH and mixed with
a triethanolamine assay mixture containing NADH, ATP, phosphoglycerate
mutase, 3-phosphoglycerate phosphokinase, and
glyceraldehyde-3-phosphate dehydrogenase. The conversion of NADH to NAD
was initiated by addition of phosphoglycolate, and the amount of
2,3-BPG in the extracts was calculated from the amount of NADH
consumed, using the absorbance at 340 nm to quantitate NADH consumption.
Deformability and Stability Measurements--
After the desired
treatment, packed RBCs were suspended in 4% polyvinylpyrrolidone
(Mr 360,000) in PBS and examined by the ektacytometry, a laser diffraction method described elsewhere (29). In
brief, suspended cells were subjected either to increasing osmolality
(from 50 to 500 mosmol/kg) at constant shear stress or to constant
osmolality at increasing shear stress (0-250 dynes/cm2),
and the axial ratio of the deformed cells was quantitated by laser
diffraction and designated as their deformability index (DI). A
measurement of DI as a function of osmolality and shear stress can
provide information on both the hydration of the cell and the
flexibility of the membrane.
For stability measurements, packed RBCs were lysed in 40 volumes of 10 mM sodium phosphate (pH 7.4) and pelleted at 20,000 × g. After centrifugation, the lysed cells were resealed by
adding 20 volumes of PBS and incubating for 45 min at 37 °C. The
resealed ghosts were washed one time in PBS, resuspended in a 35%
dextran-phosphate buffer, and analyzed by ektacytometry at constant
high shear stress (785 dynes/cm2), as described above. The
decline in deformability with time derives from the mechanical
fragmentation of the membranes into nondeformable vesicles. The
half-time for this shear-induced fragmentation to reach completion can
be used as a measure of the membrane's mechanical stability (30).
Filterability Measurements--
Analysis of the rate of
filtration of an RBC suspension through a highly uniform nickel mesh
filter was conducted by a gravity-based, vertical tube method (31) that
measures the rate of passage of erythrocytes through the filter as a
function of hydrostatic pressure (Tsukusa Sokken Co., Tokyo, Japan).
The pore diameter of the nickel mesh used in this study was 4.6 µm.
From a height (pressure)-time curve obtained during the filtration
analysis, a pressure-flow rate relationship can be determined. The
filtration was started at a pressure of 150 mm H2O, and the
percentage of the flow rate (ml/min) of the RBC suspension (0.1%
hematocrit) relative to that of the suspending medium at 100 mm
H2O was taken as the index of RBC filterability.
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RESULTS |
A variety of observations in the literature,
stemming primarily from studies on purified membrane components,
have suggested that the glycophorin C-protein 4.1 bridge to the
membrane skeleton might be physiologically regulated (22, 24, 32-34).
However, except for the impact of protein kinase C phosphorylation on
this bridging function (35), none of the potential regulatory pathways has been validated in intact erythrocytes. As a tool for investigating the significance of the glycophorin C-protein 4.1- spectrin/actin bridge to membrane mechanical properties, we have undertaken to characterize methods that might sever this bridge in intact
erythrocytes. Although not all of the hypothetical mechanisms could be
validated in situ, those that were confirmed (or newly
discovered) will be briefly characterized below, since they reveal much
about the possible regulation of the glycophorin C-protein
4.1-spectrin/actin bridge in vivo. Where such treatments
also shed light on the function of the glycophorin C-protein 4.1 bridge, they will be further evaluated for their impacts on cell
stability and deformability.
Effect of Elevated 2,3-Bisphosphoglycerate--
In a previous
study (22), we demonstrated that 2,3-BPG inhibited protein 4.1 binding
to stripped inside-out erythrocyte membrane vesicles. More detailed
studies with antibodies to band 3 and glycophorin C further established
that protein 4.1 binding to both membrane-spanning proteins was
specifically prevented by BPG. In an effort to test the impact of
elevated 2,3-bisphosphoglycerate on protein 4.1's bridging function
in situ, we incubated RBCs with PEP, a membrane-permeable
glycolytic substrate that is rapidly metabolized to
2,3-bisphosphoglycerate and lactate in situ (21). Calibration of this method in whole erythrocytes reveals that cytoplasmic BPG levels can be elevated in direct proportion to the
concentration of phosphoenolpyruvate added (Fig.
1A). By 25 mM
external PEP, intracellular BPG rises to 9.3 mM,
essentially twice its normal value.

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Fig. 1.
A, effect of external PEP on the
cytosolic concentration of 2,3-BPG. RBCs were incubated at 37 °C for
1 h in the absence or presence of PEP. Cellular 2,3-BPG content
was then determined, as described under "Experimental Procedures."
Each point represents the average ± S.D. of three
determinations. B, immunoblot analysis of the glycophorin C
content of erythrocyte membrane skeletons derived from PEP-treated
cells. After incubation at the indicated PEP concentrations, membrane
skeletons were isolated, analyzed by SDS-PAGE, and immunoblotted with
antibodies to glycophorin C and actin. Anti-actin antibody staining was
conducted to ensure even loading of protein in each gel lane.
Densitometric results were always normalized to the intensity of the
actin band in the same gel lane. Glycophorin C retained in control
membrane skeletons constitutes ~70% of the glycophorin C present in
the parent intact membranes.
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To evaluate the influence of BPG on the protein 4.1-glycophorin C
linkage in situ, PEP loaded RBCs were injected into a 1% solution of the nondenaturating detergent,
C12E8, and their membrane skeletons were
isolated on a sucrose density gradient. As shown in Fig. 1B,
at low PEP concentrations (0-10 mM), where the total intracellular BPG content was
6 mM, little change in
glycophorin C retention in the membrane skeletons was observed.
However, as PEP concentration was elevated, cytosolic BPG increased and
glycophorin C retention in the membrane skeletons was strongly
hindered. By 9 mM BPG (25 mM PEP), only 48 ± 8% (mean ± S.D., n = 3) of the original
glycophorin C remained in the membrane skeletons. Because the protein
4.1 content of these skeletal preparations was essentially unaltered
(data not shown), we conclude that BPG can sever the glycophorin
C-protein 4.1 bridge without displacing protein 4.1 from the spectrin skeleton.
Unfortunately, PEP loading was also observed to induce
hemoglobin oxidation (probably due to depletion of NADH via the lactate dehydrogenase reaction), leading to hemichrome deposition on the membrane (data not shown). Because hemichrome precipitation can independently rigidify the membrane (36), evaluation of the treated
cell's mechanical properties was not pursued. Nevertheless, the
biochemical data do suggest that endogenous changes in BPG, such as
those that occur during oxygenation/deoxygenation (37), might have the
potential to regulate the protein 4.1-glycophorin C bridge in
vivo.
Effect of Ca2+ Loading--
Previous studies
from our laboratory (24) and others (38) have shown that
Ca2+ plus calmodulin can block protein 4.1 binding to
glycophorin C. In fact, a recent crystallographic structure of the
N-terminal 30-kDa domain of protein 4.1 reveals that calmodulin
associates at a central site on protein 4.1 and that calmodulin's
occupancy of this site should obstruct protein 4.1's interaction with
glycophorin C, p55, and band 3 (39). To determine whether
Ca2+ influx might regulate the glycophorin C-protein 4.1 bridge in vivo, fresh erythrocytes were treated with a
Ca2+ ionophore (1 µM A23187) and equilibrated
with various concentrations of Ca2+ using
Ca2+-EGTA buffers. As shown in Fig.
2A, increasing intracellular
Ca2+ concentration indeed releases glycophorin C from its
attachment to the membrane skeleton. Although no change in skeletal
composition could be detected up to 0.1 mM
extracellular Ca2+, by 0.3 mM Ca2+
a 14% (n = 4) decline in glycophorin C content
was observed, and by 1 mM Ca2+, glycophorin C
retention in the membrane skeletons was reduced by 75%
(n = 2). Curiously, adducin binding was affected
similarly to glycophorin C, whereas the band 3 and protein 4.1 contents in the membrane skeletons remained essentially unchanged. We conclude from these data that Ca2+ can indeed modulate the
glycophorin C-protein 4.1 bridge in situ, as predicted by
studies in simplified systems (24, 38), but that measurable changes
only occur when intracellular Ca2+ concentrations exceed
those experienced by healthy cells.

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Fig. 2.
A, immunoblot analysis of erythrocyte
membrane skeletons derived from Ca2+- plus A23187-treated
cells. Erythrocytes were incubated for 30 min with Me2SO
(solvent control) or 1 µM A23187 in buffer (125 mM NaCl, 3 mM KCl, 10 mM glucose, 1 mM MgCl2, 16 mM HEPES, pH 7.4)
containing increasing concentrations of calcium established with
calcium chelate buffers. Membrane skeletons were then isolated and
analyzed by SDS-PAGE, followed by immunoblotting with anti-glycophorin
C, anti-actin, anti-adducin, anti-band 4.1, or anti-band 3 antibodies.
The anti-actin antibody staining was conducted to ensure even loading
of protein in each gel lane. B, evaluation of the
deformability of Ca2+- plus A23187-treated cells.
Erythrocytes were incubated in the standard HEPES buffer containing 90 mM KCl and 40 mM NaCl (instead of 125 mM NaCl and 3 mM KCl) to prevent
Ca2+-induced RBC dehydration that stems from activation of
the Gardos channel (40). Osmotic deformability was then measured by
subjecting the RBCs to a moderate shear stress while increasing osmotic
pressure. C, evaluation of the filterability of
Ca2+- plus A23187-treated cells. RBCs were diluted to 0.1%
hematocrit in the above high K+ HEPES buffer and allowed to
flow through a 4.6-µm pore-sized nickel mesh filter. The height of
the column of suspended erythrocytes was recorded on-line as a function
of time, and the filtration rate was derived assuming first order
kinetics.
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Analysis of the mechanical properties of the Ca2+-loaded
erythrocytes was complicated by unwanted side effects of
Ca2+ on several unrelated red cell systems. Even when
internal and external [K+] were balanced to prevent
Ca2+-activated K+ efflux and cell shrinkage
(40, 41), Ca2+ stimulation of RBC phospholipase C (42, 43),
the phospholipid scramblase (44), the transglutaminase (45) (note the
protein 4.1 near the top of the gel in Fig. 2A that was
presumably cross-linked through
-glutamyl-
-lysine bridges by the
RBC transglutaminase), and adducin release (Fig. 2A) either
preceded or coincided with the Ca2+-induced release of
glycophorin C. Thus, although the maximum deformability index
(DImax) and filterability of the K+ balanced
erythrocyte suspensions declined by 15% (Fig. 2B) and 44%
(Fig. 2C) at 500 µM Ca2+,
respectively, in comparison to the DImax and filterability
of normal erythrocytes, it was not possible to assign the decline to a
rupture of the protein 4.1 bridge. In fact, since no changes in
deformability or filterability were observed at 0.3 mM
Ca2+, where protein 4.1 release from glycophorin C had
already begun (Fig. 2A), the absence of any causal
relationship between the two processes would seem more plausible.
Effect of Oxygen Content--
During our search for physiological
modulators of the glycophorin C-protein 4.1 bridge, we reasoned that
RBCs might be capable of responding to entrapment in the
microvasculature by becoming more flexible. Based on this speculation,
we examined the impact of RBC deoxygenation on the integrity of the
glycophorin C-protein 4.1 linkage. Deoxygenated RBCs were generated by
bubbling N2 through the cell suspension, and membrane
skeletons were isolated by plunging the deoxygenated cells directly
into C12E8 detergent solution before any
reoxygenation could occur. As seen in Fig.
3A, less glycophorin C was
retained in membrane skeletons from deoxygenated cells than oxygenated
cells. Densitometric analysis, in fact, indicates that glycophorin C's
content was reduced 30 ± 16% (n = 4) following
O2 removal. Restoration of O2 by washing the
cells in O2-saturated buffer also restores the glycophorin
C content to its normal level (Fig. 3A), demonstrating that
O2-mediated rupture of the 4.1 bridge is fully reversible.
To further confirm that RBC oxygenation can regulate protein 4.1's
bridging function, RBCs were treated with 10 mM dithionite
to chemically consume the erythrocyte's O2, and the
extracted membrane skeletons were again examined for glycophorin C
retention. As expected (Fig. 3A), dithionite also severs the
glycophorin C-protein 4.1 linkage.

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Fig. 3.
A, immunoblot analysis of erythrocyte
membrane skeletons derived from deoxygenated RBCs. Erythrocytes were
bubbled with nitrogen gas or treated with 10 mM sodium
dithionite for 5 to 30 min at 37 °C to promote deoxygenation. For
reoxygenation studies, previously deoxygenated erythrocytes were washed
several times in oxygenated buffer. Membrane skeletons were then
isolated and analyzed by SDS-PAGE, followed by immunoblotting with
anti-glycophorin C and anti-actin antibodies. The anti-actin antibody
staining was conducted to ensure even loading of protein in each gel
lane. Sodium sulfate was used as a negative control for sodium
dithionite, since it does not alter O2 content. Nitrogen
purging for periods as short as 5 min induced measurable changes in
glycophorin C retention, whereas bubbling for >30 min caused no
additional changes. B, effect of deoxygenation on the
filterability of RBCs. RBCs at 0.1% hematocrit were deoxygenated for
1 h and allowed to flow through a 4.6-µm pore-sized nickel mesh
filter as described in Fig. 2. Filtration rates were recorded on-line
by monitoring the height of the RBC suspension as a function of
filtration time.
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Analysis of the mechanical properties of the dithionite-treated cells
was unfortunately hindered by the known generation of hemichromes
during dithionite treatment (46). Simple deoxygenation of the cells,
however, was not compromised by any known limitation or side effect;
therefore, normally deoxygenated cells were examined for changes in
filterability and deformability. As displayed in Fig. 3B,
deoxygenation similar to that achieved in Fig. 3A causes no
change in the rate of RBC filtration through a 4.6-µm nickel mesh
filter. This result suggests that, although the availability of
O2 may modulate the integrity of the 4.1 bridge,
penetration of the deoxygenated erythrocytes through a narrow pore is
not altered. In a previous study, examination of deoxygenated cells in
an ektacytometer revealed no reproducible changes in deformability (47). We, therefore, doubt that deoxygenated cells have improved rheological properties over oxygenated cells.
Effect of pH--
Because of the abundance of anion transporters
(band 3) in the erythrocyte membrane, hydroxide can rapidly equilibrate
across the membrane, leading to facile changes in intracellular pH.
During RBC transit through exercising muscles and the spleen, serum pH can decline to <6.8, especially when normal blood flow is retarded, raising the question whether pH might be exploited to regulate membrane
mechanical properties in vivo. To test whether the protein 4.1 bridge might be subject to pH regulation, RBCs were equilibrated at
various external pHs, and examined for changes in membrane skeletal
interactions, as described above. Although no change in glycophorin C
content could be detected at pH values above 7.4, as pH was decreased
below neutrality, the glycophorin C bridge gradually dissolved. By an
external pH of 5.5, 45 ± 11% (n = 3) of attached
glycophorin C molecules could be readily extracted from the membrane
skeletons (Fig. 4A).
Significantly, the largest change in glycophorin C extractability was
observed between pH 6.8 and 6.0 (Fig. 4B), where pH changes
might occur under conditions of stress. These data suggest that the
glycophorin C-4.1 bridge to the membrane skeleton might be modulated by
pH.

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Fig. 4.
A, immunoblot analysis of erythrocyte
membrane skeletons derived from RBCs incubated for 30 min at 37 °C
at the indicated pHs. After incubation, membrane skeletons were
isolated and analyzed by SDS-PAGE, followed by immunoblotting with
anti-glycophorin C and anti-actin antibodies. The anti-actin antibody
staining was conducted to ensure even loading of protein in each gel
lane. B, densitometric analysis of glycophorin C retention
in membrane skeletons derived from RBCs incubated at various pHs. The
band intensities of glycophorin C and actin from three independent
Western blots were measured by densitometry. The ratio of glycophorin
C/actin in control skeletons at pH 7.4 was set at 100%, and all other
ratios are reported as a percentage of this value.
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To determine whether the observed rupture of the glycophorin C-protein
4.1 bridge to the membrane skeleton affects the deformability or
filterability of the cells, RBCs were equilibrated at various pH levels
and examined in both the ektacytometry and nickel mesh filtration
apparatus. As shown in Fig.
5A, a decrease in external pH
promotes a minor decline in cell hydration (as indicated by the right
shift in the osmotic ektacytometry scans; see also Ref. 27), but no
significant change in DI at optimum hydration. This result suggests
that the intrinsic deformability of the membrane is not altered by
partial rupture of the protein 4.1 bridge. Significantly, a similar
conclusion is also supported by data on red cell filterability (Fig.
5B). Thus, except for a very modest decrease in
filterability due to the aforementioned hydration, no significant
decline in filtration rate was observed. It is, therefore, plausible
that the pH-induced dissociation of glycophorin C and protein 4.1 occurs with few or no mechanical consequences.

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Fig. 5.
Evaluation of the deformability and
filterability of RBCs equilibrated at various pHs. Erythrocytes
were incubated at the indicated pHs for 3 h at 37 °C, and
osmotic deformability was measured by subjecting the RBCs to a moderate
shear stress while increasing osmotic pressure (A).
Alternatively, equilibrated cells were further diluted to 0.1%
hematocrit and allowed to flow through a 4.6-µm pore-sized nickel
mesh filter. The height of the erythrocytes suspension was recorded as
a function of filtration time as the cells were forced by hydrostatic
pressure though the nickel mesh filter (B).
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Effect of Stimulation of Protein Kinase
C--
Activation of protein kinase C in human erythrocytes can be
induced by insulin and perhaps other hormones (48, 49). Stimulation of
erythrocyte protein kinase C leads to phosphorylation of three prominent membrane skeletal proteins: adducin, dematin, and
protein 4.1 (26, 50, 51). Phosphorylation of protein 4.1 has been specifically shown to inhibit its association with stripped inside-out erythrocyte membrane vesicles (34), suggesting that phosphorylation might regulate the glycophorin C-protein 4.1-spectrin/actin bridge in situ. To explore this possibility, RBCs were incubated in
0.1 µM PMA, an activator of protein kinase C, and the
polypeptide content of the derived membrane skeletons was examined as
described above. As noted by Gratzer and colleagues (35), PMA
promotes release of glycophorin C from membrane skeletons (Fig.
6A). In our hands, the
glycophorin C content was reduced 58 ± 13% (n = 4), and adducin levels were also somewhat diminished. Although lower concentrations of PMA promoted less dissociation, higher concentrations exerted no additional effect (data not shown). Furthermore, the availability of Ca2+ seemed to have no
impact on membrane skeleton protein composition, indicating that the
PMA-stimulated dissociation of glycophorin C and
-adducin was caused
by a Ca2+- independent protein kinase C isoform (Fig.
6A).

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Fig. 6.
A, immunoblot analysis of erythrocyte
membrane skeletons derived from PMA-treated cells. Erythrocytes were
incubated with Me2SO (control) or 0.1 µM PMA
dissolved in Me2SO in isotonic HEPES buffer containing
either 2 mM calcium (+calcium) or 100 µM EGTA ( calcium) for 30 min. Membrane
skeletons were then isolated and analyzed by SDS-PAGE followed by
immunoblotting with anti-glycophorin C, anti-adducin, anti-actin,
anti-glycophorin A, anti-band 3, or anti-protein 4.1 antibodies. The
anti-actin antibody staining was conducted to ensure even loading of
protein in each gel lane. B, effect of PMA-induced
phosphorylation on the retention of glycophorin C in membrane
skeletons. Erythrocytes were incubated with Me2SO
(control), 3 µM staurosporine, or 0.2 µM
calyculin A for 30 min, and then further incubated with
Me2SO, 0.1 µM 4 -PDD, or 0.1 µM PMA for 30 min, as indicated in the figure. Membrane
skeletal fractions were then isolated and analyzed by SDS-PAGE,
followed by immunoblotting with anti-glycophorin C and anti-actin. The
anti-actin antibody staining was conducted to ensure even loading of
protein in each gel lane.
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To confirm that dissociation of glycophorin C and
-adducin was a
consequence of their phosphorylation, the influence of several modulators on both processes was also compared. First, RBCs were metabolically labeled with 32PO4 and the impact
of PMA, 4
-PDD (an inactive analog of PMA), staurosporine (a protein
kinase inhibitor), and calyculin A (a phosphatase inhibitor) on the
stimulated incorporation of 32PO4 into adducin,
4.1, and 4.9 in the presence or absence of calcium was examined.
Importantly, 0.1 µM PMA induced the phosphorylation of
the above proteins, whereas 4
-PDD did not, regardless of the availability of extracellular Ca2+ (Fig.
7A). And as expected,
staurosporine inhibited the PMA-induced phosphorylation, whereas
calyculin A enhanced it (Fig. 7B). To verify that these
phosphorylation changes were indeed responsible for the observed
changes in protein interactions, RBCs incubated with the same
kinase/phosphatase inhibitors were again stimulated with PMA and
examined for changes in skeletal protein composition (Fig.
6B). In agreement with the phosphorylation studies,
staurosporine completely blocked the effect of PMA on the dissociation
of glycophorin C, whereas calyculin A enhanced it. As above, 4
-PDD
had no effect on retention of glycophorin C in the membrane
skeletons.

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Fig. 7.
Phosphorylation of membrane skeletal proteins
by PMA. A, erythrocytes were metabolically labeled with
32PO4 and treated for 30 min in the presence of
calcium (+calcium) or EGTA ( calcium) with
Me2SO (DMSO), 0.1 µM 4 -PDD (the
inactive analog of PMA) or 0.1 µM PMA. B,
erythrocytes were incubated with Me2SO, 3 µM
staurosporine, or 0.2 µM calyculin A for 30 min, and then
further incubated with Me2SO, 0.1 µM
4 -PDD, or 0.1 µM PMA for an additional 30 min, as
indicated in the figure. Skeletal fractions were prepared and collected
by ultracentrifugation and subjected to SDS-PAGE. Phosphorylation was
visualized using a Cyclone phosphorimager.
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By restaining the above blots with antibodies to other components of
the membrane skeletons, it was also possible to demonstrate that band
3, protein 4.1, and glycophorin A remained unaffected by PMA treatment
(Fig. 6A). Thus, as with the other modulators of the
glycophorin C bridge, only glycophorin C and possibly adducin respond
with a change in skeleton association. These data would suggest that
regulation by protein kinase C is not random, but instead is focused on
the bridging interaction that is modulated by many other erythrocyte variables.
To determine whether the aforementioned PMA-induced changes in protein
interactions exert an impact on red cell deformability, RBCs were
treated with PMA and subjected to increasing osmolality during shear
stress. As revealed by the ektacytometry, no significant changes in the
DI either as a function of osmolality (Fig.
8A) or shear stress (Fig.
8B) were observed in response to PMA, even for incubation
periods up to 2 h. Although higher concentrations of PMA (above 3 µM) did lead to decreases in DI (data not shown), we
attribute these changes to nonspecific effects, since maximal phosphorylation is attained at 0.1 µM PMA (26) and higher
concentrations of PMA do not further enhance protein phosphorylation or
dissociation of glycophorin C or
-adducin from the skeletons (data
not shown).

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Fig. 8.
Evaluation of the deformability of PMA
treated RBCs by ektacytometry. Erythrocytes were incubated with
Me2SO, 0.1 µM 4 -PDD, or 0.1 µM PMA for 30 min. A, osmotic deformability
was then measured by subjecting the RBCs to a moderate shear stress
while increasing osmotic pressure. B, whole cell
deformability was measured by subjecting isotonic RBCs to increasing
shear stress. C, membrane stability was examined by
subjecting resealed ghosts to a constant high shear stress and
evaluating the rate of membrane fragmentation by monitoring the
decrease in deformability index with time. The various scans were
obtained on membranes derived from Me2SO
(DMSO)-treated control cells (thin
line), 0.1 µM 4 -PDD-treated cells
(medium line), and 0.1 µM
PMA-treated cells (thick line).
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In order to obtain a more sensitive measure of the effect of PMA
on membrane mechanical properties, resealed ghosts were prepared from
PMA-treated or untreated cells and examined for their mechanical stability during exposure to increasing shear stress. As seen in Fig.
8C, the half-time of shear-induced fragmentation of ghosts prepared from cells incubated with PMA was identical to that from cells
incubated with 4
-PDD (inactive analog) and control cells incubated
in buffer. These data confirm that RBCs treated with PMA retain their
normal deformability and stability, despite the release of part of
their glycophorin C and adducin from the membrane skeletons.
In a final attempt to identify a mechanical consequence of the
PMA-induced release of glycophorin C and adducin from the membrane skeletons, we examined the effect of PMA on the filterability of RBCs
through a 4.6-µm nickel mesh. Following exposure to 0.1 µM PMA for 30 min at 37 °C, where the dissociation of
glycophorin C and
-adducin was previously shown to be maximal, the
filtration rate of the treated cells was found to decrease by less than
5% (Fig. 9). After a much longer
incubation time (2 h), an average decrease in filterability of ~13%
(compared with RBCs treated with the same concentration of 4
-PDD)
was observed (Fig. 9). However, since the longer incubation time
exerted no further effect on retention of glycophorin C or
-adducin
in the membrane skeletons, such a decrease in filterability may not be
related to the integrity of membrane-to-skeleton bridges. More likely,
the decrease in filterability could derive from other unrelated effects
of protein kinase C, such as loss of phospholipid asymmetry (52) or
increase in ion channel activity (53).

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Fig. 9.
Effect of PMA on the rate of RBC filtration
through a 4.6-µm nickel mesh filter.
Erythrocytes were incubated with Me2SO (DMSO),
0.1 µM 4 -PDD, or 0.1 µM PMA for 30 min,
1 h, or 2 h. Cells were then further diluted to 0.1%
hematocrit and allowed to flow through a 4.6-µm pore-sized nickel
mesh filter. Filtration rates were recorded on-line as described in
Fig. 2.
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DISCUSSION |
The human erythrocyte is commonly considered biologically
inactive, essentially unable to respond to signals from within and outside the cell with meaningful changes in cellular properties. We
have, however, demonstrated that fresh intact erythrocytes do indeed
sense changes in 2,3-bisphosphoglycerate (a modulator of
hemoglobin-O2 affinity), calcium, O2 levels,
intracellular pH, and protein kinase C activity, and respond to these
changes by altering the number of bridges connecting glycophorin C to the membrane skeleton. Although each of these membrane rearrangements could admittedly be entirely fortuitous, collective consideration of
the data may suggest otherwise. Thus, none of the above modulators affects the band 3-ankyrin-spectrin bridge and none of the stimulants alters the content of protein 4.1 in the membrane skeletons. In fact,
except for minor changes in adducin retention, all of the changes are
specific for the glycophorin C-protein 4.1 bridge. Furthermore, the
most prominent modulatory effects appear to be situated near the
physiological ranges of the above stimulants and several of the
stimulants might be most prominent in erythrocytes retarded in
deoxygenated tissues. Although we cannot yet assign a function to the
modulations, we nevertheless hypothesize that the ability to regulate
the integrity of the glycophorin C bridge to the junctional complex
might somehow improve the fitness of the circulating erythrocyte.
The most unanticipated observation from this study was that
dissociation of glycophorin C from the membrane skeletons does not
impact membrane deformability, stability, or whole cell filterability. Several explanations of this finding are worth considering. First, the
glycophorin C bridge may not be highly important to membrane mechanical
properties. Although protein 4.1-deficient erythrocytes are abnormally
shaped and mechanically unstable, reconstitution of the spectrin-actin
binding fragment of protein 4.1 into these cells restores normal
behavior (19). Since the glycophorin C binding domain is not contained
within this fragment, one can easily argue that the glycophorin C
bridge to the junctional complex is unimportant to membrane stability.
In this scenario, the instability of glycophorin C-deficient membranes
might simply be attributed to the concomitant partial deficiency in
protein 4.1 (54).
Second, the lack of impact of glycophorin C dissociation
could derive from the short duration of our treatments. Thus, release of glycophorin C from the spectrin skeleton could enable only slow
weakening of other interactions more critical to membrane stability.
Although we extended our investigations of membrane mechanical
properties for at least 2 h following treatment, red blood cells
must circulate for ~120 days in vivo; hence, the true significance of the glycophorin C interaction may only emerge when a
cell is stressed for longer periods of time.
A third possible explanation for the absence of obvious mechanical
consequences is that complete release of glycophorin C was never
achieved. Whether part of the protein 4.1 population is inaccessible to
each type of regulation or whether other components render the retained
population of glycophorin C less extractable cannot be ascertained from
the data, but clearly not all copies of glycophorin C are subject to
similar modulation. Perhaps the extractable fraction of glycophorin C
was already contributing little to membrane stability.
Fourth, it should not be ignored that ektacytometry and nickel mesh
filtration could easily be insensitive to the mechanical changes
induced by rupture of the protein 4.1 bridge. Thus, PMA treatment has
been shown to delay the onset of the discocyte to echinocyte transition
upon ATP depletion or Ca2+ entry (55) and to directly cause
erythrocyte morphological changes (56), albeit at concentrations much
higher than those required to induce maximal protein phosphorylation.
Based on these observations, it would be surprising if PMA did not
modulate some mechanical properties of the membrane. Whether such
mechanical changes only occur at excessive PMA concentrations or
whether our current techniques are too insensitive to detect them must obviously await further investigation.
Finally, the glycophorin C-protein 4.1 bridge could serve a function
unrelated to membrane mechanical properties. Instead, the tether to the
membrane could simply position the spectrin/actin skeleton closer to
the bilayer where it could better interact with other membrane
components such as metabolic enzymes, ion channels/transporters,
adhesion receptors, or phospholipid flippases/scramblases, etc. The
impact of changes in BPG, Ca2+, O2, pH, and
protein kinase C activity would then presumably be manifested in the
regulation of these other membrane functions. It will be important to
evaluate whether bilayer-to-skeleton tethers can indeed participate in
the regulation of nonstructural membrane functions.