From the
The functions of major intrinsic protein (MIP) of lens are still
unresolved; however the sequence homology with channel-forming integral
membrane protein (CHIP) and other Aquaporins suggests that MIP is a
water channel. Immunolocalizations confirmed that Xenopus oocytes injected with bovine MIP cRNA express the protein and
target it to the plasma membrane. Control oocytes or oocytes expressing
MIP or CHIP exhibited small, equivalent membrane currents that could be
reversibly increased by osmotic swelling. When compared with
water-injected control oocytes, the coefficient of osmotic water
permeability ( P
Major intrinsic protein (MIP)
MIP was the first identified member of an ancient family of membrane
proteins from diverse organisms that now includes more than 20 members.
Several MIP family members from animal and plant tissues were shown to
function as water-selective membrane channels and are now referred to
as the ``Aquaporins'' (Chrispeels and Agre, 1994). The
purification and cDNA cloning of the 28-kDa channel-forming
integral membrane protein, CHIP (Denker et al.,1988; Preston and Agre, 1991), a MIP homolog from red
cells and renal proximal tubules, permitted the first demonstration of
a molecular water channel (Preston et al.,1992).
CHIP is now designated Aquaporin-1 (AQP1), and cDNAs encoding four
other Aquaporins have subsequently been isolated from diverse mammalian
tissues (Fushimi et al.,1993; Ishibashi et
al.,1994; Ma et al., 1994; Echevarria et
al., 1994; Hasegawa et al.,1994; Jung et
al.,1994b; Raina et al.,1995).
The
hourglass model was recently proposed to merge structural and
functional features of the Aquaporins (Jung et al.,1994a). The model describes two tandem repeats with the first and
second half of the molecule oriented at 180° to each other, and
each half contains the sequence asparagine-proline-alanine (NPA) in
intracellular loop B and extracellular loop E. In the hourglass model,
it was predicted that loop B and loop E fold back into the membrane,
forming a single water-selective pore. The striking sequence homology
with the Aquaporins suggests that MIP may also be involved in water
movement through the lens fiber cell membranes. In this report, we
investigated the water transporting capacities of MIP using the
Xenopus oocyte expression system, and water channel properties
qualitatively similar to the Aquaporins were observed.
The
chimeric protein, MIP-CHIP (loop A), contains the first exofacial loop
(A) of CHIP (amino acids 34-51) in place of the first exofacial
loop of MIP (amino acids 33-43) and was constructed with an
82-base pair insertional-substitutional oligonucleotide primer (not
shown) in a site-directed mutagenesis reaction. All mutations were
confirmed by enzymatic nucleotide sequencing (U. S. Biochemical Corp.).
Capped RNA transcripts were synthesized in vitro using T3
RNA polymerase with XbaI-digested MIP, CHIP, or mutant
expression vector DNA, and the RNA was purified as described (Yisraeli
and Melton, 1989).
On-line formulae not verified for accuracy
Unlike all of the known
Aquaporins, MIP does not contain a potential N-glycosylation
site in either loop A or loop C, and no glycosylation of MIP was
detected when the polypeptide was expressed in oocytes
(Fig. 2 B). The MIP-CHIP (loop A) construct contains the
CHIP glycosylation site at residue asparagine 42 in loop A. Despite the
presence of a potential glycosylation site, oocytes expressing this
construct still did not exhibit an increase in
P
Oocytes
expressing MIP-CHIP-1 and CHIP-MIP-1 chimeras did not exhibit a
significant increase in P
These results show that MIP exhibits a low P
Even though the cDNA encoding MIP was cloned more than a
decade ago (Gorin et al., 1984), the physiological roles of
the protein are still not understood. Detailed studies of the
homologous proteins MIP (Ehring et al., 1990), Nodulin-26
(Weaver et al., 1994), and preliminary studies with
CHIP
Soon after the discovery that CHIP is a molecular water
channel (Preston et al., 1992), several labs evaluated MIP and
other homologous proteins for osmotic water permeability. One group
reported that MIP is not a water channel (Verbavatz et al.,
1994), but apparently these investigators used the same techniques with
which they failed to detect osmotic water permeability of AQP3 (Ma
et al., 1994), a protein found by two other groups to exhibit
high P
A molecular explanation for why MIP
is a weaker water transporter than the other Aquaporins was sought
since the amino acid sequences of MIP and the Aquaporins are strikingly
similar (Preston and Agre, 1991; Reizer et al., 1993))
(Fig. 10). MIP was expressed well in oocytes and was targeted to
the plasma membrane (Fig. 2). Neither the phosphorylation sites nor the
other residues in the carboxyl-terminal cytoplasmic domain of MIP
appeared to function as restraints (Figs. 6 and 8), and several
different residues and domains from CHIP were spliced into MIP without
increasing the P
A mutation has been identified in mice that may
provide insight into other potential functions of MIP, since these mice
develop cataracts prior to birth (Muggleton-Harris et al.,
1987). The cat mouse mutation results in lower abundance of
MIP mRNA with the major transcript being truncated, and MIP was not
detectable in lens by immunocytochemistry (Shiels and Griffin, 1993).
The mutation is expressed as a dominant trait and has been mapped to
the distal end of chromosome 10 (Muggleton-Harris et al.,
1987), coincident with the MIP locus (Griffin and Shiels, 1992).
Mutations in genes encoding structural proteins usually produce
dominantly inherited disorders, whereas mutations in transporters such
as the CFTR are recessively inherited. Consistent with this, mutations
in the AQP2 gene were recently identified in homozygotes and a compound
heterozygote with severe nephrogenic diabetes insipidus, while the
heterozygous relatives were unaffected (Deen et al., 1994; van
Lieburg et al., 1994). Careful histological analysis of the
early stages of disease in the cat mouse may provide clues to
the critical function of MIP, which is the first defect leading to the
development of cataracts in this animal model.
We gratefully thank A. Hartog for performing
immuno-cytochemistry of MIP in oocytes and Dr. R. J. M. Bindels for
help in formulating a computer routine to analyze swelling curves of
oocytes. We also thank James Hall, Richard Mathias, Paul Lampe, and
David Beebe for valuable discussions.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) of MIP oocytes was
increased 4-5-fold with a low Arrhenius activation energy, while
the P
of CHIP oocytes increased >
30-fold. To identify structures responsible for these differences in
P
, recombinant MIP proteins were
expressed. Analysis of MIP-CHIP chimeric proteins revealed that the
4-kDa cytoplasmic domain of MIP did not behave as a negative regulator.
Individual residues in MIP were replaced by residues conserved among
the Aquaporins, and introduction of a proline in the 5th transmembrane
domain of MIP raised the P
by 50%. Thus
oocytes expressing MIP failed to exhibit ion channel activity and
consistently exhibited water transport by a facilitated pathway that
was qualitatively similar to the Aquaporins but of lesser magnitude. We
conclude that MIP functions as an Aquaporin in lens, but the protein
may also have other essential functions.
(
)
is a
26-kDa protein expressed exclusively in lens fiber cells where it
comprises over 60% of the membrane protein. The cDNA encoding MIP was
isolated from a bovine lens cDNA library, and hydrophobicity plots
predicted that MIP is an integral membrane protein with cytoplasmic
amino and carboxyl termini and six bilayer-spanning domains (Gorin
et al.,1984). MIP reconstituted into liposomes
exhibits voltage-dependent channels permeable to ions and small
molecules that may be closed by Ca
and calmodulin
(Nikaido and Rosenberg, 1985; Girsch and Peracchia, 1985; Peracchia and
Girsch, 1989; Shen et al.,1991). In planar lipid
bilayers, MIP forms single channels of various conductances (up to 3
nanosiemens) and a weak selectivity for anions (Ehring et
al.,1990); Nodulin-26, a homologous protein from soy
bean root nodules, was recently shown to behave similarly (Weaver
et al.,1994). In spite of these studies with
reconstituted MIP protein, studies of MIP in native membranes or
expressed in oocytes have failed to confirm a physiologic function for
MIP. For example, although MIP was initially considered to be the
gap-junction protein, immunological, biochemical, and
electrophysiological studies failed to identify electric coupling of
lens fiber cells via MIP (Swenson et al.,1989).
Plasmid DNA Mutagenesis, DNA Sequencing, and in Vitro
RNA Synthesis
Standard molecular procedures were used (Sambrook
et al., 1989). The bovine MIP coding sequence (Gorin et
al., 1984) flanked 5` and 3` by Xenopus -globin gene
untranslated sequences (Swenson et al., 1989) was transferred
into pBluescript II KS (Stratagene) following digestion with
HindIII and XbaI. The CHIP expression vector was
constructed as described (Preston et al., 1992). These
constructs served as templates for site-directed mutagenesis reactions
using the Muta-Gene phagemid in vitro mutagenesis kit
(Bio-Rad). lists the mutants used in this study. The
locations of mutations within the MIP polypeptide are illustrated in
Fig. 1
.
Figure 1:
Membrane topology of MIP showing sites
targeted for mutagenesis and domain exchanges in this study. SPLICE 1 is the splice site for MIP-CHIP-1 and CHIP-MIP-1;
SPLICE 2 is the splice site for MIP-CHIP-2 and
CHIP-MIP-2. The proline introduced in CHIP at position Asp-131 is
indicated with #. NPA motifs are indicated with asterisks.
Filled symbols indicate amino acids that are
conserved in more than half of the members of the MIP family from
bacteria, yeast, plants and animals (Reizer et al.,
1993).
The chimeric protein vectors CHIP-MIP-1 and MIP-CHIP-1
were constructed by inserting a BamHI restriction site at
Val-112 of MIP and Thr-120 of CHIP, resulting in the insertion of the
amino acids aspartic acid and proline. MIP(V112Bam) and CHIP(T120Bam)
were digested with BamHI, the 5`-half of CHIP was ligated to
the 3`-half of MIP (CHIP-MIP-1), and the 5`-half of MIP was ligated to
the 3`-half of CHIP (MIP-CHIP-1). The chimeric protein vectors
MIP-CHIP-2 and CHIP-MIP-2 were constructed using the megaprimer
polymerase chain reaction method (Barik and Galinski, 1991) exchanging
at Arg-226 of MIP and Arg-234 of CHIP, using the following antisense
primers: MIP-CHIP-2, 5`-GTCTGTGAGGTCACTGCTG-CGAGGGAAGAGGAGAAAG-3`;
CHIP-MIP-2, 5`-CTCAGAAACACTCTTGAGC-CGTGGGGCCAGGATGAAG-3`.
Preparation of Oocytes and Measurement of
Pf
Female Xenopus laevis were anesthetized on ice, and
stage V and VI oocytes were removed and prepared (Lu et al.,
1990). The day after isolation, oocytes were injected with either 50 nl
of water or 0.5-25 ng of cRNA in 50 nl of water. Injected oocytes
were maintained for 2-3 days at 18 °C prior to osmotic
swelling, membrane isolation, or voltage clamp experiments. Oocyte
swelling was performed at 22 °C following transfer from 200
mos
M (osm) to 70 mos
M (osm
, CHIP) or either 70 or 20 mos
M (osm
, MIP) modified Barth's solution diluted
with water. Sequential oocyte images were digitized at 5-s intervals
for a total of 1 min, and the volumes of the sequential images were
calculated as described (Preston et al., 1993). The change in
relative volume with time,
d( V/ V
)/ dt, was fitted
by computer to a quadratic polynomal, and the initial rates of swelling
were calculated. The osmotic water permeability
( P
, µm/s) was calculated from osmotic
swelling data between 5 and 10 s, initial oocyte volume
( V
= 9
10
cm
), initial surface area ( S = 0.045
cm
), and the molar ratio of water ( V
= 18 cm
/mol)(Zhang et al., 1990)
using the formula
Oocyte Membrane Isolation and Immunoblot
Analysis
Total oocyte membranes (Preston et al., 1993)
and plasma membranes (Wall and Patel, 1989) were isolated from groups
of 4-30 oocytes, solubilized in 1.25% (w/v) SDS at 60 °C for
10 min, electrophoresed into 12% SDS-polyacrylamide gels (Laemmli,
1970), transferred to nitrocellulose (Davis and Bennett, 1984),
incubated with a 1:10,000 dilution of anti-MIP antibody, or a 1:1000
dilution of anti-CHIP antibody (Smith and Agre, 1991), and visualized
using the ECL Western blotting detection system (Amersham Corp.).
Molecular weights were determined relative to the mobility of
prestained SDS-PAGE standards (Bio-Rad).
Anti-MIP Antibody
A synthetic peptide
corresponding to the 15 carboxyl-terminal amino acids of bovine MIP
(PEVTGEPVELKTQAL) (Gorin et al., 1984) was coupled to keyhole
limpet hemocyanin. Rabbits were injected with 400 µg of conjugated
synthetic peptide mixed with Freund's complete adjuvant. After 4
weeks, and every 3 weeks thereafter, the rabbits were boosted with 200
µg of conjugated synthetic peptide mixed with incomplete
Freund's adjuvant. The produced anti-MIP antiserum was tested for
specificity and cross-reactivity by an enzyme-linked immunosorbent
assay.
Immunolocalization of MIP in Xenopus
Oocytes
Oocytes were frozen for immunofluorescence microscopy in
Tissue-Tek mounting medium (Miles Inc.). Sections of 5 µm were cut
by cryostat, collected on gelatin-coated slides, and fixed at room
temperature for 5 min in 1% (w/v) periodate-lysine-paraformaldehyde
fixative (McLean and Nakane, 1974). Sections were washed 3 times with
TBS (0.9% NaCl, 25 m
M Tris, pH 7.4) and incubated overnight at
4 °C with anti-MIP antibody at a dilution of 1:1,000 in TBS
containing 10% (v/v) goat serum. After 3 washes with TBS, the sections
were incubated for 1 h at 37 °C with affinity-purified fluorescein
isothiocyanate-labeled goat-anti-rabbit IgG (Sigma Immuno Chemicals) at
a dilution 1:50 in TBS containing 10% (v/v) goat serum. After 3 more
washes in TBS, the sections were embedded in Mounting Medium (Sigma)
and analyzed by immunofluorescence microscopy.
Electrophysiology
Studies were carried out as
described (Preston et al., 1992) using a two-microelectrode
voltage clamp with Clampex software. Water- and cRNA-injected oocytes
were voltage-clamped at a holding membrane potential of -70 mV,
repolarized to -90 mV, and depolarized to +50 mV in 20 mV
step intervals. The membrane potential was returned to the holding
potential between each step.
Immunolocalization
Oocytes from X. laevis have provided the standard system for expression of water channels
and measurement of their functional activity by a swelling assay (see
``Experimental Procedures''). Expression of MIP and insertion
of the protein into the oocyte plasma membrane was studied by
immunofluorescence microscopy of frozen sections. Oocytes injected with
MIP cRNA showed a strong anti-MIP immunolabeling of the plasma
membrane, whereas the cytoplasm showed only weak immunostaining (Fig.
2 A). Immunoblot analysis of isolated oocyte plasma membranes
also showed a single strong signal at 27 kDa, confirming that MIP is
targeted to the plasma membrane (Fig. 2 B).
Figure 2:A, immunofluorescence microscopy of control oocytes and oocytes
expressing MIP. Frozen sections were prepared from Xenopus oocytes injected with 50 nl of water or 50 nl of water containing
10 ng of MIP cRNA and probed with anti-MIP antibody (see
``Experimental Procedures''). Note strong labeling of plasma
membrane and negligible labeling of cytoplasm of the oocyte expressing
MIP. B, immunoblot of total protein ( TP, equivalent
of 0.2 oocytes) and plasma membranes ( PM, equivalent of 15
oocytes) from oocytes expressing MIP. SDS-polyacrylamide gel
electrophoresis immunoblot was incubated with a polyclonal antibody
directed against the carboxyl terminus of
MIP.
Conductance
It was reported that MIP reconstituted
into planar lipid bilayers forms channels with high conductance (Ehring
et al., 1990). The hypothesis that MIP is an ion channel was
tested by measuring the conductance of oocyte membranes expressing MIP
with a two-microelectrode voltage clamp. In the absence of osmotic
swelling, no differences in conductance were measured in oocytes
injected with water, MIP cRNA, or CHIP cRNA (Fig. 3). The oocytes
were placed in hypotonic medium, and, after being osmotically swollen
to a similar size, membrane currents were again measured. Oocytes
injected with water, MIP cRNA, or CHIP cRNA exhibited a similar
increase in conductance. After replacing oocytes in isotonic buffer to
cause shrinkage to the initial volume, the conductance decreased again
to the initial values, although the shrinking process for
water-injected oocytes was too slow for analysis (not shown).
Figure 3:
Membrane
ion conductance. Voltage clamp was used to measure currents at voltages
from -90 mV to +50 mV of water-injected control oocytes,
oocytes injected with 25 ng of MIP cRNA, or oocytes injected with 0.5
ng of CHIP cRNA. The currents were determined in 200 mos
M modified Barth's solution. The oocytes were swollen by
replacing modified Barth's solution with a hypotonic medium, and
current measurements were repeated. Control oocytes were placed in
HO, and the current was measured after 30 min. Oocytes
expressing MIP and CHIP were placed in modified Barth's solution
diluted from 200 to 70 mos
M, and currents were measured after
20 and 30 s. Oocytes were shrunk back to their normal volume by
replacing the hypotonic medium with isotonic modified Barth's
solution, and currents in oocytes expressing MIP and CHIP were measured
after 8 min.
Water Permeability of MIP
Osmotic water
permeability ( P) of Xenopus oocytes was measured in hypotonic medium 3 days after injection
with 50 nl of water or 10 ng of MIP or CHIP cRNA. Water-injected
control oocytes swelled minimally ( P
= 10 ± 3.2 µm/s). Oocytes injected with MIP
cRNA displayed a 4-5-fold increase in water permeability above
the basal level of control oocytes ( P
= 45 ± 8.2 µm/s). A more than 30-fold increase
in P
was exhibited by oocytes injected
with the same amount of CHIP cRNA ( P
= 351 ± 43 µm/s, Fig. 4, A and
B). Although the P
of
MIP-injected oocytes was only
15% of CHIP-injected oocytes, the
MIP-injected oocytes had significantly greater
P
than the control oocytes ( p < 0.05, Fig. 4 A), a difference that was
consistently reproduced in each of 14 independent experiments.
Figure 4:A, increased osmotic water
permeability of Xenopus oocytes expressing MIP and CHIP.
Oocytes were injected with water or 10 ng of in vitro transcribed cRNA encoding MIP or CHIP. After 72 h, Ps
were determined by video microscopic measurement of the rate of
swelling after transfer to hypotonic medium. Shown are the means
± S.D. for 8 oocytes. B, time-dependent osmotic
swelling of oocytes injected with water or 10 ng of cRNA encoding MIP
or CHIP. Shown are the means from five traces ±
S.E.
Water
transport through aqueous channels is characterized by a low Arrhenius
activation energy, which is equivalent to diffusion of water in bulk
solution ( E 4 kcal/mol), whereas
diffusion of water through lipid membranes is characterized by a high
value ( E
> 10 kcal/mol) (Finkelstein,
1987). Water permeability of oocytes injected with 25 ng of MIP cRNA
was measured at multiple temperatures; consistent with facilitated
water transport, the activation energy was low
( E
= 3.9 kcal/mol ± 1.4
kcal/mol, average of 4 experiments, Fig. 5). Indicative of
diffusional water movement, the activation energies of water-injected
oocytes were measured in two experiments ( E
= 10 and 18 kcal/mol).
Figure 5:
Arrhenius activation energy ( E)
of osmotic water permeabilities of control oocytes and oocytes
expressing MIP. The E was determined by measuring the P at 10, 20, and 30 °C. Control oocytes were injected with water
and MIP oocytes were injected with 25 ng of MIP cRNA. In this
experiment, each point represents 7-10 control oocytes or
11-15 MIP oocytes.
Chimeric MIP-CHIP Proteins
Domains of MIP and CHIP
that might be of importance for the specific functional characteristics
of the two proteins were exchanged, and the functional consequences
were measured (, Fig. 1). The MIP-CHIP (loop A)
chimera represents a MIP molecule with residues 33-43 in loop A
replaced by the residues 34-51 from the loop A of CHIP. The
MIP-CHIP-1 and CHIP-MIP-1 chimeras contain the amino-terminal half of
one molecule and the carboxyl-terminal half of the other spliced at a
site in loop C where the tandem repeats are normally joined. The
MIP-CHIP-2 and CHIP-MIP-2 chimeras are spliced at a site in the
carboxyl-terminal cytoplasmic domains of the molecules. When membranes
of oocytes expressing each of these constructs were analyzed by
immunoblot, polypeptides of the anticipated size were identified, and
the levels of expression were comparable with oocytes expressing native
MIP and CHIP proteins (not shown).
, and immunoblots revealed a 27-kDa
polypeptide but not higher molecular mass bands, indicating that
N-glycosylation had still not occurred (not shown).
above
water-injected controls, suggesting that these chimeric proteins cannot
form functional water channels. Oocytes expressing MIP-CHIP-2 also
exhibited P
s similar to control oocytes.
In contrast, oocytes expressing CHIP-MIP-2 were found to have
P
s similar to oocytes expressing
wild-type CHIP (Fig. 6), indicating that the carboxyl-terminal
cytoplasmic domain of MIP does not interfere with the
water-transporting domains of CHIP.
Figure 6:
Osmotic water permeability of oocytes
expressing chimeric MIP-CHIP proteins. P was determined from
oocytes injected with water or 10 ng of cRNA encoding MIP, CHIP, or the
indicated MIP-CHIP chimeric proteins. Shown are the means ± S.D.
of five oocytes. *, p < 0.05 compared with water-injected
control oocytes.
Single Amino Acid Substitutions
Unlike CHIP, the
increase in water permeability induced by MIP expression is not blocked
by HgCl. A putative mercury-sensitive cysteine residue was
introduced into MIP at position 181, which conforms to the
mercury-sensitive cysteine 189 in CHIP, but the substitution MIP(A181C)
did not confer mercury sensitivity to MIP (Fig. 7). The cysteine
at position 14 in the amino-terminal domain of MIP was replaced by a
valine, since other Aquaporins do not contain a cysteine at this
position, but the P
of C14V mutant MIP
was not different from wild-type MIP (Fig. 8 A). MIP contains a
proline in the second extracellular loop (residue 123 in loop C), a
site where other members of the MIP family do not contain this residue.
Introducing a proline at this position in CHIP (D131P) also did not
change the P
(Fig. 8 A).
Figure 7:
Osmotic water permeability and
Hg inhibition of oocytes expressing CHIP, MIP, or MIP
(A181C). Oocytes were injected with 1 ng of CHIP cRNA or 10 ng of MIP
cRNA or 10 ng of MIP(A181C) cRNA. P was determined without
pretreatment ( open bars) or after 5 min in buffer
containing 3 m
M HgCl
, followed by swelling in the
presence of HgCl
( solid bars). Shown are
the means ± S.D. of five oocytes.
Figure 8:
Osmotic water permeability of oocytes
expressing MIP and CHIP mutants. A, oocytes were injected with
water or 25 ng of cRNA encoding MIP, MIP(C14V), MIP(V160P), MIP(S243V,
S245E), or 5 ng of cRNA encoding CHIP, CHIP(D131P), or CHIP(P169A). The
P was measured 3 days after injection. Shown are the means
± S.D. *, p < 0.05 compared to water-injected
oocytes, **, p < 0.05 compared with wild-type MIP.
B, immunoblot comparison of total protein ( TP,
equivalent of 0.2 oocytes) or plasma membranes ( PM, equivalent
of 20 oocytes) from oocytes expressing MIP or the indicated MIP
mutants. SDS-PAGE immunoblot was incubated with a polyclonal antibody
specific for the carboxyl terminus of MIP.
All known Aquaporins except AQP4 contain a proline in the 5th
transmembrane segment; no proline exists in the 5th transmembrane
domain of MIP. When a proline was introduced in MIP (V160P), the
substitution reproducibly enhanced the Pby 50 ± 20% relative to the P
of oocytes expressing wild-type MIP (Fig. 8 A).
Notably, the amount of MIP protein expressed in oocytes was not
different after injection of equal amounts of MIP cRNA and MIP(V160P)
cRNA (Fig. 8 B). Although not large, this increase in
P
was confirmed in each of five different
experiments. A ``gain of function'' mutation suggests that
the P
of MIP can be increased by
introducing subtle conformational changes in the molecule.
Nevertheless, replacing the proline in CHIP at this position (P169A) to
the corresponding residue of MIP had no measurable influence on the
P
of CHIP (Fig. 8 A).
value that can be increased by introducing subtle changes in the
molecule. One explanation may be that MIP needs to undergo an
activation or structural rearrangement to function as a water channel,
whereas CHIP is constitutively in the activated state. Two sites in the
carboxyl-terminal cytoplasmic domain of MIP are phosphorylated in
vivo (Lampe and Johnson, 1990). Since these sites are not
conserved in other members of the MIP family, the sites may play a role
in regulation of MIP function. Mutation of the two putative
phosphorylation sites to the corresponding residues in CHIP (S243V,
S245E) slightly decreased the P
(Fig.
8 A), but the amount of protein expressed was comparably
reduced as compared with wild-type MIP by immunoblot (Fig.
8 B). The possibility of stretch-activation was also
considered, but if it exists, it was not reproduced by simple increase
in volume, since the rate of osmotic swelling did not increase with
time (Fig. 9). Thus, if an activation step confers CHIP-level
water permeability on MIP, the identity of this step remains unknown.
Figure 9:
Time-dependent osmotic swelling of oocytes
expressing MIP. 3 days after injection of 25 ng of MIP cRNA, oocytes
were transferred to 20 mos
M modified Barth's solution,
and swelling was recorded until the oocyte burst ( X). Shown
are the means from 7 traces ± S.E.
(
)
revealed large voltage-dependent
conductances when reconstituted into planar lipid bilayers.
Nevertheless, the magnitude of the currents in bilayers containing MIP
was far above the conductances measured in normal lens (Mathias et
al., 1979, 1991). Therefore, while the planar lipid bilayer
studies are highly reproducible, their relevance to normal lens
physiology remains uncertain. Moreover, the studies reported here (Fig.
3) and preliminary studies of other investigators (Kushmerick et
al., 1994) failed to confirm ion conductance by MIP expressed in
oocytes.
comparable with the other
Aquaporins (Ishibashi et al., 1994; Echevarria et
al., 1994). The studies reported here document that oocytes
expressing MIP exhibit osmotic water permeabilities 4-5-fold
above control oocytes (Fig. 4) with activation energies identical
to the Aquaporins (Fig. 5). These observations are supported by
preliminary studies of other investigators who also found an increase
in P
of oocytes expressing frog MIP
(Kushmerick et al., 1994) or bovine MIP (Chandy et
al., 1995). MIP may contribute to the maintenance of lens
transparency by enhancing uptake of intercellular water by adjacent
lens fiber cells. The narrow geographic separation of lens fiber cells
(which contain MIP) and lens epithelial cells (which contain CHIP)
suggests functional cooperativity. Osmotic gradients provide the
driving force for Aquaporin-mediated water transport in kidney and most
other tissues (Nielsen et al., 1993a, 1993b). It is likely
that hydrostatic forces move water through CHIP in endothelium of the
proximal capillary bed (Nielsen et al., 1993b), and a related
process may occur through MIP when the lens shape is rapidly altered by
contraction of muscles in the ciliary body to provide fine focus of the
corneal image upon the retina.
(Figs. 6-8).
Despite introduction of an N-glycosylation site, MIP-CHIP
(loop A) was still not glycosylated, and introduction of the potential
mercurial inhibition site in MIP (A181C) failed to confer sensitivity
(Fig. 7). Likewise, when a cysteine was introduced into the
mercury-insensitive AQP4, the recombinant protein also failed to
demonstrate mercury-inhibitable P
(Jung
et al., 1994b), implying that MIP and AQP4 are structurally
different from CHIP near the extracellular side of the aqueous pore.
Nevertheless, substitution of proline in the 5th bilayer-spanning
domain yielded a 50% increase in P
(Fig. 8). This intriguing gain of function suggests that
simple alterations in the bilayer-spanning domains can alter the
conformation of MIP to more closely resemble CHIP. No loss of function
was detected in the corresponding substitution (P169A) in CHIP;
however, a small decrease in a high P
should be more difficult to measure than a comparable increase in
a low value. An alternative explanation may be that MIP requires a
specific membrane organization, such as formation of orthogonal arrays,
or a particular membrane environment that may be poorly reproduced in
the oocyte expression system. For example, oocytes do not have plasma
membranes with wavy junctions similar to lens fiber cells (Zampighi
et al., 1989), and if this is important to the function of
MIP, the water permeability studies may yield spuriously low values in
oocytes.
Figure 10:
Evolutionary tree of MIP and the
mammalian Aquaporins. The percentage of amino acid identity between MIP
and the five mammalian Aquaporins is
indicated.
Similar to other bilayer-spanning proteins, MIP may have
multiple physiological functions, and it is possible that the primary
role of MIP is not water transport. The red cell band 3 protein is the
membrane anion exchanger (AE1), a cytosolic regulator of glycolytic
enzyme activity, and the structurally important attachment site for
ankyrin on the membrane (for review, see Low (1986)). MIP may also have
a structural function, since the protein has been shown to enhance
adhesion with membranes containing negatively charged phospholipids
(Michea et al., 1994). Also, it is known that some proteins
are expressed in lens where their function is unrelated to their
functions in other tissues ( e.g. crystallins, for review, see
Piatigorsky and Wistow (1989) and De Jong et al. (1994)). Thus
the extremely high expression of MIP in lens may be far above the level
needed for water permeability, since the abundance may be needed for an
unrelated function.
Table: Site-specific mutations, insertional
mutations, and chimeric constructs of MIP and CHIP
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.