1 Program in Cell Biology and Biochemistry, The Hospital for Sick Children, and Biochemistry Department, University of Toronto, Toronto, Ontario, Canada, M5G 1X8; and 2 Institut de Pharmacologie, Lausanne, Switzerland CH-1005
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ABSTRACT |
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The epithelial Na+ channel (ENaC) plays a key role in the regulation of Na+ and water absorption in several epithelia, including those of the distal nephron, distal colon, and lung. Accordingly, mutations in ENaC leading to reduced or increased channel activity cause human diseases such as pseudohypoaldosteronism type I or Liddle's syndrome, respectively. The gain of ENaC function in Liddle's syndrome is associated with increased activity and stability of the channel at the plasma membrane. Thus understanding the regulation of channel processing and trafficking to and stability at the cell surface is of fundamental importance. This review describes some of the recent advances in our understanding of ENaC trafficking, including the role of glycosylation, ENaC solubility in nonionic detergent, targeting signal(s) and hormones. It also describes the regulation of ENaC stability at the cell surface and the roles of the ubiquitin ligase Nedd4 (and ubiquitination) and clathrin-mediated endocytosis in that regulation.
epithelia sodium channel; glycosylation; ubiquitin; Nedd4; endocytosis; WW domain; Liddle's syndrome
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INTRODUCTION |
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THE AMILORIDE-SENSITIVE EPITHELIAL
Na+ channel (ENaC) is an apically located channel expressed
primarily in polarized epithelia in the distal nephron, lung, distal
colon, and other organs (26). It is composed of three
related subunits, ,
, and
(9, 11, 47, 52, 53,
84), arranged in a stoichiometry of 2
:1
:1
(23, 44; see,
however, Ref. 74 for another view). Expression of all
three ENaC subunits is needed for full channel activity, although
expression of the
-subunit alone or
- or
-subunit combinations leads to the generation of small currents as well (9, 11, 24). Each ENaC chain is composed of two
transmembrane domains, a large ectodomain separating them that contains
numerous potential N-linked glycosylation sites and two
short NH2 and COOH termini (10, 63, 75). The
extracellular domain represents more than half of the mass of the ENaC
subunits and contains two conserved cystein-rich domains. The COOH
terminus of each subunit contains two proline-rich regions
(65), the second of which also includes a highly conserved
sequence now known as the PY motif (xPPxY; where x is any amino acid, P
is proline, and Y is tyrosine) (70, 77).
ENaC plays a critical role in the regulation of Na+
and fluid absorption and, hence, in the regulation of blood volume and blood pressure. Accordingly, mutations in ENaC have been genetically linked to two disorders affecting fluid reabsorption in the distal nephron. The first is pseudohypoaldosteronism type I (PHA-1), a
salt-wasting nephropathy caused by loss-of-function mutations in either
-,
-, or
-ENaC that results from reduced channel opening
(13, 29, 80). PHA-1-like disease is also recapitulated in
knockout mice models lacking either
- or
-ENaC or expressing a
reduced dose of
-ENaC, all of which suffer from an effective reduction in ENaC activity (4, 37, 54), as seen in PHA-1 patients. The second, Liddle's syndrome, is a hereditary (or sporadic) form of arterial hypertension (7) resulting from excessive ENaC activity. Liddle's syndrome is caused by deletion or mutations of
the PY motifs of
- or
-ENaC (30, 31, 38, 72, 81, 87). These mutations lead to elevated activity of ENaC when it
is expressed heterologously in Xenopus laevis
oocytes (69), an elevation caused by an increase in both
channel number and openings at the cell surface (24).
In this review, we will focus on recent advances in our understanding of ENaC trafficking and regulation of its stability and the implications they have on the functioning of this channel under physiological and pathological conditions.
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ENAC TRAFFICKING TO THE CELL SURFACE |
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In recent years, several groups have focused their studies
on characterizing ENaC processing and trafficking, primarily in A6
cells and in cells that express ENaC heterologously [e.g. X. laevis oocytes, Cos cells, HEK-293 cells, and Madin-Darby canine kidney (MDCK) epithelial cells]. In general, it appears that ENaC processing is inefficient, with ~1% of channels synthesized in the
endoplasmic reticulum (ER) making it to the cell surface when they are
expressed in X. laevis oocytes (82) and an
estimated ~20% in A6 cells, which express X. laevis ENaC
(xENaC) endogenously (86). Moreover, ENaC
presence at the cell surface is almost undetectable in kidney tight
epithelia that are not stimulated by aldosterone, as no channel
activity is observed in cortical collecting ducts from rats with low
plasma aldosterone levels (57), and ENaC subunits cannot
be detected at the apical membrane of the distal nephron by
immunohistochemistry under similar conditions (48, 50). An
important determinant of the efficiency of ENaC maturation is the
assembly of its chains in the ER. ENaC assembly likely occurs very
early during its processing in the ER, as suggested by
coimmunoprecipitation experiments that demonstrate association of ENaC
chains in both glycosylated and unglycosylated forms (2). Although biochemical (2, 15, 24) and functional (11, 39) data have demonstrated that the expression of the
-subunit alone, or a combination of
- or
-subunits, can
lead to the appearance of functional channels at the cell surface, the
efficiency of maturation of such "partial" channels is extremely
low. This coincides with enhanced ubiquitination and proteasomal
degradation of ENaC chains when expressed individually (78,
82) (Fig. 1). Moreover, although
ectopic expression of the isolated
-subunit leads to exclusive
lactacystin-sensitive (proteasomal) degradation, expression of all
three ENaC subunits results in the appearance of an ENaC pool that is
sensitive to lysosomal inhibitors (78), suggesting that
this is a more mature pool that enters the lysosomes, possibly after
arrival at the plasma membrane and endocytosis. Thus assembly of all
three ENaC chains leads to increased stability of the complex and
likely increased efficiency of delivery to the plasma membrane,
although, even with all three subunits assembled together, the
maturation efficiency of this channel is quite low.
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A consistent observation in several studies is the short intracellular half-life of the ENaC protein, even when all three subunits are coexpressed. Typically, the total cellular pool of ENaC turns over rapidly, with a reported half-life of ~40-120 min in cultured cells (51, 78, 86) and a somewhat longer half-life (3.5-4 h) in X. laevis oocytes (82); the latter apparent increased stability may reflect the lower temperature in which the oocytes were grown. This rapid turnover may have important implications for the regulation of ENaC function. The stability of ENaC at the cell surface, a more important factor, is discussed in a separate section below.
Glycosylation
A very useful tool in studying maturation and trafficking of transmembrane proteins from the ER to the Golgi (Fig. 1) is tracing their Asn (N)-linked glycosylation pattern. At the ER, transmembrane proteins acquire core glycosylation, which is sensitive to N-glycosidase-F (PNGaseF). This core glycosylation is then trimmed at the ER to become high mannose and, on transport to the Golgi apparatus and maturation, is replaced with complex glycosylation at the medial-Golgi compartment, which is now resistant to endoglycosidase H (Endo-H). The Endo-H-resistant pool is often used as a marker to follow the mature transmembrane protein. However, one of the major difficulties (and a source of great frustration) in studying ENaC trafficking and maturation is the apparent lack of a detectable Endo-H-resistant pool of this channel (10, 63, 75, 82; Hanwell D, Saleki R, Ishikawa T, and Rotin D, unpublished observations). This lack of a detectable Endo-H-resistant pool of cellular ENaC suggested that either this channel does not acquire complex glycosylation or that the very small fraction of ENaC that actually makes it to the cell surface is complex glycosylated but is below our detection limit. Our recent work, however, suggests that the cell surface pool of ENaC expressed in epithelial MDCK cells and analyzed by surface biotinylation is Endo-H sensitive (Hanwell D, Saleki R, Ishikawa T, and Rotin D, unpublished observations). This suggests that ENaC is able to traffic to the cell surface without acquiring complex N-linked sugars. This is in contrast to the ENaC relative FaNaCh, in which a large fraction of the synthesized channel become Endo-H resistant during maturation (16). Although uncommon, there have been several proteins reported to traffic normally to the cell surface without the acquisition of complex N-linked glycosylation. Noted examples include the anion exchanger AE1 (27), the Torpedo AChR (8), and the Shaker K channel (67), although, in the latter two, a lack of complex glycosylation was seen when they were expressed in X. laevis oocytes (which nevertheless resulted in the expression of functional channels at the cell surface). In contrast to previous reports suggesting that the mature ENaC is stripped of its N-linked sugars (60, 61), our work demonstrates that the surface pool of ENaC is glycosylated in a pattern similar to that for the ER pool and exhibits PNGaseF and Endo-H sensitivity (Hanwell D, Saleki R, Ishikawa T, and Rotin D, unpublished observations), hinting that, at least in epithelial MDCK cells, an immature pattern of glycosylation persists in ENaC during its trafficking to the cell surface.The role of glycosylation of transmembrane proteins has received great
attention, and it appears that in some cases it is involved in
vectorial (e.g., apical) targeting, whereas in others it does not
appear to be so (reviewed in Ref. 88). The role of ENaC
glycosylation in ENaC trafficking is presently obscure. All three ENaC
subunits possess numerous potential N-linked glycosylation sites at their ectodomains (10), and it is clear that all
subunits are glycosylated in cells. All the glycosylation sites are
utilized in -ENaC, and mutating some or all of these sites does not
seem to affect channel activity (10, 75), suggesting that
at least sugar modification of that subunit does not affect proper
trafficking to and insertion of the channel in the plasma membrane.
Solubility in Nonionic Detergent
Recent work in Cos and HEK-293 cells heterologously expressing ENaC has shown that the channel is transformed during its processing from a nonionic detergent (Triton X-100)-soluble form in the ER [or possibly in the cytosol (82)] to a Triton X-100-insoluble form during trafficking to the cell surface and has suggested that the mature ENaC is detergent insoluble (60, 61). The acquisition of such nonionic detergent insolubility is often proposed to be mediated by either an association with lipid rafts or with the cytoskeleton or by the formation of a macromolecular oligomeric complex. Lipid rafts are cholesterol- and sphingolipid-rich microdomains within membranes often associated with targeting (mainly apical) of proteins sequestered in them (73). Our recent floatation assays with ENaC suggest, however, that ENaC expressed in MDCK cells is not associated with lipid rafts (Hanwell D, Saleki R, Ishikawa T, and Rotin D, unpublished observations). This conclusion is also supported by the observation that lysis of ENaC-expressing cells in octylglucoside at high pH (treatment that solubilizes caveolae-associated proteins) failed to solubilize ENaC (60). Thus it appears that ENaC is not utilizing lipid rafts to traffic to the cell surface.Several reports have proposed interactions between ENaC and
cytoskeletal proteins, including -spectrin (65, 90) and
actin (5, 40). However, although it is quite likely that
cytoskeletal interactions are involved in stabilizing ENaC at the cell
surface (as seen for numerous other transmembrane proteins) and
possibly in regulating its activity, it is not known whether these
interactions cause the detergent insolubility of this channel, as the
addition of the cytoskeletal disrupters cytochalasin, nocodazole, or
colchicine in one study failed to alter ENaC solubility
(61). Thus so far it is not clear what factors are
involved in mediating the acquisition of nonionic detergent
insolubility during maturation of ENaC or what fraction of ENaC
channels actually acquire such insolubility.
Targeting Signals
The determinants that control apical targeting of ENaC are not yet known. Earlier work has demonstrated retention of the COOH terminus ofOther Regulatory Hormones and Proteins
Vasopressin (antidiuretic hormone; ADH) increases ENaC activity by binding to a V2 receptor and activating adenylate cyclase. The vasopressin effect is mediated by cAMP and activation of protein kinase A; the nature of this phosphorylation has not yet been elucidated. A number of observations suggest that cAMP acts by translocating ENaC from a cytoplasmic pool into the apical membrane, but this issue still remains controversial (20, 43). Similar to aldosterone (see below), vasopressin also increases the abundance of ENaC, but to a lesser extent (17, 19). The stimulation of ENaC activity at the cell surface by vasopressin seems different from that of aldosterone (see below) because both effects are synergistic.Patch-clamp experiments in rat renal cortical collecting ducts
indicated that variations in plasma aldosterone levels, induced by
changes in dietary salt intake, modify the abundance of active ENaC in
the apical plasma membrane (57). No apical channel
activity could be detected in animals that had low plasma aldosterone
and were fed a high-salt diet, whereas increasing plasma aldosterone levels by restriction of Na+ intake dramatically increased
the number of functional apical channels. The physiological relevance
of this regulation has been quantitatively assessed recently by
measurements of the amiloride-sensitive Na+ currents
recorded in cortical collecting tubule cells of rats that were
subjected to short-term salt deprivation and had their urinary
Na+ excretion measured (25). This study showed
that the activation of ENaC current in response to a two- to threefold
increase in plasma aldosterone levels can account for the reduced
Na+ excretion under these conditions. This regulation of
ENaC activity in the distal nephron is likely important for the
day-to-day variations in urinary Na+ excretion. In situ
immunohistochemical studies in rodent kidney clearly showed that
changes in plasma levels of aldosterone affect the intracellular
distribution of ENaC subunits (48, 50). An increase in
plasma aldosterone level associated with dietary salt restriction
causes a large increase in intracellular abundance of -ENaC and a
redistribution of ENaC subunits from the cytoplasm to the apical
membrane. The induction of
-ENaC expression and its shift from a
cytoplasmic to an apical membrane pool in the distal nephron can be
observed as early as 2 h after aldosterone injection
(49). The molecular and cellular mechanisms underlying these effects remain to be elucidated. Besides the increase in
-ENaC
mRNA levels, aldosterone induces or represses the synthesis of many
other transcripts in principal cells of the distal nephron (aldosterone-induced and aldosterone-repressed transcripts; see Ref.
64). These aldosterone-induced transcriptional events are likely to play an important role in ENaC regulation. For instance, the
expression of the serum and glucocorticoid-induced kinase (sgk)
(85) rapidly increases in the distal nephron in response to aldosterone activity (14, 49, 55). The sgk protein is expressed in the cytoplasm of the principal cells expressing ENaC (49). When coexpressed with ENaC in X. laevis
oocytes, sgk stimulates ENaC activity (14, 55) by
increasing the number of ENaC channels at the cell surface. It has been
proposed that this increase is mediated by an augmented insertion of
channels at the plasma membrane rather than by a reduction in
endocytosis. (3). Taken together, the effects of sgk on
ENaC in X. laevis oocytes and the in vivo expression of sgk
in the distal nephron suggest that sgk is involved in the regulation of
cell surface expression of ENaC during the early phase (<4 h) of
aldosterone stimulation (49).
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ENAC STABILITY AT THE CELL SURFACE |
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Regulation of cell surface stability of ENaC has very important
implications for the control of channel function under physiological and pathological (e.g., Liddle's syndrome) conditions and has received
much attention in recent years. The half-life of ENaC at the plasma
membrane has been estimated from either its activity (amiloride-sensitive Na+ currents) in X. laevis
oocytes after brefeldin A (BFA) treatment, which inhibits ER-to-Golgi
transport of proteins, or from direct measurement of cell surface ENaC
protein using cell surface biotinylation, also after the addition of
BFA or blockers of protein synthesis. In either case, the fraction of
ENaCs that is recycled to the cell surface after internalization has
not been determined. The half-life of (heterologously expressed) ENaC
at the cell surface of X. laevis oocytes after BFA treatment
was reported to be ~2-3.5 h (71, 78). In mammalian
MDCK cells, this value was between 1 and 2 h (Hanwell D, Saleki R,
Ishikawa T, and Rotin D, unpublished observations), which is quite
comparable considering the higher temperature in which these cells are
grown relative to that for X. laevis oocytes. A recent study
using A6 cells proposed a much longer half-life of xENaC and
suggested that cell surface stability of - and
-ENaC (at least
24 h) is much greater than that of
-ENaC (6 h)
(86). It is presently difficult to understand, however,
the discoordinate internalization of the ENaC chains and the suggestion
that they may traffic to and from the plasma membrane independently,
considering the various studies demonstrating that ENaC chains assemble
very early on in the ER. Thus most studies suggest that ENaC at the
cell surface is turned over quite rapidly. This rapid turnover is
greatly inhibited, however, in
- or
-ENaC chains bearing
Liddle's syndrome mutations in their PY motifs, with several-fold
increased stability of the channel at the plasma membrane (24,
70, 71, 76). Presently, there are two models, not mutually
exclusive, that attempt to explain this increased stability:
1) decreased binding of ENaC to the ubiquitin ligase Nedd4
and 2) loss of an internalization signal. These models are discussed below in some detail.
Nedd4 and Its Regulation of Cell Surface Stability of ENaC
Nedd4 is a ubiquitin protein ligase composed of a C2 domain, three or four WW domains, and a ubiquitin ligase (E3) Hect domain (45). Ubiquitination of proteins serves to tag them for degradation, usually by the proteasome (34). In some cases, however, especially of transmembrane proteins, ubiquitination appears to tag proteins for endocytosis and lysosomal degradation (35, 66). The E3 Nedd4 catalyzes the third and final step in the ubiquitination cascade and is responsible for attaching ubiquitin moieties onto lysine residues of target proteins.The C2 domain of Nedd4 has been demonstrated to bind to membranes in a calcium-dependent manner and to localize Nedd4 to the apical membrane in polarized MDCK cells (59). This apical membrane targeting is achieved by a calcium-dependent association of the Nedd4-C2 domain with annexin XIIIb (58), a protein enriched in apical rafts (46), which thus recruits Nedd4 to these rafts and to the apical membrane.
WW domains are protein-protein interaction modules that bind
proline-rich sequences, and in the case of Nedd4, seem to prefer binding to PY motifs (xPPxY). Earlier work has demonstrated that the
Nedd4-WW domains bind to the PY motifs of ENaC and that mutations in
the PY motif of -ENaC, which cause Liddle's syndrome (31, 38,
72, 81) and lead to elevation of channel activity
(70), also abrogate binding to the Nedd4-WW domains
(77). More recently, the solution structure of the third
WW domain of Nedd4 in a complex with the PY motif of
-ENaC was
solved by NMR (42) (Fig. 2). It reveals several interesting features that also help explain the
failure of the WW domain to bind the Liddle's syndrome mutations. The
actual region in ENaC that binds the WW domain includes not only the PY
motif itself but also COOH-terminal residues and thus encompasses the
sequence pPPnYdsL, where P (proline), Y (tyrosine), and L (leucine)
indicate the residues that contact the domain. The sequence pPPxYxxL is
highly conserved in all ENaC subunits in all species. The two prolines
that precede the tyrosine form a polyproline type II helix and are
accommodated in an XP groove in the WW domain (42) (Fig.
2), as seen for other WW domains (36, 83) and for SH3
domains (89). What was unexpected and unique (so far) to
the Nedd4-WW:ENaC-PY motif complex is the sharp turn formed by the YxxL
region (Fig. 2) and the strong interactions of both tyrosine and
leucine with the WW domain (42). In accordance, Snyder et
al. (76) had demonstrated that mutation of leucine, similar to mutation of the prolines and tyrosine of the PY motif (70, 76), leads to elevation of ENaC activity. Thus these studies suggest that our definition of the PY motif should be extended
to PPxYxxL, which includes, in the case of
-ENaC, COOH-terminal sequences to the original "core" PY motif (PPxY). They also suggest the existence of Liddle's syndrome mutations in the leucine of the
extended PY motif, which have not yet been described but should be
searched for. Moreover, these studies have some important biological implications regarding endocytosis of ENaC, as described below.
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The interaction between Nedd4-WW domains and ENaC-PY motifs prompted
the obvious investigations into the physiological relevance of such an
association. Moreover, Nedd4 is expressed in numerous tissues and cell
types that express ENaC, such as the principal cells of the cortical
and outer medullary collecting ducts in the distal nephron and lung
airway and distal epithelia (18, 21, 79). Several recent
reports have indeed demonstrated that Nedd4 is a suppressor of ENaC
that regulates the number of channels at the cell surface (1, 22,
28, 33). This function of Nedd4 requires the presence of intact
PY motifs, to allow binding to Nedd4-WW domains, and accordingly,
mutations in the PY motif that cause Liddle's syndrome also impair the
suppressive effect of Nedd4 on ENaC (1, 28). Moreover,
suppression by Nedd4 also required its Hect domain to be active,
indicating that ENaC ubiquitination by this E3 ligase is involved
(1). Ubiquitination of ENaC was previously demonstrated to
regulate its cell surface stability (78), and mutation (to
arginine) of key conserved lysine residues located at the
NH2 termini of - and
-ENaC led to both impaired
channel ubiquitination and increased stability of the channel at the
plasma membrane (78). Thus, like several other
transmembrane proteins, including numerous yeast receptors and
permeases (which are regulated by the Nedd4 ortholog Rsp5p; reviewed in
Ref. 66), ENaC stability at the cell surface is regulated
by ubiquitination. Of the Nedd4 isoforms that affect ENaC, it appears
that those isoforms (or Nedd4-like proteins) possessing four WW domains
have a stronger suppressive effect on ENaC than those possessing three
WW domains (1, 32, 41). This could be due to increased
affinity and avidity of interactions between Nedd4 and ENaC because of
the presence of the "extra" WW domain. Taken together, it appears
that, in response to elevated calcium levels, Nedd4 translocates to the
apical membrane by association of its C2 domain with annexin XIIIb. It
then binds the PY motifs of ENaC by its WW domains, thus allowing
ubiquitination of the channel by the Hect domain, leading to
endocytosis and degradation of ENaC. This process is at least partially
impaired in Liddle's syndrome, causing increased retention of ENaC at
the cell surface and, hence, increased Na+ (and fluid)
absorption in the distal nephron, resulting in hypertension.
Endocytosis of ENaC
An alternative explanation for the increased cell surface retention of ENaC in Liddle's syndrome was put forward by Shimkets and colleagues (71) and Snyder and colleagues (76), proposing that the PY motifs ofEndocytosis of ENaC may not rely solely on its extended PY motif, and
other sequences may be involved as well. A recent report has suggested
the presence of an endocytic region in the NH2 terminus of
r-ENaC, which includes residues 47-67 (12).
Curiously, this region includes Lys47 and
Lys50, previously demonstrated to be targets
for ENaC ubiquitination (78). However, point mutation of
these lysines to arginines was only effective in stabilization of ENaC
at the cell surface in the context of Lys
Arg mutations of a critical
cluster of lysine residues (K6, 8, 10, 12, 13) in
-ENaC
(78). Thus it is not presently clear whether the role of
residues 47-67 of
-ENaC in endocytosis involves channel ubiquitination.
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CONCLUDING REMARKS |
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Despite difficulties in studying ENaC trafficking due to the low abundance of this protein in native tissues and the lack of an apparent Endo-H-resistant pool of the channel, recent published and unpublished work is beginning to shed some light on how this channel is processed and travels to the cell surface, although, clearly, our understanding of these processes is rudimentary. Similarly, our understanding of ENaC stability at the cell surface is very basic. However, the interaction of the channel with the ubiquitin ligase Nedd4 both structurally and functionally, as well as the described role of clathrin-mediated endocytosis in ENaC internalization, provides insight into at least some of the processes involved in regulation of channel stability at the plasma membrane. This knowledge is important for understanding both the physiological regulation of ENaC and its impaired regulation in diseases such as Liddle's syndrome.
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ACKNOWLEDGEMENTS |
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The work of D. Rotin and L. Schild was supported by the Human Frontier Science Program, the Medical Research Council/Canadian Institutes of Health Research, and the Canadian Cystic Fibrosis Foundation.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Rotin, Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, ONT, Canada M5G 1X8 (E-mail: drotin{at}sickkids.on.ca).
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