Current Genomics, 2003, 4, 109-121
1
CFTR and MDR: ABC Transporters with Homologous Structure but
Divergent Function
Douglas B. Luckie*,1, John H. Wilterding2, Marija Krha1 and Mauri E. Krouse3
1
Department of Physiology and Lyman Briggs School, Michigan State University, East Lansing, MI 48824-1101, USA;
3
Department of Biology, Olivet College, Olivet, MI 48828, and Cystic Fibrosis Research Laboratory, Stanford
University, Stanford, CA 94305-2130
2
Abstract: While a gene family by definition will have homologies in sequence, the “functional genomics” or
characterization of the functionality of siblings can reveal significant differences. One gene family that shares an ATPbinding cassette (ABC) sequence motif is comprised of members named “ABC Transporters.” Until 1989, the members of
this family were primarily ATPases that pumped compounds out of cells and many were linked to multidrug resistance
(MDR). Hence, at that time the function of the members of the ABC gene family appeared equally homologous to their
structure. Since then the discovery of new members, like the cystic fibrosis transmembrane conductance regulator (CFTR)
Cl- channel and the sulfonylurea receptor (SUR), increased diversity and the homologous structure seemed to no longer
link to homologous function. In this mini-review we will introduce two parallel investigations, separated by 10 years,
where researchers re-examined the functionality of the human ABC transporters, CFTR and MDR, under the hypothesis
that the structurally similar ABC transporters must have similarities in function.
INTRODUCTION
“Structural genomics,” the cloning and sequencing of
genes, has revolutionized biology and generated a credible
classification of gene families and subgroups. Although a
gene family by definition will have sequence homologies,
the “functional genomics,” or characterization of functionality, of siblings can reveal significant differences and diverge
from the common expectation that form and function are
tightly linked.
One gene family that shares an ATP-binding cassette
(ABC) sequence motif is so named “ABC Transporters.”
Mutations of genes in this family have been linked to various
clinical maladies including: ALD gene-adrenoleukodystrophy, SUR gene-diabetes, CFTR gene-cystic fibrosis, MDR
gene-multidrug resistance in cancer [1]. For many years the
genetically related ABC transporters appeared uniformly to
split ATP and use that energy to “pump” substrates upstream
across cell membranes. After the discovery of a new ABC
transporter gene in 1989, the cystic fibrosis transmembrane
conductance regulator (CFTR), perspectives began to
change. Since structure is aligned with function, the finding
that CFTR was not a pump, but, in fact, a Cl- channel, led
investigators to re-examine the activity of other ABC
transporters.
In 1992, the function of the multidrug resistance protein
(the MDR gene product is also called P-glycoprotein) was
revised. New data indicated that it might be “bifunctional.”
The transporter, believed to split ATP and pump
chemotherapy drugs out of cancer cells, was also found to be
tightly linked to volume-activated Cl- channel activity. In
fact, in an article by Valverde et al. (1992) [2], investigators
used the known homology between MDR and CFTR to
propose that MDR was a Cl- channel, as well as a drug
pump. As a result, the study of Cl- channel activity of MDR
became an active subfield in ABC gene research. Similarly,
although the CFTR gene was found to be a cAMP-activated
Cl- channel, the possibility that it might be responsible for
other processes has often been tested [3]. Recent studies
suggest that CFTR may be bifunctional (both a Cl- channel
and Cl-/HCO3- exchanger) and this has now become an
important new subfield in CF research in 2002 [4-18].
In the early 1990s, the authors began an NIH-funded
investigation entitled “Bifunctionality of ABC Transporters”
and became participants in these two parallel subfields where
structural and functional genomics of human CFTR and
MDR played a central role. In this mini-review we will first
introduce some members of the ABC Transporter family and
then focus on research into the potential “bifunctionality” of
both CFTR and MDR. Lastly, we will suggest an
evolutionary hypothesis which may explain how these genes
evolved and examine what evolutionary theory can, and
cannot tell us about the functional outcomes of the
evolutionary process. This mini-review is by no means a
comprehensive examination of the fields, multidrug
resistance, cystic fibrosis, or evolutionary biology but
instead attempts to highlight some events from the past that
may inform the present/future.
What is an ABC Transporter?
*Address correspondence to this author at the Cystic Fibrosis Research
Laboratory, 2100 Biomedical and Physical Sciences Bldg., Michigan State
University, East Lansing, MI 48824, USA; Tel: 517-355-6475, Ext. 1311;
Fax: 517-355-5125; E-mail: [email protected]
1389-2029/03 $41.00+.00
The term, “ABC Transporter” was first applied to unite
the relations of this gene family, by Christopher Higgins in a
1992 review [19]. Members of this superfamily have various
©2003 Bentham Science Publishers
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Current Genomics, 2003, Vol. 4, No. 3
genomic similarities but the one that has proven central is the
presence of an “ATP-binding cassette” (ABC) motif. An
ABC transporter typically contains two transmembrane
domains (TMDs) and two nucleotide (ATP) binding domains
(NBD’s) (Fig. (1)). Each NBD contains the ABC motif
where two sequences common to all ATP-binding proteins,
the Walker A and B domains, are combined with a third, C
motif, specific to this family. One ABC gene often contains
all four domains (two TMD and two NBD) on one long
peptide, however, the four domains can also be expressed
from separate genes that then assemble at the membrane to
form the fully functioning unit (Fig. (1)).
Early in 2001, Michael Dean and colleagues published
two comprehensive reviews of the ABC transporter family
tree [20, 21]. Dean et al. took advantage of the latest
sequence information from the international and private
genome projects and generated the most up to date family
classification indicated in Table (1) [20, 21]. While this
mini-review only deals with human ABC transporters, for
further reference we direct readers to reviews on non-human
ABC Transporters [22], the evolution of the ABC family
[23] and its walker domains [24]. For the purposes of this
review we will focus on the human MDR and CFTR
Luckie et al.
members of this family found in two separate groups, the
“ABCB” and “ABCC” subfamilies of the ABC Transporters
(Table (1)).
ABCB Subfamily (MDR and Siblings)
The ABCB subfamily is composed of seven known
transporters (Table (1)) and the multidrug resistance (MDR)
or P-glycoprotein (PGY1) gene is identified as gene ABCB1.
The terminology “P-glycoprotein” or “MDR” can sometimes
be used interchangeably to describe the gene, RNA or the
protein depending on the context, and the name ABCB1 adds
a third identity. For simplicity, in this review we will refer to
it as MDR (Fig. (1)). The MDR (ABCB1) gene is normally
only expressed in liver cells and those involved in the blood
brain barrier and is thought to be involved in protecting cells
from toxic agents [25, 26]. It was originally discovered as an
over-expressed protein in some drug-resistant tumor cell
lines. The ABCB transporters appear to be responsible for
movement of molecules across these cellular membranes and
in particular, MDR, appears to pump toxic chemotherapy
drugs out (efflux) of cells [25, 26], although the mechanism
is uncertain [27]. Investigators found that when a tumor cell
Fig. (1). Illustration of ABC Transporter domain structure. Prototypical ABC transporter tertiary/quartenary structure contains four protein
domains. Two transmembrane domains (TMD) in the lipid bilayer and two nucleotide binding domains (NBD) in the cytoplasm. A. In some
cases (i.e. CFTR, MDR) all domains are encoded by one gene and expressed as a single protein. B. In other cases domains are encoded by
separate genes and assemble spontaneously to form the final functional unit.
CFTR and MDR: ABC Transporters with Homologous Structure
Table 1.
Current Genomics, 2003, Vol. 4, No. 3
List of Human ABC Genes, Chromosomal Location, and Function
Symb
Alias
Location
Expression
Function
ABCA1
ABC1
9q31.1
Ubiquitous
Cholesterol efflux onto HDL
ABCA2
ABC2
9q34 2
Brain
Drug resistance
ABCA3
ABC3, ABCC
16p13.3
Lung
ABCA4
ABCR
1p22.1–p21
Rod photoreceptors
ABCA5
17q24
Muscle, heart, testes
ABCA6
17q24
Liver
ABCA7
19p13.3
Spleen, thymus
ABCA8
17q24
Ovary
ABCA9
17q24
Heart
ABCA10
17q24
Muscle, heart
ABCA12
2q34
Stomach
ABCA13
7p11–q11
Low in all tissues
N-retinylidiene-PE efflux
ABCB1
PGY1, MDR
7p21
Adrenal, kidney, brain
Multidrug resistance
ABCB2
TAP1
6p21
All cells
Peptide transpor
ABCB3
TAP2
6p21
All cells
Peptide transport
ABCB4
PGY3
7q21.1
Liver
PC transport
7p14
Ubiquitous
ABCB5
ABCB6
MTABC3
2q36
Mitochondria
Iron transport
ABCB7
ABC7
Xq12–q13
Mitochondria
Fe/S cluster transport
ABCB8
MABC1
7q36
Mitochondria
12q24
Heart, brain
ABCB9
ABCB10
MTABC2
1q42
Mitochondria
ABCB11
SPGP
2q24
Liver
Bile salt transport
ABCC1
MRP1
16p13.1
Lung, testes, PBMC
Drug resistance
ABCC2
MRP2
10q24
Liver
Organic anion efflux
ABCC3
MRP3
17q21.3
Lung, intestine, liver
Drug resistance
ABCC4
MRP4
13q32
Prostate
Nucleoside transport
ABCC5
MRP5
3q27
Ubiquitous
Nucleoside transport
ABCC6
MRP6
16p13.1
Kidney, liver
CFTR
ABCC7
7q31.2
Exocrine tissues
Chloride ion channel
ABCC8
SUR1
11p15.1
Pancreas
Sulfonylurea receptor
ABCC9
SUR2
12p12.1
Heart, muscle
ABCC10
MRP7
6p21
Low in all tissues
ABCC11
16q11–q12
Low in all tissues
ABCC12
16q11–q12
Low in all tissues
Xq28
Peroxisomes
ABCD1
ALD
VLCFA transport regulation
3
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Current Genomics, 2003, Vol. 4, No. 3
Luckie et al.
(Table 1) contd….
Symb
Alias
Location
Expression
ABCD2
ALDL1, ALDR
12q11–q12
Peroxisomes
ABCD3
PXMP1, PMP70
1p22–p21
Peroxisomes
ABCD4
PMP69, P70R
14q24.3
Peroxisomes
ABCE1
OABP, RNS4I
4q31
Ovary, testes, spleen
ABCF1
ABC50
6p21.33
Ubiquitous
ABCF2
7q36
Ubiquitous
ABCF3
3q25
Ubiquitous
Function
Oligoadenylate binding protein
ABCG1
ABC8, White
21q22.3
Ubiquitous
Cholesterol transport
ABCG2
ABCP, MXR, BCRP
4q22
Placenta, intestine
Toxin efflux, drug resistance
ABCG4
White2
11q23 5 59
Liver
ABCG5
White3
2p21 17
Liver, intestine
Sterol transport
2p21 17
Liver, intestine
Sterol transport
ABCG8
Data provided by Michael Dean [6]; PBMC= peripheral blood mononuclear cells; VLCFA= very long chain fatty acids.
over-expressed MDR, it became resistant to a wide variety of
chemotherapy drugs, hence the terminology “multidrug
resistance”. The expression of MDR can be significant from
the a clinical perspective, since resistant cancers quickly
become fatal.
unregulated insulin secretion [34]. The remaining ABCC
family members are nine MRP-related genes associated with
a different form of multidrug resistance and appear to pump
glutathione conjugates across the cell membrane [35].
Other members of this subfamily transport phosphatidylcholine (PC) and bile salts (ABCB4 and B11), aid
immune cells by tranporting antigens into their ER (ABCB2
and B3 (TAP)), as well as, can manage iron flux in the
lysosome (ABCB9) or the mitochondria (ABCB6, B7, B8,
and B10)[20, 21]. Failures in these processes (impairing the
immune system) are significant and the clinical relevance of
this gene family continues with members in the ABCC
subgroup which includes CFTR.
ABC TRANSPORTERS 1992: “IS MDR BIFUNCTIONAL?”
ABCC Subfamily (CFTR and Siblings)
The ABCC subfamily contains 12 transporters (Table
(1)). Two of these members do not fit well under the
definition that ABC transporters are “pumps.” The CFTR
(ABCC6) protein is unique among ABC proteins in that it is
a cAMP-regulated chloride ion channel [28]. Cystic fibrosis
is a disease caused by mutations in the gene for CFTR (Fig.
(1)) [29-32]. In cystic fibrosis (CF), the disease pathology in
many organs is attributed primarily to impaired Clconductance in apical membranes of wet epithelia [33]. The
ABCC8 gene (SUR1) is another ABC transporter that is not
a pump. SUR1 is a high affinity receptor for the drug
sulfonylurea [34]. Sulfonylureas are widely used to increase
insulin secretion in patients with non-insulin-dependent
diabetes. These drugs bind to the ABCC8 protein that closes
a potassium channel (Kir) and triggers insulin secretion. If
there is a mutation in the SUR1 gene the secretion of insulin
is disturbed. In fact, the gene was identified as the locus for
familial persistent hyperinsulinemic hypoglycemia of
infancy, an autosomal recessive disorder characterized by
Animal cells lyse (explode) due to osmotic swelling if
they are exposed to a hypotonic conditions for too long.
Cells respond to the dangers of swelling by activating
channels that allow the efflux of osmolytes and inorganic
ions (especially Cl-) followed by water. This important
process is known as regulatory volume decrease (RVD) and
one ubiquitous and well-studied pathway for osmolyte
release involves the “volume sensitive osmolyte and anion
channel” (VSOAC) [36]. Identifying and cloning swelling
channels has been a long-term effort in the field that is yet to
yield a convincing result. Yet in 1992, a report in the journal
Nature suggested that the molecular identity of VSOAC was
finally found.
Is MDR Both a Drug Pump and a Swelling-activated ClChannel?
In 1992, the multidrug-resistance MDR protein, thought
to be an ATPase that pumped hydrophobic drugs, was
hypothesized by Miguel Valverde and Christopher Higgins
to also serve as a swelling-activated chloride channel [2].
The stimulus to link VSOAC and MDR rose from using
homologous structure to predict homologous function
(form=function). Investigators began with an examination of
domain similarities between MDR and the recently cloned
cystic fibrosis protein (CFTR). When the evidence that
CFTR was an intrinsic Cl- channel became unequivocal [3739], it was suggested that MDR might also be a Cl- channel,
and have dual roles as ion channel and efflux pump [2].
CFTR and MDR: ABC Transporters with Homologous Structure
Valverde and Higgins tested their hypothesis by
comparing Cl- currents in cells that did or did not express
MDR. They found that swelling-activated Cl- currents
occurred only in cells when MDR protein was expressed,
therefore concluding that MDR was a swelling-activated Clchannel (VSOAC) [2]. This was followed by detailed studies
from the same investigators that confirmed and extended the
original work [40-44] as well as developed elegant models to
explain how this bifunctionality worked [45]. They had
shown that MDR functioned alternately as either a Clchannel or an efflux pump, with the two functional states
being mutually exclusive [40].
Current Genomics, 2003, Vol. 4, No. 3
5
expression and Cl- currents were characterized through a
battery of tests including rhodamine efflux, cell volume
measurements and whole cell patch clamp. In the end they
reported that “The rates of induction, biophysical properties
and magnitude of Cl- conductance were indistinguishable
between control and corresponding multidrug-resistant cells”
[60]. During this period, numerous studies using many cell
types and methods for manipulating MDR protein expression
and for assessing channel activity [61-70] also reported the
lack of a correlation between MDR expression and the
presence of VSOAC currents. All of them concluded that the
MDR protein was not the VSOAC channel. It is now clear
that MDR is not bifunctional (at least in this way).
Why was this Finding Controversial?
Epilogue
These reports generated a great deal of interest [46] and
to this day many scientists believe that the story ended there,
however, other laboratories still needed to confirm their
work. Experienced electrophysiologists also found the
findings quite surprising, since they experienced large
swelling currents throughout their studies [47] even though
MDR expression was not ubiquitous. This “disconnect” was
immediately recognized by scientists who studied VSOAC
swelling currents and a number of groups went to work to
test MDR for VSOAC activity [48-56].
Is MDR Expression Related to VSOAC Activity?
One of the first publications to report a close examination
of MDR for swelling channel activity was that of Rasola et
al. (1994) [57]. Throughout 1992, they carefully tested four
epithelial cell lines (LoVo/H LoVo/Dx; 9HTEo- 9HTEo/Dx) both for the expression of MDR-1 gene and for the
presence of volume-sensitive Cl- currents. They found
greatly elevated expression of MDR protein in Dx,
(doxorubicin-selected) lines but Cl- current was unchanged.
Like Rasola, our laboratory (Krouse, Luckie and Wine at
Stanford) felt that the question of whether MDR was a Clchannel, was too important to ignore. Given that the
techniques of patch clamp electrophysiology are extremely
good at determining whether a protein is a channel or not, it
seemed like a relatively simple question to test. In October
of 1993, we presented our findings at the North American
Cystic Fibrosis Conference [58] reflecting that we saw no
evidence to prove that MDR could be a channel. However,
Christopher Higgins, Deborah Gill and Stephen Hyde
presented enough new evidence [pers. comm., 1993] to
support their hypothesis and we were convinced to go back
to the lab and conduct further experiments. After numerous
additional trials we felt certain our data was solid and
published our work [59].
Perhaps the most comprehensive study published on this
topic was that of Ehring et al. 1994 [60]. This well designed
study from Michael Cahalan’s laboratory was published in
The Journal of General Physiology at the end of 1994, after
a dozen reports had debated the issue since Rasola et al.
[57]. Ehring studied control and MDR1-transfected NIH-3T3
cells selected in colchicine, as well as control and
doxorubicin-selected myeloma cells [60]. Both MDR
Today Higgins and Valverde agree that MDR is not
VSOAC or likely any channel yet they continue their
investigations into the possibility that MDR may yet be
shown to regulate VSOAC and thus strive to find the
molecular identity of the ubiquitous swelling activated Clchannel [44, 71]. One line of evidence that supports the idea
that MDR regulates VSOAC, is that in recent years the
structurally homologous ABC proteins CFTR and SUR have
been shown to regulate other channels [71]. It seems
apparent that after the molecular identity of VSOAC is
found, the mechanism by which MDR could alter the activity
of VSOAC may be clarified.
ABC TRANSPORTERS 2002: “IS CFTR BIFUNCTIONAL?”
Cystic fibrosis (CF) is caused by mutations in CFTR, an
apical membrane anion channel involved in epithelial fluid
secretion and salt absorption [29-32]. The most important
aspects of CF’s pathophysiology arise from alterations in
extracellular fluids, such as, premature activation of
pancreatic enzymes, increased thickness of mucus and
consequent ductal blockage and irritation [28].
Manifestations of CF in some organs have been difficult to
be attributed solely to a defect in Cl- conductance [28] and
various investigators have documented changes in other
properties as well [3]. For years investigators have been
aware that HCO 3- secretion in the CF pancreas was impaired
[72]. Peter Durie and others in the 1980’s [73-75]
demonstrated that the CF pancreas has both chloride and
bicarbonate secretion defects. Yet recently the question has
been extended to the aspects of bicarbonate conductance
associated with other organs impaired in the disease, in
particular the lung [76-79]. Secretion of HCO3- (as well as
H+) may be critical to pH and various cell studies have
yielded evidence that pH homeostasis is dependent upon
CFTR function [80-84]. Studies have focused on pH as well
as the capacity of CFTR to conduct bicarbonate ions [77, 8588] or alter bicarbonate transport through other pathways
[85, 87, 88-92], and in late 2001, Paul Quinton convened an
“International Symposium on HCO 3- and Cystic Fibrosis” on
the topic [7-18, 93-94].
As a result, the topic, “CFTR and bicarbonate,” has
become a hot subfield for those who study ABC Trans-
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Current Genomics, 2003, Vol. 4, No. 3
porters [7-18, 93-94] and the question of bifunctionality has
also raised its head for the CFTR in 2002, as it did for MDR
in 1992.
Is CFTR Both a Cl- Channel and HCO3-/Cl- Exchanger?
In a series of recent papers the research group of Shmuel
Muallem has tightly linked apical exchange of HCO3- and Clto CFTR [16, 78, 84, 90] and present the novel idea (as well
as convincing data) that much of cystic fibrosis may really
be a result of defective bicarbonate transport [76]. In a recent
report in the journal Nature, Choi et al., (2001) [76]
examined a variety of CFTR mutants and recorded HCO3movement with pH sensitive dye (BCECF). They found that
several mutants known to cause the disease exhibited normal
Cl- conductance but impaired HCO3- transport. These results
suggested that HCO3- flux might be more relevant to CF
status than Cl- movement. If that wasn’t revolutionary
enough, Muallem’s group, in their latest paper [95] have
suggested that CFTR is both a Cl-/ HCO3- exchanger and a
chloride channel. While they found that HCO3- efflux from
cells is dependent on activated CFTR, they found no
evidence that HCO3- anion moves through the pore of the
CFTR channel [76, 95].
Luckie et al.
HCO3- ions would efflux from epithelia [99] one might
expect CFTR to effect extracellular pH (pHo).
Does CFTR Activity Effect Extracellular pH?
Abnormalities in pH have been seen in the CF field
before but never assumed to be critical beyond the pancreas
[81-82, 89, 102-103]. In recent experiments in our own
laboratory (Luckie & Wilterding at Michigan State)[104] we
found that C127 cells expressing CFTR alkalinized the
extracellular solution after forskolin stimulation whereas
non-CFTR expressing cells did not. Using the same cell line
(C127) Zegarra-Moran et al. (2001) [105] built upon
previous work [106], and also found that CFTR regulates the
HCO3- pathway. Recently, Jayaraman et al. (2002) has also
examined the question of pHo and found otherwise [107].
They used an in situ fluorescence method to measure the
ionic composition, pH and viscosity of freshly secreted fluid
from human submucosal airway glands and found;
“(Compared to controls) Neither [Na+] nor pH differed in
gland fluid from CF airways” [107]. Thus, more studies need
to be carried out to clarify if epithelial pHo does change (and
in which organs) in cystic fibrosis.
Can HCO3- Move Through the CFTR Pore?
Why is this Finding Controversial?
These reports have generated a great deal of interest [4,
96], however, other laboratories still need to confirm their
work. Experienced electrophysiologists also find these
findings quite surprising, since some of them have recorded
bicarbonate conductance through what they assumed to be
the pore of the CFTR channel [39, 80-81, 97-100]. Even
earlier data from the Muallem laboratory may even oppose
their own hypothesis but putting that aside let’s just examine
this general model. In evaluating whether CFTR is both a Clchannel and anion exchanger, many questions immediately
come to mind, but let us review the basics first: (i) Does
HCO3- secretion require CFTR?, (ii) Does CFTR activity
effect extracellular pH? (iii) Can HCO3- move through the
CFTR pore?
Does Bicarbonate Secretion Require CFTR?
Ballard et al. (1999) [85] found that in pig submucosal
glands both Cl- and HCO3- secretion are dependent on
CFTR. In these whole trachea preparations, because CFTR
can conduct both Cl- and HCO3-, there is no need to invoke
the presence of a Cl-/ HCO3- exchanger. However, Clarke
and Harline (1998) [87] found in mouse intestine a CFTRdependent Cl -/ HCO 3- exchange. This could be due to CFTR
upregulating the expression of an exchanger, regulating a
separate exchanger, or being an exchanger itself. In CFT-1
lines Wheat et al. (2000) [101] found AE activity and
expression of DRA (a protein implicated in the apical anion
exchange) to be upregulated in CFTR expressing cells. They
suggested that CFTR Cl- channel activity and direct
interaction with DRA both serve to direct HCO3- movement.
These reports link bicarbonate exchange to CFTR
activity and thus given that at physiological conditions
CFTR was shown to be a chloride channel in bilayer
experiments by Christine Bear [39] and since then various
investigators have examined HCO3- conductance through
CFTR [97-100]. Linsdell et al. 1997 [97] performed an
elegant single channel study with CHO cells and determined
that bicarbonate’s permeability through CFTR is 14-25% of
chloride’s. Later, Machen’s group [99] measured the
permeability of CFTR to bicarbonate using single channel
techniques in transfected NIH 3T3 or C127 cells [80], bovine
tracheal cells [79] and Calu-3 cells [100]. They reported
evidence that bicarbonate does go through CFTR and that
the permeability of bicarbonate is ~25% of that of chloride.
In Ussing chamber experiments with Calu-3 cells (short
circuit) Krouse et al. [108] presented evidence that 50%-90%
of the bicarbonate current is going through open CFTR.
Lazrak A et al. (2002) found CFTR dependent HCO3currents in rat fetal distal lung epithelial (FDLE) cells [5]
and Sun et al. (2002) found it in bovine corneal endothelial
cells [6].
While several research groups have reported evidence
that HCO 3- can travel through the CFTR pore, in addition to
Choi et al. inability to detect it, Devor et al. (1999) [88]
found that bicarbonate secretion was dependent upon CFTR
but might not go through the CFTR pore; moreover Reddy
and Quinton (2002) [14] have found conditions where CFTR
exhibits Cl- conductance without HCO3- in the sweat gland
[14]. Perhaps this question of whether CFTR is a HCO3-/Clexchanger-, is too important to ignore.
Epilogue or Prologue?
The powerful effects of these findings have been
mitigated somewhat by commentaries in the recent reviews
[4, 96] and in particular Jeffrey Wine provides data that at
CFTR and MDR: ABC Transporters with Homologous Structure
least one of the mutations (R1070Q), reported by Choi et al.
as “pancreatic sufficient,” is not so, thus breaking the perfect
correlation with bicarbonate movement and pancreatic
sufficiency [96]. More work is needed to determine the
significance of the relationship between cystic fibrosis and
bicarbonate movement. Even if CFTR is not truly a
bifunctional protein, given that the CFTR has a “velcro-like”
PDZ domain [16] that can bind to other transporters with
PDZ regions, it’s interaction with NHE and other AEs along
the apical membrane might create not only a bifunctional but
a multifunctional complex that could implicate “HCO3conductance defects” as a potentially universal problem in
the multi-organ pathology which is cystic fibrosis.
CONCLUSION: RETURN TO CURRENT GENOMICS
It is interesting to see how the functional aspects of these
ABC gene products have evolved in the literature. When first
cloned they were assumed to be analogous transporters, just
like other ABC proteins. When CFTR was found to be
different (a Cl- channel), MDR was immediately tested for
Cl- conductance. Although CFTR and MDR have since been
shown to be different, in the light of the phylogenetic
similarities in this gene family, what lessons concerning
functional genomics can be learned from this?
The phylogeny of the ABC transporter family has
previously been characterized [20, 23]. The ubiquity of ABC
cassette motif and walker domains in the human genome
suggests the widespread utility of this sequence in many
proteins [20, 21]. A potential hypothesis is that the genetic
homology shared by ABC transporters was a result of gene
evolution by exon-shuffling [109-110]. Briefly, in this
model, new novel proteins are assembled in part from preexisting subunits that are mixed and matched with others to
form genes that assume new and novel functions [111-113].
If this is so, it might be attractive to assume that since a new
gene in question shares one or more subunits found in other
related genes [114, 115], its protein might share analogous
properties with those related proteins.
Indeed, as we have seen, the pairing of MDR and CFTR
with other ABC transporters lead to the early, and partly
inaccurate assumption as to the functional aspects of MDR
and CFTR [2, 40-45]. However, as we have seen, the
situation is far more complex, but these results should not be
viewed as surprising or discouraging. Studies of exon 9
shuffling in CFTR support Walker A likely went through a
series of duplications, amplification and dispersion
throughout the human genome in evolution [24]. If a
mechanism such as exon-shuffling did in fact give rise to
new proteins, it would bring a well characterized domain
(like the Walker A domain) into a new association with other
disparate flanking sequences [116]. The molecular
interactions (folding, bonding interactions, hydrophobicity,
etc.) in this new environment may change the functional
nature of the domain [117]. For example, an examination
phylogenetic tree of ABC transporters reveals that CFTR
(ABCC7), a Cl- channel has greatest sequence homology to
SUR (ABCC8), a sulfonylurea receptor. Therefore, while the
known function of one gene can be useful in generating
hypotheses as to the functional nature of another gene to
Current Genomics, 2003, Vol. 4, No. 3
7
which it share homologous domains, this should be done so
with caution.
In the realm of functional genomics then, a credible
phylogeny of gene families may only have limited
application in developing a meaningful taxonomy to aid in
the study of the origin of a gene. For example, Martinoia et
al. (2002) [22], found that while being structurally-related,
ABC transporters in plants in fact served diverse
physiological functions. As a result, domain homologies may
provide researchers with reasonable starting points, but
ultimately they may imply little, if anything definitive
concerning the actual function. Interestingly the examination
of the large-scale organization of cell components into
higher order complexes called “modules” appears to be an
interesting new area of study [118]. Even in this, at the more
macroscopic level, surprisingly, one particular protein can
serve distinctly different functions in separate parts of the
same cell [119]. The whole depends on process neighboring
proteins and ultimately it’s “context.”
ACKNOWLEDGEMENTS
The authors thank Jeffrey Wine, Paul Quinton, Errett
Hobbs, Joseph Maleszewski and Seth Hootman for helpful
discussions. This work was supported by a Cystic Fibrosis
Foundation Grant (98I0) and National Institute of Diabetes,
Digestive and Kidney Disease Grant DK09203 to D.B.
Luckie.
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