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Choline Transport for
Phospholipid
Synthesis
Choline is an essential
nutrient for all cells
because it plays a
role in the synthesis of
the membrane
phospholipid components
of the cell membranes,
as a methyl-group donor
in
methionine metabolism as
well as in the synthesis
of the
neurotransmitter
acetylcholine. Choline
deficiency affects the
expression of genes
involved in cell
proliferation,
differentiation,
and apoptosis, and it
has been associated with
liver dysfunction
and cancer. Abnormal
choline transport and
metabolism have
been implicated in a
number of
neurodegenerative
disorders
such as Alzheimer’s and
Parkinson’s disease.
Therefore, the
study of choline
transport and the
characteristics of
choline
transporters are of
central importance to
understanding the
mechanisms that underlie
membrane integrity and
cell signaling
in such disorders.
Kinetic studies with
radiolabeled choline and
inhibitors distinguish
three systems for
choline transport: (i)
low-affinity facilitated
diffusion, (ii)
high-affinity,
Na+-dependent
transport, and (iii)
intermediate-affinity,
Na+-independent
transport.
It is only recently,
however, that the
proteins having
transport
characteristics of at
least one of these
systems have
been identified. They
include (i) polyspecific
organic cation
transporters (OCTs) with
low affinity for
choline, (ii)
high-affinity
choline transporters
(CHT1s), and (iii)
intermediate-affinity
choline transporter-like
(CTL1) proteins. CHT1
and CTL1 but
not OCT transporters are
selectively inhibited
with hemicholinium-
3 and essentially
display characteristics
of specialized
transporters for
targeted choline
metabolism. CHT1 is
abundant
in neurons and almost
exclusively supplies
choline for
acetylcholine
synthesis. The focus
here is more on
newly-discovered
CTL1 choline
transporters. They are
expressed in different
organisms and cell
types, apparently not
for the biosynthesis of
acetylcholine but for
the production of the
most abundant
metabolite of choline,
the membrane lipid
phosphatidylcholine.
Exp Biol Med
231:490–504, 2006
Key words: choline;
phospholipids; choline
transport; CTL1
Importance of Choline
In 1998, choline was
identified as an
essential nutrient
for humans by the
National Academy of
Sciences, United
States (1). Choline is a
quaternary amine
(trimethyl-bhydroxy-
ethylammonium) (2) and
its concentration in
plasma has a
physiological range of
10–50 lM (3). Choline
is predominantly
utilized for the
synthesis of essential
lipid
components of the cell
membranes,
phosphatidylcholine
(PC) and sphingomyelin,
and for the production
of potent
lipid mediators such as
platelet-activating
factor and
lysophosphatidylcholine
(4). Quantitatively, PC
is the most
important metabolite of
choline and accounts for
approximately
one-half of the total
membrane lipid content
(5).
Similarly to folate,
choline is a source of
labile methyl
groups (6). In the liver
and kidney mitochondria,
choline is
oxidized to betaine
(trimethylglycine),
which then enters the
one-carbon cycle and
serves as a methyl donor
in the
remethylation of
homocysteine to
methionine, to
ultimately
generate the methylation
agent
S-adenosylmethionine
(SAM; Ref. 7). Finally,
choline is best known
for its key
role in neurons as the
precursor of the
neurotransmitter
acetylcholine (8, 9).
Acetylcholine could also
be synthesized
and released from
nonneuronal cells;
however, its role
outside of neurons is
not clearly defined
(10).
The adequate intake
level for choline is 550
mg/day for men and 425
mg/day for women (11).
Choline contentvaries considerably
among common foods (12)
and dietary
choline supplementation
could improve multiple
body
functions, including
cognition (13),
learning, and memory
(14, 15). Therefore,
choline is an essential
dietary component
for the normal
functioning of
organisms. Here, we
focus on specific
aspects of choline
research in recent
years,
including choline
deficiency and choline
metabolic pathways
and transport, with
emphasis on the emerging
new
families of transporters
necessary for the
production of PC,
a lipid essential for
the structural integrity
and functioning
of cell membranes.
Choline Deficiency
Healthy individuals meet
or exceed the adequate
intake
levels for choline (16);
however, certain
population groups
are at risk for choline
deficiency. These
include pregnant
and lactating women,
infants, cirrhosis
patients, and patients
depending on parenteral
nutrition (17). Dietary
choline
deficiency in animal
models causes
spontaneous carcinoma
of the liver and
generally increases
sensitivity to
carcinogens
(18). It is the only
type of nutritional
deficiency known
to have such deleterious
effect (19). A gradual
reduction in
choline supplementation
initially causes
apoptosis in rat
hepatocytes (20, 21);
however, when cells
adapt to a low
choline intake, they
become resistant, and a
tumorigenic
transformation is
frequently observed
(21). Apoptosis
induced by choline
deficiency is
p53-independent and is
linked to TGFb1
signaling and generation
of reactive
oxygen species, which
disrupt the membrane
potential and
eventually lead to
mitochondrial
dysfunction and cell
death
(21, 22). Adaptive
responses to deleterious
effects of
choline deficiency
include increased
transcription of the
proto-oncogenes c-myc,
c-Ha-ras and c-fos, and
increased
levels of
proinflammatory
cytokines such as TNF-a
(23).
Furthermore, it has
recently been shown that
choline
deficiency affects the
expression of 1000 genes
in neural
precursor cells, with
one-third of the genes
involved in the
cell proliferation,
differentiation,
methyl-group metabolism,
and apoptosis (24),
demonstrating the
essentiality of choline
for proper cell
function.
Because it is
predominantly utilized
for membrane PC,
choline has a
significant impact on
liver function and
systemic lipid
metabolism. In
experimental animals as
well
as in humans (25–28),
choline deficiency
results in the
formation of
‘‘nonalcoholic fatty
liver,’’ associated with
accumulation of hepatic
lipids and an increased
sensitivity
to inflammation (29). In
knockout mouse models
lacking
liver CTP:phosphocholine
cytidylyltransferase
(Pcyt1),
which is the major
regulatory enzyme in PC
synthesis,
hepatic PC decreases and
an accumulation of
triglycerides in
the liver is observed.
At the same time, plasma
lipids as well
as plasma lipoprotein
content become reduced
(30). It is
well established that
homocysteine levels are
elevated after
a methionine load in
choline-deficient
individuals (26). The
interaction of choline
with other nutrients in
one-carbon
metabolism and
homocysteine production
is becoming an
increasingly important
issue to be examined
more actively
in the future (31). The
close relationship
between choline
and folate/methionine
metabolism offers an
opportunity for
improved assessment of
human susceptibility to
choline
status, development of
nonalcoholic fatty
liver, and
development of cancer.
The risk assessment
based on
genetic variations may
offer novel preventive
strategies for
similar diseases. The
relationship between
folate deficiency
and polymorphisms of the
5,10-methylenetetrahydrofolate
reductase (MTHFR) gene
is well-known; it
resulted in
widespread
supplementation of food
products with folate to
prevent development of
birth defects (32).
Choline deficiency
is not related to the
variations in the MTHFR
gene;
however, the
polymorphism of
5,10-methylenetetrahydrofolate
dehydrogenase (MTHFDH)
is a significant risk
factor.
When placed on a
choline-deficient diet,
carriers of the most
common allele,
MTHFDH1958A, become
depleted of
choline faster than
noncarriers (27).
During pregnancy, fetal
plasma choline levels
are
several-fold higher
compared with choline
levels in
maternal plasma, which
may result in a
depletion of
maternal choline stores
(29), implicating the
importance of
choline for the
developing fetus. In a
normal pregnancy,
plasma choline levels
rise constantly
throughout the
gestation period (33),
coinciding with elevated
homocysteine
levels (34), which
indicates that the high
fetal demand
for choline may
stimulate an increased
choline synthesis in
the maternal liver (34).
Choline supplements
during
pregnancy have been
shown to improve
lifelong memory
in rats (35). Adult
offspring of mothers
that had received
choline supplements were
more adept at tasks
involving
spatial and temporal
memory and attention
(35–37), and
these behavioral effects
persisted beyond the age
of 2 years.
These data suggest that
choline has a permanent
developmental
effect on brain
organization and
function (1).
Furthermore, consuming
foods high in choline
during
pregnancy may reduce the
risk of neural tube
defects; this
is attributed mainly to
the role of choline as a
methyl-group
donor (38). More on
choline deficiency and
the relationship
between choline, the
one-carbon cycle, and
lipid metabolism
is discussed in the
following sections on
choline
metabolic pathways and
transport.
Choline Metabolic
Pathways
The majority of cellular
choline is
phosphorylated by
choline kinase (CK) to
phosphocholine, to which
CTP can
then be added by Pcyt1
to yield CDP-choline.
The
formation of PC results
from the reaction of
CDP-choline
with diacylglycerol
(DAG), catalyzed by
CDP-choline:
DAG
cholinephosphotransferase
(CPT; Fig. 1). This
pathway
is known as the Kennedy
(CDP-choline) pathway
for
de novo synthesis of PC
and is essential for the
formation of
membrane PC in all
nucleated cells. Since
its discovery in the
1950s by Eugene Kennedy,
this pathway has been
extensively studied, and
most aspects of its
regulation are
well established. In
recent years, focus is
more on cloning
and characterization of
genes participating in
the pathway
(39–43), with emphasis
on the rate-regulatory
gene Pcyt1
(44–46); the Kennedy
pathway is not
considered to be
regulated by other steps
in the pathway, nor by
the rate of
choline transport.
During evolution, the
liver has retained a
backup
pathway for choline
production from the
second most
abundant membrane
phospholipids,
phosphatidylethanolamine
(PE; Fig. 1), to provide
this essential
metabolite when
dietary choline is
limited, as, for
example, during
starvation,
embryonic development,
pregnancy, or lactation.
PE is
transformed to PC in a
three-step methylation
by SAM,
which is catalyzed by
phosphatidylethanolamine-N-methyltransferase
(PEMT). The PE
methylation pathway has
been
extensively studied by
Dr. Dennis Vance and his
collaborators
(for reviews see Refs.
47, 48). More recently,
his
laboratory developed a
PEMT knockout mouse
model,
providing valuable data
on the role of this
pathway in the
liver, PC synthesis,
lipoprotein secretion,
and bile and
homocysteine production
(42, 49).
Liver
choline/phospholipid and
methionine/homocysteine
pathways are
interrelated by their
need for methyl
groups provided by SAM
(Fig. 1). When PE
methylation is
blocked, as it is in
PEMT knockout mice (PEMT
/ ), plasma
homocysteine is 50%
lower than in wild-type
PEMTž/ž
mice (50). On the other
hand, mice having Pcyt1a
conditionally deleted in
macrophages have
elevated plasma
homocysteine because of
an increased methylation
of PE, to
compensate for the
dysfunctional
CDP-choline pathway
(49). When fed a
choline-deficient diet,
the PEMT / mouse
dies because of hepatic
depletion of PC. When
fed a regular
diet, the mouse is
normal, because the PEMT
/ liver can
utilize dietary choline
via the CDP-choline
pathway. The
PEMT / animals develop
an anomalous lipoprotein
profile
and a ‘‘fat-liver’’
phenotype when fed a
high-fat/highcholesterol,
‘‘Western-style’’ diet
(51). Surprisingly,
doubleknockout
PEMT / /MDR2 / mice
(MDR2 is a hepatic
Pglycoprotein
that exports PC into
bile) survive on a
cholinedeficient
diet (52). In these
animals, choline is
recycled via
increased PC metabolism
and increased flux
through the
CDP-choline pathway, as
well as by suppression
of choline
oxidation to betaine
(Fig. 1). Therefore, it
becomes apparent
that PEMT / mice die on
a choline-deficient diet
because
these pathways do not
compensate the PC drain
into bile.
Together, these
invaluable studies
strongly demonstrate the
importance of proper
metabolic balances
between choline,
SAM, PE, and PC in liver
function, and make the
pathways
(shown in Fig. 1) for
their regulation
significant risk factors
for development of
‘‘fatty-liver’’
steatosis,
hyperlipoproteinemia,
hyperhomocyteinemia, and
cardiovascular disease.
Importance of Choline
Transport
Separately from choline
metabolism, disturbed
choline
transport has been
proposed to play an
important role in
multiple clinical
manifestations,
particularly in
neurological,
muscular, and
immunological disorders
(53, 54) and in
various cancers,
including breast and
ovarian cancer (55,
56). However, choline
transport has been
studied predominantly
in neurodegenerative
disorders, particularly
in
Alzheimer’s disease.
Alzheimer’s disease is
manifested
with the specific loss
of cholinergic neurons,
and choline
transport is an
attractive target for
drug development to
treat
the disease (57). It is
now clear that choline
is provided to
cholinergic neurons by a
high-affinity transport
system,
which controls the
formation of
acetylcholine (57) and
as
such is of the greatest
interest in this
disorder (58). In
addition, a low-affinity
choline transport system
unrelated to
cholinergic transport
has often been irregular
in the
erythrocytes of
Alzheimer’s disease
patients (59). A reduced
choline level in the
cerebrospinal fluid of
Parkinson’s
disease and Huntington’s
disease patients is a
result not only
of impaired choline
transport but also of a
modified
phospholipid output (60,
61). A lower
phospholipid output
could result in membrane
damage, in addition to
the
disturbances in
neurotransmitter
production that are very
common in neurological
disorders. In
Friedreich’s ataxia,
the PC molecules
incorporated into the
membrane of certain
cells are abnormal in
that they contain a low
percentage of
linoleic acid (62).
Emerging new data
(discussed below)
suggest that choline
carriers that
participate in PC
metabolism, not only in
neuronal cells but in
most cells,
are distinct from
cholinergic carriers.
These cells typically
have a medium to low
affinity for choline and
could be
choline-specific,
polyspecific for various
organic cations, or
both. Therefore, besides
the independent
regulation of
intracellular choline
utilization for PC
production in the
Kennedy pathway and in
liver PE methylation,
complex
transport systems for
choline to enter and/or
exit specialized
cells are required in
order to adjust free
choline with specific
cellular demands for
choline, which, when
disturbed, could
have serious
implications for
membrane fluidity and
function in all tissues.
Systems for Choline
Transport
Choline is a positively
charged quaternary amine
and
requires a
protein-mediated
mechanism to effectively
pass
the membrane lipid
barrier. The mechanisms
underlying
choline transport have
not been completely
elucidated;
however, three
protein-mediated and
thus saturable uptake
systems, following
Michaelis-Menten
kinetics, are well
documented (63–68). The
first mechanism is the
facilitated
diffusion that has an
apparent Km of 10 lM of
choline,
driven by a choline
concentration gradient
as described in
red blood cells (64).
The second is a
high-affinity, Naž- and
energy-dependent
transport system with a
Km of 0.5–3 lM
that is sensitive to
inhibition by low
concentration of
hemicholinium-3 (HC-3,
Ki of 1–3 lM; Ref. 65).
This
‘‘active transport’’
system is coupled to the
biosynthesis of
acetylcholine and is
primary to neuronal
tissues (65). The
third, also ‘‘active
transport,’’ system,
which is somewhat
lower in affinity for
choline (Km & 20–200
lM), is
ubiquitously
distributed, and less
effectively inhibited by
HC-3 (Ki & 20–200 lM)
than the neuronal system
(66–68).
This system operates in
most cells as a means of
choline
uptake for the purpose
of phospholipid
synthesis.
Many organs, such as
lung, brain, placenta,
kidney, and
liver have acquired one
or a combination of
several choline
transport systems, which
allow them to fulfill
specific
functions. The alveoli
of the lungs are lined
with pulmonary
surfactant, which is a
PC phospholipid-protein
complex that
promotes lung stability
(69). The physiological
activity of
the surfactant is
largely attributable to
the unique PC
component of the
surfactant, disaturated
phosphatidylcholine
(69). In all other cells
in the lung, the major
source of
PC is the Kennedy
(CDP-choline) pathway,
which requires
the circulating choline
to be transported into
the lung by the
alveolar epithelium. It
has been suggested that
this transcellular
transport process may be
rate-limiting for the
biosynthesis of
pulmonary PC (70, 71).
In addition, alveolar
type II epithelial cells
have developed two types
of ‘‘active’’
choline transport
mechanisms to regulate
the pulmonary
choline content (68):
The first is a
high-affinity,
Naždependent
system (Km of 1.5 lM; Ki
of 1.7 lM for HC-3)
resembling the neuronal
transport, and the
second is a
Nažindependent
system that has a lower
affinity for choline (Km
of 19 lM; Ki of 12 lM
for HC-3), resembling
the cholinespecific
but nonneuronal
transport. There is also
a third,
very slow residual
transport, which is
presumed to be by
passive diffusion.
The central nervous
system requires choline
for the
synthesis of membrane
phospholipids and for
acetylcholine.
In the brain, choline
that is transported via
the ‘‘highaffinity’’
system is destined for
acetylcholine synthesis,
whereas the
‘‘lower-affinity’’
system supplies choline
for
phospholipid synthesis
(72). The ranges of
affinity for these
systems could vary and
depend on the region of
the brain in
which they are located,
suggesting that a
combination of
choline carriers could
be involved. Because the
brain cannot
synthesize de novo
choline, choline uptake
from extracellular
fluids is essential
(73). Choline enters the
brain from
the systemic circulation
by a saturable transport
at the
blood-brain barrier
(BBB) at a rate
proportional to changes
in the blood choline
concentration, thus
representing a
facilitated diffusion
process (72). Human
keratinocytes
express a
Naž-independent,
polyspecific choline
transport
system similar to the
choline carrier in the
BBB and
intestinal epithelial
cells. This choline
carrier has relevance
both for the
biosynthesis of skin
lipids and for the
uptake of
cationic drugs into the
keratinocytes, as it
does at the BBB
(28).
The importance of
placental choline has
been demonstrated
in studies in which
mouse embryos showed
growth retardation and
developmental defects of
the neural tube and
face during neurulation
when either choline
uptake or
metabolism was inhibited
(7). The placenta
possesses a
choline transport
mechanism, which is
essential because a
growing fetus has to
derive all its metabolic
choline
requirements from its
mother via the placenta
(74). It has
both a passive diffusion
system and an active
carriermediated
process of low affinity
with a Km of 350 lM
choline (75). Passive
diffusion is the least
physiologically
relevant type of choline
transport, considering
positivelycharged
choline molecules and
the blood concentration
of
choline, which is in the
10–50 lM range. More
relevant is
the active choline
transport, as present at
the apical side of
BeWo and JEG-3
epithelial cells of the
human placenta
(76). The uptake is
Naž-independent and
saturable, with a
Km of 108 lM and 206 lM,
respectively. Several
cationic
drugs, including
diphenhydramine,
etilefrine, clonidine,
ranitidine, and
butylscopolamine
interact with this
transport
system and could be
instrumental in the
drug’s transferring
from the maternal blood
to the fetal circulation
(76).
All ingested choline and
free choline generated
by
phospholipid metabolism
enters the hepatic
circulation,
making the liver, where,
as discussed earlier,
there are very
active biochemical
pathways for choline
metabolism, a
significant ‘‘sink’’ for
choline (Fig. 1). The
liver contains a
transport system
resembling the
‘‘low-affinity’’
facilitated
diffusion, but multiple
systems are also
detectable under
different conditions
(77). Studies of rat
liver plasma
membrane vesicles have
demonstrated the
presence of a
basolateral transport
for both choline and
organic cations
(77). It seems that the
murine transporter
organic cation
transporter (OCT) 1 is
responsible for the most
of the
hepatic choline and
cationic drug
circulation (78). At
present, it is unknown
whether a
choline-specific
transport
is operating in the
liver or whether
different systems could
be present at different
membrane sites.
Renal tubular transport
of choline is of
importance
because it maintains the
plasma choline
concentration
within relatively narrow
limits by employing both
net
secretion and
reabsorption (79). When
choline is presented
to the kidney in excess
of a species-specific
threshold
concentration, it will
be excreted from the
kidney into the
urine (80). If the
plasma concentration is
below this level,
choline will be
reabsorbed into the
kidney, not excreted
(80). Membrane transport
of choline is elevated
in renal
failure in erythrocytes
and cerebral tissue, but
the origins
and clinical importance
of that are unknown.
(53). OCT1,
for example, is
localized in the
basolateral membrane of
renal proximal tubules,
in intestinal
enterocytes, and in the
sinusoidal membrane of
hepatocytes, where it
probably
mediates the efflux of
many organic cations,
including
choline. The substrate
specificity of rat OCT1
is for more
hydrophilic (type 1)
cations like tetra-ethyl
ammonium and
choline. Quinine,
quinidine, and cyanine
863, which have
been classified as type
2 cations, are not
eliminated with rat
OCT1 (81). The secretion
(efflux) of organic
cations by
renal proximal tubule is
electrogenic,
facilitated diffusion,
and it is associated
with basolateral uptake
of type 1 organic
cations. These processes
are also believed to
support
electroneutral organic
cation/organic cation
exchange. An
extensive presentation
of renal polyspecific
transport of
organic cations is
described by Wright and
Dantzler (82).
Choline Transporters
Different families of
proteins that have
either been
demonstrated or thought
to play a role in
choline transport
have been isolated in a
variety of species. The
most
prominent are (i) the
high-affinity choline
transporter family
(CHT), (ii) the family
of OCTs, and (iii) the
choline
transporter–like family
(CTL). They exhibit
varying affinities
not only for choline
transport but also for
transport of a
number of other organic
cations (Table 1).
Studies of the
effect of various
organic cation
inhibitors revealed that
hemicholinium-3 is the
most effective inhibitor
of choline
uptake. Although CHT
transporters are mostly
important for
choline transport for
acetylcholine synthesis
rather than for
lipid synthesis, we want
to include a brief
summary of these
high-affinity transport
systems to complete the
picture of
choline transport.
Comprehensive reviews of
CHT are
presented by Ferguson
and Blakely (83) and
Sarter and
Parikh (84).
High Affinity
Choline-Specific
Transporters
Cholinergic
neurotransmission plays
an important role
in regulation of many
physiological functions
(85–88). It is
well known that basal
forebrain cholinergic
neurons are
involved in learning and
memory processes, and
that
hypoactivity of the
cholinergic system is
responsible for
the cognitive deficit in
Alzheimer’s disease (4).
Naždependent
and HC-3-sensitive
choline transport is
very
active in presynaptic
terminals, and it is
mediated by a
highaffinity
choline transporter.
After many years of
searching,
this transporter, named
CHT1, has recently been
successfully
cloned, first from
Caenorhabditis elegans
(cho-1) and
rat (rCHT1; Ref. 89),
and subsequently from
humans
(hCHT1; Refs. 90, 91),
Limulus (LchCoT; Ref.
92), and
mouse (mCHT1; Ref. 93).
Based on the cDNA
sequence,
there is a high degree
of conservation among
CHT1
proteins, and their
closest structural
similarity is with the
Naž-dependent glucose
transporter SGLT (89).
hCHT1 is
located on chromosome
2q12. The entire gene is
&25 kb
long and contains 9
exons. hCHT1 protein is
made of 580
amino acids with a
predicted molecular mass
of 63 kDa and
could form 13
transmembrane domains
(90, 91, 94). CHT1
mRNA expression is
restricted to the brain
regions rich in
cholinergic neurons,
like the forebrain,
striatum, brain stem,
and spinal cord (89–93).
Recently, however, CHT1
transcripts
have been also detected
in nonneuronal cells,
but the
function of CHT1 outside
of neurons is not known
(95–97).
Now it is clear that
CHT1 provides choline
for the
synthesis of
acetylcholine in all
cholinergic neurons (92–
94). Functional
expression of rat CHT1
in Xenopus oocytes
or COS-7 cells produces
typical high-affinity
choline uptake
that is identical with
the high-affinity
choline uptake in
synaptosomes: (i) Km for
choline of 2 lM, (ii)
inhibition by
CH-3 with Ki of 2–5 nM,
and (iii) Naž and Cl ion
dependence (94). Apart
from these cholinergic
transport
characteristics, the
regulation of CHT
expression is becoming
increasingly important.
It has been shown that
CHT1
could be regulated by
neuronal depolarization
(98–102),
second messengers
(103–105), and acute
drug treatments
(106–108). CHT1 contains
serine and threonine
residues for
regulation of function
and surface expression
by protein
kinase C (PKC; Refs. 92,
94) and protein
phosphatase 1/2A
(PP1/PP2A; Ref. 92). It
seems a very common
phenomenon
that PKC and PP1/PP2A
regulate
neurotransmitter
transporter
proteins via rapid
changes in transporter
surface
expression and
phosphorylation (109).
Recently it was
reported that prostate
apoptosis response-4
(Par-4), a leucine
zipper protein that
plays an important role
in neuronal
dysfunction and cell
death in
neurodegenerative
disorders
such as Alzheimer’s
disease (88, 110–116),
inhibits CHT1-
mediated choline uptake
activity and reduces the
cell surface
expression of CHT1 by
directly interacting
with CHT1 (88).
The importance of the
biological function of
CHT1 has been
further confirmed by the
fact that disruption of
CHT1 in
mice is lethal to the
neonate (117).
Low-Affinity
Polyspecific Organic
Cation
Transporters
The transport of
endogenous organic
cations, clinical
drugs, and exogenous
toxic cations is
generally mediated by
the family of OCTs also
known as the solute
carrier 22
family of proteins
(118). This family is
comprised of five
members, including OCT1,
OCT2, OCT3 (119–126),
OCTN1 (127), and OCTN2
(128), and has been
subject to
ongoing intensive
research since the
initial cloning of OCT1
from a rat kidney
library in 1994 (119).
OCT1 is expressed
in the liver, kidney,
and small intestine in
rodents (119, 121,
129, 130); however, in
humans, it is mainly
expressed in the
liver (120). OCT2 has
substrate specificity
similar to that of
OCT1, but its expression
is limited to the kidney
and
specific regions in the
brain (122, 131). OCT3
is expressed
most abundantly in the
placenta and moderately
in the
intestine, heart, and
brain (Table 1; Ref.
132).
The functional
characterizations from
different species
strongly suggest that
OCT1, OCT2, and OCT3
facilitate
polyspecific cationic
transport (119–121, 128,
131–133).
Regarding their
physiological role in
choline transport,
however, the data are
less extensive and the
information
varies in different
experiments. In
transiently transfected
human embryonic kidney
(HEK) 293 cells, choline
was
transported exclusively
by OCT1, but not by OCT2
(134).
When expressed in
Xenopus oocytes, both
OCT1 and OCT2
could facilitate choline
uptake with low
affinity, that is, Km
of 346 lM for rOCT1, 441
lM for rOCT2, and 102 lM
for
hOCT2 (135). OCT-1 and
OCT2-mediated choline
uptake
could be inhibited by
cimetidine (136) and
larger, more
hydrophobic cations like
the antiarrhythmics
quinine and
quinidine (81, 135,
137). Disprocynium 24
and decynium
22, the most potent
inhibitors recognized so
far, inhibit
OCT2 with Ki values
around 10 nM (134, 138),
and
corticosterone, another
potent inhibitor,
inhibits OCT2 with
Ki values around 200 nM
(137). Disprocynium 24,
decynium 22, and
corticosterone are less
effective with
OCT1 (134, 137). The
major driving force for
choline
transport via OCT1 and
OCT2 is the membrane
potential (135, 139). An
in vivo study indicates
that OCT2 plays an
active role in brain
choline homeostasis and
that choline is
not a substrate for OCT3
(135).
A most recent summary of
the OCT1 and OCT2 family
is shown in Table 1. The
genes of OCT1 and OCT2
have
been mapped on
chromosome 6q26 in human
(138, 140).
Rat and mouse forms,
rOCT1 and mOCT1(Lx1),
have been
localized on chromosome
1q11-q12 and chromosome
17A1,
respectively (121, 141).
The primary structure of
rOCT1
and a tentative model of
its membrane topology
predict 12
transmembrane domains
(TMDs) and three
potential
glycosylation sites
located in the large
hydrophilic loop
between the first and
second transmembrane
domains
(reviewed in reference
133). Some data indicate
the
existence of
substrate-binding
pockets in rOCT1 (142)
and
rOCT2 (143). There are
four potential protein
kinase C
phosphorylation sites on
the large intracellular
loop between
TMD6 and TMD7 (133).
OCT1 knockout mice (OCT
/ mice) are healthy and
fertile (118). OCT /
mice have reduced
hepatic uptake and
intestinal excretion of
various organic cations,
and although
OCT1, OCT2, and OCT3
have overlapping
substrates, the
loss of OCT1 is not
compensated for by
upregulation of
either OCT2 or OCT3.
Interestingly, the
levels of radiolabeled
[14C] choline in the
livers of OCT / mice
were
unaffected compared to
those in the livers of
wild-type
mice, suggesting that an
efficient choline
transport system
other than the OCT type
exists in the liver
(118). A
deficiency in OCT2 and a
combined deficiency in
OCT1
and OCT2 had no obvious
effect on the physiology
of mice
(144, 145), but in
OCT1/2 / mice, renal
secretion of TEA
was completely abolished
(144), which implies
that OCT1
and OCT2 together are
essential for renal
secretion of small
organic cations;
however, their role in
choline transport is
not known (144). OCTN1
and OCTN2 have been
identified in human and
rat (127, 128, 146,
147). However, choline
is
not a substrate of OCTN1
and OCTN2 (128, 147).
Choline-Specific
Transporter-Like
Proteins
The choline
transporter–like
proteins of the CTL
family
are comprised of the
five genes, CTL1-CTL5,
with CTL1
being the main member of
the family (148). In
2000,
O’Regan et al. (149)
first cloned and
characterized Torpedo
marmorata CTL1 (tCTL1)
and rat CTL1 (rCTL1).
Subsequently
the human form was
cloned by Dr.
Stockinger’s
laboratory (hCTL1/CDw92;
Ref. 150), and we have
cloned
the mouse form, mCTL1
(151). It is interesting
that the
tCTL1 was isolated by
complementation of a
yeast strain
defective in choline
transport and that hCTL1
was cloned
from a cDNA expression
library using a
monoclonal
antibody for the human
antigen CDw92. CDw92 has
a role
in immune cell function
as part of an
auto-regulatory
signaling loop that
controls expression and
maintenance of
IL-10 production in
dendritic cells (150).
The function
assigned to CDw92 as a
choline transporter has
been based
on the cDNA sequence
similarity to rCTL1 and
other family
orthologs.
Mouse CTL2 and CTL4 cDNA
have been initially
identified in the
Genbank libraries as
members of this family
(149); however, the
mouse form of CTL1 was
not detected,
suggesting that this
form could be rare or
not present in the
mouse. We searched for
this rare form and were
able to
successfully clone the
mCTL1 cDNA from the
mouse
embryonic fibroblasts,
to fully characterize
the mouse gene
that is localized at
chromosome 4B2. Thus, we
added a new
member to the CTL1
family of proteins that
is a choline
transporter abundantly
expressed in the mouse
skeletal
muscle, the function of
which would be in
transporting
choline for phospholipid
synthesis and/or choline
recycling
at the neuromuscular
junctions. The protein
sequence
alignment of mouse, rat,
and human CTL1 is shown
in
Figure 2. They are all
transmembrane proteins
containing
10 highly conserved
TMDs, possibly with
intracellular Nand
C-termini. Figure 3
shows that mCTL1 protein
contains
three potential
N-glycosylation sites
(N134GSA137,
N179ISC182, N506STN509)
and seven protein kinase
C
phosphorylation sites
(S12SK, T86HR, T151SK,
S209KE,
S268 PK, S513AK,
S628RK), five of which
are distributed
within the N-terminal
domain. Additionally
(not shown),
there is one
cysteine-rich region
(C473ARC476MLKSC
481IC483C484LWC487LEKC491)
and one
cadmium-resistance
transporter domain
between L220 and F341,
covering
the membrane domains 3,
4, and 5.
The hCTL1 gene is
located at chromosome
9q31.2, the
rCTL1 gene at chromosome
5q24, and the mCTL1 gene
at
chromosome 4B2. (Table
1). tCTL1 mRNA is
present
throughout the central
nervous system and along
the electric
nerves (152); mCTL1 RNA
is expressed in testis,
brain,
heart, and skeletal
muscle, but the mCTL1
protein is
abundantly present only
in the skeletal muscle
(Fig. 4; Ref.
151). hCTL1 protein is
expressed as two major
polypeptides
of 50 and 23 kDa in a
variety of tissues,
including brain,
heart, small intestine,
kidney, liver, lung,
skeletal muscle,
pancreas, spleen, ovary,
and testis (Fig. 4; Ref.
153). Rat
rCTL1a mRNA is expressed
in the ileum and colon,
whereas rCTL1b mRNA is
expressed exclusively in
the
brain (148, 149). Both
isoforms of rCTL1 were
recently
functionally expressed
and identified as an
intermediateaffinity,
Naž-independent choline
transport system in N18
neuroblastoma cells
(148) and in astrocytes
(154).
Two different splice
variants have been
identified in rat
(rCTL1a and rCTL1b) and
humans (hCTL1a and
hCTL1b;
Refs. 148, 149, 153).
The hCTL1 gene spans
over 194 kb at
chromosome 9q31.2 and
consists of 17 exons and
16 introns
(Fig. 5). The last two
exons, 16 and 17, are
alternatively
spliced in the two
isoforms hCTL1a and
hCTL1b; hCTL1a
is made of exons 1–16,
whereas hCTL1b uses
exons 1–15
and exon 17, resulting
in distinct 39 UTRs in
both isoforms
(153). The alternative
splicing produces
proteins with
differing C-terminal
peptides, A651SGASSA657
peptide in
hCTL1a and L651KKR654
peptide in CTL1b.
Noteworthy,
KKXX is a short
carboxy-terminal signal
that plays a crucial
role for the
localization in the
endoplasmic reticulum.
The
existence of KKXX motif
for hCTL1b would support
the
identification of the
additional site for
choline transport at
the endoplastic
reticulum (148, 153,
155). Our human tissue
expression profiles
indicate that the hCTL1a
mRNA is
highly expressed in
placenta and brain and
the hCTL1b
mRNA is most abundant in
the liver (153). The
significance
of both hCTL1 isoforms
has yet to be
discovered.
The function of CTL1 as
a choline transporter
has
recently been disputed.
CTL1 was initially
identified to
restore choline
transport in a triple
yeast mutant strain (ctr
ise URA3D), which is
deficient in both
choline transport and
neosynthesis under
selective conditions
(149, 156). Furthermore,
expression of tCTL1 in
Xenopus oocytes
increased
sodium-independent
high-affinity choline
uptake
(156). However, Zufferey
et al. reported that the
loss of
PNS1, a CTL1 homolog in
yeast, does not impair
choline
transport, and that
overexpression of both
PNS1 and tCTL1
in transport-deficient
mutants did not restore
choline uptake
in the yeast strain
(157).
In our laboratory, we
demonstrated a 2.5-fold
increase
in choline transport by
overexpression of mCTL1
in Cos-7
cells (151). We also
identified hCTL1 to be
the predominant
choline transporter in
THP-1 cells and
demonstrated that
choline uptake is high
in monocytic cells and
severely
reduced after PMA
treatments as a result
of diminished
hCTL1 protein
trafficking to the
plasma membrane (Fig.
6).
Our choline transport
studies using
[3H]-choline revealed
that choline uptake in
THP-1 cells was almost
completely
inhibited by HC-3,
whereas OCT-specific
inhibitors had no
effect on choline
transport. The transport
was of intermediate
affinity for choline (Km
¼ 68 6 12 lM),
PMAdependent
and CTL1-related,
because we strongly
excluded OCT and CHT1 as
possible transporters
(for recently
published data, see ref.
158).
Three recent studies
have given further
strong support
to the idea that CTL1 is
in fact a choline
transporter, as
suggested by O’Regan’s
studies on rCTL1 and our
studies
on mCTL1. Inazu et al.
(154) identified a
Naž-independent,
saturable choline
transport system in rat
astrocytes with an
intermediate affinity
for choline (Km¼37.7
lM), which was
likely CTL1.
Interestingly, they
reported an impaired
choline transport with a
decrease in pH, which
may give
an indication of a
possible proton
dependency of CTL1-
mediated choline
transport. Fujita et al.
(159) report a
Naždependent,
saturable choline
transport in mouse
neurons of
the cerebral cortex with
an affinity for choline
of Km¼26.7
lM. The only member of
the OCT group expressed
in these
cells is OCT3, which
does not have choline as
a substrate.
Although CHT1, which is
expressed by these mouse
neurons, likely
accounted for the
Naž-dependent choline
transport, CTL1 is most
likely responsible for
the Nažindependent
choline transport
observed in this study.
Both
of these recent studies
imply that CTL1 could
also play an
important role in the
choline transport of
neuronal cells.
Choline transport by
CTL1 has also recently
been associated
with amyloid-b-peptide
(160), a fragment of
amyloid
precursor protein that
shows an altered
metabolism in
Alzheimer’s disease. A
long-term treatment of
NG108–15
cells with
amyloid-b-peptide
resulted in a
downregulation of
choline uptake by CTL1.
Although CTL1 mRNA
levels did
not change and the
amyloid-b-peptide
therefore did not
affect gene expression,
the reduced choline
uptake may be
caused by changes in
mechanisms downstream of
gene
expression. Importantly,
similarly to our studies
with THP-1
monocyte-macrophages
cells (158), protein
kinase C could
be involved in the
regulation of CTL1,
because inhibition of
PKC increases choline
uptake, whereas after
PKC activation
choline uptake is
impaired. Recently, both
CTL1 and CHT
mRNA expression has been
shown to be upregulated
by
leukemia inhibitor
factor, which further
supports a regulation
of CTL1 expression by
PKC (161). Therefore,
these
data suggest that CTL1
may be involved in the
pathogenesis
of Alzheimer’s disease,
which further underlines
its
importance in the human
metabolism.
Interestingly, we also
demonstrated that hCTL1
protein
is overexpressed in the
skeletal muscle of
muscular
dystrophy patients
(153). The CTL1 promoter
contains a
binding site for the
MyoD transcription
factor, which
typically regulates
muscle myogenesis by
maintaining fast
muscle fiber phenotypes
as well as other
regulatory
functions (162). The
remarkable regenerative
capacity of
skeletal muscle depends
largely on the number of
available
satellite cells and
their proliferative
capacity (163). An
increased macrophage
accumulation can also be
observed in
the diseased or injured
muscle, with the extent
of macrophage
accumulation correlating
with protein clearance
following injury and the
efficiency of
regeneration (164).
The increase of hCTL1
protein in muscular
dystrophy is a
sign that the hCTL1 gene
is responsive to
processes
common to degenerated
muscle, and it could
represent a
target for improving
muscle function.
Conclusions and
Prospects
With the identification
of three kinds of
choline
transport
systems—facilitated
diffusion (64),
low-affinity
(68), and high-affinity
(65)—and the
identification of three
families of choline
transport proteins—CHT,
OCT, and
CTL—it is now possible
to further elucidate the
role of
choline transport in
health and disease, and
to identify
possible practical
applications in the
treatment of disorders
associated with abnormal
choline metabolism. It
is crucial to
further investigate the
regulation of choline
transporters at
the protein and RNA
level and to clarify the
role that protein
kinase C may play,
considering the fact
that all choline
transporters contain
highly conserved
potential PKC
phosphorylation sites.
The identification of
choline binding
sites and the precise
mechanism of choline
uptake of the
different transporters,
as well as the
crystallization and
identification of their
tertiary structures,
will present a
further challenge in the
future.
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