Cardiovascular Research 33 Ž1997. 272–283
Review
The effect of membrane cholesterol content on ion transport processes in
plasma membranes
E.M. Lars Bastiaanse, Karin M. Hold,
¨ Arnoud Van der Laarse
)
Department of Cardiology, UniÕersity Hospital, Building 1, C5-P24, Rijnsburgerweg 10, 2333 AA Leiden, Netherlands
Received 10 May 1996; accepted 3 September 1996
Abstract
Cholesterol is a prominent component of mammalian plasma membranes and is one of the factors that determine membrane function.
In this review the effects of cholesterol content on transport processes in biological membranes are summarized. Membrane cholesterol
affects a variety of membrane proteins, including ion channels, transporters, and receptors. Present concepts concerning the mechanistic
basis of lipid-induced modulation of transport protein function range between two extremes: modulation by bulk properties, or by specific
interactions. Interest in bulk properties has been focussed mainly on membrane fluidity. The fluidity of biomembranes is diminished
particularly by enrichment with cholesterol. As a change in membrane composition alters the environment in which the proteins are
dissolved, any process which depends on membrane protein function may be affected by alterations in membrane composition, such as a
change in cholesterol content. This review emphasizes the inhibitory effect of cholesterol enrichment on all membrane ATPases studied,
and the stimulating effect of cholesterol enrichment on most other membrane transport proteins. Together with the intriguing feature that
the cholesterol content of plasma membranes is considerably higher than that of subcellular membranes, there is ample evidence for a
significant role of plasma membrane cholesterol in transmembrane protein function.
Keywords: Cholesterol; Sarcolemma; Ion transport; Ion channels
1. Introduction
Epidemiological studies have indicated that elevated
plasma levels of cholesterol are associated with an increased risk of coronary heart disease w1,2x. However,
cholesterol is an essential component of biological membranes and a precursor in the synthesis of steroid hormones
and bile acids w3–5x. This review focusses on the multiple
roles of cholesterol with regard to membrane function.
All biological membranes, including the plasma membrane, have a common basic structure w3,6x consisting of
assemblies of lipid and protein molecules held together by
non-covalent interactions. The most abundant lipids in
plasma membranes are phospholipids and cholesterol. The
cholesterol molecule is mostly hydrophobic, whereas the
3b-hydroxyl group is polar. This amphipathic character
causes cholesterol to be surface-active, allowing interaction with phospholipid molecules by cholesterol’s polar
)
Corresponding author. Tel. q31 715263704; Fax q31 715226567.
hydroxyl group to the polar head of the phospholipid,
while the hydrophobic steroid ring is oriented parallel to,
and buried in, the hydrocarbon chains of the phospholipid
bilayer w3,6x. This lipid bilayer provides the basic structure
of the membrane and serves as a relatively impermeable
barrier to most water-soluble molecules.
Cells maintain their cholesterol homeostasis Žsee Fig. 1.
through a regulated balance between endogenous biosynthesis, receptor-mediated uptake of cholesterol from circulating lipoproteins, such as low-density lipoprotein ŽLDL.,
and cellular release of cholesterol to circulating lipoproteins, such as high-density lipoprotein ŽHDL. w3,4,7x.
Cholesterol is synthesized from small carbon units via a
series of reactions that take place in the cytosol and in the
membranes of the endoplasmic reticulum w8x. Generally,
the activity of the b-hydroxy-b-methylglutaryl-coenzyme
A ŽHMG-CoA. reductase reaction is the rate-limiting step
in de noÕo cholesterol synthesis. The activity of HMG-CoA
Time for primary review 21 days
0008-6363r97r$17.00 Copyright q 1997 Elsevier Science B.V. All rights reserved.
PII S 0 0 0 8 - 6 3 6 3 Ž 9 6 . 0 0 1 9 3 - 9
E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
Fig. 1. Schematic representation of cholesterol supply, intracellular
metabolic pathways, and cholesterol removal from the cell. 1 s apo B,E
ŽLDL. receptor-mediated uptake of cholesterol-rich LDL; 2 s hydrolysis
of LDL in the lysosomes leading to the formation of cholesteryl esters
ŽCE. and free cholesterol ŽCHS.; 3 s de novo synthesis of cholesterol; 4
s esterificationrhydrolysis cycle of cholesterol; 5 s translocation of
cholesterol to the membrane; 6 s HDL-mediated ‘reverse’ cholesterol
transport.
reductase is regulated at several points including transcription and translation, phosphorylation, activation of
sulfhydryl groups, and protein degradation. Cellular
cholesterol content is the major factor in the regulation of
HMG-CoA reductase activity. By negative feedback control, HMG-CoA reductase activity is repressed when cellular cholesterol content exceeds the demand w8,9x.
It has become increasingly evident that cholesterol is
not randomly distributed in either artificial or biological
membranes, but is organized into structural and kinetic
domains or pools w5,10x. The physiological importance of
these domains is poorly understood. It is suggested that
several membrane proteins are selectively localized in
cholesterol-rich domains Že.g., the acetylcholine receptor.
or in cholesterol-poor domains Že.g., the sarcoplasmic
reticulum Ca2q-ATPase.. Consequently the structure and
properties of the different domains rather than of the bulk
lipid may selectively affect the function of membrane
proteins located in the different domains.
Another intriguing observation is that the cholesterol
content of plasma membranes is much higher than that of
subcellular membranes. The way in which this difference
is maintained still presents an enigma w5,11x, but it could
be indicative of a distinctive role of cholesterol in plasma
membranes, necessary to communicate between intracellular and extracellular spaces.
An important characteristic of the membrane is its fluid
character. Since the introduction of the fluid mosaic model
w12x, attempts have been made to quantify membrane lipid
fluidity and to relate membrane fluidity to other membrane
characteristics. The fluorescent probe, 1,6-diphenyl-1,3,5-
273
hexatriene ŽDPH. w13x has been used most frequently, but
nowadays a wealth of other probes and methods, including
electron spin resonance ŽESR. and nuclear magnetic resonance ŽNMR. w6x, is available to sense the fluidity of
membranes. For an ordinary liquid such as water or oil,
fluidity is defined as the reciprocal of viscosity w14x.
However, the lipid bilayer differs from a macroscopic fluid
in several aspects. It is a fluid with stringent boundary
conditions, essentially two-dimensional in nature, and
anisotropic. Moreover, the viscoelastic properties of the
membrane cannot easily be separated from those of the
whole cell and its microscopic environment w13x.
As applied to membranes, the term ‘fluidity’ is used to
describe the motional freedom of lipid-soluble molecular
probes within a lipid bilayer w13,14x. Besides temperature,
fatty acid composition, and protein content, the fluidity of
a membrane is determined by its cholesterol content w15x.
It has been demonstrated that the higher the cholesterol
content in membranes, the lower its fluidity, and vice versa
w16–19x.
Membranes do not only confine compartments, they
also determine the nature of all communication between
the interior and exterior of the cell. This communication
includes transport of ions or molecules across the membrane, and is accomplished by the action of specific transport proteins. In addition, receptors and enzymes are localized in or on membranes, and are involved in mediating
the communication between the interior and exterior of the
cell.
Changes in the molar ratio of cholesterolrphospholipid
of cell membranes induced by a change in cholesterol
content affect a number of important membrane properties,
including permeability, transport functions, membrane enzyme activities, the availability of membrane components
as substrates, conformation of membrane proteins, and
exposure of proteins w5,20,21x. These alterations are mediated either by the change in membrane cholesterol content
itself or by a concomitant change in membrane fluidity.
This review deals with the influence of cholesterol on
plasma membrane transporters. We have confined ourselves mainly, but not exclusively, to ion transporters
located in the plasma membrane of myocytes. Proposed
mechanisms by which cholesterol may affect the different
transporters are presented.
2. Calcium
The calcium ion acts both as an electrical and chemical
signal. This dual role is facilitated by its high transmembrane gradient ŽwCa2q xerwCa2q x i f 10 000.. Alterations in
the cytosolic free calcium concentration triggered by
chemical or electrical signals regulate many intracellular
processes such as muscle contraction, activation of enzymes, and membrane ionic conductance w22–24x.
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E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
Intracellular free calcium concentration can be increased by: Ži. increased Ca2q influx through transmembrane channels; Žii. decreased NaqrCa2q exchange activity in the direct mode Ži.e., Ca2q efflux.; Žiii. decreased
sarcolemmal ŽCa2q,Mg 2q .-ATPase activity; Živ. release of
Ca2q from internal stores, andror diminished calcium
uptake capacity of these stores; or Žv. any combination of
the previous items Ži to iv.. In cardiac and smooth muscles
the main subcellular structures capable of accumulating
calcium are the sarcoplasmic reticulum ŽSR. and the mitochondria. Calcium efflux is accomplished by sarcolemmal
ŽCa2q,Mg 2q .-ATPase and by NaqrCa2q exchange. Like
the sarcolemma, the SR membrane contains a
ŽCa2q,Mg 2q .-ATPase which is responsible for calcium
uptake in the SR.
2.1. Ca 2 q channel
Several studies have demonstrated that an increase in
cholesterol content of plasma membranes leads to increased Ca2q flux through the Ca2q channel in plasma
membranes w25–28x.
Enrichment of arterial smooth muscle cell membranes
with cholesterol was accompanied by a selective increase
in sensitivity to the vasoconstrictor action of norepinephrine, but not to vasoconstriction mediated by histamine,
serotonin or KCl w25,26x. Since this increase in sensitivity
was reversed by verapamil, Tulenko et al. w27x concluded
that excess membrane cholesterol in arterial smooth muscle cells increased Ca2q influx through the Ca2q channel
under norepinephrine stimulation. Gleason et al. w28x and
Bialecki et al. w29x investigated the relation between membrane cholesterol content, 45 Ca2q movements across the
plasma membrane, intracellular Ca2q concentration
ŽwCa2q x i . and membrane fluidity in smooth muscle cells
and isolated plasma membranes. Enrichment of vascular
smooth muscle cells with cholesterol Žleading to an increase in free cholesterol content of 52%. is associated
with an increase in wCa2q x i of about 34% w28x. If the
procedure to enrich vascular smooth muscle cells with
cholesterol was performed in the presence of a calcium
antagonist Žeither nifedipine, verapamil, or diltiazem., the
rise in wCa2q x i was attenuated by 25–50%, whereas the
calcium antagonist alone slightly reduced wCa2q x i by 6–
13%. Thus, Gleason et al. w28x hypothesized that
‘‘cholesterol enrichment alters lipid dynamics in the arterial smooth muscle plasma membrane and results in the
appearance of a new, or an unmasking of an otherwise
silent, dihydropyridine-sensitive calcium channel.’’ This is
consistent with the findings of Strickberger et al. w30x who
reported that in cholesterol-fed rabbits the intracellular
calcium content of aortic segments is increased 4–8-fold
compared to that of aortic segments of rabbits fed a normal
chow.
Similar results Ži.e., an increased cholesterol content of
the plasma membrane is associated with an increased
wCa2q x i , and vice versa. were also found in smooth muscle
Fig. 2. Change in the fluorescence ratio ŽF340 rF380 . of fura-2, reflecting
wCa2q x i concentration, due to sarcolemmal cholesterol depletion by treatment with cholesterol-free liposomes ŽFCrPL s 0., or due to sarcolemmal cholesterol enrichment by treatment with cholesterol-rich liposomes
ŽFCrPL s 2., compared to control Žcultures treated with solute.. Due to
the wide variability of the starting ratio values Ž1.08"0.45; mean"s.d..,
the data are presented as percentage changes in the ratio. ) P - 0.05 vs.
control. From w32x Žwith permission..
cells of bovine arteries w31x, rat heart myocytes w32x,
platelets w33x, monocytes w34x, and erythrocytes w35x. The
study performed with erythrocytes showed that Ca2q influx through the Ca2q channel was increased by 100% if
the membrane cholesterol content was increased by 80%,
and was reduced by 47% if the membrane cholesterol
content was reduced by 45% w35x. If cultured neonatal rat
cardiomyocytes were incubated for 5 h with cholesterol-rich
or cholesterol-free liposomes having a molar free cholesterolrphospholipid ratio of 2 and 0, respectively, the molar
free cholesterolrphospholipid ratio of the cells changed
from a baseline value of 0.328 " 0.017 to 0.538 " 0.081
Žwith cholesterol-rich liposomes. and to 0.257 " 0.040
Žwith cholesterol-free liposomes. w32x. Subsequent determination of the fluorescence steady-state anisotropy with the
probe TMA-DPH Ž1-Ž4-trimethylammonium-phenyl.-61,3,5-hexatriene. in cholesterol-loaded and in cholesteroldepleted cardiomyocytes gave values 8.0% higher and
4.2% lower Žcorresponding to diminished and augmented
membrane fluidity, respectively. than in untreated cardiomyocytes, respectively. Enrichment of the sarcolemma
of cultured cardiomyocytes with cholesterol was associated
with a raised wCa2q x i , and cholesterol depletion of the
sarcolemma was associated with diminished wCa2q x i ŽFig.
2.. Since the rise in wCa2q x i induced by cholesterol loading
of the cells was abolished by the presence of verapamil
throughout and after the incubation of the cells with
cholesterol-rich liposomes, Bastiaanse et al. argued that
cholesterol enrichment of cardiomyocytes causes activation of Ca2q channels either by opening more channels per
myocyte or by inducing a higher conductance per channel
w32x. Renaud et al. w36x demonstrated that if the cholesterol
content of chicken embryonic ventricular cells in culture
was reduced by mevinolin, a HMG-CoA reductase in-
E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
hibitor, spontaneous electrical activity and contractions
were suppressed. The number of tetrodotoxin binding sites
Žrepresentative of the number of Naq channels. was unchanged as was the number of nitrendipine binding sites
Žrepresentative of the number of L-type Ca2q channels..
However, treatment of the cells with mevinolin did not
alter the activation characteristics of the Naq channels, but
completely abolished the activation of the Ca2q channels
normally induced by high Kq, isoproterenol or Bay K8644.
2.2. (Ca 2 q,Mg 2 q )-ATPase in sarcolemma and sarcoplasmic reticulum
O rtega and M as-O liva w 37 x m easured the
ŽCa2q,Mg 2q .-ATPase activity of a sarcolemmal preparation isolated from hearts of New Zealand white rabbits.
Cholesterol incorporation into the sarcolemma was
achieved by in vivo and in vitro procedures. Cholesteroldepleted membranes were obtained in vitro after incubation of the sarcolemma preparation with inactivated plasma.
The ŽCa2q,Mg 2q .-ATPase was inhibited by an increase of
the sarcolemmal cholesterol content, whereas the withdrawal of small amounts of cholesterol from the membranes had a considerable stimulatory effect. Changes in
ŽCa2q,Mg 2q .-ATPase activity due to an altered membrane
cholesterol content were fully reversible w37x.
Ortega and Mas-Oliva w38x and Mas-Oliva and Santiago-Garcia w39x suggested that cholesterol interacts with
the sarcolemmal calcium pump in a direct manner, thereby
inhibiting its enzymatic activity. They demonstrated that
this inhibition could be, at least in part, explained by a
complete loss of the pump’s sensitivity to calmodulin w38x.
It is possible that by interaction with the boundary lipid of
ATPase, cholesterol might alter the amount of water associated with the enzyme in such a way that the intermolecular hydrogen bonds of the protein are altered, leading to a
modification of the protein structure w39x. Since the K m of
sarcolemmal ŽCa2q,Mg 2q .-ATPase for ATP in different
membrane preparations is independent of the sarcolemmal
cholesterol content, it is likely that specific regions of the
enzyme, such as the low-affinity site for ATP, are not
affected by the inclusion of cholesterol in the membrane
w38,39x. The reversible modification of the enzyme caused
by cholesterol impairs the catalytic activity of the ATPase
Ži.e., reduces the Vmax ., but—at the same time—renders
the ATPase resistant to thermal inactivation w39x. The
effects might be mediated by a more general mechanism
probably related to the overall change in the tertiary
structure of the ATPase. Apparently, a restriction in the
freedom of movement of the ATPase interferes with conformational changes occurring during the catalytic cycle,
and moreover renders the enzyme less susceptible to thermal inactivation. Likewise, the lower wCa2q x i in cholesterol-depleted cardiomyocytes described by Bastiaanse et
al. w32x was ascribed by them to the activation of sarcolemmal Ca2q-ATPase.
The sarcoplasmic reticulum ŽSR. is a useful membrane
275
system to investigate the influence of cholesterol on intrinsic membrane proteins. ŽCa2q,Mg 2q .-ATPase is the major
intrinsic protein of the membrane and is responsible for the
active transport of calcium ions into the SR. Two calcium
ions are taken up into purified SR vesicles for each ATP
molecule hydrolyzed w40x.
Warren et al. w41x have proposed a general theory
concerning the interaction of membrane lipids with the
intrinsic membrane protein ŽCa2q,Mg 2q .-ATPase of the
SR and the biochemical consequences of this interaction.
The theory postulates that about 30 phospholipid molecules
interact directly with the hydrophobic surface of the ATPase spanning the bilayer, and form the minimal lipid
environment required to maintain full ATPase activity of
the protein. Cholesterol is considered to be excluded from
the phospholipid annulus Ži.e., the direct environment of
the protein in the SR membrane., which is relatively poor
in cholesterol. This would suggest that cholesterol is unable to modulate the enzyme activity in the SR. Moreover,
it has been suggested that the annulus protects the enzyme
from changes in bulk membrane fluidity caused by the
incorporation of cholesterol into the membrane. To force
cholesterol to interact directly with the ATPase, it was
necessary to use high concentrations of the ionic detergent,
cholate, which strips phospholipid from the annulus. The
activity of the ŽCa2q,Mg 2q .-ATPase is unaffected by the
presence of cholesterol if 30 or more phospholipid
molecules per ATPase molecule are preserved in the annulus. Below this value, cholesterol inhibits ATPase activity
by replacing phospholipid in the annulus, and equimolar
amounts of cholesterol and phospholipid in the annulus
completely and reversibly inhibit ATPase activity w41x. In
vivo, however, the phospholipid annulus of SR Ca2qATPase normally excludes cholesterol, rendering the enzyme relatively insensitive to changes in SR cholesterol
content.
Madden et al. w40x examined the biochemical evidence
which supports the annulus hypothesis. They found that
increasing amounts of cholesterol caused a proportional
decrease in enzyme activity. This result is in contrast with
the observation reported by Warren et al. w41x. Apart from
the method of reconstituting the enzyme system with
cholesterol, another major difference between the studies
of Warren et al. w41x and Madden et al. w40x concerns the
method of membrane preparation. Madden et al. w40x
showed that in vesicles of SR with or without supplemented cholesterol prepared in the presence of a protease
inhibitor Žphenylmethyl-sulphonylfluoride. and dithiothreitol, the ATPase is in a tightly coupled state in that
enzyme activity is geared to the transport of calcium into
the vesicles. The effect of membrane cholesterol on enzyme activity in these preparations is obscured by the fact
that the rate-limiting step of the process is likely to be the
efflux of Ca2q from the vesicles. Collapsing the Ca2q
gradient in coupled preparations with a calcium ionophore
allows an expression of maximal catalytic action and
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E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
unmasks the inhibitory effect of cholesterol on the
ŽCa2q,Mg 2q .-ATPase. The results of Johansson et al. w42x
confirm the findings of Warren et al. w41x. Johansson et al.
w42x showed that under conditions in which the ATPase
activity in SR vesicles is fully uncoupled from Ca2q
movements or in which the ATPase activity is coupled to
calcium accumulation, cholesterol had no effect on ATPase activity.
Simmonds et al. w43x used a fluorescence quenching
method to measure relative binding constants of hydrophobic compounds such as cholesterol and phospholipids to
the phospholipidrprotein interface. Cholesterol is capable
of displacing only negligible quantities of phospholipid
from the annulus around the ATPase, as the relative binding constant of the ATPase for cholesterol compared to
that for phosphatidylcholine is 0.1 or less. These authors
supported the concept that phospholipid molecules interact
with this transmembrane protein more effectively than
does cholesterol. Thus, while the sarcolemmal
ŽCa2q,Mg 2q .-ATPase activity is inhibited by sarcolemmal
cholesterol enrichment Žand vice versa. w37–39x, the
ŽCa2q,Mg 2q .-ATPase of sarcoplasmic reticulum appears
to be insensitive to changes in membrane cholesterol content w41–43x, likely due to the inability of cholesterol
molecules to become incorporated into the annulus around
the ATPase molecule.
2.3. Na qr Ca 2 q exchange
By the process of NaqrCa2q exchange calcium ions
run in either direction across the membrane depending on
the transmembrane Naq and Ca2q gradients and on the
membrane potential. NaqrCa2q exchange is electrogenic;
the stoichiometry of the sarcolemmal exchange is three
Naq ions per Ca2q ion w44x.
Kutryk and Pierce w45x demonstrated that incorporation
of cholesterol into isolated cardiac sarcolemmal vesicles
resulted in a significant stimulation of NaqrCa2q exchange by up to 48% over control values. This stimulation
was specific to NaqrCa2q exchange as the activities of
ŽCa2q,Mg 2q .-ATPase and ŽNaq,Kq .-ATPase were depressed by cholesterol enrichment w45x. The observation
that oxidation of membrane cholesterol by treatment of
cardiac sarcolemmal vesicles with cholesterol oxidase
completely eliminated NaqrCa2q exchange activity suggests that cholesterol is intimately associated with the
NaqrCa2q exchange protein w45,46x.
These results indicate that the NaqrCa2q exchange
protein is surrounded by a cholesterol-rich annulus which
modulates its activity.
3. Potassium and sodium
In this section the ŽNaq,Kq .-ATPase and the Ca2q-dependent Kq channel, both involved in the maintenance of
transmembrane Naq and Kq gradients, are discussed.
3.1. (Na q,K q )-ATPase
The ŽNaq,Kq .-ATPase is responsible for the energy-dependent transmembrane transport of Naq and Kq ions.
When energized by intracellular ATP, the pump transports
two Kq ions inwardly and three Naq ions outwardly w47x,
leading to hyperpolarization.
Claret et al. w48x have shown that a 20–25% depletion
in cholesterol of erythrocyte membranes caused substantial
modifications of the kinetic parameters of the ŽNaq,Kq .ATPase: the apparent affinity for internal Naq was decreased and the maximal translocation rate was increased,
whereas the apparent affinity for external Kq remained
unchanged. It is not known whether these effects are a
reflection of a direct interaction of cholesterol molecules
or are mediated indirectly by a change in the packing of
phospholipids Ži.e., a change in membrane fluidity.. The
observation by Giraud et al. w47x that the Naq and Kq sites
of the ŽNaq,Kq .-ATPase of erythrocyte membranes were
affected differently by the lipid order of the leaflet in
which the cation sites were located, suggests that the Naq
and Kq sites are located in different lipid environments.
In membranes of human erythrocytes w49x, guinea pig
erythrocytes w50x, rat liver cells w51x, rabbit kidney outer
medulla cells w52x and bovine kidney basolateral cells w53x,
a supranormal cholesterol content Žby 50–100% higher
than normal values. inhibits the ŽNaq,Kq .-ATPase activity
by 30–70%. This inhibitory effect of cholesterol is likely
caused by a diminished molecular motion or conformational freedom of the enzyme in the bilayer, limited by the
increase in molecular order Ž‘freezing’. of the phospholipid acyl chains in the membrane.
3.2. Ca 2 q-dependent K q-channel
The Ca2q-dependent Kq channel opens in response to a
raised wCa2q x i . Patch-clamp techniques and fluorescence
polarization analysis with DPH were used to study whether
Ca2q-dependent Kq channel kinetics and membrane fluidity depended on the cholesterol content of the plasma
membranes of cultured rabbit aortic smooth muscle cells
w54x. Mevinolin, a potent inhibitor of endogenous cholesterol biosynthesis by inhibition of HMG-CoA reductase,
was used to deplete the cholesterol content of the plasma
membrane. Treatment of smooth muscle cells with mevinolin led to an increase in membrane fluidity Žcorresponding to a decrease in fluorescence anisotropy of 31%. and
to a ninefold increase in the probability of the Ca2q-dependent Kq channels being open Ž Po ., suggesting a modulatory role of membrane composition and lipid dynamics on
the function of this channel. Direct evidence for the role of
cholesterol was obtained in experiments with cholesterol
enrichment of cell membranes and isolated membrane
patches w54x. After the membranes had been enriched with
cholesterol, membrane fluidity was decreased Žcorresponding to an increase in fluorescence anisotropy of 36%. and
E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
Po was decreased by 50%. Thus, the conductance of the
Ca2q-dependent Kq channel is inversely related to the
membrane cholesterol content.
A direct interaction of cholesterol with the ion channel
is unlikely since cholesterol was shown to be absent in the
annulus and located mainly in the bulk w5,15x. Several
interventions designed to vary membrane fluidity were
reported to affect ion channel function in general and
Ca2q-dependent Kq channel function in particular.
Bregestovski et al. w55x described elevated activity of the
Ca2q-dependent Kq channel from human aorta by increasing the lateral motion of membrane lipids using 2-decenoic
acid. Thus, the conductance of the Ca2q-dependent Kq
channel appears to be sensitive to membrane fluidity.
4. Miscellaneous
4.1. Gap junctions
A gap junction is a special kind of solute transporter
which mediates intercellular communications. The gap
junction channels permit the exchange of ions and of small
hydrophilic molecules of up to 1000 Da. By providing a
low-resistance pathway for conduction of the action potential, gap junctions serve to synchronize electrical, and
thereby contractile, events in the heart w56,57x.
Gap junctions are localized in plasma membrane domains rich in cholesterol Ži.e., the lipid environment of the
gap junctions has a higher cholesterol content than the
extrajunctional plasma membrane.. This phenomenon may
indicate that cholesterol plays a role in gap junction assembly, structure and function w58x.
The effect of cholesterol on gap junction assembly and
function was studied in Novikoff hepatoma cells by incubating the cells in medium containing serum supplemented
with cholesterol w58–60x. Depending on the cholesterol
concentration, gap junction assembly and permeability were
either stimulated or inhibited. If cellular cholesterol content was increased by about 50% of control levels, maximal stimulation was observed: a seven-fold increase in the
number of aggregated gap junction particles, and a threefold increase in gap junction plaque area. The rate of
Lucifer yellow transfer between cells was increased. At
higher cholesterol content, gap junctional formation was
negatively affected, or was even lower than in control cells
w59x.
In human smooth muscle cells, cholesterol supplementation resulted in an increase in gap junctional communication to levels of 130% compared to control values w61x.
In a study on gap junctional conductance of neonatal rat
cardiomyocytes, different fluorescent probes were used to
assess the fluidity of the various domains: TMA-DPH for
bulk fluidity and DHE Ždehydroergosterol. to assess the
fluidity of cholesterol-rich domains w62x. These probes
allow differentiation between effects of lipophilic sub-
277
stances on bulk fluidity and on the fluidity of cholesterolrich domains. The uncoupling action of 1-heptanol Ž2 mM.
Ži.e., the decrease of gap junctional current to zero. was
associated with increased bulk membrane fluidity and decreased fluidity of the cholesterol-rich domains in which
the gap junctions reside. Thus, gap junctional conductance
of neonatal rat cardiomyocytes is affected by the fluidity
of cholesterol-rich domains, rather than by fluidity of the
bulk lipids w62x.
4.2. Receptors
Several receptors can be recognized as transporters of
ions Ži.e., upon stimulation they transport ions across the
membrane.. Barnett et al. w63x used chick atrial myocytes
cultured in medium supplemented with either 6% fetal calf
serum ŽFCS. which represents control conditions, 6%
lipoprotein-deficient serum ŽLPDS. to stimulate cellular
cholesterol synthesis, or 6% LPDSq 30 mM mevinolin to
inhibit cellular cholesterol synthesis. Under control conditions Žin mediumq FCS. isoproterenol stimulated adenylate cyclase activity by 128% over basal. The number of
b-adrenergic receptors assayed by w 125 Ixpindolol binding
was 24 fmolrmg protein. If incubated in medium with
LPDS, isoproterenol-stimulated adenylate cyclase activity
was only 35% over basal, and the number of b-adrenergic
receptors was reduced by 50% to 12 fmolrmg protein.
Blockade of cholesterol synthesis by addition of mevinolin
restored the isoproterenol-stimulated adenylate cyclase activity to a value of 118% over basal, in combination with a
number of b-adrenergic receptors of 28 fmolrmg protein.
Moreover, atrial myocytes incubated in medium with LPDS
had lower Žby 70%. levels of a s-subunits of the stimulatory guanine nucleotide binding protein ŽGs ., which couples the receptor–agonist interaction to adenylate cyclase
stimulation, compared to cells incubated in medium with
FCS. Blockade of cellular cholesterol synthesis partially
reversed the effects of LPDS on the levels of a s-subunits,
and increased the relative amounts of a s-subunits to 73%
of that in FCS. Thus, stimulation of cellular cholesterol
synthesis blunted the responsiveness of atrial myocytes to
b-adrenergic stimulation. However, the responsiveness of
muscarinic receptors was increased in these cells under
conditions of stimulated cellular cholesterol synthesis w64x.
Increased cholesterol synthesis of the cells was associated
with a two-fold increase in the number of muscarinic
receptors which bind agonist with high affinity, and a
two-fold increase in the levels of the a-subunits of Go and
Gi . Addition of mevinolin was associated with a reduction
in the number of muscarinic receptors to a value equal to
that observed under control conditions Žmedium with FCS;
180 fmolrmg protein..
In view of these results, the findings of Dalziel et al.
w65x and Criado et al. w66x are worth mentioning here.
Dalziel et al. w65x demonstrated that in reconstituted vesicles of electroplax tissue from Torpedo californica choles-
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E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
terol was necessary to maintain Naq flux through the
acetylcholine receptor channel. In these experiments
cholesterol appeared to have no effect on the ligand binding properties of the acetylcholine receptor, nor on its
ability to undergo affinity transitions Ži.e., a shift from a
low-affinity to a high-affinity binding state for the agonist..
They suggested that cholesterol may be involved in modulating the ion channel protein itself without affecting the
agonist-binding properties of the receptor. However, in a
study on the acetylcholine receptor isolated from the electroplax tissue of Torpedo marmorata and incorporated into
vesicles of pure synthetic lipids, Criado et al. w66x found
that exogenous cholesterol is essential for maintenance of
agonist-induced state transitions of the acetylcholine receptor in lipid vesicles.
5. Integrative view
Present concepts concerning the mechanisms of lipidinduced modulation of transport protein function range
between two extremes: modulation by bulk properties, or
by specific interactions w14x. Interest in bulk properties has
been focused mainly on membrane fluidity. As mentioned
in Section 1, the fluidity of biomembranes is diminished
particularly by cholesterol enrichment. Little is known
about the nature and specificity of interactions of the
cholesterol molecule with specific protein sites.
Table 1 presents an overview of the effects of cholesterol enrichment and cholesterol depletion on several transporters in the membrane. A striking point is that cholesterol enrichment has an inhibitory effect on all membrane
ATPases studied, whereas most other membrane transport
proteins are stimulated by an increased membrane cholesterol content.
The ATPases listed in Table 1 are so-called E1rE2-type
cation transporters, and are related by mechanistic similarities as well as amino acid homologies w67x. One of their
characteristics is the conformational change that takes
place during the transport process of the ions w67,68x. The
fluidity of the lipid environment may affect the conformational change during the transport cycle, which is a key
element of the function of these transporters, since a large
free energy barrier requires a change in protein conformation to enable transport. It is likely that an increase in the
rigidity of the membrane, as a result of an increased
cholesterol content, hinders the conformational change of
the protein. This is consistent with the proposal of MasOliva and Santiago-Garcia w39x who stated that the restriction in freedom of movement interferes with the conformational changes of the sarcolemmal Ca2q-ATPase during the
catalytic cycle.
Besides interference in protein conformational changes
by membrane rigidity, other mechanisms cannot be excluded. In fact, the similar catalytic units of the ATPases
studied could also be a common target for direct interac-
tions of cholesterol with the enzyme, causing diminished
activity.
The acetylcholine receptors are activated by an elevated
cholesterol content of the membrane ŽTable 1.. A generally
accepted theory to explain this phenomenon is not yet
available. In this respect, the theory of Trudell about the
way anesthetics affect ion channel conductance is worth
mentioning w69x. This theory implies that there is a lateral
phase separation in the region of a protein–lipid interface,
and this phase separation is destroyed by anesthetics. For
the function of Naq channels, for instance, the conversion
of high-volume fluid-phase lipids in the annulus into the
low-volume solid phase of the membrane lipids surrounding the annulus allows the Naq channel to expand. Anesthetic molecules fluidize the entire bilayer, including the
annulus region, thereby preventing, partly or completely,
the conformation change Žexpansion. of the protein to
allow Naq ions to pass w69,70x. Cholesterol may have an
effect opposite to that of anesthetics Ži.e., a stabilisation of
the phase separation by an anti-fluidizing action..
To elucidate the effect of the annulus on the function of
the transport protein, the physical characteristics of lipid
domains must be examined. This is achieved by using
structurally distinct spin probes and fluorescent probes.
Probes that closely approximate intrinsic membrane components would be expected to partition differentially among
the various lipid domains, thereby revealing the heterogeneous structure of biological membranes w71x. If this approach is combined with enrichment Žor depletion. of
cholesterol, more detailed knowledge of the effect of fluidity changes Žas a result of changes in cholesterol content.
on membrane function is likely to emerge.
5.1. Cell dysfunction
Maintenance of ion homeostasis by the sarcolemma is
necessary for proper cell function. In heart cells many
processes are controlled or affected by particular ion
species, including the control of membrane potential, cell
contraction processes, synthesis reactions, and signalling
of receptor stimulation. Thus, any process which depends
on membrane protein function may be affected by alterations in membrane composition Že.g., due to a change in
cholesterol content.. Two pathophysiologic processes—
hypercholesterolemia and ischemiaranoxia—will be discussed.
In hypercholesterolemia, membrane cholesterol content
of vascular cells, such as endothelial cells, macrophages,
and smooth muscle cells, is increased. There are several
reasons for considering an increased membrane cholesterol
content of vascular cells as essential in atherosclerosis. An
increased sarcolemmal cholesterol content of vascular
smooth muscle cells, as will occur in atherosclerotic arteries, was proposed to increase the number of active Ca2q
channels, leading to increased basal and norepinephrinestimulated calcium uptake w25,26,28x. In atherosclerotic
q
q
q
y
y
y
no effect
no effect
no effect
q
y
y
y
y
q
q
y
y
q
q
45
Ca2q uptake
Intracellular Ca2q ion concentration
Intracellular calcium content
Enzyme assay
Enzyme assay
Enzyme assay 1
Enzyme assay 2
Enzyme assay 3
Enzyme assay 4
45
2q
Naq
uptake
i -dependent Ca
Enzyme assay
Ouabain-sensivite Naq efflux
Ouabain-sensitive 86 Rbq uptake
Single channel conductance
Aggregated gap junction particles
Dye transfer
Aggregated gap junction particles
w 125 IxPindolol binding sites
w 3 HxQNB binding sites
Agonist-stimulated 86 Rbq influx
w26–28,31,35x
w28,31–33x
w29,30x
w31,37–39,45x
w40x
w41x
w41x
w42x
w43x
w45x
w45,49,51–53x
w50x
w25x
w54x
w59x
w59,61x
w60x
w63x
w64x
w65x
2
If cholesterol if forced to replace phospholipids in the annulus of the ATPase.
If the phospholipid annulus is complete.
3
If SR was prepared in the presence of a thiol-reducing agent.
4
In fully-uncoupled ATPase.
SL s sarcolemma; SR s sarcoplasmic reticulum; AchR s acetylcholine receptor; QNB s quinuclidinyl benzilate.
1
b-Adrenergic receptor
Muscarinic receptor
Naq flux AchR
Ca2q-dependent Kq channel
Gap junction
NaqrCa2q exchange in SL
ŽNaq,Kq .-ATPase
ŽCa2q,Mg 2q .-ATPase in SL
ŽCa2q,Mg 2q .-ATPase in SR
Ca2q channel in SL
q
y
y
y
y
y
q
q
?
q
?
y
y
Function is
increased Žq.
or decreased Žy.
w37–39x
w45x
w45,53x
w47,48x
w49x
w54x
w63x
w64x
w65x
45
2q
Naq
uptake
i -dependent Ca
Enzyme assay
Apparent affinity for internal Naq
Enzyme assay
Single channel conductance
w 125 IxPindolol binding sites
w 3 HxQNB binding sites
Agonist-stimulated 86 Rbq influx
w35,36x
w32,34x
Reference
Enzyme assay
45
Ca2q uptake
Intracellular Ca2q ion concentration
Measurement
If membrane cholesterol content is decreased
Reference
Function is
increased Žq.
or decreased Žy.
Measurement
If membrane cholesterol content is increased
Table 1
The effect of membrane cholesterol content on transport proteins, ATPases and receptors
E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
279
280
E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
arteries of rabbits fed a rabbit chow supplemented with 1%
cholesterol, smooth muscle cell calcium levels were reported to be elevated. Thus, in hypercholesterolemia the
increased membrane cholesterol content of and increased
calcium entry into vascular smooth muscle cells will promote calcium-dependent atherogenetic cell processes, such
as migration, proliferation, and stimulus–secretion coupling w30x. Several findings indicate that the cholesterol-induced increase in vascular smooth muscle cell calcium
content is a crucial, if not imperative, event in atherogenesis: Ži. calcium antagonists inhibit serum-stimulated proliferation and DNA synthesis in cultured vascular smooth
muscle cells w72x; Žii. calcium antagonists inhibit atherosclerotic plaque formation in animals w73x and retard the
progression of minimal atherosclerotic lesions in human
coronary arteries, defined as less than 20% in stenosis
diameter on the angiogram made before randomization to
nicardipine w74x; Žiii. a characteristic feature of advanced
atherosclerosis is the presence of calcium deposits in the
vascular wall. These extracellularly localized calcium deposits are considered to be the remains of calcium-overloaded vascular smooth muscle cells following their migratio n , p ro life ra tio n , lip id a c c u m u la tio n a n d
necrosisrapoptosis w75x.
Normothermic no-flow ischemia of isolated rat hearts
for 60 min resulted in a decline in sarcolemmal cholesterol
content of 43% and a reduction in cellular cholesterol
content of 34% w76x. In cultured neonatal rat cardiomyocytes, 60 min of anoxia caused a decrease in cellular
cholesterol content of 18% w77x while 60 min of metabolic
inhibition induced by a combination of 2-deoxyglucose
and NaCN caused a decrease in cellular free cholesterol
content of 22% w19x. In the latter study, a decrease in the
free cholesterol concentrations was significant after 15 min
of metabolic inhibition already ŽFig. 3.. Fig. 4, taken from
the same study, shows that the decrease in fluorescence
Fig. 3. Cellular free cholesterol content during control incubation Žhatched
bars. and metabolic inhibition Žopen bars. presented as percentage baseline value Ž100%s 75.2"8.6 nmol free cholesterol per mg protein. in
neonatal rat cardiomyocytes in culture. During the first 90 min of
metabolic inhibition, cellular free cholesterol content decreased by about
30%. Values represent means"s.e., ns 4. ) P - 0.05 vs. control incubation at corresponding time points. From w19x Žwith permission..
Fig. 4. The decrease in fluorescence steady-state anisotropy using TMADPH during metabolic inhibition of neonatal rat cardiomyocytes in
culture is correlated linearly with the decrease in cellular free cholesterol
content at corresponding time points. From w19x Žwith permission..
steady-state anisotropy using TMA-DPH observed in the
first 90 min of metabolic inhibition is correlated with the
decrease in cellular free cholesterol content at corresponding time points, suggesting that the increase in sarcolemmal fluidity which occurs early after the onset of metabolic
inhibition, may be due to loss of cholesterol from the
sarcolemma w19x. The decrease of sarcolemmal cholesterol
preceded the onset of irreversible cell damage w19,77x. The
occurrence of irreversible cell death is primarily caused by
ATP depletion and its consequences, such as loss of Kq
ions and accumulation of Naq, Ca2q and Hq ions. The
subsequent destruction of the cellular architecture includes
the degradation of cytoskeletal and membrane components.
Cellular calcium overload to resting wCa2q x i levels of 2
mM or higher will lead to severe mitochondrial calcium
overload causing dissipation of mitochondrial inner membrane potential, impairment of oxidative phosphorylation,
and the inability to regenerate a sufficiently high cytoplasmic phosphorylation potential to drive active calcium
transport systems of the plasma membrane and SR w78x.
The mitochondrial calcium overload is considered to be
the consequence of the formation of a non-specific ‘channel’ regulated by Ca2q w79x or a Ca2q-activated pore which
is opened by complete oxidation of the mitochondrial
glutathione redox couple w80x. Does sarcolemmal cholesterol content determine the duration of the reversible phase
of cell injury during ischemiaranoxia? The time-dependent decrease of membrane cholesterol may indicate a
degradative action operating at the membrane level. In our
laboratory, it has been shown that the tolerance to anoxia
of cultured cardiomyocytes is dependent on the cholesterol
content of the sarcolemma w81x. The higher the initial
cholesterol content of the sarcolemma, the higher was the
tolerance to anoxia, and vice versa ŽFig. 5.. Where does
the cholesterol go during ischemiaranoxia after it has left
the sarcolemma? Rouslin et al. demonstrated that although
whole tissue cholesterol in canine myocardial tissue made
E.M.L. Bastiaanse et al.r CardioÕascular Research 33 (1997) 272–283
281
content is on several other transport proteins, for instance
transporters located in mitochondrial membranes.
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Fig. 5. Change in time to reach half-maximal release of lactate dehydrogenase Ž DLDH 50 % . is correlated with percentage change in cellular free
cholesterol content Ž DFC content., in cultures of neonatal rat cardiomyocytes incubated under anoxic conditions. Values are compared with
those of control cultures. Free cholesterol content was changed by
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line: y s 0.80Xy8.6, r s 0.91, P - 0.001. From w81x Žwith permission..
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6. Conclusions
Several transport proteins are affected by the cholesterol
content of the membranes in which the proteins are located. The effect of cholesterol may be mediated by membrane fluidity or by the cholesterol molecules themselves,
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