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Biophys J, November 2002, p. 2702-2704, Vol. 83, No. 5
*Department of Biophysical Chemistry, Umeå University, SE-901 87 Umeå, Sweden; Department of Biochemistry and Molecular
Pathology, Northeastern Ohio Universities' College of Medicine,
Rootstown, Ohio 44272 USA
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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The pulsed field gradient NMR method for measuring self-diffusion has been used for a direct determination of the lateral diffusion coefficient of cholesterol, fluorine labeled at the 6-position, for an oriented lamellar liquid-crystalline phase of dimyristoylphosphatidylcholine (DMPC)/cholesterol/water. It is found that the diffusion coefficients of DMPC and cholesterol are equal over a large temperature interval. The apparent energy of activation for the diffusion process (58 kJ/mol) is about the same as for a lamellar phase of DMPC/water, whereas the phospholipid lateral diffusion coefficient is approximately four times smaller in the presence of cholesterol.
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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There is still no consensus, despite over 70 years of study, of the role played by cholesterol in biological
membranes. The more recent suggestions propose that cholesterol is
involved in the formation of domains in the membrane, usually referred
to as lipid rafts (Simons and Ikonen, 1997
), and that it contributes to
the formation of caveolae (Andersson, 1998; Gustavsson et al., 1999).
In these latter flask-like invaginations in the plasma membranes,
various signaling functions of the cell are initiated such as the
signal transduction pathways activated by insulin binding to its
receptor. These receptors are localized in caveolae microdomains
(Gustavsson et al., 1999; Parpal et al., 2001), which require the
presence of cholesterol in the membrane. Depletion of cholesterol
results in the disappearance of the caveolae structure (Gustavsson et
al., 1999). Another very interesting idea propounded by McConnell
(Radhakrishnan and McConnell, 2000) is that cholesterol may form
condensed complexes with phospholipids (Anderson and McConnell, 2001)
in the plasma membrane that serve a regulatory function with respect to
intracellular cholesterol transport and biosynthesis. With the aim of
further probing the functions of cholesterol in lipid bilayers we have
started a NMR spectroscopic investigation of the lateral diffusion of
both the cholesterol and phospholipid components in macroscopically
aligned bilayers. In this brief communication we present, for the first
time, a direct determination of the lateral diffusion coefficient
(DL) of cholesterol in a lipid
bilayer. The great advantage with the pulsed field gradient NMR method
(Lindblom and Orädd, 1994) is that it is nonperturbing and in
most cases does not need any labeling, although for this study of
cholesterol diffusion we have used a simple monofluorinated derivative.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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To obtain a resolvable NMR signal from cholesterol we have used
a singly fluorinated cholesterol (6FCH) in which the proton at the
6-position of the ring system has been replaced by a
19F (Kauffman et al., 2000). The oriented samples
were prepared according to a method similar to the one described in
Kurtze et al. (2000). Weighed dry powders of
dimyristoylphosphatidylcholine (DMPC) (Larodan, Malmö, Sweden)
and 6FCH, giving a composition of 38 mol % 6FCH, was dissolved in
methanol, and 1-propanol was then added to a final solution composition
of 1:4 in methanol:1-propanol. The total concentration of DMPC and 6FCH
was 10 mg/ml. This choice of solution provides suitable conditions for
good wetting of the glass plates in the sample preparation. A 13-µl
volume of the solution was applied to each of 35 glass plates (2.5 × 14 mm2), and the solvent was allowed to
evaporate. The last traces of solvent were removed under high vacuum
for at least 8 h. This procedure resulted in a thin film covering
the whole area of the glass plates. The glass plates were then stacked
on top of each other and placed in a glass tube with a square cross
section. The tube was placed in humid atmosphere at 30°C for 5 days.
During this time the glass plates became transparent as the hydrated lamellar phase was formed. To ensure the presence of excess water, some
extra water were added and the sample tube was then sealed with wax in
both ends and kept in a humid atmosphere until measurement. A sample
with only DMPC/water was produced in the same way.
The orientation was checked by inspection of the samples in crossed polarizers where extensive dark areas indicated that large portions of the samples were oriented with the bilayers along the glass plates. The orientation was also checked by 31P-NMR from which it was estimated that more than 85% of the samples was oriented along the glass plates (Fig. 1).
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The sample was placed in a diffusion probe equipped with a goniometer
stage that allows the orientation of the sample to be adjusted from
outside the magnet. When the angle between the bilayer normal and the
main magnetic field (B0) was close to
the magic angle (54.7°), significant narrowing of the proton NMR
signal from the water and DMPC was observed. This is a consequence of the reduction of the dipole-dipole interactions, and the sample then
regains some of the characteristics of an isotropic sample (Lindblom
and Orädd, 1994). In particular, the signal from a sample
oriented at the magic angle can be refocused to produce a spin-echo,
which is required in the pulsed field gradient NMR method of diffusion
measurements. The refocused proton signal was used to adjust the sample
to the magic angle.
The stimulated spin-echo pulse sequence (Tanner, 1970) was used for
both 19F and 1H diffusion
experiments. The 90° pulse width was 11 µs, the short delay was 5 ms, and the long delay was 20 ms. Both delays were kept at a minimum
due to the rapid relaxation of the fluorine NMR signal. The gradient
pulse duration was 3 ms, the gradient strength was varied between 0.45 and 8.9 T/m, and the diffusion coefficient was obtained from a
nonlinear fit of the Fourier-transformed peak amplitudes according to
the Stejskal-Tanner equation (Stejskal and Tanner, 1965). The obtained
diffusion coefficients were finally multiplied by the factor 1.5 to
compensate for the fact that the measured diffusion is along the
z axis, whereas the lipid motion occurs along the bilayers
oriented with their normal at 54.7° with respect to this axis
(Lindblom and Orädd, 1994).
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RESULTS AND DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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The choice of 6FCH was made on the basis of monolayer studies,
which indicated that fluorine substitution at the 6-position has a
negligible effect on the physico-chemical properties of cholesterol
(Kauffman et al., 2000). In addition, we have shown by deuterium NMR
that the effect of 10 mol % 6-fluorocholesterol on the orientational
ordering at all positions of the alkyl chains in chain-perdeuterated
(d54) DMPC bilayers is identical to the effect of
an equivalent amount of cholesterol (P. Westerman and S. Prosser,
unpublished results). However, the disadvantage of the 6-position is
that the T2 relaxation of the fluorine is fast, because the rigid ring structure gives the fluorine a highly restricted motion. Therefore, extensive signal averaging was necessary due to the
weak echo signal. The low signal-to-noise level gives an estimated
error in the diffusion coefficient for cholesterol of ±5%, whereas
the error in DL of DMPC is much smaller.
Fig. 2 shows Arrhenius plots of
DL for DMPC and 6FCH in the mixed
DMPC/6FCH sample along with DL for the
DMPC sample. We also include earlier published values of
DL in a DMPC sample with 20 wt % water (Kuo and Wade, 1979). As seen in the figure, the temperature dependence is similar to our values, but the values of Kuo and Wade is
lower than ours. This is most probably a consequence of the low water
content in the sample. In the temperature interval 30-60°C,
DL for DMPC and
DL for 6FCH) are equal for the mixed sample, with an apparent energy of activation of 58 kJ/mol. This contrasts with the lateral diffusion of the phospholipid
(DL) in DMPC/water bilayers, in which
the activation energy is comparable (49 kJ/mol), but
DL is approximately four times larger.
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The most interesting observations in this study are that 1) cholesterol
and DMPC exhibit the same lateral diffusion coefficients over the whole
temperature interval investigated, 2) the addition of cholesterol to
the phospholipid bilayer results in a decrease of the translational
diffusion of the phospholipid, and 3) the apparent energy of activation
of the diffusion process is the same for both components as well as for
DMPC in a pure lipid/water system. Many workers have reported the
second observation before, for saturated phospholipids in both
monolayers and bilayers (Cullis, 1996; Kuo and Wade, 1979; Rubenstein
et al., 1979; Vaz et al., 1979). In a previous study, more than 20 years ago, we did not observe this (or any) effect of cholesterol on
unsaturated lipid diffusion (Lindblom et al., 1981). Whether this is
due to the unsaturation of the acyl chains of the phospholipids or due
to a much lower accuracy in the measurements with the old lower-field NMR spectrometer used at that time, needs additional investigation.
At present we can give only a qualitative explanation to the observed
decrease in the lipid diffusion upon addition of cholesterol. It is
well known that cholesterol has a condensing effect on the lipid
packing in lipid bilayers (Leathes, 1925), and it seems reasonable to
assume that a decrease in the phospholipid lateral diffusion may arise
from such an effect. The fact that both components have the same
diffusion coefficient indicates that most probably the translational
diffusion is bestowed by some kind of cooperative mechanism. This is in
line with our previous observation on studies of the effect of
different additives to a cubic liquid-crystalline phase composed of
monooleoylglycerol, where it was found that, depending on the additive,
the diffusion coefficient could either increase or decrease (Eriksson
and Lindblom, 1993). Thus, the addition of a short-chained amphiphile,
characterized by a relatively rapid diffusion, will result in an
increase in the lipid diffusion, and interestingly enough the overall
diffusion coefficients of both components will be the same. For a
long-chained compound, the opposite will prevail. Finally, the straight
line obtained in the Arrhenius plot in Fig. 2 indicates that there are
no phase transitions or domain or complex formations occurring in the
temperature interval investigated. If complexes or domains were present
in the bilayer one would expect to see the effect of some kind of change of their number or sizes. However, recently published results indicate the presence of condensed complexes at lower temperatures (Radhakrishnan and McConnell, 2002), and we are therefore extending our
future investigations toward lower temperatures as well as to several
different cholesterol concentrations.
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ACKNOWLEDGMENTS |
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The technical support of Marcia Malmer (Northeastern Ohio Universities' College of Medicine) is gratefully acknowledged.
This work was supported by grants from The Swedish Research Council.
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FOOTNOTES |
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Address reprint requests to Dr. Greger Orädd, Department of Biophysical Chemistry, Umeå University, SE-901 87 Umeå, Sweden. Tel.: 46-90-786-5367; Fax: 46-90-786-7779; E-mail: greger.oradd{at}chem.umu.se.
Submitted May 13, 2002, and accepted for publication June 27, 2002.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Biophys J, November 2002, p. 2702-2704, Vol. 83, No. 5
© 2002 by the Biophysical Society 0006-3495/02/11/2702/03 $2.00
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) and DMPC (
) in the mixed DMPC/6FCH sample and
DL of DMPC in the DMPC sample (
). The
obtained apparent energy of activation is 58 kJ/mol for the mixed
sample and 49 kJ/mol for the DMPC sample. Also shown are values
of DL in a DMPC sample with 20 wt % water
taken from Kuo and Wade, 1979