Originally published In Press as doi:10.1074/jbc.M106618200 on October 26, 2001
J. Biol. Chem., Vol. 277, Issue 2, 1249-1254, January 11, 2002
Unique Effects of Different Fatty Acid Species on the Physical
Properties of the Torpedo Acetylcholine Receptor
Membrane*
Silvia S.
Antollini and
Francisco J.
Barrantes
From the Instituto de Investigaciones Bioquímicas de
Bahía Blanca and UNESCO Chair of Biophysics and Molecular
Neurobiology, B8000FWB Bahía Blanca, Argentina
Received for publication, July 13, 2001, and in revised form, September 28, 2001
 |
ABSTRACT |
To study the effects produced by free fatty acids
(FFA) on the biophysical properties of Torpedo marmorata
nicotinic acetylcholine receptor-rich native membranes and to
investigate the topology of their binding site(s), fluorescence
measurements were carried out using the fluorescent probe Laurdan
(6-dodecanoyl-2-(dimethylamino) naphthalene) and ADIFAB, an
Acrylodan-derivatized intestinal fatty acid-binding protein. The
generalized polarization (GP) of the former probe was used to learn
about the physical state of the membrane upon FFA binding. Saturated
FFA induced a slight increase in GP, whereas
cis-unsaturated fatty acids decreased GP. Double bond
isomerism could also be distinguished; oleic acid (18:1cis) induced a net disordering effect, whereas elaidic acid
(18:1trans) produced no changes in GP. The changes in the
efficiency of the Förster energy transfer from the protein to
Laurdan brought about by addition of FFA, together with the distances
involved in this process, indicate that all FFA studied share a common
site at the lipid-protein interface. However, despite being located at the same site, each class of FFA differs in its effect on the physical
properties of the membrane. These data lead us to suggest that it is
the direct action of FFA at the lipid-protein interface, displacing
essential lipids from their sites rather than changes in bulk
properties such as membrane fluidity that accounts for the effect of
FFA on the acetylcholine receptor membrane.
 |
INTRODUCTION |
The nicotinic acetylcholine receptor
(AChR)1 is an integral
membrane protein deeply embedded in the postsynaptic region of muscle,
electrocytes, and nerve cells. Experimental evidence from various
groups including ours substantiates the notion that the function of
this rapid ligand-gated channel is influenced by its lipid
microenvironment (see reviews in Refs. 1-3). Although the occurrence
of specific interactions between the transmembrane region of the AChR
and adjacent lipid molecules in the membrane has been reported (4-6),
the exact nature of these interactions has not been clearly
established. The first shell of lipids around the AChR, the so-called
annular lipid (7, 8), exhibits distinct characteristics, such as a
higher degree of order and a lower mobility than the bulk lipids. This
specialized region of the membrane has received particular attention as
the likely candidate domain where modulation of AChR function by lipids
occurs (7, 9).
The presence of both cholesterol and negatively charged
phospholipids (10-16) has been shown to be necessary for proper
AChR-mediated ion translocation in vitro. Various hypotheses
were postulated to explain this functional dependence, because these
lipids may modify the biophysical properties of the lipid annulus
and/or the bulk lipid bilayer (12, 18, 19).
A second possibility is the occurrence of specific sites for certain
lipids at the lipid-facing surface of the AChR, substantiated by the
work of several groups (20-25). Upon interacting with these sites,
lipids would stabilize the secondary structure of the AChR transmembrane segments (12, 26, 27). Recently, Baenziger et
al. (28) proposed a model of how lipid composition modulates the
function of the AChR, suggesting that membrane fluidity or some other
bulk property of the membrane modulates the equilibrium between the
resting and desensitized states of AChR. They also suggested that in
addition to this indirect effect, the AChR has a specific requirement
for anionic lipids (such as phosphatidic acid) binding to a specific
site on the AChR or exerting a less specific charge effect on AChR conformation.
Previous studies from several laboratories demonstrate that free fatty
acids (FFA) inhibit the ion flux mediated by the AChR in
vitro (29, 30) or in vivo (31). Analysis of
single-channel electrophysiological data argues for a mechanism
compatible with noncompetitive inhibition of the AChR. From these
studies the conclusion was drawn that the effect of FFA is related to
their hydrophobic character, but the exact mechanism of FFA action is still not clear.
To further investigate the possibility that these compounds exert their
effect on AChR at the lipid-protein interface and to investigate the
nature of these effects on the biophysical property of the AChR-rich
membrane, we carried out fluorescence studies using the fluorescent
probes Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) and ADIFAB,
an Acrylodan-derivatized intestinal fatty acid-binding protein. Laurdan
possesses an exquisite sensitivity to the phase state of the membrane.
The physical origin of Laurdan spectral properties resides in its
capacity to sense the polarity and the molecular dynamics of dipoles in
its environment because of the effect of dipolar relaxation processes
(32, 33). The principal dipoles sensed by Laurdan in the membrane are
water molecules. When no relaxation occurs, high Laurdan GP values
result, indicative of low water content in the hydrophobic/hydrophilic
interface region of the membrane. Thus, GP values depend on the extent
of water penetration allowed by the local membrane packing and hence provide a direct report on the AChR membrane environment.
In the present work, we have exploited the advantageous spectroscopic
properties of Laurdan to study the effect of FFA with different
structure in the native membrane in which the AChR protein is embedded.
Complementary studies with ADIFAB were also performed to determine the
partition coefficient of the different fatty acids in the membrane.
This information enabled us to compare the effects caused by fatty
acids at the same effective concentration in the membrane. We found
that the carbon chain length of fatty acids, as well as the number of
double bonds and their stereochemical configuration, are important
determinants of the unique effects of the different FFA species on the
physical properties of the AChR-rich membrane. Furthermore, changes in
the efficiency of the energy transfer from the protein to Laurdan,
brought about by the addition of exogenous FFA, revealed the presence
of sites for FFA at the lipid-protein interface in the native AChR
membrane. Preliminary data have been presented in abstract form
(34).
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EXPERIMENTAL PROCEDURES |
Materials
Torpedo marmorata specimens were obtained from
the Mediterranean coast off Alicante, Spain. They were killed by
pithing, and the electric organs were dissected and stored at
70 °C until further use. Laurdan and ADIFAB were purchased from
Molecular Probes (Eugene, OR). All other drugs were obtained from Sigma.
Methods
Preparation of AChR-rich Membranes
Membrane fragments rich in AChR were prepared from the electric
tissue of T. marmorata as described previously (35).
Specific activities in the order of 2.0-2.8 nmol
-bungarotoxin
sites/mg protein were obtained. The orientation of AChR in vesicles was measured as described by Hartig and Raftery (36) by determining the
total toxin-binding sites in the presence of Triton X-100 and the right
side out toxin-binding sites in the absence of detergent (36) as in
previous work from our laboratory (37).
For fluorescent measurements, AChR-rich membranes were suspended in
buffer A (150 mM NaCl, 0.25 mM
MgCl2, and 20 mM HEPES buffer, pH 7.4) at a
final concentration of 50 µg protein/ml (0.2 µM). The
optical density of the membrane suspension was kept below 0.1 to
minimize light scattering.
Preparation of Fatty Acid Solutions
Sodium salts of FFA were dissolved in buffer A with a bath
sonicator. In same cases we first prepared a sodium salt FFA stock solution in 4 mM NaOH, and aliquots were diluted to final
concentrations with buffer A. FFA were dissolved in ethanol (in all
cases the amount of ethanol added to the samples was kept below
0.5%).
Fluorescence Measurements
All fluorimetric measurements were performed in an SLM model
4800 fluorimeter (SLM Instruments, Urbana, IL) using a vertically polarized light beam from a Hannovia 200-W mercury/xenon arc
obtained with a Glan-Thompson polarizer (4-nm excitation and emission
slits) and 10 × 10-mm quartz cuvettes. Emission spectra were
corrected for wavelength-dependent distortions. The
temperature was set at 20 °C with a thermostated circulating water
bath (Haake, Darmstadt, Germany).
Laurdan Measurements--
Laurdan was added to AChR-rich
membrane samples from an ethanol solution to give a final probe
concentration of 0.6 µM. The amount of organic solvent
was kept below 0.2%. The samples were incubated in the dark for 60 min
at room temperature. Excitation GP (exGP) (32, 33) was calculated as follows.
|
(Eq. 1)
|
where I434 and
I490 are the emission intensities at the
characteristic wavelength of the gel phase (434 nm) and the liquid crystalline phase (490 nm), respectively. Excitation GP values were
obtained from emission spectra obtained with an excitation wavelength
of 360 nm.
Förster Resonance Energy Transfer (FRET)
Measurements--
The energy transfer efficiency (E)
in relation to all other deactivation processes of the excited donor
depends on the sixth power of the distance between donor and acceptor.
According to Förster's theory (38), E is given by the
following equation.
|
(Eq. 2)
|
where r is the intermolecular distance and
Ro is a constant parameter for each
donor-acceptor pair, defined as the distance at which E is
50%. E can be calculated as follows
|
(Eq. 3)
|
where
and
D are the fluorescence quantum
yields of donor in the presence and absence of the acceptor,
respectively, and I and ID are the
corresponding emission intensities in any given measurement. Here
I and ID correspond to the maximal
intrinsic protein emission intensity, which is 330 nm.
When E was measured in the presence of exogenous FFA, a
further correction was introduced to compensate for any modification of
the intrinsic fluorescence of Trp by any other quenching mechanism induced by FFA.
|
(Eq. 4)
|
where Ecorr is the experimentally
determined value of E corrected by the quenching of the
intrinsic fluorescence by the FFA. E(+Laurdan)
and E(
Laurdan) values were calculated using
Equation 3 in the presence of FFA, with or without Laurdan, respectively.
Partition Coefficient (Kp)
Measurements--
Kp was calculated with the
following expression.
|
(Eq. 5)
|
where [FFA]m and [FFA]a are the
concentrations of fatty acid in the membrane phase and in the aqueous
phase, respectively, and Va and Vm are the
corresponding volumes of the aqueous and membrane phases, respectively.
[FFA]m is defined as [FFA]T
[FFA]a, where [FFA]T is the added FFA
concentration. Experimentally, the Kp values were obtained using ADIFAB, which responds to FFA binding with a shift in
fluorescence emission from 432 nm in the apoform to 488 nm in the
holoform. As a consequence, [FFA]a can be determined from the
ratio of the fluorescence intensity at 488 nm to that at 432 nm (39),
according to the following expression.
|
(Eq. 6)
|
where R is the measured ratio of 488 to 432 nm
intensities, Ro is this ratio with no FFA
present, Rmax is the value when ADIFAB is
saturated with FFA, and Q = IF(432)/Ib(432), where
IF(432) and Ib(432) are
the ADIFAB intensities with zero and saturating concentrations of FFA,
respectively. On the basis of the numerical analysis of FFA titration
data, Q and Rmax values were found to be 19.5 and 11.5, respectively (40). These values are constant for all
FFA. Kd is the dissociation constant for FFA (ADIFAB-FFA
ADIFAB + FFA), calculated from a plot obtained by titration of ADIFAB with FFA, after linearization according to a
logarithmic form of Equation 6 (Hill plot). Kd
values were obtained using the following expression.
|
(Eq. 7)
|
where A = Q(R
Ro)/(Rmax
R) and [ADIFAB] was 0.2 µM.
Determination of the Effective FFA Concentration in Native
Torpedo AChR-rich Membrane--
The [FFA]T for each
addition was converted to its effective concentration inside the
membrane ([FFA]e) using the following equation.
|
(Eq. 8)
|
where CL = Kp/{1 + (Kp
1)
L [L]},
L is the lipid molar volume, and [L] is the lipid
concentration in the cuvette, which increases for each addition of FFA.
A value of 0.95 dm3/L
(dipalmitoylphosphatidylcholine fluid phase) was assumed because AChR-rich membranes are in a liquid fluid phase at 20 °C (41).
Data Analysis
Intergroup comparisons were done by impaired t test.
Statistical significance was accepted as p < 0.05.
 |
RESULTS |
Partition of Free Fatty Acids in AChR-rich Membranes--
To
compare the effects caused by the presence of different exogenous FFA
on the native AChR-rich membrane from T. marmorata, we first
measured their partition coefficient. There are several methods for
measuring partition coefficients of lipids in membranes, but the clear
advantage of using ADIFAB is that it is not necessary to physically
separate free and membrane-bound fatty acid, as is the case when using
radiolabeled fatty acids, a method more prone to error. ADIFAB, an
Acrylodan-tagged intestinal fatty acid-binding protein (39, 40), is a
suitable fluorescent indicator for the measurement of FFA concentration
in the 1 nM to 20 µM range. The detection of
FFA by ADIFAB is based on a change in the position of the Acrylodan
fluorescent tag relative to the nonpolar binding pocket of the protein
when the latter is occupied by a fatty acid. ADIFAB undergoes a marked
spectral shift upon FFA binding, allowing the determination of FFA
concentrations from the ratio of the fluorescence intensities of bound
and unbound forms, measured at about 488 and 432 nm, respectively. In
the example shown in Fig. 1, the decrease
in intensity at 432 nm and the corresponding increase at 488 nm of the
ADIFAB spectrum is apparent upon titration of AChR-rich membrane from
T. marmorata with arachidonic acid.

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Fig. 1.
Changes in ADIFAB fluorescence emission
spectrum by titration of AChR-rich membrane from T. marmorata with arachidonic acid. The dotted
line corresponds to the spectrum of ADIFAB in its apoform (maximum
at 432 nm); the solid lines are the spectra of ADIFAB in the
presence of increasing amounts (up to 20 µM, from
top to bottom) of arachidonic acid. A diminution
of the emission maximum at 432 nm and an increase of a second emission
maximum at 488 nm is apparent.
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|
Using Equations 6 and 5 we obtained the values of the dissociation
constant (Kd) and partition coefficient
(Kp) for the different fatty acids and ADIFAB in
aqueous solution and in the presence of Torpedo AChR-rich
membranes, respectively (Table I). The
calculated Kp values allowed us to classify the
fatty acids into three different groups: (i) highly hydrophobic fatty
acids, such as 20:0 and 18:0; (ii) less hydrophobic fatty acids, such
as 18:1cis and 18:1trans, and (iii) more
hydrophilic fatty acids, such as 18:2, 18:3, 20:4, and 22:6.
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Table I
Fatty acid-ADIFAB dissociation constants (Kd) and fatty
acid-native Torpedo AChR-rich membrane partition coefficients
(Kp)
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|
The experimentally determined Kp values of the fatty
acids in native AChR-rich membrane were subsequently used to transform
the nominal concentration added in the cuvette into an effective
concentration in the membrane for each fatty acid (see "Experimental
Procedures").
Variations of Laurdan GP Values Caused by the Addition of Free
Fatty Acids to AChR-rich Membranes of T. marmorata--
To study the
modification of the physical properties of the Torpedo
native membrane induced by the presence of FFA, we exploited the
amphiphilic fluorescence probe Laurdan's exquisite sensitivity to the
phase state of the membrane. We have previously used the so-called GP
of Laurdan as a sensitive tool to measure the physical state of
Torpedo native AChR-rich membrane (25, 41).
In the present work we investigated the possible modification of the
polarity of both the AChR belt and bulk lipid regions by fatty acids.
Laurdan GP values were obtained by direct excitation (at 360 nm) or
under FRET conditions, using the intrinsic membrane fluorescence as
donor (excitation at 290 nm) and Laurdan molecules as acceptor. In a
previous work we characterized this system and showed that the GP
values obtained under FRET conditions exhibit higher absolute GP values
than those obtained by direct excitation of the probe, indicating that
the microenvironment of the AChR has lower polarity than the bulk lipid
(41).
The changes in GP values caused by different FFA could be compared
using the effective concentration of each fatty acid inside the
membrane, calculated using their Kp values (Fig. 2). We found that addition of FFA to
Torpedo native membrane changed GP values, albeit to
different extents, in a manner dependent on the chemical structure of
the fatty acids.

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Fig. 2.
Laurdan excitation GP using direct excitation
of the probe (360 nm) (a) or FRET conditions (290 nm)
from the protein emission in T. marmorata AChR-rich
membrane (b) in the presence of increasing
concentrations of stearic acid ( ), arachidic acid ( ), oleic acid
( ), linoleic acid ( ), linolenic acid ( ), arachidonic acid
( ), and docosahexaenoic acid ( ). The abscissa
shows effective fatty acid concentration in the membrane, calculated
using the Kp of each FFA and Equation 8 (see
"Experimental Procedures"). Each point is the average of at least
four independent measurements (see text for statistics).
|
|
Unsaturated fatty acids decreased GP values, whereas saturated ones
caused a small increase. Statistically significant differences were
observed between different fatty acids: 18:0-18:1cis
(p < 0.005); 18:1-18:2 (p < 0.001);
18:2-18:3 (p < 0.001); 18:2-20:4 (p < 0.005); 18:2-22:6 (p < 0.001); 18:3-20:4
(p < 0.001); 18:3-22:6 (p < 0.001);
and 20:4-22:6 (p < 0.025), the exception being
18:0-20:0 (p > 0.05). In a fatty acid, each double
bond induces a kink of nearly 30° in the acyl chain (42). Thus, a
fatty acid with several double bonds undergoes noticeable changes in
the direction of its main molecular axis, resulting in an increase in
the cross-sectional area of the hydrocarbon chain over the minimum
found in a saturated fatty acid. The decrease in the GP values induced
by unsaturated FFA indicates an increase in polarity within the Laurdan
microenvironment. This in turn is a clear reflection of the disordering
of the bilayer caused by the kinked chains of unsaturated fatty acids,
which increases the content of water molecules in the membrane. In
contrast to unsaturated FFA, saturated FFA induce only a small increase in GP and thus a slight decrease in polarity and hence in the amount of
water in the membrane. In other words, saturated fatty acids induce a
slight ordering effect in the membrane, probably because of their rigid
linear structure.
The effect of fatty acid isomerization on GP was studied next. A
notable difference was observed in the effect caused on Laurdan GP by
18:1cis (oleic acid) and 18:1trans (elaidic acid)
(Fig. 3). Whereas oleic acid decreased GP
values, elaidic acid caused no changes. Again, the difference in
molecular structure of these fatty acids is a plausible explanation for
this behavior, because as stated above, cis-double bonds
induce a kink in the chain, whereas trans-double bonds
produce no change in chain direction (Fig. 3, inset).

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Fig. 3.
Net variation in Laurdan GP
( GP) in T. marmorata
AChR-rich membrane obtained between GP values in the presence of
20 µM oleic acid
(18:1cis) or elaidic acid (18:1trans)
on the one hand and GP values with addition of only buffer on the
other. Striped and black bars correspond to
the direct excitation or FRET conditions, respectively. Each point is
the average of at least four independent measurements. *, statistically
significant differences with respect to control values and with respect
to 18:1trans (in both cases p < 0.001); **,
nonsignificant differences. Inset, molecular structure of
oleic and elaidic acids.
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|
Similar Sites for Fatty Acids at the AChR-Lipid Interface--
In
a previous work we were able to discriminate between distinct sites for
phospholipids and sterols, both accessible to fatty acids, at the
lipid-protein interface in Torpedo native membrane, by
measuring the decrease of E between the membrane intrinsic fluorescence (tryptophan residues) and Laurdan upon addition of different exogenous lipids (25). Here we measured changes of E between AChR and Laurdan, brought about by the addition of
FFA of different chain length and degree of saturation. The addition of
FFA decreased the efficiency of FRET in a
concentration-dependent manner. The maximal diminution of
E achieved by all fatty acids was very similar (~ 50%)
(Fig. 4). This suggests that all fatty acids, independently of their physical characteristics, bind to similar
sites at the lipid-protein interface in the Torpedo
AChR-rich native membrane.

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Fig. 4.
Decrease in normalized FRET efficiency
(E) between intrinsic fluorescence in native T. marmorata AChR-rich membrane and Laurdan in the presence of
maximal concentrations (20 µM) of
stearic acid (18:0), arachidic acid (20:0), oleic acid
(18:1cis), elaidic acid (18:1trans),
linoleic acid (18:2), linolenic acid (18:3), arachidonic acid (20:4),
and docosahexaenoic acid (22:6). The E values obtained
in the presence of fatty acid were normalized with respect to the
corresponding E value in its absence (100%). Each point
corresponds to the average ± S.D. of four determinations.
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|
To corroborate whether different FFA bind to similar sites, we
conducted an additional series of experiments using the following strategy. We added first a given FFA, and, after obtaining the maximal
decrease of E, we added a second FFA, with similar or different structural characteristics, to the sample. If different FFA
bind to different sites, E should further decrease when a second FFA is added, whereas no additional decrease of E
would occur if the two FFA compete for the same site. Fig.
5 (a and b) shows
one such series of experiments. When, for example, 20:0 was added
first, Laurdan GP increased. When a second FFA was added, changes in GP
depended on its chemical structure; 18:0 caused a further
increase in GP, whereas 18:1 and 20:4 decreased
GP values to the same extent as they did by themselves separately. The
differences between GP values obtained after addition of different
fatty acids were in all cases statistically significant. Only in the
case of the saturated fatty acids 18:0 and 20:0 did we find
statistically nonsignificant differences. The fact that Laurdan GP was
affected under FRET conditions (Fig. 5b) is a clear
indication that the second FFA partitioned well into the membrane and
localized in the AChR-vicinal region. The decrease of E
obtained with the first fatty acid (saturated or unsaturated) remained
constant in the presence of a second fatty acid, independently of its
physical characteristics (Fig. 5, c-f).

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Fig. 5.
Net variation of Laurdan GP using direct
excitation of the probe (a) and FRET conditions
(b) first in the presence of increasing concentrations
of arachidic acid (20:0, ) and then in the presence of stearic acid
(18:0, ), oleic acid (18:1, ), or arachidonic acid (20:4,
). c and d, normalized energy transfer
efficiency for the AChR/Laurdan pair in T. marmorata
membranes. Two different conditions are shown here: increasing
concentrations of 20:0 up to 20 µM (c) and
increasing concentrations of 20:4 up to 20 µM
(d). Subsequently, a second fatty acid was added up to a
concentration of 20 µM. c, 20:0 ( ), 20:4
( ), 18:1 ( ), and 18:0 ( ) were added. d, 20:4 ( ),
18:1 ( ), and 20:0 ( ) were added. e and f,
final values of FRET E attained upon addition of a second
fatty acid. The differences between experimental conditions were
statistically not significant. These results correspond to the
averages ± S.D. of at least four independent measurements.
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|
 |
DISCUSSION |
In the present work, we used fluorescence techniques to study the
modifications of the physical properties of native Torpedo AChR-rich membrane by the addition of fatty acids with different structural characteristics. We first determined the partition coefficient of different FFA using an Acrylodan-labeled fatty acid-binding protein, ADIFAB. This enabled us to calculate the effective concentration of the FFA in the membrane. Quantification of
Laurdan GP yielded information on the physical state of the bulk and
belt-lipid region of the AChR-rich membrane in the presence of known
concentrations of FFA. Changes in FRET efficiency induced by fatty
acids made possible the measurement of fatty acid-protein interactions
within short distances of the probe Laurdan.
The FFA partition coefficients, determined using the extrinsic
fluorescent properties of ADIFAB, made apparent relatively pronounced
differences in Kp between different fatty acids
(Table I). Although FFA are highly hydrophobic compounds, their
physicochemical properties have a common origin: their carbon atoms
confer hydrophobicity, and the double bonds reduce hydrophilicity in
the molecule (42). We have attempted to relate these two opposite
effects in the same molecule by using an empirical algorithm that we
have coined the actual hydrophobicity coefficient (AHC).
|
(Eq. 9)
|
We calculated this coefficient for all FFA studied and
correlated it with their partition coefficient (Fig.
6). Using this approach, it is observed
that the tendency of different FFA to partition in the membrane follows
the sequence: 20:0 > 18:0 > 18:1 > 18:2
20:4
22:6 > 18:3. According to this order, FFA can be classified into
three different groups: (i) high AHC fatty acids, such as
20:0 and 18:0; (ii) fatty acids of intermediate AHC, such as
18:1cis and 18:1trans; and (iii) low
AHC fatty acids, such as 18:2, 18:3, 20:4, and 22:6.

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Fig. 6.
Correlation between
hydrophobicity/hydrophilicity of fatty acids and their Kp in
Torpedo native membrane.
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All fatty acids tested decreased the efficiency of the energy transfer,
E, between the membrane intrinsic fluorescence and Laurdan.
In a previous work (25), we interpreted this diminution as an increase
in the distance between donor and acceptor resulting from the
displacement of Laurdan molecules from the AChR lipid microenvironment
caused by the exogenously added lipid (Fig.
7). We were also able to characterize
distinct sites for phospholipids, sterols, and oleic acid. Here, we
investigated whether the sites to which oleic acid binds are the same
as those to which other fatty acids bind. The fact that a similar
decrease in E was observed for all FFA is a strong
indication that this is indeed the case. Further evidence in support of
the above hypothesis is that the addition of a second fatty acid did
not produce a further decrease of the E caused by the first
fatty acid.

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Fig. 7.
Schematic diagram illustrating a
cross-section of the membrane containing Laurdan molecules in the belt
and bulk lipid regions, showing the displacement of Laurdan molecules
([hatched circle]) from the AChR microenvironment
by exogenously added fatty acids ( ).
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The most striking finding of the present work is that although all
fatty acids displaced a fatty acid analog such as Laurdan to almost the
same extent (Fig. 4), different fatty acids perturbed the physical
properties of native Torpedo AChR-rich membranes quite
differently, in a manner that strongly depended on their structure
(Fig. 2). Thus, saturated fatty acids ordered the membrane, whereas
unsaturated fatty acids disordered it. Applying a simple algebraic
rearrangement of the changes in GP made it possible to classify FFA
effects.
|
(Eq. 10)
|
where GPfinal and
GPinitial are the GP values obtained at the same
fatty acid effective concentration in the membrane and before addition
of the fatty acid, respectively, and GPg and
GPlc are the larger and smaller GP values
obtained for a pure gel and liquid crystalline phase, respectively.
GPg (0.61) and GPlc
(
0.19) were experimentally measured at 20 °C in multilamellar
DOPC and dipalmitoylphosphatidylcholine liposomes, which are in
the liquid crystalline and gel phases, respectively.
Applying Equation 10, the changes induced by FFA ranged from ordering
(positive values of FFA effect) to disordering (negative values of FFA
effect) effects and followed the sequence: 20:0 (0.016) > 18:0
(0.005) > 18:1cis (
0.047)
18:2 (
0.046)
20:4 (
0.047)
22:6 (
0.047) > 18:3 (
0.056). Furthermore,
whereas 18:1cis (oleic acid) induced a net disordering
effect, 18:1trans (elaidic acid) produced no change
whatsoever in GP values (Fig. 3). This enables us to conclude that FFA
carbon chain length as well as the number of double bonds and their
stereochemical configuration are important determinants of the unique
effects of the different FFA on the physical properties of the
Torpedo AChR-rich membrane.
In living BC3H-1 cells in culture, fatty acids exert inhibitory effects
on AChR channel activity (31). These effects are also observed in
membrane patches excised from the cell and are therefore not mediated
by signal transduction pathways that require soluble factors such as
nucleotides and Ca2+. Thus, fatty acids appear to regulate
the action of the AChR channel directly, much as they regulate the
action of several purified enzymes and other channels (43). In chick
ciliary neurons and in
7 neuronal type AChR heterologously expressed
in Xenopus oocytes, saturated FFA or with only one double
bond had little effect on ACh-mediated currents, whereas FFA with two
or three double bonds produced partial inhibitory effects but less
effectively than arachidonic acid (44). The latter FFA has also been
reported to inhibit ACh-mediated currents in bullfrog sympathetic
neurons (17). The mechanism by which 20:4 or other fatty acids inhibit nicotinic transmission is unknown. To account for these inhibitory effects on AChR function, binding of fatty acid to a site(s) at the
lipid-AChR interface and/or perturbation of the local receptor microenvironment have been suggested.
Structurally different fatty acids (i) all appear to alter AChR
function in a similar manner, in terms of single channel open channel
durations at the level of resolution achieved (31); (ii) occupy
equivalent sites at the lipid-protein interface, in fact the same sites
as cholesterol and phospholipids (25); and (iii) induce changes in the
fluidity of the AChR lipid microenvironment that clearly depend on
their chemical structure. Thus, an explanation of fatty acid action
mediated only through fluidity changes is difficult to sustain, because
different fatty acids induce changes in membrane fluidity of different
sign and magnitude; some in fact induce no change at all. We suggest
that it is the direct action of FFA in displacing essential lipids from
their sites at the lipid-protein interface and not changes in bulk
properties such as membrane fluidity that is responsible for the
inhibitory effect of FFA on AChR function.
 |
FOOTNOTES |
*
This work was supported in part by grants from the
Universidad Nacional del Sur, the Agencia Nacional de Promoción
Científica (Fondo Nacional de Ciencia y Técnica),
Argentina, the Ministerio de Salud, Argentina, Fogarty International
Center Research Collaboration Award, National Institutes of Health
Grant 1-RO3-TW01225-01, and Antorchas/British Council (to F. J. B.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. E-mail:
rtfjb1@criba.edu.ar.
Published, JBC Papers in Press, October 26, 2001, DOI 10.1074/jbc.M106618200
 |
ABBREVIATIONS |
The abbreviations used are:
AChR, nicotinic
acetylcholine receptor;
AHC, actual hydrophobicity
coefficient;
FFA, free fatty acid(s);
FRET, Förster resonance
energy transfer;
GP, generalized polarization.
 |
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