Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4329-4333, September 1978

Biophysics

Immobilized lipid in acetylcholine receptor-rich membranes from Torpedo marmorata (spin label/electron spin resonance/fluorescence quenching/interstitial lipid/cholinoreceptors)

DEREK MARSH* AND F. J. BARRANTESt Departments of * Spectroscopy and t Molecular Biology, Max-Planck-Institut fur Biophysikalische Chemie, 3400 Gbttingen-Nikolausberg, W. Germany

Communicated by Manfred Eigen, June 29,1978

ABSTRACT

The lipid environment of acetylcholine receptor-rich membranes from Torpedo marmorata has been studied with spin labels. The electron spin resonance spectra of both stearic acid and steroid probes in the membranes revealed an immobilized lipid component, in addition to the fluid component which is found in aques bilayer dispersions of the extracted lipids. The spin labels also cause a differential paramagnetic quenching of the intrinsic protein fluorescence of the membranes, which is sensitive to the action of cholinergic ligands and follows a modified Stern-Volmer law. Electron spin resonance difference spectroscopy shows that the protein-associated lipid is immobilized with respect to rotation both around and perpendicular to the long molecular axis, with correlation times TR 50-70 ns. The proportion of lipid in the immobilized component is greater than calculated for a single boundary layer around the protein and corresponds more closely to the total interstitial lipid occupying the area between densely packed protein units in acetylcholine receptor-rich membranes. The nicotinic acetylcholine receptor (AcChoR) is an integral membrane protein (1) involved in the control of cation permeability at the postsynaptic membrane. Fragments of such membranes having more than 50% of their protein as AcChoR and still retaining functional properties can be prepared from the electric organs of fish (2, 3). The AcChoR, integrated in its natural membrane environment, occurs mostly as densely packed particles of about 90 A in diameter, occasionally ordered in two-dimensional arrays (2, 4). The AcChoR in these clustered arrays has been shown to have little if any lateral mobility (5) and very restricted rotational motion (6). The suggestion has been made that the anchoring of the AcChoR is involved in the stabilization of the developing synapse (7). The above considerations raise the question of whether the lipids in the AcChoR-rich membranes are involved in the immobilization of the receptor in its packed configuration or in the stability of the two-dimensional lattice. There is evidence from other membrane systems (8, 9) that a specifically immobilized boundary layer of lipids surrounds large, integral membrane proteins and that these lipids form the interface between the protein and other membrane constituents. On the other hand, studies with acylcholine, active site-directed, spin labels have led to a somewhat different picture of the immediate environment of the AcChoR, characterized by its high

membrane proteins, because it was not present in bilayers formed with the extracted lipids. In addition, paramagnetic quenching of the intrinsic fluorescence with the same spin labels indicates that at least a certain population of the lipid is in contact with the proteins and is influenced in its quenching properties by the presence of cholinergic effectors. MATERIALS AND METHODS AcChoR-rich membranes were prepared from the electric organ of T. marmorata according to Cohen et al. (11). The a-[3H]cobrotoxin binding capacity was assayed by the Millipore filtration method (12). Typical preparations had specific activities of 1 nmol of a-toxin per mg of protein. Total lipid extracts were made from lyophilized membranes with CHC13/CH30H (2:1 vol/vol). The stearic acid spin labels 2-(3-carboxypropyl)-(4,4-dimethyl-2-tridecyl-3-oxazolidinyloxy [1(12,3)], 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl-3-oxazolidinyloxy [I(5,10)] and 2-(14-carboxytetradecyl)-2-ethyl-4,4- dimethyl-3-oxazolidinyloxy' [I(1,14)], and the androstanol spin label 17 fl-hydroxy-4,4'-dimethylspiro-[ 15-a-androstan- 3,2'-oxazolidin]3'-yloxyl (II) were obtained from Syva, Palo Alto, CA. Membrane suspensions in Torpedo Ringer's solution (11) were labeled for ESR at a level of approximately 0.01-0.02 mg of label/mg of protein from a concentrated spin label solution in ethanol. The total amount of ethanol added was less than 1% (vol/vol). The membrane suspension was then centrifuged and the pellet was resuspended in a minimal volume of Torpedo Ringer's solution. Bilayer dispersions of the extracted lipids were prepared from a CHCI3/CH30H (2:1 vol/vol) solution of lipids plus 1% (wt/wt) spin label. The solution was dried down with a nitrogen gas stream, placed under vacuum for at least 5 hr, and then dispersed in Torpedo Ringer's solution at a concentration of approximately 50 mg/ml by Vortex mixing at 300-400. ESR samples were contained in sealed, 1-mm (internal diameter) glass capillaries accommodated within standard 4-mm quartz ESR tubes containing silicon oil for thermal stability. ESR measurements were made with a Varian E-12 9 GHz spectrometer equipped with a nitrogen gas-flow temperature regulation system. Temperatures were measured with a thermocouple placed just above the cavity within the quartz ESR tube. ESR spectra were digitized on paper tape [1000 points/ 100 gauss (1 gauss = 10-4 tesla)] by using a Hewlett Packard 345OB/2547A/2753A data collection system and processed on a PDP 11/34 minicomputer with Tektronix 4006 display.

fluidity (10). In this study we have examined the total lipid environment of the AcChoR-rich membrane fragments from Torpedo marmorata with spin labeled lipids. Electron spin resonance (ESR) spectra of these spin labels revealed a highly immobilized population of lipid in addition to the normal fluid bilayer. The strongly immobilized component interacts specifically with the

Abbreviations: AcChoR, acetylcholine receptor; ESR, electron spin resonance; I(12,3), 2-(3-carboxypropyl)-(4,4-dimethyl-2-tridecyl-3oxazolidinyloxy; 1(5,10), 2-(10-carboxydecyl)-2-hexyl-4,4-dimethyl3-oxazolidinyloxy; 1(1,14), 2-(14-carboxytetradecyl)-2-ethyl-4,4dimethyl-3-oxazolidinyloxy; II, 17,B-hydroxy-4,4'-dimethylspiro[15-a-androstan-3,2'-oxazolidin]-3'-yloxyl.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solelv to indicate this fact. 4329

Proc. Nati. Acad. Sci. USA 75 (1978)

Biophysics: Marsh and Barrantes

4330 0

N-0

0

I (12,3)

HO

0

I (5,10)

HO O-N

0

0

I(1,14)

HO 0-N

0

OH

II

0

0

For fluorescence measurements, membrane fragments (70 nM in a-toxin sites, 60,gg protein/ml) were suspended in 0.1 M phosphate buffer (pH 8.0) in 7 X 7 mm quartz cuvettes and the spin labels, dissolved in ethanol, were added in 2-5-,gl aliquots. The concentration of ethanol was kept below 1%, and

the small reduction of fluorescence caused by the solvent, as determined in control experiments, was subtracted from each experimental reading. The intrinsic fluorescence of the membrane proteins was recorded as the total spectrum above 320 nm, excitation at 297 nm, by using an apparatus designed in this laboratory (13). RESULTS The ESR spectra of the stearic acid spin labels I(m,n) and the steroid label II in AcChoR-rich membranes and in aqueous bilayers of the extracted lipids are given in Fig. 1. In all three cases the extracted lipid spectra consist of a single-component spectrum characteristic of the spin label undergoing rapid anisotropic motion in a single, fluid lipid bilayer environment (14). In contrast, all three membrane spectra contain a second, well-resolved component, in addition to the fluid bilayer component, whose large anisotropy is characteristic of a high degree of immobilization (small amplitude or rate of motion) of the lipid molecules on the ESR time scale (15). This strongly immobilized component is most clearly seen at the high-field and low-field extremes of the membrane spectra and is indicated by the arrows in Fig. 1. Because of the different motional properties of the three spin labels in the fluid lipid bilayer, the immobilized lipid component is optimally resolved at different temperatures for the three different labels. Thus Fig. 1 shows not only that the immobilized lipid regions are accessible to both fatty acid and steroid labels, but also that these immobilized regions exist over a relatively wide temperature range; from below 00 to temperatures greater than 340. The motional characteristics of the immobilized regions can better be seen from the difference spectra obtained by subtracting out the fluid bilayer component from the membrane spectra using the digitized spectra from the extracted lipid bi-

FIG. 1. ESR spectra of lipid spin labels in AcChoR-rich membranes from T. marmorata and aqueous bilayer dispersions of the extracted lipids. The upper spectrum of each pair is from the membrane and the'lower from the lipid bilayers. Upper two spectra: spin label I(1,14) at -4°; middle two spectra: spin label I(5,10) at 340; lower two spectra: spin label II at 140.

layers. As can be seen in Fig. 2, a reasonably good endpoint is obtained in such subtractions yielding an ESR difference spectrum that is characteristic of a strongly immobilized spin label environment (15). Strongly immobilized spectra such as those of Fig. 2 lie in the slow motion regime of ESR and the motional correlation times of the spin labels can be estimated from the linewidth of the high-field outermost peak, AHh, or of the low-field outermost peak, AH1, or from their separation Azz, as compared with the corresponding values AHhR, AH1R, ARzz expected for rigidly immobilized spin labels. The empirical equations, TR = a (1 Azz/ARzz)b and TR = am' (AHm/AHmR - l)b'm, are used where the following values for the calibration constants have been obtained from spectra simulations: ah' = 2.12 X 10-8 S; bh' -0.778; a,' = 1.15 X 10-8 s; bl' = -0.943; a = 5.4 X 10-10 s and b = -1.36 (16). Correlation times for the spin labels in the immobilized regions obtained by this analysis are given in Table 1. Because the direction of the maximum hyperfine splitting lies along the axis of the stearic acid spin labels I(m,n), but perpendicular to the long axis of the steroid spin label II, the outer extrema in the spectra of the former give the correlation time for angular rotation of the long axes ('TR) whereas those of the latter give the correlation time for rotation around the long molecular axis (-RIR)R To obtain further information regarding the location of the lipid environments sensed by the spin labels, we studied the quenching of the intrinsic fluorescence of the AcChoR membranes by the spin labels. Classical Stern-Volmer plots of the quenching (not shown) displayed a downward curvature which is indicative of a heterogeneous population of fluorophores, not all of which are accessible to the spin labels in the lipid phase. In such cases of heterogeneity-i.e., different chromophores with varying quenching constants-it is only possible to obtain

Proc. Natl. Acad. Sci. USA 75 (1978)

Biophysics: Marsh and Barrantes

30-

4331

,'

A

2' b0-

*,*

F,

'' .~~~-

.

,,

AF

0-

,-'*--S,

AS vI

0

0.1

0.2

0, 3

Fo AF

FIG. 2. ESR difference spectra of lipid spin labels in AcChoR-rich membranes, obtained by subtracting the bilayer spectra of Fig. 1 to yield the immobilized lipid component. Upper spectrum: spin label II in membranes at 14°; middle spectrum: spin label I(1,14) in membranes at -4°; lower spectrum: spin label 1(5,10) in membranes at 340. parameters from the quenching data. Fig. 3 gives the modified Stern-Volmer plot (19) that corresponds to the

average

0.2 0.1 1/[Nitroxidel, ;MMI

FIG. 3. Modified Stern-Volmer plots (19) of the quenching of the intrinsic fluorescence in AcChoR-rich membranes by lipid nitroxide spin labels. Curves correspond to quenching by the following labels: (A) 1(12,3) (0); I(12,3) after preincubation of the membranes with 20MuM suberyldicholine for 50 min (0); I(5,10) (A). (B) I(1,14) (a); 1(1,14) plus suberyldicholine as in A (x); II (0).

equation Fo/AF

=EfiKQjQ]

+

(z

KQ4/ Xft KQt)

in which AF is the change in fluorescence due to the addition of the quencher at a concentration [Q], KcQ are the quenching

of the individual chromophores, and fi are their fractional fluorescence contributions to the total emission, Fo. By analogy with the simple situation in which a fraction, fa, of the fluorophores are accessible to Q and all have the same quenching constant, KQ, whereas the remainder are inaccessible (K = 0), it is possible to define an "effective" fraction of accessible fluorophores, fa(app), given by:

constants

E ftKQ/ E KQt and an "effective" quenching constant for the fraction, KQ(ap) = 2;KQ,. Only the quenching data from the low nitroxide concentration range were included in the calculations. At low fa(app)

=

Table 1. Rotational correlation times of lipid spin labels in the immobilized lipid regions of AcChoR-rich membranes from T. marmorata

Spin label I(5,10) I(1,14) II

Temperature, TR, nst

34

TR, ns* 50

46

12

-4 14

34 45

41

45 47

CC

73

TR, nst

AHR and AHh were obtained from spectra of an extensively delipidated sample of cytochrome oxidase (17). A z values, corrected for polarity, were obtained from ref. 18. * Deduced from the low-field linewidth, AHIL. t Deduced from the high-field linewidth, AHh. t Deduced from the outer splitting, Azz.

concentrations the labels are likely to be strongly partitioned into the membrane, although detailed intercomparisons will depend on the different partition coefficients of the various labels. It is noteworthy that preincubation of the AcChoR-rich membranes with the potent agonist suberyldicholine (20) leads to a change in the quenching parameters (Fig. 3 and Table

2).

DISCUSSION The ESR experiments clearly show the existence of a population of immobilized lipid, quite distinct from the fluid lipid bilayer lipid, in the AcChoR-rich membranes. This immobilized lipid exists over a wide temperature range and is preferentially segregated about the membrane proteins since it is not found in Table 2. Apparent quenching constant, KQ(app), and apparent fraction of available fluorophores, fa(app), for quenching by nitroxide spin labels in AcChoR-rich membrane fragments* fa(app) KQ(app) Spin label 0.39 6.5 X 103 1(12,3) 0.33 5.5 X 103 I(12,3) + subt 0.20 7.1 X 103 1(5,10) 0.20 8.5 X 103 I(1,14) 0.20 2.3 X 104 I(1,14) + subt 0.10 1.5 X 104 II * The apparent parameters, as described in the text, were obtained from the linear region of the modified Stern-Volmer plot (19) (Fig. 3), where fa(app) = 1/intercept and KQ(app) = intercept/slope. t Samples preincubated with the cholinergic agonist suberyldicholine (sub) as described in Fig. 3.

4332

Proc. Natl. Acad. Sci. USA 75 (1978)

Biophysics: Marsh and Barrantes

bilayers of the extracted lipids.t Previous workers (10) using spin-labeled acyl-cholines to probe the immediate environment of the AcChoR binding site, concluded that the spin

aqueous

labels were immobilized only because of their tight binding to the protein and not because of the lipid. However, our results with spin labels which definitely probe the lipid environment clearly reveal that the lipid close to the protein is immobilized. It is possible that the above acylcholines may not directly sense the lipid environment because the AcChoR recognition site, located in the 40,000-dalton subunit (2, 3) may protrude well out into the aqueous phase (4). The results with the steroid and stearic acid spin probes show that the protein-associated lipid is immobilized with respect to motions both around and perpendicular to the long molecular axes of the lipid molecules, respectively. From Table 1 it is apparent that the rotational correlation times of the immobilized lipid, TRII and TR 1 are in the order of 50-70 ns and 30-50 ns respectively-i.e., approximately 50-100 times longer than is typically found in fluid lipid bilayer membranes (21, 22). It thus may be anticipated that the rate of lateral translational motion in these immobilized regions will also be a factor of 50-100 times slower-i.e., of a frequency of -105 s- (23, 24). Thus the lipid has sufficient rotational freedom to effect intermolecular communication, but is sufficiently translationally hindered to remain associated with the protein for a functionally significant time period. Protein-associated, immobilized lipid has been demonstrated in partially delipidated systems (8, 9, 17) and its existence was also inferred in membranes (25, 26), but here it has been directly observed in a natural, nonreconstituted membrane. From the lipid/protein stoichiometry of the immobilized lipid associated with cytochrome oxidase (8) or the Ca2+-ATPase from sarcoplasmic reticulum (9) it has been suggested that it forms a single boundary layer shell of lipid around the protein. In the present instance it is found that the amount of lipid associated with the immobilized spectra of Fig. 2 is considerably more than would be required to form a single boundary shell around the particles revealed in negative-contrast electron micrographs of AcChoR-rich membranes (2, 4). Instead, the amount of immobilized lipid corresponds rather closely to the total area of negative contrast in the AcChoR-rich regions. It thus seems possible that the immobilized lipid corresponds to the total interstitial lipid population in these regions of the membrane.§ If this were the case, the immobilized lipid could provide the medium of intercommunication between the receptor units. The fluorescence quenching experiments provide additional information on the interaction of the spin-labeled lipids with the membrane protein. It is seen from Fig. 3 and Table 2 that the quenching efficiency of the various probes follows the sequence: 1(12,3) > 1(5,10) > I(1,14) > II. These differential effects correlate with the known locations of the nitroxide groups of the various positional isomers within the membrane (29) and, although the additional effects of the polarity of the environBecause the immobilization is sensed by two very different types of probes, namely the stearic acid and the steroid spin labels, it is un-

likely that it arises from a preferential binding of the probes, but rather the probes reflect the behavior of the bulk membrane lipid in this immobilized region. Preliminary experiments with phospholipid spin labels also similarly reveal the immobilized lipid

component. § In this respect there may be similarities with the purple membrane of Halobacterlum halobium in which the protein is packed in wellordered arrays. Both systems have a similarly high protein content:

75% by weight measured for the purple membrane (27) and 75-80% calculated for the lattice regions of the AcChoR membrane (4). However, even in the purified crystalline purple membranes a fluid component has been observed in addition to the immobilized lipid

population (28).

ment cannot be neglected, the results are consistent with a preferential localization of the accessible tryptophan chromophores relative to the membrane surface, in agreement with previous studies in which quenching from the aqueous phase was employed (30). A paramagnetic quenching mechanism requires that quencher and fluorophore lie within approximately 5 A of each other. Thus, the values of fa(app) in Table 2 would imply that the majority of the fluorophores accessible from the lipid phase are located close to the apolar-polar interface of the membrane, at or above the C5 region of the phospholipid alkyl chains. The existence of paramagnetic quenching by the lipid spin labels also demonstrates that they sense the lipid regions in immediate contact with the membrane protein, presumably the immobilized lipid population observed by ESR. The fact that the quenching constants are of the same order or greater than those for spin-labeled fatty acids or steroids binding to bovine serum albumin (31) further supports this view. More importantly, the sensitivity of the quenching parameters to the presence of the potent agonist suberyldicholine implies that the spin labels sense the lipid environment of the AcChoR. It would not be surprising if many of the agents known to modify the state of the AcChoR in the membrane either by altering its affinity towards cholinergic ligands or by modulating its gating properties were to exert their pharmacological effect by interacting within the immediate vicinity of the AcChoR itself, and possibly via the highly immobilized lipids reported here. We would like to thank Mrs. Annelies Zechel for her excellent technical assistance and W. Maschke for the computer programs for data manipulation. 1. Barrantes, F. J. (1975) Biochem. Biophys. Res. Commun. 62, 407-414. 2. Changeux, J.-P., Benedetti, L., Bourgeois, J.-P., Brisson, A., Cartaud, J., Devaux, P., Grunhagen, H. H., Moreau, M., Popot, J.-L., Sobel, A. & Weber, M. (1976) Cold Spring Harbor Symp.

Quant. Biol. 40,211-230. 3. Hucho, F., Bandini, G. & Suarez-Isla, B. A. (1978) Eur. J. Biochem. 83, 335-340. 4. Ross, M. J., Klymkowsky, M. W., Agard, D. A. & Stroud, R. M. (1977) J. Mol. Biol. 116,635-659. 5. Axelrod, D., Ravdin, P., Koppel, D. E., Schlessinger, J., Webb, W. W. & Elson, E. L. (1976) Proc. Natl. Acad. Sci. USA 73, 4594-4598. 6. Rousselet, A. & Devaux, P. F. (1977) Biochem. Biophys. Res. Commun. 78,448-454. 7. Changeux, J.-P. & Danchim, A. (1977) Nature (London) 264, 705-712. 8. Jost, P. C., Griffith, 0. H., Capaldi, R. A. & Vanderkooi, G. (1973) Proc. Nati. Acad. Sci. USA 70,480-484. 9. Hesketh, T. R., Smith, G. A., Houslay, M. D., McGill, K. A., Birdsall, N. J. M., Metcalfe, J. C. & Warren, G. B. (1976) Biochemistry 15, 4145-4151. 10. Bienvenue, A., Rousselet, A., Kato, G. & Devaux, P. F. (1977) Biochemistry 16, 841-848. 11. Cohen, J. B., Weber, M., Huchet, M. & Changeux, J.-P. (1972) FEBS Lett. 26,43-47. 12. Olsen, R. W., Meunier, J.-C. & Changeux, J.-P. (1972) FEBS Lett. 28,96-100. 13. Rigler, R., Rabl, C. R. & Jovin, T. M. (1974) Rev. Sci. Instr. 45,

580-588. 14. Knowles, P. F., Marsh, D. & Rattle, H. W. E. (1976) Magnetic Resonance of Biomolecules (Wiley, London), pp. 185-187,

189-197,258-271. 15. Smith, I. C. P., Schreier-Muccillo, S. & Marsh, D. (1976) in Free Radicals in Biology, ed. Pryor, W. A. (Academic, New York), Vol. 1, pp. 149-197. 16. Freed, J. H. (1976) in Spin Labeling, ed. Berliner, L. J. (Academic, New York), chapt. 3.

Biophysics: Marsh and Barrantes 17. Marsh, D., Watts, A., Maschke, W. & Knowles, P. F. (1978) Biochem. Biophys. Res. Commun. 81,397-402. 18. Griffith, 0. H., Dehlinger, P. J. & Van, S. P. (1974) J. Membr. Biol. 15, 159-192. 19. Lehrer, S. S. (1971) Biochemistry 10,3254-3263. 20. Barrantes, F. J. (1976) Biochem. Biophys. Res. Commun. 72, 479-488. 21. Schindler, H. & Seelig, J. (1973) J. Chem. Phys. 59, 18411850. 22. Schindler, H. & Seelig, J. (1974) J. Chem. Phys. 61, 29462949. 23. Trauble, H. & Sackmann, E. (1972) J. Am. Chem. Soc. 94, 4499-4510. 24. Scandella, C. J., Devaux, P. F. & McConnell, H. M. (1972) Proc. Nati. Acad. Sci. USA 69,2056-2060.

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25. Stier, A. & Sackmann, E. (1973) Biochim. Biophys. Acta 311, 400-408. 26. Trauble, H. & Overath, P. (1973) Biochim. Biophys. Acta 307, 491-512. 27. Oesterhelt, D. & Stoeckenius, W. (1971) Nature (London) New Biol. 233, 149-152. 28. Chignell, C. F. & Chignell, D. A. (1975) Biochem. Biophys. Res. Commun. 62, 136-143. 29. Schreier-Muccillo, S., Marsh, D. & Smith, I. C. P. (1976) Arch. Biochem. Biophys. 172, 1-11. 30. Barrantes, F. J. (1978) J. Mol. Biol., in press. 31. Morrisett, J. D., Pownall, H. J. & Gotto, A. M. (1975) J. Biol. Chem. 250,2487-2494.

Immobilized lipid in acetylcholine receptor-rich membranes from Torpedo marmorata.

Proc. Natl. Acad. Sci. USA Vol. 75, No. 9, pp. 4329-4333, September 1978 Biophysics Immobilized lipid in acetylcholine receptor-rich membranes from...
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