ELSE V IE R Molecular Brain Research 27 (1994) 333-336

MOLECULAR BRAIN

RESEARCH

S h o r t c o m m u n i c a t i o n

Correlation between [125I]calmodulin binding and lipid fluidity in synaptic plasma membranes: effects of ethanol

and other short-chain alcohols

Z a f a r I q b a l **, P a u l Y. Sze *

Department of Pharmacology and Molecular Biology, The Chicago Medical School, North Chicago, 1L 60064-3095, USA

Accepted 13 September 1994

Abstract

Ethanol inhibits [a25I]calmodulin binding to synaptic plasma membranes from rat brain, and this inhibition is correlated in a conentration-dependent manner with the increase of membrane fluidity, as determined by diphenylhexatriene fluorescence polarization. Moreover, several short-chain alcohols that increase membrane fluidity are also effective inhibitors of [azSI]calmodulin binding. These data support the notion that ethanol inhibits calmodulin binding by increasing lipid fluidity of the synaptic membranes.

Keywords: Ethanol; Alcohol; Alcoholism; Calmodulin; Membrane fluidity; Synaptic plasma membrane

Ethanol produces extensive alterations in neuronal plasma membrane function, and the list includes adenylate cyclase [8], phospholipase A 2 [11], (Na +, K÷)-activated ATPase [13], CaZ+-activated ATPase [10], voltage-dependent Na ÷ and Ca 2÷ channels [6,14], opiate receptors [29], fl-adrenergic receptors [30], 5- HT 3 receptors [15], nicotinic ACh receptors [1], GABA A receptors [19], and two types of glutamate receptors: NMDA and kainate receptors [7,31]. The mechanism underlying these diverse biochemical changes appears intriguing. To date, there is no evi- dence that ethanol, a simple polar aliphatic molecule, acts as a selective effector for particular proteins. For more than a decade, the notion has been pursued that ethanol acts on plasma membranes primarily by in- creasing membrane fluidity [2,21,24], although the gen- erality of this mechanism has been questioned [4,22]. We have recently shown that ethanol in vitro inhibits calmodulin (CAM) binding to synaptic plasma mem- branes (SPM) from rat brain, and that in animals

* Corresponding author. Fax: (1) (708) 578-3268. ** Present address: Department of Neurology, Northwestern Uni-

versity Medical School, Chicago, IL 60611-3008, USA.

0169-328x/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0169-328X(94)00214-2

chronically treated with ethanol, the SPM are more resistant to the inhibitory effect of ethanol on CaM binding [26]. In Arrhenius analysis, CaM binding to SPM exhibits a biphasic function in temperature-de- pendency [9] and ethanol lowers the transition temper- ature in the biphasic function [26], suggesting that ethanol inhibits CaM binding by increasing lipid fluid- ity of the membranes. The present study is aimed to provide a direct correlation between the alteration of membrane fluidity and the inhibition of CaM binding.

SPM were prepared by the procedure described previously [9,26]. Male Sprague-Dawley rats (180-200 g) were supplied by a commercial breeder (Sasco King Animal Labs., Oregon, WI). Briefly, the cerebral cortex was homogenized in 5 vol. of 0.32 M sucrose/10 mM Tris-HCl, pH 7.4. After separation of nuclei and other cellular debris, the P2 pellet was resuspended in the homogenization buffer and placed on a discontinuous gradient of 0.85 M, 1.0 M, and 1.2 M sucrose. After centrifuging at 90,000 × g for 90 min, the synaptosomal fraction was removed from the interface between the 1.0 M and 1.2 M sucrose, diluted with 4 vol. of 0.25 M sucrose/10 mM Tris-HCl, pH 7.4, and pelleted. The synaptosomes were lyzed by resuspending the pellet in 10 mM Tris-HC1, pH 7.4. The osmotically lyzed synap-

334 Z. lqbal, P.Y. Sze / Molecular Brain Research 27 (1994) 333-336

tosomes were layered on a discontinuous sucrose gradi- ent (0.85 M, 1.0 M, and 1.2 M) and centrifuged at 90,000 × g for 90 min. The band containing the SPM fraction at the interface between 1.0 and 1.2 M sucrose was removed and diluted with 5 vol. of 10 mM Tris-HCl, pH 7.4. SPM were then pelleted at 15,000 × g for 20 rain. The purity of the SPM preparations was moni- tored by 5'-nucleotidase, a plasma membrane marker, as previously described [28].

Equilibrium binding of [t25I]CaM to SPM was deter- mined by the precedure used in our previous studies [9,26]. SPM were washed twice with 2 mM EGT A (in 50 mM Tris-HC1, pH 7.4) to deplete endogenously bound CaM. The EGTA-washed membranes (10-12 /~g protein) were equilibrated with ethanol (or another alcohol) by preincubation at 37°C for 15 min in 190 p.1 of binding buffer (50 mM Tris-HC1, pH 7.4, 0.1 mM CaC12, 2 mM dithiothreitol, and 2 mg/ml bovine serum albumin). [125I]CaM (87 mCi/mg; NEN Research Products, Boston, MA) in 10 Izl was added to produce a final concentration of 50 nM. The binding of the labeled ligand to the membranes was allowed to reach equilibrium at 37°C (15 min). The membranes were separated by filtration on a Whatman G F / C filter and washed twice with 3 ml of ice-cold binding buffer. Radioactivity bound to the membranes was deter- mined. Non-specific binding was determined in the presence of 500-fold unlabeled CaM. Radioactivity due to non-specific binding was less than 5% of total bind- ing.

Lipid fluidity of the membranes was assessed by fluorescence polarization of a lipid probe, 1,6-di- phenyl-l ,3,5-hexatriene (DPH), according to the method of Shinitzky and Barenholz [23]. This method has been extensively used in previous studies to deter- mine the effects of ethanol on membrane fluidity [5,6,12,20]. SPM were suspended in 3 ml of 50 mM Tris-HCl, pH 7.4 (30 /xg /ml ) and maintained at 37°C. A 1.5 /xl aliquot of DPH (Molecular Probes, Eugene, OR), dissolved in tetrahydrofuran, was added to the membrane suspension, yielding a fluorophore concen- tration of 0.5/xg/ml. The degree of fluorescence polar- ization was recorded when the steady state was reached (20 min with constant stirring at 37°C). An SLM 8000 spectrofluorometer (SLM Instruments, Urbana, IL) was used to measure fluorescence intensity parallel (Iii) and perpendicular ( I x ) to the polarization phase of the excitation beam. The excitation wavelength was 360 nm and the emission wavelength 430 nm. The degree of fluorescence polarization (P ) was calculated accord- ing to the following equation [23]:

P = (Ill + I±)/(I,l- I±) Ethanol (or another alcohol) was then added, and the degree of fluorescence polarization was again deter- mined when polarization reached a new steady state.

r"

i:5

0 "6 c: 0

:.E c'-

100

80

60

40

20

/(300)

{150) F (200)

(100) e /

/ / • (25)

. . . . . . . . i . . . . . . . .

2 5 10 20 50 100

Changein DPH Fluorescence Polarization (-AP x l 0 3 )

Fig. 1. Correlation between the inhibition of [1251]CAM binding (%) and the change in fluorescence polarization ( - zIP × 103) at various ethanol concentrations. Ethanol concentration (raM) for each value is indicated by the number in parenthesis. The data on % inhibition and - zIP, respectively, are as follows (mean + S.E.M. from 6 deter- minations): at 25 mM ethanol: 17.4-+0.5%, 0.0022_+0.0001; at 50 raM: 24.5_+0.9%, 0.0034+0.0002; at 100 mM: 45.0_+ 1.5%, 0.0064_+ 0.0003; at 150 mM: 53.0_+2.5%, 0.0102_+0.0005; at 200 raM: 60.4_+ 2.5%, 0.0114 _+ 0.0003; at 300 mM: 68.4_+ 2.9%, 0.0182_+ 0.0006. Con- trol value for [125I]CaM binding (without ethanol inhibition) is 74.5 _+ 2.2 pmol/rng protein. See text for statistical analysis of the linear correlation of the points.

The decrease of fluorescence polarization (-AP) after the addition of ethanol was used as a measure of disordering of the membranes [5,6,12,20].

Fig. 1 shows the inhibition of [~2sI]CaM binding to SPM and the reduction of DPH fluorescence polariza- tion by ethanol in the concentration range of 25-300 raM. At 25 mM ethanol, the inhibition of [125I]CaM binding was marginal (17%), but the inhibition became substantial (25%) at 50 mM ethanol. At 100 raM, ethanol inhibited the binding by 45%. When ethanol reached 300 mM, the inhibition was 68%. These data on the inhibition of [lzSI]CaM binding as a function of ethanol concentration are consistent with those re- ported previously [26]. Ethanol decreased fluorescence polarization of SPM-bound DPH (i.e., increased mem- brane disorder) in a similar concentration-dependent manner. In the concentration range of 25-300 raM, the decrease of DPH fluorescence polarization by ethanol was 0.0022 at 25 mM and 0.0182 at 300 mM. These data on the increase of membrane disorder as a func- tion of ethanol concentration are in good agreement with those reported in several earlier studies using similar DPH fluorescence polarization [5,6,20] or elec- tron paramagnetic resonance (EPR) spectrometry [3,16,20]. In the log-log plot in Fig. 1 where the de- crease of DPH fluorescence polarization is plotted versus the inhibition of [t25I]CaM binding, linear re- gression analysis gives a correlation coefficient of 0.983, with a standard error of estimate of 0.049. Clearly,

Z. lqbal, P.Y. Sze / Molecular Brain Research 27 (1994) 333-336 335

iT) "0 rn

t~ 0 "5 t-- 0

t-" _=

100

80

60

40

20

(1-BuOH)~e (2-PeOH)

(2.BuOH) y / " (iso-BuOH)

(1 .PrOH) . ~

~ (t-BuOH)

o.,

, i , . . . . . , i , , , , , , ,

2 5 10 20 50 100 Relative AP

Fig. 2. Correlation between the inhibition of [125I]CaM binding (%) and the relative change in fluorescence polarization (Ap) by various alcohols (all 50 raM). Relative Ap is defined as Ap relative to the value of ethanol. The data on % inhibition and - AP~ respectively, are as follows (mean+S.E.M. from 6 determinations): ethanol (EtOH): 25.2+ 1.0%, 0.0035 +0.0002; 2-propanol (2-PrOH): 31.2+ 1.3%, 0.0055+0.0002; t-butanol (t-BuOH): 38.8:1:2.0%; 0.0074+ 0.0003; 1-propanol (1-PrOH): 52.5___ 2.3%; 0.0104+0.0005; 2-butanol (2-BuOH): 62.3+3.1%, 0.0202_+0.0008; i-butanol (&o-BuOH); 70.6 + 2.5%; 0.0285 -+ 0.0014; 1-butanol (1-BuOH): 77.4 + 2.8%, 0.0337 + 0.0016; 2-pentanol (2-PeOH): 81.9_+ 3.0%, 0.0617 + 0.0031. See Fig. 1 for control value for [125IlCaM binding (without alcohol inhibition). Statistical analysis of the linear correlation of the points is provided in the text.

there is a correlation between ethanol inhibition of [125I]CAM binding and ethanol-induced membrane dis- order.

A number of aliphatic alcohols with 3-6 carbons are known to increase membrane disorder in SPM, and the membrane disordering potency of these short-chain alcohols is higher than that of ethanol because of their increased lipophilicity [17]. If the inhibition of [t25I]CaM binding by ethanol is consequent to the perturbation of the membranes, these short-chain alco- hols should be potent inhibitors of [125I]CAM binding. This was found to be the case. Fig. 2 summarizes the effects of seven water-soluble short-chain alcohols on the inhibition of [125I]CAM binding and the reduction of DPH fluorescence polarization, as compared with those of ethanol. At 50 mM, ethanol, the least potent of the group in the membrane disordering effect, de- creased DPH fluorescence polarization by 0.0034, whereas 2-pentanol, the most potent of the group, decreased the fluorescence polarization by 0.0617, an 18-fold range between the two alcohols. The mem- brane disordering potency of the alcohols was com- pared as follows: 2-pentanol > 1-butanol >/-butanol > 2-butanol > 1-propanol > t-butanol > 2-propanol > ethanol. These data obtained by DPH fluorescence polarization are in good agreement with those obtained previously by EPR spectrometry [17]. Of particular note is the effectiveness of the seven short-chain alco- hols as inhibitors of [125I]CAM binding. Ethanol at 50

mM inhibited [125I]CAM binding by 25%, whereas 2- pentanol at the same concentration inhibited the bind- ing by as much as 82%. It is apparent from Fig. 2 that the effectiveness of the alcohols in inhibiting binding parallels the potency of these alcohols in disordering the membranes. In the log-log plot where the decrease of DPH fluorescence polarization (relative to that by ethanol) is plotted versus the inhibition of [125I]CAM binding, linear regression analysis gives a correlation coefficient of 0.969, with a standard error of estimate of 0.052.

The results of our study demonstrate a correlation between ethanol inhibition of [t25I]CaM binding and ethanol-induced membrane fluidization over a wide range of ethanol concentrations. More importantly, several other short-chain alcohols known to disorder membranes were used to manipulate membrane fluid- ity, and the increase of membrane fluidity by these alcohols was also found to produce inhibition of [125I]CAM binding. The magnitude of the inhibition is again well correlated with the potency of these alcohols in fluidizing the membranes. These results, taken to- gether with the data from Arrhenius analysis of [125I]CaM binding [26], provide evidence that ethanol inhibits [~25I]CaM binding to SPM by increasing lipid fluidity of the membranes.

Fluidization of SPM by ethanol and other short- chain alcohols has been correlated with the potency of these alcohols in inducing sleep in mice [17]. It is therefore relevant to note that the inhibition of CaM binding following membrane fluidization by ethanol is also an event correlated with the hypnotic action of ethanol. We have found that opposite to the inhibition by ethanol, CaM binding to SPM is enhanced by gluco- corticoids, and this direct action of steroids on synaptic membranes appears to involve the decrease of mem- brane fluidity [27]. In a behavioral study, co-adminis- tration of glucocorticoids in mice antagonizes the seda- tive action of ethanol, as shown by the reduction of sleep time following a hypnotic dose of ethanol [25]. Thus, the antagonistic action of glucocorticoids on the induction of sleep by ethanol is consistent with their opposite effects on CaM binding in synaptic mem- branes.

As mentioned in the introductory remarks, ethanol produces extensive alterations in neuronal membrane function, ranging from the activities of membrane- bound enzymes to the activities of ion channels and neurotransmitter receptors. To date, bilayer fluidiza- tion offers a plausible mechanism whereby the action of a simple two-carbon molecule in producing such diverse biochemical alterations can be explained. The question has recently been raised as why certain mem- brane-bound proteins, such as GABA A receptors and NMDA receptors, are affected by low concentrations of ethanol (less than 50 mM), whereas other mem-

336 Z. lqbal, P.Y. Sze / Molecular Brain Research 27 (1994)333-336

brane-bound proteins, such as voltage-dependent Ca 2 + channels, are affected only by higher conentrations of ethanol [4]. These differential responses to low and high concentrations of ethanol do not necessarily pre- clude membrane fluidization as the underlying mecha- nism. The bilayers of biomembranes consist of hetero- geneous types of phospholipids, and different phospho- lipids may have different sensitivity to the fluidizing effect of ethanol. For example, in artificial membranes composed of a single class of phospholipids, egg phos- phatidylcholine, ethanol as low as 0.4-1.6 mM was found to fluidize the lipid membranes [18], although no global alteration of lipid fluidity can be detected in whole biomembranes at these concentrations of ethanol. Therefore, it is possible that the responsivity of a membrane-bound protein to ethanol depends upon the type of lipids present in the membrane microenvi- ronment. It is also possible that membrane fluidity may not directly affect the activity of certain membrane- bound proteins, particularly those that are subjected to complex biochemical regulation, such as receptor-gated ion channels. Rather, the alteration of membrane flu- idity may perturb regulatory events that in turn affect the activity of these membrane-bound proteins. In this regard, the inhibitory effect of ethanol on membrane binding of CaM is of particular interest.

This study was supported by USPHS Grant AA07230. The technical assistance of Mary Balmer and Hanna Sidorowicz is acknowledged.

[1] Forman, S.A., Righi, D.L. and Miller, K.W., Ethanol increases agonist affinity for nicotinic receptors from Torpedo, Biochim. Biophys. Acta, 987 (1989) 95-103.

[2] Goldstein, D.B., The effects of drugs on membrane fluidity, Annu. Ret,. Pharrnacol. Toxicol., 24 (1984) 43-64.

[3] Goldstein, D.B., Chin, J.H. and Lyon, R.C., Ethanol disordering of spin-labeled mouse brain membranes: correlation with genet- ically determined ethanol sensitivity, Proc. NatL Acad. Sci. USA, 79 (1982) 4231-4233.

[4] Gonzales, R.A. and Hoffman, P.L., Receptor-gated ion chan- nels may be selective CNS targets for ethanol, Trends Pharma- col. Sci., 12 (1991) 1-3.

[5] Harris. R.A., Baxter, D.M., Mitchell, M.A. and Hitzemann, R.J., Physical properties and lipid composition of brain mem- branes from ethanol tolerant-dependent mice, Mol. Pharmacol., 25 (1984) 401-409.

[6] Harris, R.A. and Bruno, P., Effects of ethanol and other intoxi- cant-anesthetics on voltage-dependent sodium channels of brain synaptosomes, J. Pharmacol. Exp. Ther., 232 (1985) 401-406.

[7] Hoffman, P.L., Grant, K.A., Snell, LD., Reinlib, L., lorio, K. and Tabakoff, B., NMDA receptors: role in ethanol withdrawal seizures, Ann. N.Y. Acad. Sci., 654 (1992) 52-60.

[8] Hoffman, P.L. and Tabakoff, B., Ethanol and guanine nu- cleotide binding proteins: a selective interaction, FASEB J., 4 (1990) 2612-2622.

[9] Iqbal, Z. and Sze, P.Y., [12~I]Calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and Arrhenius analy- sis, Neurochem. Res., 18 (1993)897-905.

[10] Iqbal Z. and Sze, P.Y., Ethanol modulates calmodulin-depend- ent CaZ+-aetivated ATPase in synaptic plasma membranes, Neurochem. Res., 19 (1994) 475-482.

[11] John, G.R., Littleton, J.M. and Nhamburo, P.T., Increased activity of Ca2+-dependent enzymes of membrane lipid metabolism in synaptosomal preparations from ethanol- de- pendent rats, J. Neurochem., 44 (1985) 1235-1241.

[12] Johnson, D.A., Lee, N.M., Cooke, R. and Lob, H.H., Ethanol- induced fluidization of brain lipid bilayers: required presence of cholesterol in membranes for the expression of tolerance, Mol. PharmacoL, 15 (1979) 739-749.

[13] Levental, M. and Tabakoff, B,, Sodium-potassium activated adenosine triphosphatase activity as a measure of neuronal membrane characteristics in ethanol-tolerant mice, J. Pharma- col Exp. Ther., 212 (1980) 315-319.

[14] Littleton, J., Little, H. and Laverty, R., Role of neuronal cal- cium channels in ethanol dependence: From cell cultures to the intact animals, Ann. N.Y. Acad. Sci., 654 (1992) 324-334.

[15] Lovinger, D.M. and White, G., Ethanol potentiation of 5-hy- droxytryptamine 3 receptor-mediated ion current in neuroblas- toma cells and isolated adult mammalian neurons, MoL Phar- macol., 40 (1991) 263-270.

[16] Lyon, R.C. and Goldstein, D.B., Changes in synaptic membrane order associated with chronic ethanol treatment in mice, Mol. PharmacoL, 23 (1983) 86-91.

[17] Lyon, R.C., McComb, J.A., Schreurs, J. and Goldstein, D.B., A relationship between alcohol intoxication and the disordering of brain membranes by a series of short-chain alcohols, J. Pharrna- col. Exp. Ther., 218 (198t) 669-675.

[18] Michaelis, E.K., Chang, H.H., Roy, S., McFaul, J.A., and Zim- brick, J.D., Ethanol effects on synaptic glutamate receptor func- tion and on membrane lipid organization, Pharmacol. Biochem. Behav., 18 (1983) 1-6.

[19] Montpied, P., Morrow, A.L., Karanian, J.W., Ginns, E.I., Mar- tin, B.M., and Paul, S.M., Prolonged ethanol inhalation de- creases y-aminobutyric acid A receptor a subunit mRNAs in the rat cerebral cortex, Mol. Pharmacol., 39 (1991) 157-163.

[20] Perlman, B.J. and Goldstein, D.B., Genetic influences on the central nervous system depressant and membrane-disordering actions of ethanol and sodium valproate, Mol. Pharmacol., 26 (1984) 547-552.

[21] Rubin, E. and Rottenberg, H., Ethanol-induced injury and adaptation in biological membranes, Fed. Proc., 41 (1982) 2465- 2471.

[22] Samson, H.H. and Harris, R.A., Neurobiology of alcohol abuse, Trends Pharmacol. Sci., 13 (1992) 206-211.

[23] Shinitzky, M. and Barenholz, Y., Fluidity parameters of lipid regions determined by fluorescence polarization, Biochim. Bio- phys. Acta, 515 (1978) 367-394.

[24] Sun, G.Y. and Sun, A.Y., Ethanol and membrane lipids, Alco- hol Clin. Exp. Res., 9 (1985) 164-180.

[25] Sze, P.Y., Glucocorticoids antagonize the sedative action of ethanol in mice, Pharmacol. Biochem. Behau., 45 (1993) 991-993.

[26] Sze, P.Y. and lqbal, Z., Ethanol modulates [125I]calmodulin binding to synaptic plasma membranes from rat brain, J. Phar- macol. Exp. Ther., 268 (1994) 1183-1189.

[27] Sze, P.Y. and Iqbal, Z., Glueocorticoid actions on synaptic plasma membranes: Modulation of [125I]calmodulin binding, J. Steroid Biochem. Mol. Biol., 48 (1994) 179-186.

[28] Sze, P.Y. and Towle, A.C., Developmental profile of glucocorti- coid binding to synaptic plasma membrane from rat brain, Int. J. Deu. Neurosci., 11 (1993) 339-346.

[29] Tabakoff, B. and Hoffman, P.L., Alcohol interactions with brain opiate receptors, Life Sci., 32 (1983) 197-204.

[30] Valverius, P., Hoffman, P.L. and Tabakoff, B., Effect of ethanol on mouse cerebral cortical /3-adrenergic receptors, Mol. Phar- macoL, 32 (1987) 217-222.

[31] Weigh, F.F., Lovinger, D.M., White, G. and Peoples, R.W., Alcohol and anesthetic actions on excitatory amino acid- activated ion channels, Ann. N.Y. Acad. Sci., 625 (1991) 97-107.