BIOCHIMICA ET BIOPHYSICA ACTA 451

BBa 75203

T H E I N T R A M E M B R A N E LOCATION OF ALCOHOL ANESTHETICS*

H E N R Y SCHNFI D E R

Division of Biology, National Research Council of Canada, Ottawa (Canada)

(Received August 5th, I968)

SUMMARY

In order to identify the chemical nature of the membrane receptor sites involved in general anesthesia the apparent free energy of binding of methylene groups of n-alcohols in anesthetic systems was evaluated and compared with data from model systems. I t was concluded tha t the sites m a y consist either of (i) the non-polar portions of lipids, (ii) a non-polar region of a lipoprotein made up of non-polar port ions of the protein and lipid moieties or (iii) protein. However, if the site consists only of protein it is possible tha t this material undergoes a ligand-induced conformation change on binding the anesthetic. Some reasons for the use of caution in employing t empera tu re effects to identify the chemical nature of the receptor sites are outlined.

INTRODUCTION

The chenfical nature of the site of membrane action of general anesthetics is unknown. I t has been suggested to consist of protein or a p ro t e in -wa te r interface 1 :~ or lipid 5-7. I t has been shown recently tha t there is a close correlation between the anesthetic properties of phenols, aliphatic alcohols and steroids with their ability to protect erythrocytes against hypotonic hemolysis ~ suggesting tha t the chemical nature of the site of action m a y be similar. To obtain more information about its nature the apparent lree energy of adsorption per methylene group on erythrocyte membranes and in various anesthetic systems was evaluated. I t is concluded tha t either a lipid, a lipoprotein or a protein site is compatible with the data. However, if the site contains protein, this material may undergo a ligand-induced conformational change on binding the alcohol.

METHODS

The free energy of adsorption per methylene group was evaluated assuming the extent of anesthesia or protection against hemolysis is directly proport ional to the number of molecules adsorbed at the sites responsible for these phenomena. The s tandard free energy of adsorption of the alcohols at these active sites, A F °, is ~

0 ~/F ° -- --2.303 R T log K -- --2.303 t~7[" log - - - - - - (i)

(i --O)a * N.R.C.C. Publication IO452.

Biochim. Biophys. Acla, 163 (1968) 451-458

4 5 2 H. SCHNEIDER

where K is the a lcoho l - recep tor affinity constant , a the ac t iv i ty of alcohol in t i le aqueous phase, 0 the fract ion of narcosis-act ive sites occupied b y adsorbate , R the gas cons tant and T the absolute t empera ture . The s t anda rd free energy of adsorp t ion per methy lene group, A F t ~, was taken as the difference in A F ° for two homoh)gues differing in length by a single CH 2 group, hence as

ai , l , I/"1 e.303 RT log . . . . (e)

ai

where ai and a i q refer to the ac t i v i t y of the i th and i th + z homologues, respect ively. "File s t anda rd s ta te for the ah;ohol in solut ion was t aken as ti le hyt)othet ical uni t 1hole fract ion solut ion which has ti le e n t h a l p y proper t ies of the infinitely di lute s',flution. Fo r the alcohol on tile membrane the s t anda rd s ta te was taken to be tha t where 0 equals I bu t where the adsorba te has the proper t ies it would possess at an ex t r eme ly low level of surface coverage. In pract ice A F t " was evalua ted , assuming the ac t i v i t y of the alcohol equaled its mole fraction, from the leas t -mean-square slope of the l inear por t ion of p lots of the logar i thm of the mole fract ion of alcohol required t() cause anesthes ia or to inhibi t hemolysis to a given ex ten t ",,erszts the number of carbon a toms of the alcohol.

The use of mole fract ion te rms ins tead of act ivi t ies in the above eva lua t ion seems to be ]ustifiable. For C,, to C: alcohols at the concent ra t ions of in teres t ac t iv i ty coefficients for the water in b ina ry water--alcc)hol soluti(ms are a ppa re n t l y I (ref. m), hence t ha t for the alcohol component is p robab ly also very close to I. Ac t iv i ty co- efficients are not avai lable fl)r the higher alc(flaols bu t can be es t imated as also being close to u n i t y if devia t ions from ideal behavior of such a l iphat ic alnt)hiphilic com- pounds are ascr ibed to dimer format ion resul t ing from hydrophob ic bonding u. On this basis, es t imat ing the free energy of d imer isa t ion as - -6oo cal/mole per CH,_, group le, the amoun t of d imer m a y be computed as less than I °o tha t of luononler. The va l id i ty of this calculat ion is suppor t ed by d a t a for the amount of dilner and quadra lner in solu t ions of sodium dodecanoate . At at mole % of 6.5' IO ~ (36 raM) at 25 ° less than I o/,,o of this conlp~mnd is ca lcula ted to be aggrega ted ~1. Most of the d a t a employed for the alcohols in the anes the t ic and e ry th rocy te hemolysis sys tems are for shor ter chain lengths :it even lower concentra t ions , hence devia t ions from idea l i ty due to s o l u t e - s o l u t e in teract ions m a y be expec ted to be even smaller.

The presence of salt in the aqueous phase of the biological sys tems would be expec ted to increase the ac t i v i t y coefficients of the alcohols over the values in pure water. Using solubi l i ty d a t a for Jz-alc(fl~ols in water and in Ringers solut ion at 3o'-" (ref. I3) ac t iv i ty coefficients in the presence of salt could be es t ima ted as being 4-3 °o higher for bu tanol and increasing to 9.7 % for heptanol . Since AFt" is computed from a slope which involves the logar i t lnn of alcohol ac t iv i ty , ignoring the effect of salt leads to an error of 5.4 % or less, a q u a n t i t y which is considered to be insignificant in the present analysis .

The above lne thod for eva lua t ing A F t has e lements in common with tha t ot MEvEI¢ AXl) HFMm 5. I t differs from others'4, ~'~ in choosing the s t anda rd s ta te of the anes the t ic to be the hypo the t i ca l mole fract ion un i ty solut ion ins tead of the pure l iquid or solid narcot ic . Using this l a t t e r procedure results in each alcohol having a different s t a n d a r d s ta te with the result tha t i n t e rp re t a t ion of compar isons among homologues m a y become somewhat ambiguous, pa r t i cu la r ly for the lower members .

]3iochim. Biophys. Acta, i63 (i968) 451-458

I N T R A M E M B R A N E LOCATION OF A L C O H O L A N E S T H E T I C S 453

For instance, using ethanol and n-butanol as examples, it becomes necessary to interpret in molecular terms the difference in free energy in taking ethanol from pure ethanol and n-butanol from pure butanol and transferring them to the membrane at the concentration at which narcosis occurs. This is difficult to do since the two alcohols are in different molecular environments when in their pure liquid states. The use of the mole fraction unity solution coupled with the evaluation of free energies for the non-polar portions only may obviate such difficulties to a large extent and also facilitates comparisons with model systems.

R E S U L T S

Several representative curves of the logarithm of alcohol concentration required to cause a given degree of anesthesia or to inhibit erythrocyte hemolysis to a given extent as a function of the number of carbon atoms are shown in Fig. I. They are characterised by a non-linear portion for the lower members, usually up to ~z-propanol or mbutanol followed by an apparently linear portion. In some instances a second non-linear portion appears with lz-octanol or ~z-nonanol as shown in Fig. IA for the inhibition of reflex responses in tadpoles. In terms of Eqn. 2 and the assumption that the physiological or anti-hemolytic effects observed depend on 0 this behavior means that initially A F t ° varies as chain length increases then assumes a constant value and subsequently may begin to vary again. A possible reason for the variation with

10-3

10 -4

z O

l0 -s <

z 0 I - tO-6

c~ u_

10 -7 O

"lI .

~lI %- \ , \

REFLEX RESPONSE IN TADPOLES

10 . 8

50% INHIBITION OF ERYTHROCYTE HEMOLYSIS

"\:\ I r

\

\

\

NOTNHwSAYyN APT I C

B

iO -~

10 +~

EO - 4

10 -5

10 .6

10 .7

I 2 3 4 5 6 7 8 9 I0 II 12 13 I 2 5 4 5 6 7 8 9

N U M B E R OF C A R B O N A T O M S IN A L C O H O L

Fig . I. : \ . L o g a l c o h o l c o n c e n t r a t i o n r e q u i r e d t o s u p p r e s s t h e r e f l ex r e s p o n s e in t a d p o l e s a n d t o p r o d u c e a r e l a t i v e i n h i b i t i o n of 5 ° % of h y p o t o n i c h e m o l y s i s o f h u m a n e r y t h r o c y t e s . T h e d a s h e d p o r t i o n s i n d i c a t e t h e r e g i o n s t a k e n t o b e n o n - l i n e a r . B. L o g a l c o h o l c o n c e n t r a t i o n r e q u i r e d t o s u p p r e s s t h e t r a n s m i s s i o n of n e r v e i m p u l s e s in c a t s t r a v e l l i n g along d i f f e r e n t p a t h w a y s . T h e d a s h e d p o r t i o n s i n d i c a t e t h e r e g i o n s t a k e n t o b e n o n - l i n e a r .

Biochim. Biophys. Acta, 163 (1968) 451 45 s

454 H . S C H N E I D E R

the lower alcohols could be the presence of head group effects tG-ls on tim free energy of alcohol uptake. Binding of the alcohol by the receptor sites may inw)lve removal, either partially or conlpletely, of water surrounding the aliphatic chain. The polar head group may perturb tim structure of water in its innnediate vicinity for a distance equivalent to that spanned by several CH., residues with a concomitant dependance of A F t ~ upon the distance between tim head group and lnethylene groups.

The A F t ° values obtained from the linear portions are summarised in Table i for thirteen anesthetic systems. A notable fact about these systems is their diversity since they include exanlples ranging from bacterial to maxnmalian tissues or intact organisms. The A F t ~ for the inhibition of hyp~tonic hemolysis by alcohols is also included in Table I. The average of the d i " t values for the erythrocyte and anesthetic systems together is --8I 5 cal per mole of CH e groups anti 813 cal/mole for the anesthetic systems alone. The values for the anesthetic systems range from --637 to -lO8O cal/mole.

The free energy of transfer per mole of CH,, groups from water to three different non-polar environments is shown in Table Ii. These values range from

-750 to --883 cal/mole. The temperature at which the anesthesia experiments were carried out is

T A B L E 1

I?RI'E ENERGY OI r BINDING AND STANI)ARD ERROR PER MOLE OF ME'['ItYLENI~; GROUPS

N o . d e n o t e s t h e m m ~ b e r of p o i n t s u s e d t o c o m p u t e t h e s lope .

@ , s t e m - - , I l:t ' N o . T e m p .

N a r c o s i s of f r o g x e n t r i c l e TM

N a r c o s i s of t o r t o i s e h e a r t 2° R e v e r s i b l e s u p p r e s s i o n of l u m i n e s c e n c e in b a c t e r i a ~° R e v e r s i b l e s u p p r e s s i o n of r e f l ex r e s p o n s e in t a d p o l e s ~° l m n l o b i l i s a t i o n of I d a r n a c l e n a t @ l i i '-'t S u p p r e s s i o n of r e s p o n s e of

f r o g g a s t r o c n e n l i u s m u s c l e t o e l e c t r i c a l s t i n m l u s 2° N a r c o s i s of ( ; o b i o . f l u v i a l i l i s 22 G u i n e a - p i g g u t c o n t r a c t i l i t y 2a R e v e r s i b l e i n h i b i t i o n of r e s p i r a t i o n of g o o s e e r y t h r o c y t e s 2° R e v e r s i b l e s u p p r e s s i o n of

s p o n t a n e o u s c o n t r a c t i o n s of i s o l a t e d f r o g h e a r t 2" P a r a m e c i u m m o b i l i t y 2a R e v e r s i b l e s u p t ) r c s s i o n of t r a n s m i s s i o n of n e r v e i i n p u l s e s in c a t s 2a

s y n a p t i c p a t h w a y n o n s y n a p t i c p a t h w a y

]<eve r s ib l e s u p p r e s s i o n of s p o n t a n e o u s m o v e m e n t in t a d p o l e s 20 6 - d a y - o M 2 - d a y - o l d

4 e - d a y - o l d 8 3 - d a y - o l d

I n h i b i t i o n of e r y t h r o c y t e h e m o l y s i s (ref. 8; P . SV=I~MAX, p e r s o n a l c o m m u n i c a t i o n ) :

25 o,,~ i n h i b i t i o n 5 o % i n h i b i t i o n 75 o,~ i n h i b i t i o n

* Ass l l l l l ed v a l u e .

(c(d)

924 t 77 5 20';* ~82 2 ~5 ° 857 !: 27 5 2o" bI 5 -- 81 O 20 °* 794 : 8 4 18.5 °*

753 ~ 18 4 I7 ° 748 :~ 55 4 15 :~ 722 ]: 15 5 33 :~ 72I ± 7 L 4 20°*

705 :g 4 5 20°* 637 :} 29 5 2 ° ° *

924 ~ i0 4 30o 7 ° 0 ~ 14 4 30o

1080 2 18 ° 931 2 18 ° 833 ± TO 5 18:~ 792 ± 48 3 18°

857 ± 35 IO 22 ° 8o9 :~ 8 IO 22 ° 8o3 ~ I i IO 22 °

l l i o c h i m . ]3io]Shys. A c t a , I63 ( i968) 45 I 458

INTRAMEMBRANE LOCATION OF ALCOHOl. ANESTHETICS 455

TABLE II

F R E E E N E R G Y O F T R A N S F E R O F M E T H Y L E N E G R O U P S T O N O N - P O L A R E N V I R O N M E N T S

Sys t em -- zJ F t ° (cal)

Adsorption at a light petroleum-water interface 25 82o Solubility of liquid hydrocarbons (pentane to octane) in water 26 883 ~ 23 Triolein-water partition coefficients of alcohols ~7 75 ° :I- 2o

"xl'g t N ]b .

2 0 ~

25 ~ 2 5 c

* Standard error.

shown in Table I. In most ins tances where it was not c i ted in the original reference i t was assumed equal to 2o ° . Because of the differences in t empe ra tu r e among some systems, the unce r t a in ty in others, and the fact t ha t AFt ° would be a funct ion of t empera tu re , a de ta i led s ta t i s t ica l analysis of the significance of the differences in AFt ° was considered to be inappropr ia te . However , it is no t e w or thy t ha t in two in- s tances where a s imilar t empe ra tu r e was employed, the suppression of nerve impulses in cats t rave l l ing b y different pa thways , the differences in AFt ° are significant at the 1 % level.

DISCUSSION

The A F t ° values in the anes thet ic and hemolys is sys tems are close to those in model sys tems where CH 2 groups are t ransfer red from water to non-polar environ- ments . This correla t ion suggests t ha t the non-po la r residues of the alcohols in the hemolysis and in the anes the t ic sys tems are also in a non-polar envi ronment . Fu r the r - more, the s imi la r i ty of A F t ° in the anes thet ic and hemolys is systems, the l a t t e r being a p p a r e n t l y a pure ly membrane phenomenon ~8, coupled with the corre la t ion between the anes thet ic and inhibi t ion of hemolysis effects of var ious compounds ~ suppor t s the view tha t anes thes ia involves membranes 29.

Since membranes genera l ly conta in apprec iable amoun t s of l ipid 3°, the non- polar site could consist of cholesterol and of the f a t t y acid residues of m e m b r a n e phosphat ides . Ano the r poss ib i l i ty is t ha t it consists of non-po la r residues of the pro te in and l ipid componen t s of a membrane l ipoprote in . A th i rd poss ib i l i ty is t ha t the site consists en t i re ly of pro te in and is, p resumably , made up p r imar i ly of non-polar side chains. In this l a t t e r ins tance it is possible to describe a feature the recep tor molecules could be expec ted to possess. Thus, in general , where A F t ° for the combina t ion of CH 2 groups with prote ins can be de te rmined or es t imated , the values are lower t han those in anes the t ic systems, ranging from abou t - -56o cal down to about --IOO cal (refs. 31-34 ). Larger values have been found: - -65o cal for c h y m o t r y p s i n - f a t t y acid t y rosy l ester in terac t ions aS, - -65o cal for l i poxygenase -a l ipha t i c alcohol in terac t ions al and --ILOO cal for hyd roca rbon b inding to f l - lactoglobulin A (ref. 36). However , chymot ryps in undergoes a conformat iona l change on combina t ion with inhib i tors 37 and this might occur also when f l- lactoglobulin combines wi th hydroca rbons a,~. Ali- pha t i c alcohols inhibi t l ipoxygenase b y a mechanism involving non-compe ta t ive inhibi t ion, hence there is the poss ib i l i ty here too of a l igand- induced conformat iona l change. The A F t ° in such coupled sys tems would be expec ted to conta in t e rms due

B i o c h i m . B i o p h y s . Ac ta , 163 (~968)45I -458

456 u. SCHNEIDER

to hydrophob ic in terac t ions as well as to ti le l inked conformat ion change ag. Thus, for A F t ° in the biological sys tems to lie in the range of - -637 to .... Io8o cal and the b inding site be loca ted on a protein, the pro te in could be expected to undergo a l igand- induced conformat ion change.

Since the A F t ° values indicate tha t the receptor sites have a re la t ive ly high affini ty for non-polar mater ia ls , gaseous general anesthet ics , which are essential ly non-polar mater ia ls , would be expec ted to b ind at these same sites and m a y possess their physiological effects for th is reason. If these sites involve only prote ins ti le general anes thet ics m a y cause t hem to undergo a conformat ion change as suggested by o ther workersa, ~°,~ and seems plausible for the a l iphat ic alcohols. The fact tha t such s t ruc tura l changes can occur has been shown in s tudies of g lobular p r o t e i n - anes the t ic interact ionsa, ~'.

Anesthet ics such as inert gases which do not form hydrogen bonds in aqueous solut ion have been suggested to act th rough the fo rmat ion of hydra tes . In contras t , polar mater ia l s such as alcohols which can forln hydrogen bonds were suggested to act in some other way ~. The present analysis, b y emphasis ing ti le apparen t l inear dependence of anes the t ic po t ency on chain length, suggests t ha t hydrogen bond forming abi l i ty , of the alcohols at any rate, is not of pr ime impor tance .

Because the A F t ° wtlues for alcohol methylene groups are compat ib le with at least three different s t ruc tures for the membrane binding sites it is evident t ha t free energy d a t a alone cannot be used to dis t inguish between them unequivocal ly . There- fore, the quest ion of whether or not the use ()f another t he rmodynamic pa ramete r , the en tha lpy ( A H ) , would provide grea ter d i scr imina t ion was considered. I t was con- c luded tha t there m a y be a l imi ta t ion in the use of this parameter . The reason is t ha t in using e n t h a l p y da t a a compar ison nmst be made between the effects of t empera tu re on ti le po teney of the anes the t ic in a bioh)gical sys tem on the one hand and on the effects of t empe ra tu r e on a model sys tem involving the anes thet ic on t i le other. This compar ison is based ()n the assumpt ion t ha t the re levant in terac t ions in the two sys tems are sufficiently similar. Oi l - -water pa r t i t ion ing of anesthet ics has 1)een used as a model in solnc instaneesl3, H and hydrot)hobic bonding in prote ins ~'~ m a y be considered as ano ther possibi l i ty. However, the en tha lpy of solut ion of non- polar c()mpounds in water , a process which has similar t he rmodynamic pa rame te r s to tha t for t ransfer or pa r t i t ion ing between a non-polar solvent and water, m a y change sign with t empera tu re . This occurs with benzene and bu tane 45, two lnoleeules s imilar in size to some general anesthet ics , a t about 2 6 , a t empera tu re where anesthes ia exper iments have been carr ied (rot a3,a4. Moreover, AH for hydrophobic bonding has been pred ic ted to change sign as t e lupera tu re increases ~',. The t empe ra tu r e a t which the change in sign occurs in a par t i cu la r sys tem m a y be expected to depend on the na tu re of the in termolecular in terac t ions involved. Therefore, in the case of non-polar anesthet ics the anes thet ic -receptor in terac t ions over a given t empe ra tu r e in te rva l m a y be genera l ly similar to those in a par t i cu la r model vet sufficiently different to make A H differ in sign.

in the case of polar molecules such as alcohols their amphiph i l i c i ty m a y intro- duce an addi t iona l complicat ion. Molecules of this sort m a y reside in the receptor site such t ha t the hydroxyl residue is in contac t with the aqueous phase while the hydroca rbon por t ion is immersed or 'bur ied ' in a non-polar region. The en tha lpy ot in terac t ion of an alcohol with the receptor would include a con t r ibu t ion from the

B iochim, t3iophys. A cta, l() 3 (1968) 45t-458

INTRAMEMBRANE LOCATION OF ALCOHOL ANESTHETICS 457

hydroxyl group and this quanti ty and its variation with temperature would have to be determined before the effects of temperature on anesthesia could be rationalised.

I t is noteworthy in connection with the general argument presented above that there is an instance where the potency of an anesthetic passes through a minimum at about 20 ° as temperature varies from 4 to 28 ° (butyl acetate innnobilisation of Barnac l e naup l i i ) "n.

A rigorous discussion of the differences in A F t ° among the various anesthetic systems must await identification of the chemical nature of the receptors, especially since it is conceivable that they need not be the same in every tissue or organisnl. However, it is noteworthy that if the site involves the non-polar portions of membrane lipids arranged in a lamellar array the differences could depend, in part, on the nature of the fa t ty acid residues of the phosphatides. This possibility arises from the fact that the interaction of phosphatides and cholesterol in monolayers depends on the fa t ty acid components of the former ~6. Consequently, the free energy of penetration of alcohols would also be expected to depend on their nature. Another possible reason for the differences is that A F t ° is probably a composite quanti ty containing contri- butions from changes induced in the membrane structure somewhat remote from the receptor site. For instance, if the binding site is a protein, the conformation change undergone by the protein may cause changes in the arrangement of the lipids associ- ated with it. Alternately, if the site consists of lipids, changes might be induced in nearby proteins.

ACKNOWLEI)GE3IENTS

The author offers his thanks to Dr. P. SEEMAN for helpful and stimulating dis- cussions and for permission to use unpublished data. Thanks are due also to Dr. GoI~I)ON C. K~ESHECK and Dr. ARNOLI) FROESE for critically reviewing the manu- script and to Dr. D. B. WETLAUFER for helpful discussions.

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Biochim. Biophys. Acta, 163 (lq68) 451-458

4 5 ~ H. SCHNEIDEI{

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