Water and Nonelectrolyte Permeability of Lipid Bilayer Membranes ALAN FINKELSTEIN From the Departments of Physiology, Neuroscience, and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461
ABST RAC T Both the permeability coefficients (Pd's) through lipid bilayer membranes of varying composition (lecithin [L], lecithin:cholesterol [LC], and sphingomyelin:cholesterol [SC]) and the n-hexadecane:water partition coefficients (Khe's) of HzO and seven nonelectrolytes (1,6 hexanediol, 1,4 butanediol, n-butyramide, isobutyramide, acetamide, formamide, and urea) were measured. For a given membrane composition, Pd/DKhe (where D is the diffusion constant in water) is the same for most of the molecules tested. There is no extraordinary dependence of Pd on molecular weight; thus, given Pa(acetamide), Pa(1,6 hexanediol) is correctly predicted from the Khc and D values for the two molecules. The major exceptions are H20, whose value of Pa/DKhc is about 10-fold larger, and urea, whose value is about 5-fold smaller than the general average. In a "tight" membrane such as SC, Pd(n-butyramide)/Pa(isobutyramide) = 2.5; thus this bilayer manifests the same sort of discrimination between branched and straight chain molecules as occurs in many plasma membranes. Although the absolute values of the Pa's change by more than a factor of 100 in going from the tightest membrane (SC) to the loosest (L), the relative values remain approximately constant. The general conclusion of this study is that H~O and nonelectrolytes cross lipid bilayer membranes by a solubilitydiffusion mechanism, and that the bilayer interior is much more like an oil (fi la Overton) than a rubber-like polymer (h ia Lieb and Stein). INTRODUCTION
T o account for nonelectrolyte permeability, 1 physiologists have generally accepted Collander a n d B~irlund's view o f the plasma m e m b r a n e as a "lipoidal sieve" (see H 6 b e r , 1945). This concept was developed to reconcile the permeability increase o c c u r r i n g in h o m o l o g o u s series o f lipophilic molecules (such as alcohols a n d weak acids) u p o n addition o f each methyl g r o u p , with the p e r m e a bility decrease o f small hydrophilic molecules (such as urea, acetamide, a n d ethylene glycol) with increasing molecular weight. T h e plasma m e m b r a n e was viewed as a lipoidal matrix (nowadays a lipid bilayer), t h r o u g h which molecules pass via a solubility-diffusion m e c h a n i s m , a n d in which are sprinkled a q u e o u s pores that select a m o n g nonelectrolytes according to size. Recently, this "accepted" view has been challenged by Lieb a n d Stein (1971), I exclude in these considerations obviously specialized transport processes that, for e x a m p l e , discriminate between L and D a m i n o acids or recognize subtle structural differences a m o n g sugars. THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 68, 1976 " pages 127-135
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who attempt to do away entirely with the pore concept by viewing the lipoidal region not as a viscous fluid such as olive oil, but r a t h e r as a polymeric network such as rubber. Because the diffusion coefficient in such a p o l y m e r can show a striking d e p e n d e n c e on molecular weight, they a r g u e that the decrease in permeability with size seen a m o n g polar nonelectrolytes is a consequence o f this r a t h e r than o f the presence o f separate aqueous pathways. Artificial lipid bilayer m e m b r a n e s offer the possibility o f directly m e a s u r i n g the nonelectrolyte permeability o f the structure that is the lipoidal backbone o f cell m e m b r a n e s . But in spite o f n u m e r o u s investigations o f ion permeability (by electrical means) and several studies o f water permeability, little attention has been paid to nonelectrolyte permeability. Technical problems associated with either too small or too large permeability coefficients have restricted the a m o u n t o f usable data and obscured their interpretation. I u n d e r t o o k the present investigation for two reasons. T h e first, the subject o f this p a p e r , concerns the issues raised above. How well does the solubilitydiffusion model explain nonelectrolyte permeability o f lipid bilayers? Is the bilayer interior a liquid h y d r o c a r b o n milieu, or is it a rubber-like polymeric structure? Studies o f water permeability indicate the f o r m e r (Finkelstein a n d Cass, 1968), but o t h e r permeability data are n e e d e d to e n s u r e that water is not a fortuitous exception. T h e second reason concerns the action o f antidiuretic h o r m o n e on water permeability and is considered in the succeeding paper. MATERIALS
AND
METHODS
Permeability Measurements Membranes were formed by the brush technique of Mueller et al. (1963) at either 25°C 2° or 14.5°C -+ 0.5 ° across a 0.8-ram 2 hole in a 125-/~m thick Teflon partition separating two Lucite chambers. Magnetic fleas stirred both chambers continuously during the course of an experiment. Each chamber contained 3.0 ml of unbuffered (pH ~5.6) 0.1 M NaCI. The procedure for measuring the permeability coefficient (Pd) of a molecule was that described by Holz and Finkelstein (1970); in some experiments, both T H O and a ~4Clabeled solute were used, so that Pd's for both water and solute were obtained on the same membrane. An unstirred layer thickness of 122 ~tm was calculated from the observed value of 8.2 × 10-4 cm/s for Pd (n-butanol), on the assumption that butanol is so permeant that its flux is completely determined by the unstirred layers on the two sides of the membrane (Holz and Finkelstein, 1970). This was used to correct all measured Pa values; the correction was generally trivial ( Pa(propionamide) on plain lecithin bilayers, thus making formamide even more anomalous. Neither our results nor those of Galluci et al. (1971) confirm this. (The discrepancy between the data of Poznansky et al. and those of Gailuci et al. and ours, is their much smaller values for Pa(other amides).) 7 This suggests that molecules of greater molecular weight and larger cross-sectional area will have disproportionately small values of Pa/(DKhc/6x), particularly in tight membranes such as SC. This has been confirmed for codeine.
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EFFECT OF LIPID COMPOSITION AND TEMPERATURE ON PERMEABILITY Pcl'S in lecithin bilayers (L) are about eightfold larger than in lecithin:cholesterol bilayers (LC), which in turn are about eightfold larger than in sphingomyelin:cholesterol bilayers (SC); Pn's in SC bilayers are about fivefold larger at 25 than at 14.5°C. Thus, the permeability coefficient of a given molecule can vary over a several 100-fold range, as a function of phospholipid composition, cholesterol content, and temperature; undoubtedly, even larger ranges could be spanned with a greater variety of lipid mixtures and temperatures. The effects of cholesterol and temperature on the water permeability of bilayer membranes have been noted earlier (Finkelstein and Cass, 1967; Price and Thompson, 1969; Redwood and Haydon, 1969). (The permeability decrease occurring in SC membranes between 25 and 14.5°C is about twice that occurring in lecithin:cholesterol membranes for a 10°C decrement [Price and Thompson, 1969; Redwood and Haydon, 1969]. Possibly a phase transition exists near 15° in SC membranes.) The smaller Pn values in SC membranes compared to those in LC membranes is consistent with a previous observation o f a smaller Pa(H20) in sphingomyelin than in lecithin membranes (Finkelstein and Cass, 1968). Brockerhoff (1974) has predicted that sphingomyelin membranes should be less permeable than lecithin membranes, because of a belt of hydrogen bonds formed by the 3-hydroxy groups of the sphingosines. However, the longer mean chain length and the greater degree of saturation of bovine sphingomyelin than of egg lecithin may, in themselves, account for the lower permeability. In considering the effect of cholesterol, sphingomyelin vs. lecithin, or temperature reduction, the question arises: Do lower values of Pa result from lower diffusion constants in the membrane or smaller partition coefficients into the membrane? Cholesterol, for example, promotes "packing" of the lipid chains (Lecuyer and Dervichian, 1969). This could lead to lower permeabilities, through either an increased viscosity of the membrane interior (i.e. a reduction in diffusion constants) or a tightening of the membrane surface to penetration and solute accommodation (i.e. a reduction in partition coefficients). Probably both effects operate, but it is difficult to sort them out experimentally. (Studies such as those by Katz and Diamond [1974] on partition coefficients into lecithin liposomes are irrelevant to this problem. The partition coefficients needed are those into the hydrocarbon region of the bilayer; the partition coefficients determined by Katz and Diamond [1974] for polar nonelectrolytes relate to accumulation of these molecules in the polar regions near the head groups.)
SUMMARY The amount of space I have devoted to the "exceptions" to Overton's rule is disproportionate. Actually, the magnitude of these exceptions is relatively small. The main point is that the relative permeabilities of molecules up to the size of hexanediol are governed by the product of partition coefficients and free diffusion constants in bulk hydrocarbon. The absolute values of the permeability coefficients are scaled by the lipid composition. Even the anomalies (with the exception of the effect of branching), if one chooses to focus on them, remain relatively constant as the absolute values o f Pa change. Thus, the ratio of Pd (H~O) (which is anomalously high) to Pn(n-butyramide) changes only by about
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a f a c t o r o f 2 in g o i n g f r o m L C at 25 ° to SC at 14.5 °, w h e r e a s t h e a b s o l u t e v a l u e s o f t h e s e P a ' s c h a n g e by a f a c t o r o f 27 a n d 60, r e s p e c t i v e l y . I thank Regina Gluck for excellent technical assistance. This work was supported by NSF grant no. BMS 74-01139. Receivedfor publication 5 March 1976. REFERENCES
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