Biophysical properties of phospholipids. II. Permeability of phosphatidylserine liquid crystals to univalent ions.

A recent study of the diffusion of univalent ions across lamellae of swollen phosphatidylcholine "liquid crystals" demonstrated the potentialities of such structures as model phospholipid membranes of well defined molecular structure and composition 1. As a continuation of these studies, it was thought that phosphatidylserine, with its additional functional groups and net negative charge, might show additional features even more closely analogous to those found in biological membranes. Indeed several investigators have recently produced evidence implicating phosphatidylserine in a number of important physiological functions of biological membranes 2-4. The present communication reports on the effect of temperature, bivalent cations, and other substances on the diffusion of Na + or K + through smectic liquid crystals of phosphatidylserine. Following the argument elaborated upon earlier 1, it is reasonable to suppose that the ions incorporated into the liquid crystals are t rapped in the aqueous spaces between lamellae and that they diffuse across these lamellae. This investigation was undertaken in conjunction with a s tudy of the physicochemical characteristics of phosphatidylserine monolayers presented in an accompanying article 5. The preparation of beef brain phosphatidylserine has been described elsewhere 5. The method for studying the diffusion of univalent ions through liquid crystals was essentially as described 1. A solution of phosphatidylserine in chloroform was evaporated to dryness in vacuo and the material dispersed in 13o mM salt solutions containing tracer ions (22NaC1 or 42KC1 or Ka6CI) and I4. 5 mM Tris-HC1. The milky dispersion was then sonicated for 5 rain and subsequently left at room temperature for I h. Bulk-phase tracer ions were separated from those contained in the lipid particles by passage through a Sephadex G-5o column, eluted with the same tracerfree salt mixture 6. Aliquots of the "washed" lipid dispersion were then pipetted immediately into dialysis tubing (usually 4 ~moles of phosphatidylserine in 0.5 ml of salt solution) and dialysed against equimolar solutions of the same tracer-free salt. The amount of isotope appearing in the dialysate during the first hour of dialysis, was taken to represent the diffusion rate constant for a unit concentration difference of I45 mM at zero time. I t was expressed in m~equiv of ions per h per ~mole of lipid, and was referred to as self-diffusion rate. The self-diffusion rates for ions diffusing through phosphatidylserine liquid crystals are given in Table I. They differ from values obtained with phosphatidylcholine liquid crystals by showing an increased permeability to cations and decreased permeabili ty to anions. The ratios of anion to cation self-diffusion rates are also given, and it can be seen that although permeability to anion decreases as the negative charge increases in accordance with counter-ion concentration, the C1diffusion through phosphatidylserine is still some ten times greater than that of Na + or K+, even though each phosphatidylserine molecule might be expected to carry one net negative charge. I t should also be noted that self-diffusion rate for Na + is higher than that for K +, although the initial amounts of these two metals captured by phosphatidylserine liquid crystals are the same.