Phloretin modifies the conductance of artificial lipid bilayer membranes. In ... to Andersen et al., 1976a the action of phloretin can be accounted for by a re-.
Phloretin-Induced Changes in Ion Transport across Lipid Bilayer Membranes E U G E N E M E L N I K , R A M O N L A T O R R E , J A M E S E. H A L L , and D A N I E L C. T O S T E S O N From the Department of Pharmacological and Physiological Sciences, T h e University of Chicago, Chicago, Illinois 60637, and the Department of Physiology and Pharmacology, Duke University, D u r h a m , North Carolina 27710. Dr. Melnik's present address is the Shemyakin Institute of Bio-organic Chemistry, Moscow 117312, Union of Soviet Socialist Republics.
Phloretin, the aglucone d e r i v a t i v e o f phlorizin, i n c r e a s e s c a t i o n conductance and decreases anion conductance in lipid bilayer membranes. In this paper we present evidence that phloretin acts almost exclusively by altering the permeability of the membrane interior and not by modifying the partition of the permeant species between the membrane and the bulk aqueous phases. We base our conclusion on an analysis of the current responses to a square voltage pulse obtained for the lipophilic anion, tetraphenylborate, and the cation complex, peptide PV-K +. These results are consistent with the hypothesis that phloretin decreases the intrinsic positive internal membrane potential but does not modify to a great extent the potential energy minima at the membrane interfaces. Phloretin increases the conductance for the nonactin-K + complex, but above 10-5 M the steady-state nonactin-K + voltage-current curve changes from superlinear to sublinear. These results imply that, above 10-5 M phloretin, the nonactin-K + transport across the membrane becomes interfacially limited. AB ST RACT
INTRODUCTION
Phloretin is a well-known inhibitor o f electrolyte a n d nonelectrolyte t r a n s p o r t across biological m e m b r a n e s . In red blood cells, this molecule at m i c r o m o l a r concentrations strongly inhibits facilitated t r a n s p o r t o f hexoses (Lefevre, 1961; Czech et al., 1973) a n d chloride t r a n s p o r t (Wieth et al., 1973). Phloretin also alters the nonelectrolyte permeability. It increases the permeabilities of lipophilic molecules a n d decreases those o f hydrophilic solutes (Owen a n d S o l o m o n , 1972). Phloretin modifies the c o n d u c t a n c e o f artificial lipid bilayer m e m b r a n e s . I n particular it increases c a r r i e r - m e d i a t e d cation c o n d u c t a n c e s a n d decreases lipophilic anion conductances (Cass et al., 1973; A n d e r s e n et al., 1976a). A c c o r d i n g to A n d e r s e n et al., 1976a the action o f phloretin can be a c c o u n t e d for by a reduction o f the dipole potential o f the m e m b r a n e interior. T h i s potential is a p p a r e n t l y several h u n d r e d millivolts positive with respect to that o f the a q u e o u s phases. T h e s e observations a n d the m o d e o f action o f phloretin in biological m e m b r a n e s have led us to investigate f u r t h e r the m e c h a n i s m by which this comp o u n d affects ion t r a n s p o r t across m e m b r a n e s . THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 69, 1977 pages 243-25.7
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In bilayers o f well-defined composition, the use o f ion carriers and lipophilic ions as probes o f m e m b r a n e interracial potentials and the barrier to transport inside the m e m b r a n e has been very successful (McLaughlin et al., 1970; Hladky and H a y d o n , 1973; Szabo, 1974, 1976; Hall et al., 1973; A n d e r s e n and Fuchs, 1975). Interracial potentials may arise f r o m the surface charge or f r o m oriented dipoles in the m e m b r a n e or as a combination o f both effects. T h e large changes in conductance for the nonactin-K ÷ complex with varying surface charge density and ionic strength seem to agree well with those predicted by the Gouy-Chapman t h e o r y for an aqueous diffuse double layer (McLaughlin et al., 1970). On the o t h e r h a n d , the relative conductances o f the nonactin-K ÷ c o m p l e x in glycerolmono-oleate and phosphatidylcholine m e m b r a n e s are accurately predicted by the difference in their surface potentials m e a s u r e d at the air-water interfaces ( H a y d o n and Hladky, 1973). This surface potential may arise f r o m oriented water dipoles and o t h e r oriented dipoles o f the film-forming molecules (Davies and Rideal, 1963). L i b e r m a n and T o p a l y (.1969) and subsequently Le Blanc (1970) and A n d e r s e n and Fuchs (1975) have shown that lipid bilayer m e m b r a n e s are much m o r e permeable to lipophilic anions than to cations (-10S-fold). T h e s e results are consistent with high positive internal m e m b r a n e potentials that arise as a consequence o f oriented dipoles. In regulating ion t r a n s p o r t , m e m b r a n e composition can alter the surface concentration o f p e r m e a n t species or the rate at which these species are being translocated (see Szabo, 1976). Essentially, the height o f the m e m b r a n e energy barrier will control the rate o f translocation, and the d e p t h o f potential e n e r g y minima at the m e m b r a n e interfaces is related to the partition coefficient o f the p e r m e a n t species between m e m b r a n e and the aqueous phases. F r o m conductance relaxation data, obtained by voltage pulsing, both translocation rates and partition coefficients for ion carriers can be calculated separately. It is then possible to assess the relative contribution o f the two processes to the overall permeability o f the p e r m e a n t ions (Stark et al., 1971; Szabo, 1976). Using the concepts and techniques described above we have studied the effect o f phloretin on cation-carrier complexes (peptide PV and nonactin) and on a lipophilic anion (tetraphenylborate) in artificial lipid bilayer m e m b r a n e s . We have c o n f i r m e d the results o f A n d e r s e n et al. (1976a) that phloretin markedly decreases anion and increases cation conductance. T h e main objective o f this p a p e r is to assess how phloretin affects the translocation rates and the partition coefficient for the d i f f e r e n t p e r m e a n t species tested. MATERIALS
AND
METHODS
Membranes were formed in a Teflon chamber with an 18-ttm thick Teflon partition according to the technique described by Montal and Mueller (1972). The surface area of each compartment was 4 cm ~and the volume 3 ml. The membrane area was 6 x l0 -4 cm:. All membranes were formed from bacterial phosphatidylethanolamine (Supelco, Inc., Bellefonte, Pa.). The phospholipid was spread on the surface of the electrolyte solution using l0 ~tl of a solution containing 12.5 mg lipid per 1 ml pentane. Nonactin (E.R. Squibb & Son, New York), PV (Gisin and Merrifield, 1972), and tetraphenylborate sodium salt (TPhB-) (kindly supplied to us by O. S. Andersen) were used as concentrated solutions in ethanol. Samples of these solutions were added directly to both compartments. Phloretin ( I C N - K . & K. Labs., Inc., Cleveland, Ohio), unless otherwise stated,
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was a d d e d to both c o m p a r t m e n t s in ethanolic solutions of various concentrations. Control experiments showed that ethanol at the concentrations used (
