Phloretin-induced changes in ion transport across lipid bilayer membranes

Phloretin, the aglucone derivative of phlorizin, increases cation 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 permanent species between the membrane and the bulk aqueous phases. We base our conclusion on an analysis of the current responses to a senylborate, 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-5+ transport across the membrane becomes interfacially limited.     

Probing amphotericin B single channel activity by membrane dipole modifiers

The effects of dipole modifiers and their structural analogs on the single channel activity of amphotericin B in sterol-containing planar phosphocholine membranes are studied. It is shown that the addition of phloretin in solutions bathing membranes containing cholesterol or ergosterol decreases the conductance of single amphotericin B channels. Quercetin decreases the channel conductance in cholesterol-containing bilayers while it does not affect the channel conductance in ergosterol-containing membranes. It is demonstrated that the insertion of styryl dyes, such as RH 421, RH 237 or RH 160, in bilayers with either cholesterol or ergosterol leads to the increase of the current amplitude of amphotericin B pores. Introduction of 5α-androstan-3β-ol into a membrane-forming solution increases the amphotericin B channel conductance in a concentration-dependent manner. All the effects are likely to be attributed to the influence of the membrane dipole potential on the conductance of single amphotericin B channels. However, specific interactions of some dipole modifiers with polyene-sterol complexes might also contribute to the activity of single amphotericin B pores. It has been shown that the channel dwell time increases with increasing sterol concentration, and it is higher for cholesterol-containing membranes than for bilayers including ergosterol, 6-ketocholestanol, 7-ketocholestanol or 5α-androstan-3β-ol. These findings suggest that the processes of association/dissociation of channel forming molecules depend on the membrane fluidity.     

Internal electrostatic potentials in bilayers: measuring and controlling dipole potentials in lipid vesicles.

The binding and translocation rates of hydrophobic cation and anion spin labels were measured in unilamellar vesicle systems formed from phosphatidylcholine. As a result of the membrane dipole potential, the binding and translocation rates for oppositely charged hydrophobic ions are dramatically different. These differences were analyzed using a simple electrostatic model and are consistent with the presence of a dipole potential of approximately 280 mV in phosphatidylcholine. Phloretin, a molecule that reduces the magnitude of the dipole potential, increases the translocation rate of hydrophobic cations, while decreasing the rate for anions. In addition, phloretin decreases the free energy of binding of the cation, while increasing the free energy of binding for the anion. The incorporation of 6-ketocholestanol also produces differential changes in the binding and translocation rates of hydrophobic ions, but in an opposite direction to those produced by phloretin. This is consistent with the view that 6-ketocholestanol increases the magnitude of the membrane dipole potential. A quantitative analysis of the binding and translocation rate changes produced by ketocholestanol and phloretin is well accounted for by a point dipole model that includes a dipole layer due to phloretin or 6-ketocholestanol in the membrane-solution interface. This approach allows dipole potentials to be estimated in membrane vesicle systems and permits predictable, quantitative changes in the magnitude of the internal electrostatic field in membranes. Using phloretin and 6-ketocholestanol, the dipole potential can be altered by over 200 mV in phosphatidylcholine vesicles.     

Interaction of electric dipoles with phospholipid head groups. A 2H and 31P NMR study of phloretin and phloretin analogues in phosphatidylcholine membranes.

Phloretin, 4-hydroxyvalerophenone, and 2-hydroxy-omega-phenylpropiophenone are lipophilic dipolar substances that modify ionic conductances of bilayer membranes. The structural changes at the level of the head groups and the hydrocarbon chains as induced by the incorporation of phloretin and its analogues were investigated with deuterium and phosphorus nuclear magnetic resonance. Membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were selectively deuterated at the choline head group and at the hydrocarbon chains, and 2H and 31P NMR spectra were recorded with varying concentrations of dipolar agents. Incorporation of phloretin leaves the bilayer structure intact, induces only a small disordering of the hydrocarbon chains, and has no significant effect on the head-group dynamics. On the other hand, quite distinct structural changes are observed for the phosphocholine head group. While the -P-N+ dipole is oriented approximately parallel to the membrane surface for pure POPC bilayers, addition of phloretin, and to a lesser extent 4-hydroxyvalerophenone and 2-hydroxy-omega-phenylpropiophenone, rotates the N+ end of the -P-N+ dipole closer to the hydrocarbon layer. The resulting normal component of the -P-N+ dipole partly compensates the electric field of the dipolar agents. In addition to this structural change, phloretin also modifies the hydration layer at the lipid-water interface. Much less 2H2O is adsorbed to the membrane surface when the bilayer contains phloretin, 4-hydroxyvalerophenone, or 2-hydroxy-omega-phenylpropiophenone. Moreover, a rather large change in the residual phosphorus chemical shielding anisotropy argues in favor of hydrogen-bond formation between the phosphate segment and the phloretin hydroxyl groups.     

Effects of the dipolar form of phloretin on potassium conductance in squid giant axons.

The effects of phloretin on membrane ionic conductances have been studied in the giant axon of the squid, Loligo pealei. Phloretin reversibly suppresses the potassium and sodium conductances and modifies their dependence on membrane potential (Em). Its effects on the potassium conductance (GK) are much greater than on the sodium conductance; no effects on sodium inactivation are observed. Internal perfusion of phloretin produces both greater shifts in GK(Em) and greater reductions maximum GK than does external perfusion; the effect of simultaneous internal and external perfusion is little greater than that of internal perfusion alone. Lowering the internal pH, which favors the presence of the neutral species of weakly acidic phloretin (pKa 7.4), potentiates the actions of internally perfused phloretin. Other organic cations with dipole moments similar to phloretin's have little effect on either potassium or sodium conductances in squid axons. These results can be explained by either of two mechanisms; on postulates a phloretin "receptor" near the voltage sensor component of the potassium channel which is accessible to drug molecules applied at either the outer or inner membrane surface and is much more sensitive to the neutral than the negatively charged form of the drug. The other mechanism proposes that neutral phloretin molecules are dispersed in an ordered array in the membrane interior, producing a diffuse dipole field which modifies potassium channel gating. Different experimental results support these two mechanisms, and neither hypothesis can be disproven.     

The effect of phloretin on sphingolipid-containing membranes modified by syringomycin E

The effects of dipole modifier phloretin on the activity of syringomycin (SRE) channels in the lipid bilayers containing different sphingolipids, N-stearoyl-phytosphingosine (PSP) and N-stearoyl-D-erythrosphingosine (DSP), have been compared. It is shown that the addition of phloretin up to a concentration of 10 μM into solutions bathing the bilayers containing 20 mol.% PSP causes a 170-fold increase in the SRE channel-forming activity. In the case of DSP-containing membranes, a more significant (5200-fold) increase of the equilibrium number of open SRE channels is observed. The enhancement of SRE activity is accompanied by about 2-fold increase of the gating charge of SRE channels in the membranes with PSP, while in the bilayers with DSP the gating charge increases about 4-fold. The revealed differences in the parameters of SRE channels in the membranes including phloretin and PSP or DSP are accounted for by different partition coefficients of the toxin and dipole modifier between the lipid and water phases. The data suggest heterogeneity of dipole potential of the PSP-containing membranes in the presence of phloretin. This heterogeneity is due to the possibility of formation in these membranes of rafts with the dipole potential not affected by modifier.     

The Interaction of Dipole Modifiers with Polyene-Sterol Complexes

Recently, we showed that the effect of dipole modifiers (flavonoids and styrylpyridinium dyes) on the conductance of single amphotericin B (AmB) channels in sterol-containing lipid bilayers primarily resulted from changes in the membrane dipole potential. The present study examines the effect of dipole modifiers on the AmB multi-channel activity. The addition of phloretin to cholesterol-containing membranes leads to a significant increase in the steady-state AmB-induced transmembrane current. Quercetin significantly decreases and RH 421 increases the current through ergosterol-containing bilayers. Other tested flavonoids and styrylpyridinium dyes do not affect the channel-forming activity of AmB independently on the sterol composition of the bilayers. The effects obtained in these trials may instead be attributed to the direct interaction of dipole modifiers with AmB/sterol complexes and not to the effect of dipole potential changes. The presence of double bonds in the Δ7 and Δ22 positions of sterol molecules, the number of conjugated double bonds and amino sugar residues in polyene molecules, and the conformation and adsorption plane of dipole modifiers are important factors impacting this interaction.     

Effects of phlorizin and phloretin on passive and dynamic electrical properties in muscle membrane

The effects of phlorizin and phloretin on the cable properties were investigated in frog sartorius muscle by conventional cable analysis. Actions of phloretin on voltage-dependent ionic conductances were also studied by analysis of the phase plane trajectories. Both drugs evoked a significant decrease in specific membrane resistance (Rm) in chloride-containing Ringer's solution. The linear membrane capacitance increased by about 30%. On the contrary, in the presence of the non-penetrating anion, glutamate, a slight increase in Rm was induced by phlorizin. It is suggested that these drugs may increase the chloride conductance in the muscle membrane. Under the effect of phloretin the resting membrane potential remained unchanged but the amplitude of the action potential was lowered and the rate of repolarization was significantly reduced. The rate of depolarization during the "foot" of the action potential and the conduction velocity calculated from the rate constant of depolarization decreased. The maximum Na conductance was not altered by phloretin but K conductance was reduced. The time constant (tau K) reflecting the kinetic properties of K conductance was increased about seven-fold. It is suggested that great importance may be attributed to the dipole properties of these drugs in the actions presented above.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Transport of large organic ions through syringomycin channels in the membranes containing dipole modifiers

The effect of the membrane dipole potential (Phid) on a conductance and a steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate and chloride was shown. The magnitude of Phid was varied with the introduction to membrane bathing solutions of phloretin, which reduces the Phid, and RH 421, increasing the Phid. It was established that in all studied systems the increase in the membrane dipole potential cause a decrease in the steady-state number of open channels. In the systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are in an order of magnitude smaller than in systems containing sodium chloride. At the same time, the conductance (g) of single SRE-channels on the membranes bathed in NaCI solution increases with the increase in Phid, and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the Phid. The latter is due to the lack of cation/anion selectivity of the SRE-channels in these systems. The different channel-forming activity of SRE in the experimental systems is defined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

The adsorption of phloretin to lipid monolayers and bilayers cannot be explained by langmuir adsorption isotherms alone.

Phloretin and its analogs adsorb to the surfaces of lipid monolayers and bilayers and decrease the dipole potential. This reduces the conductance for anions and increases that for cations on artificial and biological membranes. The relationship between the change in the dipole potential and the aqueous concentration of phloretin has been explained previously by a Langmuir adsorption isotherm and a weak and therefore negligible contribution of the dipole-dipole interactions in the lipid surface. We demonstrate here that the Langmuir adsorption isotherm alone is not able to properly describe the effects of dipole molecule binding to lipid surfaces--we found significant deviations between experimental data and the fit with the Langmuir adsorption isotherm. We present here an alternative theoretical treatment that takes into account the strong interaction between membrane (monolayer) dipole field and the dipole moment of the adsorbed molecule. This treatment provides a much better fit of the experimental results derived from the measurements of surface potentials of lipid monolayers in the presence of phloretin. Similarly, the theory provides a much better fit of the phloretin-induced changes in the dipole potential of lipid bilayers, as assessed by the transport kinetics of the lipophilic ion dipicrylamine.     

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H+, K+ and Na+ in phospholipid vesicles

Rates of M⁺/H⁺ exchange (M⁺=K⁺, Na⁺) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M⁺ and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=ΔpH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M⁺ dissociation constant, K_M, of the M⁺ bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H⁺ bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M⁺ and an anion) by forming 1:2 complexes with valinomycin–M⁺ or nonactin–M⁺. (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K_M increases. The decreases/increases in K_M mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.     

Transport-related modulation of the membrane properties of toad urinary bladder epithelium.

Impedance analysis and transepithelial electrical measurements were used to assess the effects of the apical membrane Na+ channel blocker amiloride and anion replacement on the apical and basolateral membrane conductances and areas of the toad urinary bladder (Bufo marinus). Mucosal amiloride addition decreased both apical and basolateral membrane conductances (Ga and Gbl, respectively) with no change in membrane capacitances (Ca and Cbl). Consequently, the specific conductances of these membranes decreased without significant changes in membrane area. Following amiloride removal, an increase was obtained in the steady-state rate of sodium transport compared to values before amiloride addition. This increase was independent of the initial transport rate, suggesting activation of a quiescent pool of apical sodium channels. Chloride replacement by acetate or gluconate had no significant effects on apical or basolateral membrane capacitances. The effects of these replacements on membrane conductances depended on the anion species. Gluconate (which induces cell shrinkage) decreased both membrane conductances. In contrast, acetate (which induces cell swelling) increased Ga and had no effect on Gbl. The increase in the apical membrane conductance was due to an increase in the amiloride-sensitive Na+ conductance of this membrane. In summary, mucosal amiloride addition or chloride replacements led to changes in membrane conductances without significant effects on net membrane areas.     

Phloretin-induced changes of lipophilic ion transport across the plasma membrane of mammalian cells.

The adsorption of the hydrophobic anion [W(CO)(5)CN](-) to human lymphoid Jurkat cells gave rise to an additional anti-field peak in the rotational spectra of single cells, indicating that the cell membrane displayed a strong dielectric dispersion in the kilohertz to megahertz frequency range. The surface concentration of the adsorbed anion and its translocation rate constant between the two membrane boundaries could be evaluated from the rotation spectra of cells by applying the previously proposed mobile charge model. Similar single-cell electrorotation experiments were performed to examine the effect of phloretin, a dipolar molecule known to influence the dipole potential of membranes, on the transport of [W(CO)(5)CN](-) across the plasma membrane of mammalian cells. The adsorption of [W(CO)(5)CN](-) was significantly reduced by phloretin, which is in reasonable agreement with the known phloretin-induced effects on artificial and biological membranes. The IC(50) for the effect of phloretin on the transport parameters of the lipophilic ion was approximately 10 microM. The results of this study are consistent with the assumption that the binding of phloretin reduces the intrinsic dipole potential of the plasma membrane. The experimental approach developed here allows the quantification of intrinsic dipole potential changes within the plasma membrane of living cells.     

Membrane dipole potential modulates proton conductance through gramicidin channel: movement of negative ionic defects inside the channel.

The effect of membrane dipole potential on gramicidin channel activity in bilayer lipid membranes (BLMs) was studied. Remarkably, it appeared that proton conductance of gramicidin A (gA) channels responded to modulation of the dipole potential oppositely as compared with gA alkali metal cation conductance. In particular, the addition of phloretin, known to reduce the membrane dipole potential, resulted in a decrease in gA proton conductance, on one hand, and an increase in gA alkali metal conductance, on the other hand, whereas 6-ketocholestanol, the agent raising the membrane dipole potential, provoked an increase in gA proton conductance as opposed to a decrease in the alkali metal cation conductance. The peculiarity of the 6-ketocholestanol effect consisted in its dependence on the H(+) concentration. The experiments with the impermeant dipolar compound, phloridzin, showed that the response of proton transport through gramicidin channels to varying the membrane dipole potential did not change qualitatively if the dipole potential of only one monolayer or both monolayers of the BLM was altered. In contrast to gA proton conductance, the single-channel lifetime changed similarly with varying the membrane dipole potential, regardless of the kind of permeant cations (protons or potassium ions). The results of this study could be tentatively accounted for by an assumption that one of the rate-limiting steps of proton conduction through gramicidin channels represents, in fact, movement of negatively charged species (negative ionic defects) across a membrane.     

Interaction between the basolateral K+ and apical Na+ conductances in Necturus urinary bladder

Experimental modulation of the apical membrane Na+ conductance or basolateral membrane Na+-K+ pump activity has been shown to result in parallel changes in the basolateral K+ conductance in a number of epithelia. To determine whether modulation of the basolateral K+ conductance would result in parallel changes in apical Na+ conductance and basolateral pump activity, Necturus urinary bladders stripped of serosal muscle and connective tissue were impaled through their basolateral membranes with microelectrodes in experiments that allowed rapid serosal solution changes. Exposure of the basolateral membrane to the K+ channel blockers Ba2+ (0.5 mM/liter), Cs+ (10 mM/liter), or Rb+ (10 mM/liter) increased the basolateral resistance (Rb) by greater than 75% in each case. The increases in Rb were accompanied simultaneously by significant increases in apical resistance (Ra) of greater than 20% and decreases in transepithelial Na+ transport. The increases in Ra, measured as slope resistances, cannot be attributed to nonlinearity of the I-V relationship of the apical membrane, since the measured cell membrane potentials with the K+ channel blockers present were not significantly different from those resulting from increasing serosal K+, a maneuver that did not affect Ra. Thus, blocking the K+ conductance causes a reduction in net Na+ transport by reducing K+ exit from the cell and simultaneously reducing Na+ entry into the cell. Close correlations between the calculated short-circuit current and the apical and basolateral conductances were preserved after the basolateral K+ conductance pathways had been blocked. Thus, the interaction between the basolateral and apical conductances revealed by blocking the basolateral K+ channels is part of a network of feedback relationships that normally serves to maintain cellular homeostasis during changes in the rate of transepithelial Na+ transport.     

Transport of large organic ions through syringomycin channels in membranes containing dipole modifiers

The effect of the membrane dipole potential (φ _d ) on conductance and the steady-state number of functioning channels formed by cyclic lipodepsipeptide syringomycin E (SRE) in bilayer lipid membranes made from phosphocholine and bathed in 0.4 M solution of sodium salts of aspartate, gluconate, and chloride was shown. The φ _d value varied with the introduction of phloretin to membrane bathing solutions, which reduces φ _d and RH 421, which increases φ _d . It was established that, in all studied systems, an increase in the membrane dipole potential caused a decrease in the steady-state number of open channels. In systems containing sodium salts of aspartate (Asp) or gluconate (Glc), changes in the number of functioning channels are one order lower than those of systems that contain sodium chloride. At the same time, the conductance (g) of single SRE channels in the membranes bathed in NaCl solution increases with increase in φ _d and in the systems containing NaAsp or NaGlc the conductance of single channels does not depend on the φ _d . The latter is due to the lack of cation/anion selectivity of the SRE channels in these systems. The different channel-forming activity of SRE in the experimental systems is determined by the gating charge of the channel and the partition coefficient of the dipole modifiers between the lipid and aqueous phases.     

Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.

The electrostatic potentials associated with cell membranes include the transmembrane potential (delta psi), the surface potential (psi s), and the dipole potential (psi D). psi D, which originates from oriented dipoles at the surface of the membrane, rises steeply just within the membrane to approximately 300 mV. Here we show that the potential-sensitive fluorescent dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6- naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS) can be used to measure changes in the intramembrane dipole potential. Increasing the content of cholesterol and 6-ketocholestanol (KC), which are known to increase psi D in the bilayer, results in an increase in the ratio, R, of the dye fluorescence excited at 440 nm to that excited at 530 nm in a lipid vesicle suspension; increasing the content of phloretin, which lowers psi D, decreases R. Control experiments show that the ratio is insensitive to changes in the membrane's microviscosity. The lack of an isosbestic point in the fluorescence excitation and emission spectra of the dye at various concentrations of KC and phloretin argues against 1:1 chemical complexation between the dye and KC or phloretin. The macromolecular nonionic surfactant Pluronic F127 catalyzes the insertion of KC and phloretin into lipid vesicle and cell membranes, permitting convenient and controlled modulation of dipole potential. The sensitivity of R to psi D is 10-fold larger than to delta psi, whereas it is insensitive to changes in psi S. This can be understood in terms of the location of the dye chromophore with respect to the electric field profile associated with each of these potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Cu2+-o-phenanthroline on gastric (H+ + K+)-ATPase. Evidence for opening of a closed anion conductance by S-S cross-linkings

Effects of the S-S cross-linking reagent, Cu2+-o-phenanthroline (CuP), on salt conductances of gastric vesicle membranes in which the (H+ + K+)-ATPase is present were studied. CuP caused a dose-dependent increase in the KCl conductance of the vesicle membrane. The increase of the KCl conductance caused by 10 microM CuP was completely prevented by 0.3 mM ATP or 0.3 mM adenyl 5'-yl imidodiphosphate and partially prevented by ADP. The NaCl conductance was also increased by the CuP reaction. However, CuP has no effect on the K2SO4 conductance. Pretreatments of vesicles with 0.1 mM 4-acetoamide-4'-isothiocyanostilbene-2,2'-disulfonate, an anion channel inhibitor, completely blocked the effect of CuP. Thus, these effects of CuP are ascribable to the increase in the anion conductance of the vesicle membrane produced by S-S cross-linking. Furthermore, tyrosine-tyrosine cross-linking with tetranitromethane also increases the anion conductance. Probable roles of the opening of the closed anion channel of the ATPase were discussed in regard to the acid secretory mechanism of gastric mucosa.     

Intramembrane molecular dipoles affect the membrane insertion and folding of a model amphiphilic peptide.

The relationship between the dipole potential and the interaction of the mitochondrial amphipathic signal sequence known as p25 with model membranes has been studied using 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octyl-amino)-6-naphthyl]viny l] pyridinium betaine (di-8-ANEPPS) as a fluorescent probe. The dipole potential of phosphatidylcholine membranes was modified by incorporating into the bilayer the sterols phloretin and 6-ketocholestanol (KC), which decrease and increase the dipole potential, respectively. The results derived from the application of a dual-wavelength ratiometric fluorescence method for following the variation of the membrane dipole potential have shown that when p25 inserts into the lipidic bilayer, a decrease in the dipole potential takes place. The magnitude of this decrease depends on the initial value of the dipole potential, i.e., before interaction with the peptide. Thus, when KC was incorporated into the bilayer, the decrease caused by the membrane insertion of p25 was larger than that caused in PC membranes. Alternatively, in the presence of phloretin, the decrease in the potential caused by the peptide insertion was smaller. Complementary studies involving attenuated total reflectance-Fourier transform infrared spectroscopy of the peptide membrane interactions have shown that modification of the dipole potential affects the conformation of the peptide during the course of its interaction with the membrane. The presence of KC induces a higher amount of helicoidal structure. The presence of phloretin, however, does not appear to affect the secondary structure of the peptide. The differences observed in the dipole potential decreases caused by the presence of the peptide with the PC membranes and phloretin-PC membranes, therefore, must involve differences in the tertiary and, perhaps, quaternary conformations of p25.