Membrane surface-charge titration probed by gramicidin A channel conductance.

We manipulate lipid bilayer surface charge and gauge its influence on gramicidin A channel conductance by two strategies: titration of the lipid charge through bulk solution pH and dilution of a charged lipid by neutral. Using diphytanoyl phosphatidylserine (PS) bilayers with CsCl aqueous solutions, we show that the effects of lipid charge titration on channel conductance are masked 1) by conductance saturation with Cs+ ions in the neutral pH range and 2) by increased proton concentration when the bathing solution pH is less than 3. A smeared charge model permits us to separate different contributions to the channel conductance and to introduce a new method for "bilayer pKa" determination. We use the Gouy-Chapman expression for the charged surface potential to obtain equilibria of protons and cations with lipid charges. To calculate cation concentration at the channel mouth, we compare different models for the ion distribution, exact and linearized forms of the planar Poisson-Boltzmann equation, as well as the construction of a "Gibbs dividing surface" between salt bath and charged membrane. All approximations yield the intrinsic pKain of PS lipid in 0.1 M CsCl to be in the range 2.5-3.0. By diluting PS surface charge at a fixed pH with admixed neutral diphytanoyl phosphatidylcholine (PC), we obtain a conductance decrease in magnitude greater than expected from the electrostatic model. This observation is in accord with the different conductance saturation values for PS and PC lipids reported earlier (, Biochim. Biophys. Acta. 552:369-378) and verified in the present work for solvent-free membranes. In addition to electrostatic effects of surface charge, gramicidin A channel conductance is also influenced by lipid-dependent structural factors.     

Ultraviolet flash photolysis of gramicidin-doped lipid bilayers

We have examined the rate of gramicidin channel conductance inactivation by ultraviolet photolysis using 0.1 millisecond light flashes. The lower limit on the channel photolysis reaction rate has been reduced by four orders of magnitude over previous observations. Monoolein/hexadecane bilayers formed in 1.0 M KCl were doped with (1-3) x 10(6) gramicidin A' channels and exposed to a broad-spectrum light flash. The flash reduced membrane conductance abruptly by approx. 16%. Following the flash, a further slow reduction of approx. 3% was observed followed by a slow recovery of approx. 4%. The post-flash decay and recovery may be due to slow chemical reactions, conformational relaxations, or changes in the equilibrium between aqueous, lipid-bound, and channel-forming dimerized gramicidin. Under our experimental conditions, gramicidin M was insensitive to light flashes compared to gramicidin A', demonstrating that for gramicidin A' the photolysis mechanism depends specifically on the tryptophan side-chain. Flash photolysis of a membrane containing a small population of channels (approx. 30) indicated that the decay is due to the sudden inactivation of several channels. The recovery appears to result from insertion of normal channels into the membrane. Flash photolysis of single-channel membranes showed that the flash causes abrupt, complete channel inactivation.     

Interaction of ions and water in gramicidin A channels: streaming potentials across lipid bilayer membranes

For very narrow channels in which ions and water cannot overtake one another (single-file transport), electrokinetic measurements provide information about the number of water molecules within a channel. Gramicidin A is believed to form such narrow channels in lipid bilayer membranes. In 0.01 and 0.1 M solutions of CsCl, KCL, and NaCl, streaming potentials of 3.0 mV per osmolal osmotic pressure difference (created by urea, glycerol, or glucose) appear across gramicidin A-treated membranes. This implies that there are six to seven water molecules within a gramicidin channel. Electroosmotic experiments, in which the water flux assoicated with current flow across gramicidin-treated membranes is measured, corroborate this result. In 1 M salt solutions, streaming potentials are 2.35 mV per osmolal osmotic pressure difference instead of 3.0 mV. The smaller value may indicate multiple ion occupancy of the gramicidin channel at high salt concentrations. Apparent deviations from ideal cationic selectivity observed while attempting to measure single-salt dilution potentials across gramicidin-treated membranes result from streaming potential effects.     

Activity of toxins produced by Pseudomonas syringae pv. syringae in model and cell membranes

We studied effects of toxins produced by a bacterium Pseudomonas syringae pv. syringae on the conductance of bilayer lipid membranes (BLM). The used toxins were as follows: syringopeptin 22A (SP22A), syringomycin E (SPE), syringostatin A (SSA), syringotoxin B (STB), and methylated syringomycin E (CH3-SRE). All toxins demonstrated channel-forming activity. The threshold sequence for toxin activity was SP22A > SRE approximately equal to SSA > STB > CH3-SRE, and this sequence was independent of lipid membrane composition, and NaCl concentration (pH 6) in the membrane bathing solution (in the range of 0.1-1.0 M). This sequence correlated with relative bioactivities of toxins. In addition, SRE demonstrated a more potent antifungal activity than CH3-SRE. These findings suggest that ion channel formation may underlie the bioactivities of the above toxins. The properties of single ion channels formed by the toxins in BLMs were found to be similar, which points to the similarity in the channel structures. In negatively charged membranes, bathed with diluted electrolyte solutions (0.1 M NaCl), the channels were seen to open with positive transmembrane potentials (V) (from the side of toxin addition), and close with negative potentials. In uncharged membranes the opposite response to a voltage sign was observed. Increasing the NaCl concentration up to 1 M unified the voltage sensitivity of channels in charged and uncharged membranes: channels opened with negative V, and closed with positive V. With all systems, the voltage current curves of single channels were similarly superlinear in the applied voltage and asymmetric in its sign. It was found that the single channel conductance of STB and SSA was higher than that of other toxin channels. All the toxins formed at least two types of ion channels that were multiple by a factor of either 6 or 4 in their conductance. The results are discussed in terms of the structural features of toxin molecules.     

The study of volt-ampere characteristics of ionic channels formed by gramicidin A

A method of measurement of the non-linearity coefficient of volt-ampere characteristics of the type i(U) approximately = U(1 + beta U2) has been developed for ionic channels formed by gramicidin A, using the third harmonic of the membrane current. The shape of the volt-ampere characteristics (VA) of ionic channels formed by gramicidin A did not depend on the antibiotic concentration in the membrane. The coefficient beta of non-linearity of VA of membranes modified by gramicidin A depended on electrolyte concentration "c" and it increased proportionally with the lg c from -17 V-2 at 0.03 mol/l KC1 to 8 V-2 at 3.4 mol/l KCl, and it was zero at co = 0.3 - 1 mol/l KCl. Egg lecithin and glycerol monooleate (GMO) membranes differ in their co values. The substitution of K+ for Li+ of the membrane solvent (n-heptane for n-hexadecane) did not influence the value of beta; the same applied for GMO membranes without any solvent. In a number of membranes, spontaneous change of the non-linearity coefficient with time observed after the membrane formation, as well as jumps of the non-linearity coefficient at a practically unchanged membrane conductivity. An analysis of some theoretical models of the ion transport through the channel has shown that, at voltages above 200 mV, these models provide rather small values of beta, or extremely high VA non-linearity.     

Calcium-dependent association of annexins with lipid bilayers modifies gramicidin A channel parameters

In order to examine whether calcium-dependent binding of annexin to acidic phospholipids could change the lipid bilayer environment sufficiently to perturb channel-mediated transmembrane ion-transport, gramicidin A channel activity in planar lipid bilayers was investigated in the presence of calcium and annexins II, III or V. The experiments were performed with membranes consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine in 300 mM KCl solution buffered to pH 7.4 and with either 0.1 or 1 mM calcium added to the solution. Annexin (1 microM) was subsequently applied to the cis side of the membrane. All three annexins (II, III and V) when tested at 1 mM calcium decreased the gramicidin single-channel conductance. Annexins II and III increased the mean lifetime of the channels whereas annexin V seemed to have no influence on the mean lifetime. Since the lifetime of gramicidin A channels is a function of the rate constant for dissociation of the gramicidin dimer, which is dependent on the physical properties of the lipid phase, binding of annexins II and III seems to stabilize the gramicidin channel owing to a change of the bilayer structure.     

Formation of ion channels by a negatively charged analog of gramicidin A.

O-pyromellitylgramicidin is a derivative of gramicidin in which three carboxyl groups are introduced at the terminal hydroxyl end of the peptide. Experiments with artificial lipid membranes indicate that this negatively charged analog forms ion-permeable channels in a way similar to that of gramicidin. If O-pyromellitylgramicidin is added to only one aqueous solution, the membrane conductance remains small, but increases by several orders of magnitude if the same amount is also added to the other side. In accordance with the dimer model of the channel, the membrane conductance under symmetrical conditions is proportional to the square of the aqueous concentration of O-pyromellitylgramicidin over a wide range. The ratio lambdaPG/lambdaG of the single-channel conductance of O-pyromellitylgramicidin to that of gramicidin is close to unity at high ionic strength, but increases more than fivefold at smaller ionic strength (0.01 M). This observation is explained in terms of an electrostatic effect of the fixed negative charges localized near the mouth of the channel. In a mixture of O-pyromellitylgramicidin and gramicidin, unit conductance steps of intermediate size are observed in addition to the conductance steps corresponding to the pure compounds, indicating the formation of hybrid channels. Hybrid channels with preferred orientation may be formed if small amounts of gramicicin and O-pyromellitylgramicidin are added to opposite sides of the membrane. These hybrid channels show a distinct asymmetry in the current-voltage characteristic.     

Calcium-ganglioside interactions and synaptic plasticity: effect of calcium on specific ganglioside/peptide (valinomycin, gramicidin A)-complexes in mixed mono- and bilayers.

A controlled exchange of calcium between the extracellular space (mM Ca2+) and the neuroplasm (microM Ca2+) is considered to be an essential prerequisite for almost every stage of neuronal activity. Our research interest is focused on those compounds, which due to their physico-chemical properties and localization within the synaptic membrane might fulfill the task as neuromodulators for functional synaptic proteins. Because of this specific binding properties towards calcium and their peculiar interactions with calcium in model systems gangliosides (amphiphilic sialic acid containing glycosphingolipids) are favorite candidates for a functional involvement in synaptic transmission of information. In this study we used monolayers to investigate the molecular packing and surface potential at the air/water interface, the interaction of gangliosides with the depsipeptide valinomycin (= monovalent ion carrier), and its influenceability by calcium. Furthermore we looked at calcium effects on the single channel conductance and mean channel life-time of the monovalent ion channel gramicidin A in mixed PC/ganglioside bilayers. In pure ganglioside monolayers the addition of 0.01 mM Ca2+ induces monolayer condensation, a rise in collapse pressure (= higher film stability), a shift of phase transition (= change of conformation), and a more negative head group potential (change of electric properties). In mixed ganglioside-valinomycin monolayers the addition of Ca2+ causes phase separation and/or aggregate formation between the ganglioside and the peptide. Single channel conductance fluctuations as well as mean channel life-time were analyzed for gramicidin A incorporated into binary mixed black lipid membranes of negatively charged gangliosides (GM1, GD1a, GT1b, GMix) and neutral lecithin (DOPC) in different molar ratios. At monovalent electrolyte concentrations up to < 250 mM CsCl the single channel conductance was significantly larger in the negatively charged mixed DOPC/ganglioside membranes than in the neutral DOPC membrane. Additionally, in the presence of gangliosides the mean channel life-time is increased. The addition of calcium (0.05 mM) induced a reduction of single channel conductance of gramicidin A in DOPC- and mixed DOPC/ganglioside membranes. These physico-chemical data in connection with new electromicroscopical evidences for a precise localization of calcium, a calcium pump (Ca(2+)-ATPase), a clustered arrangement of gangliosides in synaptic terminals, and biochemical results with regard to activatory nature of exogenous gangliosides for neuronal protein phosphorylation and ATPases, support the hypothesis of a modulatory function of gangliosides in synaptic transmission.     

Pore forming activity of the major outer membrane protein of Rhodobacter capsulatus in lipid bilayer membranes

Porin of the outer membrane of Rhodobacter capsulatus St. Louis (ATCC 23782) was isolated and reconstituted into lipid bilayer membranes. The porin was obtained either by the sodium dodecyl sulfate treatment of cell envelopes (SDS-porin) or by saline extraction of whole cells (NaCl-porin). Nanomolar concentrations of both porin preparations resulted in a strong conductance increase of the lipid bilayer membranes by many orders of magnitude. At small protein concentrations the conductance increased in a stepwise fashion, the average single channel conductance being about 0.35 nS in 0.1 M KCl for SDS-porin and NaCl-porin as well. The single channel conductance was a linear function of the specific conductance of the aqueous phase. The results were consistent with the assumption that the porin formed large water-filled transmembrane channels in the membrane. From the average value of the single channel conductance in 0.1 M KCl an effective channel diameter of about 1.5 nm was estimated for both types of porins.Abbreviations EDTA ethylenediamine tetraacetic acid - SDS sodium dodecyl sulfate     

Comparison of gramicidin A and gramicidin M channel conductance dispersities

To explore the possible role of Trp side chains in gramicidin channel conductance dispersity, we studied the dispersity of gramicidin M (gM), a gramicidin variant in which all four tryptophan residues are replaced with phenylalanine residues, and its enantiomer, gramicidin M(-) (gM(-)), and compared them to that of gramicidin A (gA). The conductances of highly purified gM and gM(-) were studied in alkali metal solutions at a variety of concentrations and voltages, in seven different types of lipid, and in the presence of detergent. Like gA channels, the most common gM channel conductance forms a narrow band. However, unlike gA channels, where the remaining 5-30% of channel conductances are broadly distributed below (and slightly above) the main band, in gM there is a narrow secondary band with <50% of the main peak conductance. This secondary peak was prominent in NaCl and KCl, but significantly diminished in CsCl and RbCl. Under some conditions, minor components can be observed with conductances yet lower than the secondary peak. Interconversions between the primary conductance state and these yet lower conductance states were observed. The current-voltage relations for both primary and secondary gM channel types have about the same curvature. The mean lifetime of the secondary channel type is below one third that of the primary type. The variants represent state deviations in the peptide or adjacent lipid structure.     

Small iminium ions block gramicidin channels in lipid bilayers.

Guanidinium and acetamidinium, when added to the bathing solution in concentrations of approximately 0.1M, cause brief blocks in the single channel potassium currents from channels formed in planar lipid bilayers by gramicidin A. Single channel lifetimes are not affected indicating that the channel structure is not modified by the blockers. Guanidinium block durations and interblock times are approximately exponential in distribution. Block frequencies increase with guanidinium concentration whereas block durations are unaffected. Increases in membrane potential cause an increase in block frequency as expected for a positively charged blocker but a decrease in block duration suggesting that the block is relieved when the blocker passes through the channel. At low pH, urea, formamide, and acetamide cause similar blocks suggesting that the protonated species of these molecules also block. Arginine and several amines do not block. This indicates that only iminium ions which are small enough to enter the channel can cause blocks in gramicidin channels.     

Effects of calcium on the gramicidin A single channel in phosphatidylserine membranes

In phosphatidylserine membranes the decrease in the conductance of the gramicidin A single channel caused by calcium is attributed to a reduction of surface potential and to a direct blocking of the pore (Apell et al. 1979).The aim of this paper is to make a, quantitative evaluation of these two effects. We recorded the conductance of gramicidin single channels in 100 mM KCl in the presence of different amounts of CaCl₂, MgCl₂ or TEACl.The ionic activities at the channel mouth were calculated using the Gouy-Chapman-Stern theory. Our experiments showed that even when the K⁺ activity at the channel mouth was estimated to be the same, the single channel conductance was lower if divalent cations were present. This effect is attributed to a blocking action of these ions.Abbreviations PS phosphatidylserine - TEA tetraethylammonium     

The structure of the gramicidin A transmembrane channel

Gramicidin A is a linear polypeptide antibiotic that facilitates the diffusion of monovalent cations across lipid bilayer membranes by forming channels. It has been proposed that the conducting channel is a dimer which is in equilibrium with nonconducting monomers in the membrane. To directly test this model in several independent ways, we have prepared and purified a series of gramicidin C derivatives. All of these derivatives are fully active analogs of gramicidin A, and each derivative has a useful chromophore esterified to the phenolic hydroxyl of tyrosine #11. Simultaneous conductance and fluorescence measurements on planar lipid bi-layer membranes containing dansyl gramicidin C yielded four conclusions: (1) A plot of the logarithm of the membrane conductance versus the logarithm of the membrane fluorescence had a slope of 2.0 ± 0.3, over a concentration range for which nearly all the gramicidin was monomeric. Hence, the active channel is a dimer of the nonconducting species. (2) In a membrane in which nearly all of the gramicidin was dimeric, the number of channels was approximately equal to the number of dimers. Thus, most dimers are active channels and so it should be feasible to carry out spectroscopic studies of the conformation of the transmembrane channel. (3) The association constant for dimerization is more than 1,000-fold larger in a glycerolester membrane with 26 Å-hydrocarbon thickness than in a 47 Å-glycerolester membrane. The dimerization constant in a 48 Å-phosphatidyl choline membrane was 200 times larger than in a 47 Å-glycerolester membrane, showing that it depends on the type of lipid as well as on the thickness of the hydrocarbon core. (4) We were readily able to detect 10^?14 mole cm^?2 of dansyl gramicidin C in a bilayer membrane, which corresponds to 60 fluorescent molecules per square μm. The fluorescent techniques described here should be sufficiently sensitive for fluorescence studies of reconstituted gates and receptors in planar bilayer membranes. An alternative method of determining the number of molecules of gramicidin in the channel is to measure the fraction of hybrid channels present in a mixture of 2 chemically different gramicidins. The single-channel conductance of p-phenylazo-benzene-sulfonyl ester gramicidin C (PABS gramicidin C) was found to be 0.68 that of gramicidin A. In membranes containing a mixture of these 2 gramicidins, a hybrid channel was evident in addition to 2 pure channels. The hybrid channel conductance was 0.82 that of gramicidin A. Fluorescence energy transfer from dansyl gramicidin C to diethylamino-phenylazobenzene-sulfonyl ester gramicidin C (DPBS gramicidin C), provided an independent way to measure the fraction of hybrid channels on liposomes. For both techniques the fraction of hybrid channels was found to be 2ad where a² and d² were the fractions of the 2 kinds of pure channels. This result strongly supports a dimer channel and the hybrid data excludes the possibility of a tetramer channel. The study of hybrid species by conductance and fluorescence techniques should be generally useful in elucidating the subunit structure of oligomeric assemblies in membranes. The various models which have been proposed for the conformation of the gramicidin transmembrane channel are briefly discussed.     

Ion channels formed by chemical analogs of gramicidin A.

Channel-forming peptides such as gramicidin A offer the opportunity to study the relationship between chemical structure and transport properties of an ion channel. This article summarizes a number of recent experiments with chemical analogs and derivatives of gramicidin A using artificial lipid bilayer membranes. The introduction of negative charges near the channel mouth leads to an increase in the cation transport rate. Hybrid channels consisting of a neutral and a negatively charged or of a positively and a negatively charged half-channel may be formed. The current-voltage characteristic of these hybrid channels exhibits a pronounced asymmetry.Experiments with charged derivatives of gramicidin A have been used in order to distinguish between different structural models of the dimeric channel; these studies strongly support Urry's model of a single-stranded, head-to-head associated helical dimer. In a further set of experiments gramicidin analogs with modified amino acid sequence were studied. It was found that a single substitution (tryptophan replaced by phenylalanine) leads to marked changes in the conductance of the channel. Analogs with a simplified amino acid sequence such as (L-Trp-D-Leu)7-L-Trp or L-Trp-Gly-(L-Trp-D-Leu)6-L-Trp are able to form cation permeable channels with similar properties as gramicidin A.     

Membranes of bacteria and mechanism of action of the antibiotic gramicidin S]

The cyclopeptide antibiotic gramicidin S taken at a concentration of 100--200 mkg/mg membrane protein rapidly increases the permeability of M. lysodeikticus protoplast membranes for substrates of respiratory chain and exogenous cytochromes c. Prolonged incubation of gramicidin S with protoplasts results in their lysis which is more fast at low temperatures. In contrast to natural gramicidin, a derivative of gramicidin S with acetylated amino groups does not inhibit either the micrococcus membrane dehydrogenase or the whole of respiratory chain and does not affect the osmotic barrier of protoplasts. Aliphatic diamines (at concentrations up to 0.1 M) and Ca2+ ions (10(-2) M) do not affect the functioning of the respiratory chain in isolated micrococcus membranes. Another derivative of the antibiotic with an increased distance of loaded amino groups from the cyclopeptide framework (diglycyl gramicidin S) affects the membrane in a way similar to that of natural gramicidin. Washing of gramicidin-treated membranes with NaCl enhances the inhibitory effect of the antibiotic on membrane enzymes. The data obtained suggest that in addition to ionic interactions some hydrophobic interactions also occur during gramicidin S binding to the bacterial membrane, probably at the expense of a hydrophobic peptide ring. It is assumed that gramicidin S, similar to Ca2+ and some other membranotropic agents provides for phase separation of negatively charged phospholipids from other groups of phospholipids, manifesting itself in an appearance of "frozen" sites on the membrane which destroys its barrier properties. This is due to the formation of ionic bonds of negatively charged phospholipids. Simultaneously, unlike Ca2+, gramicidin S, when interacting with membrane proteins, prevents their redistribution in more liquid parts of the membrane, which results in a situation when the respiratory enzymes become surrounded by alkyl chains with restricted motion.     

Voltage gating of VDAC is regulated by nonlamellar lipids of mitochondrial membranes

Evidence is accumulating that lipids play important roles in permeabilization of the mitochondria outer membrane (MOM) at the early stage of apoptosis. Lamellar phosphatidylcholine (PC) and nonlamellar phosphatidylethanolamine (PE) lipids are the major membrane components of the MOM. Cardiolipin (CL), the characteristic lipid from the mitochondrial inner membrane, is another nonlamellar lipid recently shown to play a role in MOM permeabilization. We investigate the effect of these three key lipids on the gating properties of the voltage-dependent anion channel (VDAC), the major channel in MOM. We find that PE induces voltage asymmetry in VDAC current-voltage characteristics by promoting channel closure at cis negative applied potentials. Significant asymmetry is also induced by CL. The observed differences in VDAC behavior in PC and PE membranes cannot be explained by differences in the insertion orientation of VDAC in these membranes. Rather, it is clear that the two nonlamellar lipids affect VDAC gating. Using gramicidin A channels as a tool to probe bilayer mechanics, we show that VDAC channels are much more sensitive to the presence of CL than could be expected from the experiments with gramicidin channels. We suggest that this is due to the preferential insertion of VDAC into CL-rich domains. We propose that the specific lipid composition of the mitochondria outer membrane and/or of contact sites might influence MOM permeability by regulating VDAC gating.     

Electrical properties and molecular architecture of the channel formed by Escherichia coli hemolysin in planar lipid membranes

A 107 kDa hemolysin from Escherichia coli is able to open pores in lipid membranes. By studying its interaction with planar phospholipid bilayers we have derived some structural information on the organization of the pore. We measured the current-voltage characteristic and the ion selectivity of the channel both in neutral membranes, made of egg phosphatidylcholine (PC) and in negatively charged membranes, made of a 1:1 mixture of PC with phosphatidylserine (PS). Experiments were performed varying both the pH and the salt concentration of the bathing KCl solution. In neutral membranes the pore is ohmic and its conductance increases almost linearly with the salt concentration. The channel is cation-selective at high pH but nearly unselective at low pH. We interpret these results in terms of a minimal model based on classical electro-diffusional theories assuming that the pore is wide and bears a negative charge at its entrances. In membranes containing the acidic lipid the current-voltage curve is non-linear in such a way to suggest that the trans (but not the cis) entrance of the pore is affected by the surface potential of the membrane. Applying our model we find that the trans and cis entrances are located, respectively, about 0.5 nm and more than 5 nm apart from the plane of the membrane. We confirmed the asymmetric disposition of the channel by enzymatic digestion of preformed pores. This was effective only when the enzyme was applied on the cis side.     

Potassium conductivity of the plasma membrane of frog photoreceptor cells

Potassium channels, uniformly distributed over the cell surface (0.5 channel/micron2) have been found in isolated fragments of plasma membranes of frog photoreceptor cells. Their conductance under symmetrical conditions (0.1 M KCl at both the membrane surfaces) is 72 +/- 6 pS for rods and 88 +/- 8 pS for cones. The channels are reversibly blocked by tetraethylammonium (TEA), Cs+, Rb+ at intra- and extracellular membrane surfaces. Half-inactivation of a channel occurs at concentrations of TEA, Cs+ and Rb+ at the intracellular surface 0.12, 2.2 and 3.9 mM, correspondingly.     

Dynamics of lipid chain attached fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) in negatively charged membranes determined by NMR spectroscopy

We have determined the average location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) attached to a C12 sn-2 chain of a phosphatidylserine (PS) analogue (C12-NBD-PS) in zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylserine (PS) host membranes. (1)H magic angle spinning nuclear Overhauser enhancement spectroscopy indicates a highly dynamic reorientation of the aromatic molecule in the membrane. The average location of NBD is characterized by a broad distribution function along the membrane director with a maximum indicating the location of the probe in the lipid/water interface of the lipid membrane. This behavior can be explained by a backfolding of the sn-2 chain towards the aqueous phase. Small differences in the distribution profiles of the NBD group along the membrane normal between PC and PS host membranes were found: in a PC host membrane, the NBD distribution has its maximum in the glycerol region; in a PS host membrane, NBD resides mostly in the upper chain region. These differences may be accounted for by packing differences in the PC versus PS host membranes. As seen by (2)H NMR order parameters, PS bilayers show a much higher packing density compared to PC membranes. Consequently, backfolding of the sn-2 chain with the NBD group attached causes a larger decrease of molecular order of the sn-1 chain in PS than in PC membranes. The broad distributions obtained for lipid chain attached NBD molecules reflect the motional freedom and molecular disorder in the liquid-crystalline lipid membrane.     

Number of water molecules coupled to the transport of sodium, potassium and hydrogen ions via gramicidin, nonactin or valinomycin.

The number of water molecules (n) coupled to the transport of cations across lipid membranes was determined in two different wats: directly from the electro-osmotic volume flux per ion, and by the use of Onsager's relation, from the open circuit streaming potential produced by an osmotic pressure difference. The results of the two approaches were in general agreement. Monoolein membranes were formed on the ends of polyethylene or Teflon tubing connected to a microliter syringe and the volume change necessary to keep the membrane at a fixed position was measured. It was necessary to make corrections for unstirred layer effects. The results for gramicidin were: n approximately 12 for 0.15 M KCl and NaCl, n approximately 6 for 3.0 M KCl and NaCl, and n approximately 0 for 0.01 M HCl. For nonactin, n approximately 4 for both 0.15 and 3.0 M KCl and NaCl. Valinomycin (for 0.15 M KCl) behaved like nonactin. It is shown that for a channel mechanism, in general, n is less than or equal to the number of water molecules in a channel that does not contain any cations. Thus, the n of 12 for the 0.15 M salts implies that the gramicidin channel can hold at least 12 water molecules. This places an important constraint on models of the channel structure. The n of 0 for HCl is consistent with a process in which protons jump along a continuous row of water molecules. The decrease of n with the 3.0 M salts may indicate that the channel becomes multiply occupied at high salt concentrations. The n of 4 for nonactin and valinomycin means that at least four water molecules are associated with the carrier . cation complex, probably in the interstices between the complex and the disordered lipid.