Effect of the dipole potential of a bilayer lipid membrane on gramicidin channel dissociation kinetics.

A technique of measuring of the light-induced transients of the gramicidin-mediated electric current across a membrane in the presence of a photosensitizer has been applied for the study of the effect of agents modifying the dipole potential of a bilayer lipid membrane (phloretin, 6-ketocholestanol, and RH421) on the processes of the gramicidin channel dissociation and formation. It is shown that phloretin, known to lower the dipole potential, decelerates the flash-induced decrease in the current, whereas 6-ketocholestanol and RH421, known to raise the dipole potential, accelerate the current decrease. It is revealed that the addition of phloretin leads to a decrease in the dissociation rate constant, whereas addition of either 6-ketocholestanol or RH421 causes an increase in this constant. Single-channel data show that phloretin brings about an increase in the lifetime of the gramicidin channels, whereas RH421 produces a more complicated effect. It is conclude that the dipole potential affects the process of channel dissociation, presumably via the influence on the movement of the dipoles of gramicidin molecules through the layer of the dipole potential drop near the membrane-water interface.     

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.     

Noncontact dipole effects on channel permeation. III. Anomalous proton conductance effects in gramicidin

Proton transport on water wires, of interest for many problems in membrane biology, is analyzed in side-chain analogs of gramicidin A channels. In symmetrical 0.1 N HCl solutions, fluorination of channel Trp(11), Trp-(13), or Trp(15) side chains is found to inhibit proton transport, and replacement of one or more Trps with Phe enhances proton transport, the opposite of the effects on K(+) transport in lecithin bilayers. The current-voltage relations are superlinear, indicating that some membrane field-dependent process is rate limiting. The interfacial dipole effects are usually assumed to affect the rate of cation translocation across the channel. For proton conductance, however, water reorientation after proton translocation is anticipated to be rate limiting. We propose that the findings reported here are most readily interpreted as the result of dipole-dipole interactions between channel waters and polar side chains or lipid headgroups. In particular, if reorientation of the water column begins with the water nearest the channel exit, this hypothesis explains the negative impact of fluorination and the positive impact of headgroup dipole on proton conductance.     

Proton conduction in gramicidin A and in its dioxolane-linked dimer in different lipid bilayers.

Gramicidin A (gA) molecules were covalently linked with a dioxolane ring. Dioxolane-linked gA dimers formed ion channels, selective for monovalent cations, in planar lipid bilayers. The main goal of this study was to compare the functional single ion channel properties of natural gA and its covalently linked dimer in two different lipid bilayers and HCl concentrations (10-8000 mM). Two ion channels with different gating and conductance properties were identified in bilayers from the product of dimerization reaction. The most commonly observed and most stable gramicidin A dimer is the main object of this study. This gramicidin dimer remained in the open state most of the time, with brief closing flickers (tau(closed) approximately 30 micros). The frequency of closing flickers increased with transmembrane potential, making the mean open time moderately voltage dependent (tau(open) changed approximately 1.43-fold/100 mV). Such gating behavior is markedly different from what is seen in natural gA channels. In PEPC (phosphatidylethanolamine-phosphatidylcholine) bilayers, single-channel current-voltage relationships had an ohmic behavior at low voltages, and a marked sublinearity at relatively higher voltages. This behavior contrasts with what was previously described in GMO (glycerylmonooleate) bilayers. In PEPC bilayers, the linear conductance of single-channel proton currents at different proton concentrations was essentially the same for both natural and gA dimers. g(max) and K(D), obtained from fitting experimental points to a Langmuir adsorption isotherm, were approximately 1500 pS and 300 mM, respectively, for both the natural gA and its dimer. In GMO bilayers, however, proton affinities of gA and the dioxolane-dimer were significantly lower (K(D) of approximately 1 and 1.5 M, respectively), and the g(max) higher (approximately 1750 and 2150 pS, respectively) than in PEPC bilayers. Furthermore, the relationship between single-channel conductance and proton concentration was linear at low bulk concentrations of H+ (0.01-2 M) and saturated at concentrations of more than 3 M. It is concluded that 1) The mobility of protons in gramicidin A channels in different lipid bilayers is remarkably similar to proton mobilities in aqueous solutions. In particular, at high concentrations of HCl, proton mobilities in gramicidin A channel and in solution differ by only 25%. 2) Differences between proton conductances in gramicidin A channels in GMO and PEPC cannot be explained by surface charge effects on PEPC membranes. It is proposed that protonated phospholipids adjacent to the mouth of the pore act as an additional source of protons for conduction through gA channels in relation to GMO bilayers. 3) Some experimental results cannot be reconciled with simple alterations in access resistance to proton flow in gA channels. Said differences could be explained if the structure and/or dynamics of water molecules inside gramicidin A channels is modulated by the lipid environment and by modifications in the structure of gA channels. 4) The dioxolane ring is probably responsible for the closing flickers seen in the dimer channel. However, other factors can also influence closing flickers.     

Effect of dipole modifiers on the kinetics of sensitized photoinactivation of gramicidin channels in bilayer lipid membranes

Photodynamic inactivation of gramicidin channels in bilayer lipid membranes induced by single flashes of the visible light in the presence of phthalocyanine has been studied. The kinetic curves of the flash-induced decrease in the gramicidin-mediated electric current are used for determination of the rate constants of formation and termination of gramicidin channels in terms of the channel dimer model. It is revealed that the kinetics of the sensitized photoinactivation of gramicidin in the membrane is altered by agents which modify the dipole potential drop at the membrane-water interface. Addition of phloretin, which is known to decrease the dipole potential drop, slows down the kinetics, whereas the addition of RH421 or 6-ketocholestanol, which increase the dipole potential drop, accelerates the kinetics. It is shown that the photoinactivation kinetics is also slowed down upon the addition of the thyroid hormone L-thyronine, which reduces the dipole potential drop similar to phloretin, as it was found earlier (M. V. Tsybulskaya, Yu. N. Antonenko, A. E. Tropsha, and L. S. Yaguzhinsky, Biofizika 29:801-805 (1984) (in Russian)). It is demonstrated that the changes in the dissociation rate constant of gramicidin dimers under the action of different dipole modifiers correlate with the changes in the dipole potential drop. It is concluded that the process of the gramicidin channel termination corresponding to the dimer dissociation is sensitive to the dipole potential drop. This conclusion is supported by the data on the effect of dipole modifiers on the lifetime of single gramicidin channels.     

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.     

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.     

Mechanism of uncoupling of oxidative phosphorylation by gramicidin

The mechanism of the uncoupling of oxidative phosphorylation in rat liver mitochondria by gramicidin and truncated gramicidin derivatives was investigated. The derivatives desformylgramicidin and des(formylvalyl)gramicidin are not expected to form head to head, dimeric, ion-conducting channels, and thus allow an evaluation of the relevance of the stimulation of transmembrane cation conductance (and the resulting collapse of the proton electrochemical gradient) to the uncoupling of oxidative phosphorylation. When assayed for the enhancement of the passive diffusion of KSCN, gramicidin was 100-fold more potent than desformylgramicidin and 50-fold more potent than des(formylvalyl)gramicidin. Yet, in a medium devoid of alkalai cations, all three compounds were nearly equally potent uncouplers at low concentrations. Moreover, this uncoupling was not associated with stimulation of cation transport or a reduction of the magnitude of the proton electrochemical potential. In the same medium, gramicidin stimulated 86Rb uptake 50-fold more than desformylgramicidin and 10 times more than des(formylvalyl)gramicidin. At higher concentrations, gramicidin induced further uncoupling, which was associated with reduction of membrane potential (and presumably with transport of alkali cations), while the truncated derivatives were considerably less effective than gramicidin in this range. Thus, with the truncated derivatives, a better separation between decoupling (i.e., uncoupling not associated with reduction of delta mu H) and uncoupling is observed. In the same medium, gramicidin, but not the truncated derivatives, strongly inhibits the formation of both the membrane potential and delta pH by the H+-ATPase. This finding suggests direct interaction of gramicidin with the H+-ATPase. The truncated derivatives stimulated the ATPase without collapsing the membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Gramicidin channels that have no tryptophan residues.

In order to understand how aromatic residues modulate the function of membrane-spanning proteins, we examined the role of the four tryptophans in gramicidin A (gA) in determining the average duration and permeability characteristics of membrane-spanning gramicidin channels; the tryptophan residues were replaced by tyrosine (gramicidin T, gT), tyrosine O-benzyl ether [gramicidin T(Bzl), gT(Bzl)], naphthylalanine (gramicidin N, gN), and phenylalanine (gramicidin M enantiomer, gM-). These analogues form channels with durations and conductances that differ some 10- and 16-fold, respectively. The single-channel conductance was invariably decreased by the Trp----Yyy replacement, and the relative conductance alterations were similar in phosphatidylcholine (DPhPC) and monoglyceride (GMO) bilayers. The duration variations exhibited a more complex pattern, which was quite different in the two membrane environments: in DPhPC bilayers, gN channels have an average duration that is approximately 2-fold longer than that of gA channels; in GMO bilayers, the average duration of gN channels is about one-tenth that of gA channels. The sequence-dependent alterations in channel function do not result from alterations in the channels' peptide backbone structure, because heterodimers can form between the different analogues and gramicidine A, and there is no energetic cost associated with heterodimer formation [cf. Durkin, J. T., Koeppe, R. E., II, & Andersen, O. S. (1990) J. Mol. Biol. 211, 221]. The alterations in permeability properties are consistent with the notion that Trp residues alter the energy profile for ion permeation through long-range electrostatic interactions.     

Asymmetric gramicidin channels: heterodimeric channels with a single F6Val1 residue.

Substitution of Val1 by 4,4,4,4',4',4'-F6Val in [Val1]gramicidin A ([Val1]gA) produces channels in which the effects of amino acid replacements on dimer stability and ion permeation are nonadditive. If only one Val1 (in a symmetric [Val1]gA channel) is substituted by F6Val, the resulting heterodimeric channels are destabilized relative to both homodimeric parent channels and the single-channel conductance of the heterodimeric channels is reduced relative to the parent channels (Russell, E. W. B., L. B. Weiss, F. I. Navetta, R. E. Koeppe II, and O. S. Andersen. 1986. Single-channel studies on linear gramicidins with altered amino acid side chains. Effects of altering the polarity of the side chain at position #1 in gramicidin A. Biophys. J. 49:673; Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. Energetics of gramicidin hybrid channel formation as a test for structural equivalence. Side-chain substitutions in the native sequence. J. Mol. Biol. 211:221-234). To understand the basis for this destabilization, we have examined further the characteristics of [F6Val1]/[Xxx1]gA heterodimers, where Xxx = Gly, Val, and Ala. These heterodimeric channels show rapid current transitions between (at least) two current levels and display asymmetric i-V characteristics. The orientation of the heterodimers relative to the applied potential was determined by asymmetric addition of the gramicidin analogs, one to each side of a preformed bilayer. The current transitions are most clearly illustrated for [F6Val1]/[Gly1]gA heterodimers, which possess two finite and well defined current levels. Based on the existence of these two conductance states and the analysis of duration and interval distributions, we conclude that the transitions between the two current levels correspond to conformational transitions in "stable" heterodimers. In the case of [F6Val1]/[Val1]gA and [F6Val1]/[Ala1]gA heterodimers, the low-conductance state is indistinguishable from zero. The two (or more) conductance states presumably correspond to different orientations of the dipolar F6Val1 side chain. The distribution between the high- and the low-conductance states varies as a function of potential in [F6Val1]/[Gly1]gA channels. These characteristics cause the [F6Val1]/nonpolar (Val, Ala, Gly)gA hybrid channels to serve as a "simple" model for understanding gating transitions in membrane-spanning channels.     

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.     

Attenuation of proton currents by methanol in a dioxolane-linked gramicidin A channel in different lipid bilayers.

The mobility of protons in a dioxolane-linked gramicidin A channel (D1) is comparable to the mobility of protons in aqueous solutions (Cukierman, S., E. P. Quigley, and D. S. Crumrine. 1997. Biophys. J. 73:2489-2502). Aliphatic alcohols decrease the mobility of H+ in aqueous solutions. In this study, the effects of methanol on proton conduction through D1 channels were investigated in different lipid bilayers and at different HCl concentrations. Methanol attenuated H+ currents in a voltage-independent manner. Attenuation of proton currents was also independent of H+ concentrations in solution. In phospholipid bilayers, methanol decreased the single channel conductance to protons without affecting the binding affinity of protons to bilayers. In glycerylmonooleate membranes, the attenuation of single channel proton conductances qualitatively resembled the decrease of conductivities of HCl solutions by methanol. However, in both types of lipid bilayers, single channel proton conductances through D1 channels were considerably more attenuated than the conductivities of different HCl solutions. This suggests that methanol modulates single proton currents through D1 channels. It is proposed that, on average, one methanol molecule binds to a D1 channel, and attenuates H+ conductance. The Gibbs free energy of this process (DeltaG0) is approximately 1.2 kcal/mol, which is comparable to the free energy of decrease of HCl conductivity in methanol solutions (1.6 kcal/mol). Apolar substances like urea and glucose that do not transport protons in HCl solutions and do not permeate D1 channels decreased solution conductivity and single channel conductance by a considerably larger proportion than methanol. Cs+ currents through D1 channels were considerably less (fivefold) attenuated by methanol than proton currents. It is proposed that methanol partitions inside the pore of gramicidin channels and delays the transfer of protons between water and methanol molecules, causing a significant attenuation of the single channel proton conductance. Gramicidin channels offer an interesting experimental model to study proton hopping along a single chain of water molecules interrupted by a single methanol molecule.     

Noncontact dipole effects on channel permeation. II. Trp conformations and dipole potentials in gramicidin A

The four Trp dipoles in the gramicidin A (gA) channel modulate channel conductance, and their side chain conformations should therefore be important, but the energies of different conformations are unknown. A conformational search for the right-handed helix based on molecular mechanics in vacuo yielded 46 conformations within 20 kcal/mol of the lowest energy conformation. The two lowest energy conformations correspond to the solid-state and solution-state NMR conformations, suggesting that interactions within the peptide determine the conformation. For representative conformations, the electrostatic potential of the Trp side chains on the channel axis was computed. A novel application of the image-series method of. Biophys. J. 9:1160-1170) was introduced to simulate the polarization of bulk water by the Trp side chains. For the experimentally observed structures, the CHARm toph19 potential energy (PE) of a cation in the channel center is -1.65 kcal/mol without images. With images, the PE is -1.9 kcal/mol, demonstrating that the images further enhance the direct dipole effect. Nonstandard conformations yielded less favorable PEs by 0.4-1.1 kcal/mol.     

Conducting gramicidin channel activity in phospholipid monolayers

Potential step amperometry (chronoamperometry) of the Tl(I)/Tl(Hg) electrochemical reduction process has been used to investigate the underlying mechanisms of gramicidin activity in phospholipid monolayers. The experiments were carried out at gramicidin-modified dioleoyl phosphatidylcholine (DOPC)-coated electrodes. Application of a potential step to the coated electrode system results in a current transient that can be divided into two regions. An initial exponential decay of current corresponds to the inactivation of monomer channel conductance and a longer time scale quasi-steady-state represents the diffusion of ions to a bimolecular surface reaction. Concentrations of monomer conducting channels are relatively low, and the results indicate that two or more forms of gramicidin are in equilibrium with each other in the layer. Aromatic/conjugated compounds incorporated into the monolayer increase the reduction current by decreasing the rate of channel inactivation and increasing the stability of the conducting channel. This effect is positively correlated with the degree of the compound's aromaticity. The anomalous influence of alkali metal ions on the reduction current is consistent with the model of gramicidin being speciated in the monolayer in more than one form. The results have implications on the lability of the peptide conformation in biological membranes and its dependence on lipid environment, solution composition, and applied potential.     

Effect of thallium ions on gramicidin-induced conductance of muscle fiber membranes

Conductance of single fibres from m. ileofibularis of Rana esculenta was studied in isotonic K2SO4 solution under constant current conditions using the double sucrose gap method. It was found that Tl+ (at concentrations 5, 10, and 20 mM) blocked K+ currents in the gramicidin channel. The decrease of K+ conductance caused by Tl+ was associated with the changes of the membrane potential. Both the decrease of K+ conductance and value of permeability ratio (PTl/PK) found from the membrane potential changes depended on Tl+ concentration in the bathing solution. No effect of Tl+ on the potassium channels was registered in the absence of gramicidin channels. The Tl+ block described here proves the existence of Tl+ ion binding within gramicidin channels of the muscle membrane and interactions among ions in the channels.     

Action of calcium channel and beta-adrenergic blocking agents in bilayer lipid membranes

The action of beta-adrenergic blockers (propranolol, exprenolol, metoprolol, sotalol, atenolol, timolol) and calcium-channel blockers (verapamil, diltiazem) on the electrical properties and fluidity of bilayer lipid membranes (BLM and liposomes) has been investigated. When antibiotic ionophore substances were used as a probe, the electrical measurements showed that many of the drugs inhibited the cation transport across the membrane facilitated by the mobile carrier valinomycin, while having no significant effect on the cation transport through channels formed by gramicidin. The ability of the drugs to decrease the carrier-dependent membrane conductance was correlated to their partition into the lipid bilayer and the magnitude of transmembrane potential induced by them. In the TEMPO ESR spectral measurements, a number of beta-adrenergic and calcium blockers showed the fluidizing effect on liposomes composed of different lipids. The drug concentration required for a detectable change in TEMPO spectra parameter (f) was rather high (0.01 M verapamil), and the variation of pH from 6.5 to 3.0 did not affect the fluidizing effect of the drugs.     

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.     

Interfacial polar interactions affect gramicidin channel kinetics

Critical to biological processes such as secretion and transport, protein-lipid interactions within the membrane and at the membrane-water interface still raise many questions. Here we examine the role of lipid headgroups in these interactions by using gramicidin A (gA) channels in planar bilayers as a probe. We show that although headgroup demethylation from phosphatidylcholine (DOPC) to phosphatidylethanolamine decreases the lifetime of gA channels by an order of magnitude in accordance with the currently accepted hydrophobic mismatch mechanism, our findings with diether-DOPC suggest the importance of the headgroup-peptide interactions. According to our x-ray diffraction measurements, this lipid has the same hydrophobic thickness as DOPC but increases gA lifetime by a factor of 2. Thus we demonstrate that peptide-headgroup interactions may dominate over the effect of hydrophobic mismatch in regulating protein function.     

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.     

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.