Tandem gramicidin channels cross-linked by streptavidin

The interaction of biotin-binding proteins with biotinylated gramicidin (gA5XB) was studied by monitoring single-channel activity and sensitized photoinactivation kinetics. It was discovered that the addition of streptavidin or avidin to the bathing solutions of a bilayer lipid membrane (BLM) with incorporated gA5XB induced the opening of a channel characterized by approximately doubled single-channel conductance and extremely long open-state duration. We believe that the deceleration of the photoinactivation kinetics observed here with streptavidin and previously (Rokitskaya, T.I., Y.N. Antonenko, E.A. Kotova, A. Anastasiadis, and F. Separovic. 2000. Biochemistry. 39:13053-13058) with avidin reflects the formation of long-lived channels of this type. Both opening and closing of the double-conductance channels occurred via a transient sub-state of the conductance coinciding with that of the usual single-channel transition. The appearance of the double-conductance channels after the addition of streptavidin was preceded by bursts of fast fluctuations of the current with the open state duration of the individual events of 60 ms. The streptavidin-induced double-conductance channels appeared to be inherent only to the gramicidin analogue with a biotin group linked to the COOH terminus through a long linker arm. Including biotinylated phosphatidylethanolamine into the BLM prevented the formation of the double-conductance channels even with the excess streptavidin. In view of the results obtained here, it is suggested that the double-conductance channel represents a tandem of two neighboring gA5XB channels with their COOH termini being cross-linked by the bound streptavidin at both sides of the BLM. The finding that streptavidin induces the formation of the tandem gramicidin channel comprising two channels functioning in concert is considered to be relevant to the physiologically important phenomenon of ligand-induced receptor oligomerization.     

Monovalent and Multivalent Binding of Streptavidin to Biotinylated Gramicidin Affects the Kinetic Properties of the Ion Channel

Biotin-avidin (or streptavidin) high affinity binding has been widely applied as a universal tool for basic research as well as diagnostic and therapeutic purposes. Here we studied the interaction of streptavidin with ionic channels formed by biotinylated gramicidin in planar bilayer lipid membranes (BLM) using the method of sensitized photoinactivation. As shown previously, the addition of streptavidin leads to a profound increase in the lifetime (tau) of gA5XB, a biotinylated analog of gramicidin A with a linker arm of five aminocaproyl groups (Rokitskaya et al. (2000) Biochemistry, 39, 13053-13058). The present study has revealed that the increase in tau is related to multivalent interaction of streptavidin with biotinylated gramicidin, i.e., to formation of a complex of streptavidin with several gramicidin channels, whereas binding of streptavidin to a single channel does not change the value of tau. A rather long linker arm attaching biotin to the C-terminus of gramicidin appeared to be required for the multivalent interaction of streptavidin with gramicidin channels, as the increase in tau was not observed with channels formed by gA2XB, the biotinylated gramicidin analog with a linker arm comprising only two aminocaproyl groups. However, the formation of a stoichiometric (1 : 1) complex of streptavidin with gA2XB apparently occurred. The multivalent interaction of streptavidin with gA5XB disappeared if biotinylated lipids were included into the diphytanoylphosphatidylcholine membrane. It is suggested that the slowing of gramicidin channel kinetics provoked by streptavidin binding is due to membrane-mediated elastic interactions between two neighboring channels.     

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double beta(5.7)-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded beta(6.3)-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.     

Sensitized photoinactivation of minigramicidin channels in bilayer lipid membranes

The method of sensitized photoinactivation based on the photosensitized damage of gramicidin A (gA) molecules was applied here to study ionic channels formed by minigramicidin (the 11-residue analogue of gramicidin A) in a planar bilayer lipid membrane (BLM) of different thickness. Irradiation of BLM with a single flash of visible light in the presence of a photosensitizer (aluminum phthalocyanine or Rose Bengal) generating singlet oxygen provoked a decrease in the minigramicidin-induced electric current across BLM, the kinetics of which had the characteristic time of several seconds, as observed with gA. For gA, there is good correlation between the characteristic time of photoinactivation and the single-channel lifetime. In contrast to the covalent dimer of gA characterized by extremely long single-channel lifetime and the absence of current relaxation upon flash excitation, the covalent head-to-head dimer of minigramicidin displayed the flash-induced current decrease with the kinetics being strongly dependent on the membrane thickness. The current decrease became slower both upon increasing the concentration of the minigramicidin covalent dimer and upon including cholesterol in the membrane composition. These data in combination with the quadratic dependence of the current on the peptide concentration can be rationalized by hypothesizing that the macroscopic current across BLM measured at high concentrations of the peptide is provided by dimers of minigramicidin covalent dimers in the double β^5.7-helical conformation having the lifetime of about 0.4 s, while single channels with the lifetime of 0.01 s, observed at a very low peptide concentration, correspond to the single-stranded β^6.3-helical conformation. Alternatively the results can be explained by clustering of channels at high concentrations of the minigramicidin covalent dimer.     

The pH-dependent induction of lipid membrane ionic permeability by N-terminally lysine-substituted analogs of gramicidin A

Insertion of charged groups at the N-terminus of the gramicidin A (gA) amino acid sequence is considered to be fatal for peptide channel-forming activity because of hindrance to the head-to-head dimer formation. Here the induction of ionic conductivity in planar bilayer lipid membranes (BLM) was studied with gA analogs having lysine either in the first ([Lys1]gA) or the third ([Lys3]gA) position. If added to the bathing solution at neutral or acidic pH, these analogs, being protonated and thus positively charged, were unable to induce ionic current across BLM. By contrast, at pH 11 the induction of BLM conductivity was observed with both lysine-substituted analogs. Based on the dependence of the macroscopic current on the side of the peptide addition, sensitivity to calcium ions and susceptibility to sensitized photoinactivation, as well as on the single-channel properties of the analogs, we surmise that at alkaline pH [Lys1]gA formed channels with predominantly single-stranded structure of head-to-head helical dimers, whereas [Lys3]gA open channels had the double-stranded helical structure. CD spectra of the lysine-substituted analogs in liposomes were shown to be pH-dependent.     

Polyanions decelerate the kinetics of positively charged gramicidin channels as shown by sensitized photoinactivation

The effects of different anionic polymers on the kinetic properties of ionic channels formed by neutral gramicidin A (gA) and its positively charged analogs gramicidin-tris(2-aminoethyl)amine (gram-TAEA) and gramicidin-ethylenediamine (gram-EDA) in a bilayer lipid membrane were studied using a method of sensitized photoinactivation. The addition of Konig's polyanion caused substantial deceleration of the photoinactivation kinetics of gram-TAEA channels, which expose three positive charges to the aqueous phase at both sides of the membrane. In contrast, channels formed of gram-EDA, which exposes one positive charge, and neutral gA channels were insensitive to Konig's polyanion. The effect strongly depended on the nature of the polyanion added, namely: DNA, RNA, polyacrylic acid, and polyglutamic acid were inactive, whereas modified polyacrylic acid induced deceleration of the channel kinetics at high concentrations. In addition, DNA was able to prevent the action of Konig's polyanion. In single-channel experiments, the addition of Konig's polyanion resulted in the appearance of long-lived gram-TAEA channels. The deceleration of the gram-TAEA channel kinetics was ascribed to electrostatic interaction of the polyanion with gram-TAEA that reduces the mobility of gram-TAEA monomers and dimers in the membrane via clustering of channels.     

Effect of Linker Length on Avidin Binding to Biotinylated Gramicidin A

Biotinylated gramicidins are an important component of the AMBRI^® “ion channel switch™” biosensor. These gramicidin A (gA) analogues have a biotin attached to the C-terminus of gA via a number of aminocaproyl linker groups (X). The structure of gA5XB has been determined in deuterated sodium dodecyl sulfate micelles and is similar to native gA and other modified gA analogues. The biotin and aminocaproyl groups were mobile and located in the aqueous phase and when avidin was added, NMR and MS studies showed that gA5XB bound more effectively to avidin than gA2XB. The length and flexibility of the linker appears to be important for biotin–avidin binding and, in the AMBRI^® biosensor, gA5XB is a more effective gated ion channel than gA2XB. The conformation and dynamics of the aminocaproyl linker groups were investigated using ²H solid-state NMR. Deuterated aminocaproyl linkers were coupled to gA and incorporated into oriented bilayers in order to analyse the order and dynamics of the aminocaproyl linker. The small ²H splittings and the T ₁ relaxation times indicated that the aminocaproyl linker is undergoing fast rotation in phospholipid bilayers. Native d ₄ -gA as well as d ₄ -gA2XB, where the ethanolamine has been deuterated, were also incorporated into oriented bilayers. Solid-state ²H NMR data showed that the addition of the linker group restricted the mobility of the ethanolamine. However, these modifications to the C-terminus of gA did not interfere with ion channel function and clarify how the biotinylated gA analogues perform in the lipid bilayer as part of the AMBRI^® biosensor.Australian Peptide Conference Issue.     

Cytochrome c decelerates channel kinetics of negatively charged gramicidin due to electrostatic interaction

The effect of cytochrome c on the kinetic properties of ion channels formed by O-pyromellitylgramicidin (OPg), the negatively charged analogue of gramicidin A (gA), in bilayer lipid membranes was studied by the method of sensitized photoinactivation. The addition of cytochrome c to both sides of the membrane caused substantial deceleration of the photoinactivation kinetics of OPg channels which expose three negative charges to the aqueous phase at both sides of the membrane. By contrast, the gA photoinactivation kinetics was unaltered by the addition of cytochrome c. Based on the sensitivity of the observed effect to the ionic strength of the bathing solution, the cytochrome c-induced deceleration of the OPg photoinactivation kinetics reflecting the increase in the OPg channel lifetime was ascribed to electrostatic interaction of positive charges of cytochrome c with negative charges of OPg that resulted in channel clustering. Formation of clusters of OPg channels was previously inferred to explain the polylysine effect on the OPg channel kinetics. The decelerating effect of cytochrome c on OPg channels was observed only at a high number of OPg channels in the membrane, thus suggesting that the interaction between cytochrome c and the charged transmembrane protein requires sufficiently high negative charge density on the surface of the membrane.     

Effect of streptavidins with varying biotin binding affinities on the properties of biotinylated gramicidin channels

The pentadecapeptide gramicidin A, which is known to form highly conductive ion channels in a bilayer lipid membrane by assembling as transmembrane head-to-head dimers, can be modified by attaching a biotin group to its C-terminus through an aminocaproyl spacer. Such biotinylated gramicidin A analogues also form ion channels in a hydrophobic lipid bilayer, exposing the biotin group to the aqueous bathing solution. Interaction of the biotinylated gramicidin channels with (strept)avidin has previously been shown to result in the appearance of a long-lasting open state with a doubled transition amplitude in single-channel traces and a deceleration of the macroscopic current kinetics as studied by the sensitized photoinactivation method. Here this interaction was studied further by using streptavidin mutants with weakened biotin binding affinities. The Stv-F120 mutant, having a substantially reduced biotin binding affinity, exhibited an efficacy similar to that of natural streptavidin in inducing both double-conductance channel formation and deceleration of the photoinactivation kinetics of the biotinylated gramicidin having a long linker arm. The Stv-A23D27 mutant with a severely weakened biotin binding affinity was ineffective in eliciting the double-conductance channels, but decelerated noticeably the photoinactivation kinetics of the long linker biotinylated gramicidin. However, the marked difference in the effects of the mutant and natural streptavidins was smaller than expected on the basis of the substantially reduced biotin binding affinity of the Stv-A23D27 mutant. This may suggest direct interaction of this mutant streptavidin with a lipid membrane in the process of its binding to biotinylated gramicidin channels. The role of linker arm length in the interaction of biotinylated gramicidins with streptavidin was revealed in experiments with a short linker gramicidin. This gramicidin analogue appeared to be unable to form double-conductance channels, though several lines of evidence were indicative of its binding by streptavidin. The data obtained show the conditions under which the interaction of streptavidin with biotinylated gramicidin leads to the formation of the double-conductance tandem channels composed of two cross-linked transmembrane dimers.     

Ionophoric activity of truncated gramicidin A analogue in phospholipid bilayers and mitochondrial and erythrocyte membranes

Ionophoric activities of an N-terminus truncated gramicidin A (gA) analogue (mini-gramicidin) and its covalent dimer were studied in planar bilayer phospholipid membranes (BLM) using macroscopic current measurements (at high concentrations of the peptides) and single-channel recordings. As with gA-induced currents, mini-gramicidin-stimulated macroscopic currents through BLM underwent sensitized photoinactivation, i.e. were suppressed after irradiation with visible light in the presence of a photosensitizer generating singlet oxygen. The sensitivity of the tested compounds to photoinactivation descended in the following order: minigramicidin dimer > mini-gramicidin monomer > gA. The data from single-channel measurements and kinetic analysis of flash-induced photoinactivation obtained at different levels of macroscopic currents suggest that mini-gramicidin and its covalent dimer induce a variety of conducting states; their ratio depends on membrane thickness. Analysis of natural (mitochondrial and erythrocyte) membranes established that ionophoric activities of mini-gramicidin and its covalent dimer depend essentially on the membrane type.     

Stabilization of O-pyromellitylgramicidin channels in bilayer lipid membranes through electrostatic interaction with polylysines of different chain lengths

Functioning of membrane proteins, in particular ionic channels, can be modulated by alteration of their arrangement in membranes. We addressed this issue by studying the effect of different chain length polylysines on the kinetics of ionic channels formed in a bilayer lipid membrane (BLM) by O-pyromellitylgramicidin carrying three negative charges at the C-terminus. The method of sensitized photoinactivation was applied to the analysis of the channel association-dissociation kinetics (characterized by the exponential factor of the curve describing the time course of the flash-induced decrease in the transmembrane current, tau). Addition of polylysine to the bathing solutions of BLM led to the deceleration of the photoinactivation kinetics, i.e. to the increase in tau. It was shown here that for a series of polylysines differing in their chain lengths, the value of tau grew as their concentration increased above a threshold level until at a certain concentration of each polylysine tau reached maximum. At higher polylysine concentrations tau began to decrease and finally became close to the control level observed in the absence of polylysine. With lengthening of the polylysine chain the maximum value of tau increased, the concentration dependence became steeper, and the threshold concentration decreased. The increase in the ionic strength of the medium shifted the concentration dependence of tau to higher polylysine concentrations and decreased the maximum value of tau. It was concluded that the increase in tau was caused by the formation of domains of O-pyromellitylgramicidin molecules induced by binding of polylysines. This can be related to functional aspects of polycation-induced sequestering of negatively charged transmembrane peptides in neutral membranes.     

A single-channel sensor based on gramicidin controlled by molecular recognition at bilayer lipid membranes containing receptor

A novel ion-channel sensor based on a membrane bound receptor and a single gramicidin channel is described, in which the binding of an analyte to the membrane bound receptor modulates the single-channel activity of gramicidin. The sensor is composed of a planar bilayer lipid membrane (BLM) containing biotin-labeled phosphatidylethanolamine as receptor for avidin and gramicidin as signal transducer. When the receptor catches an analyte (avidin or ferritin-labeled avidin (FA)) at the membrane surface, the bilayer structure is locally distorted and the gramicidin monomer/dimer kinetics is modulated in a manner that the fraction of channel opening with a short lifetime ( < or = 100 ms) to the total opening events increases. The fraction was found to increase with the concentration of avidin from 1.0 x 10(-9) to 1.0 x 10(-6) M and of FA from 1.0 x 10(-9) to 1.0 x 10(-8) M. With dinitrophenyl-labeled PE embedded as receptor in the BLM for monoclonal anti-dinitrophenyl antibody (anti-DNP), the fraction of channel openings ( < or = 100 ms) increased with the concentration of anti-DNP from 2.0 x 10(-9) to 2.0 x 10(-7) g/ml. Bovine serum albumin (BSA) and anti-BSA antibody caused no changes in the channel opening. The possible mechanism of analyte-induced modulation of single-channel activity of gramicidin is also discussed.     

Peptide-induced membrane elastic deformations decelerate gramicidin dimer-monomer equilibration

Gramicidin A (gA) is a hydrophobic pentadecapeptide readily incorporating into a planar bilayer lipid membrane (BLM), thereby inducing a large macroscopic current across the BLM. This current results from ion-channel formation due to head-to-head transbilayer dimerization of gA monomers with rapidly established monomer-dimer equilibrium. Any disturbance of the equilibrium, e.g., by sensitized photoinactivation of a portion of gA monomers, causes relaxation toward a new equilibrium state. According to previous studies, the characteristic relaxation time of the gA-mediated electric current decreases as the current increases upon elevating the gA concentration in the membrane. Here, we report data on the current relaxation kinetics for gA analogs with N-terminal valine replaced by glycine or tyrosine. Surprisingly, the relaxation time increased rather than decreased upon elevation of the total membrane conductance induced by these gA analogs, thus contradicting the classical kinetic scheme. We developed a general theoretical model that accounts for lateral interaction of monomers and dimers mediated by membrane elastic deformations. The modified kinetic scheme of the gramicidin dimerization predicts the reverse dependence of the relaxation time on membrane conductance for gA analogs, with a decreased dimerization constant that is in a good agreement with our experimental data. The equilibration process may be also modulated by incorporation of other peptides (“impurities”) into the lipid membrane.     

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.     

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.     

Chemical and Photochemical Modification of Colicin E1 and Gramicidin A in Bilayer Lipid Membranes

Chemical modification and photodynamic treatment of the colicin E1 channel-forming domain (P178) in vesicular and planar bilayer lipid membranes (BLMs) was used to elucidate the role of tryptophan residues in colicin E1 channel activity. Modification of colicin tryptophan residues by N-bromosuccinimide (NBS), as judged by the loss of tryptophan fluorescence, resulted in complete suppression of wild-type P178 channel activity in BLMs formed from fully saturated (diphytanoyl) phospholipids, both at the macroscopic-current and single-channel levels. The similar effect on both the tryptophan fluorescence and the electric current across BLM was observed also after NBS treatment of gramicidin channels. Of the single-tryptophan P178 mutants studied, W460 showed the highest sensitivity to NBS treatment, pointing to the importance of the water-exposed Trp460 in colicin channel activity. In line with previous work, the photodynamic treatment (illumination with visible light in the presence of a photosensitizer) led to suppression of P178 channel activity in diphytanoyl-phospholipid membranes concomitant with the damage to tryptophan residues detected here by a decrease in tryptophan fluorescence. The present work revealed novel effects: activation of P178 channels as a result of both NBS and photodynamic treatments was observed with BLMs formed from unsaturated (dioleoyl) phospholipids. These phenomena are ascribed to the effect of oxidative modification of double-bond-containing lipids on P178 channel formation. The pronounced stimulation of the colicin-mediated ionic current observed after both pretreatment with NBS and sensitized photomodification of the BLMs support the idea that distortion of membrane structure can facilitate channel formation.Abbreviations: AlP_cS₃, almininum trisulfophthalocyanine; BLM, bilayer lipid membrane; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidyl-glycerol; DPhPG, diphytanoylphos-phatidylglycerol; DPhPg, diphytanoylphosphatidylcholine; gA, gramicidin A; NBS, N-bromosuccinimideThis revised version was published online in August 2005 with a corrected cover date.     

The membrane interface dictates different anchor roles for "inner pair" and "outer pair" tryptophan indole rings in gramicidin A channels

We investigated the effects of substituting two of the four tryptophans (the "inner pair" Trp(9) and Trp(11) or the "outer pair" Trp(13) and Trp(15)) in gramicidin A (gA) channels. The conformational preferences of the doubly substituted gA analogues were assessed using circular dichroism spectroscopy and size-exclusion chromatography, which show that the inner tryptophans 9 and 11 are critical for the gA's conformational preference in lipid bilayer membranes. [Phe(13,15)]gA largely retains the single-stranded helical channel structure, whereas [Phe(9,11)]gA exists primarily as double-stranded conformers. Within this context, the (2)H NMR spectra from labeled tryptophans were used to examine the changes in average indole ring orientations, induced by the Phe substitutions and by the shift in conformational preference. Using a method for deuterium labeling of already synthesized gAs, we introduced deuterium selectively onto positions C2 and C5 of the remaining tryptophan indole rings in the substituted gA analogues for solid-state (2)H NMR spectroscopy. The (least possible) changes in orientation and overall motion of each indole ring were estimated from the experimental spectra. Regardless of the mixture of backbone folds, the indole ring orientations observed in the analogues are similar to those found previously for gA channels. Both Phe-substituted analogues form single-stranded channels, as judged from the formation of heterodimeric channels with the native gA. [Phe(13,15)]gA channels have Na(+) currents that are ~50% and lifetimes that are ~80% of those of native gA channels. The double-stranded conformer(s) of [Phe(9,11)]gA do not form detectable channels. The minor single-stranded population of [Phe(9,11)]gA forms channels with Na(+) currents that are ~25% and single-channel lifetimes that are ~300% of those of native gA channels. Our results suggest that Trp(9) and Trp(11), when "reaching" for the interface, tend to drive both monomer folding (to "open" a channel) and dimer dissociation (to "close" a channel). Furthermore, the dipoles of Trp(9) and Trp(11) are relatively more important for the single-channel conductance than are the dipoles of Trp(13) and Trp(15).     

Correlation analysis of electrical noise in lipid bilayer membranes: kinetics of gramicidin A channels.

If a membrane contains ion-conducting channels which form and disappear in a random fashion, an electric current which is passed through the membrane under constant voltage shows statistical fluctuations. Information on the kinetics of channel formation and on the conductance of the single channel may be obtained by analyzing the electrical noise generated in a membrane containing a great number of channels. For this purpose the autocorrelation function of the current noise is measured at different concentrations of the channel-forming substance. As a test system for the application of this technique we have used lipid bilayer membranes doped with gramicidin A. From the correlation time of the current noise generated by the membrane, the rate constants of formation (k-R) and dissociation (k-D) of the channels could be determined. In addition, the mean square of the current fluctuations yielded the single-channel conductance lambda. The values of k-R, k-D, and lambda obtained from the noise analysis agreed closely with the values determined by relaxation measurments and single-channel experiments.     

Noise analysis of the glutamate-activated current in photoreceptors.

The glutamate-activated current in photoreceptors has been attributed both to a sodium/glutamate transporter and to a glutamate-activated chloride channel. We have further studied the glutamate-activated current in single, isolated photoreceptors from the tiger salamander using noise analysis on whole-cell patch-clamp recordings. In cones, the current is generated by chloride channels with a single-channel conductance of 0.7 pS and an open lifetime of 2.4 ms. The number of channels per cell is in the range of 10,000-20,000. Activation of the channels requires the presence of both glutamate and sodium. The single-channel conductance and the open lifetime of the channel are independent of the external concentration of glutamate and sodium. External glutamate and sodium affect only the opening rate of the channels. D,L-Threo-3-hydroxyaspartate (THA), a glutamate-transport blocker, is shown to be a partial agonist for the channel. The single-channel conductance is the same regardless of whether glutamate or THA is the ligand, but the open lifetime of the channel is only 0.8 ms with THA as ligand. The glutamate-activated current in rods has a similar single-channel conductance (0.74 pS) and open lifetime (3 ms). We propose a kinetic model, consistent with these results, to explain how a transporter can simultaneously act both as a sodium/glutamate-gated chloride channel and a glutamate/sodium cotransporter.