Structural and functional crosstalk between acetylcholine receptor and its membrane environment

Nicotinic acetylcholine receptor (AChR) is a transmembrane protein belonging to the superfamily of rapid, ligand-operated channels. Theoretical models based on thermodynamic criteria assign portions of the polypeptide chains to the lipid bilayer region. From an experimental point of view, however, the relationship between the two moieties remains largely unexplored. Current studies from our laboratory are aimed at defining the structural, dynamic, and functional relationship between membrane lipids and AChR. We are particularly interested in establishing the characteristics of and differences between the lipids in each leaflet of the bilayer and the belt or “annular” lipids immediately surrounding AChR and the bulk bilayer lipids. We are also interested in determining the possible implications of lipid modifications on AChR channel properties. Toward these ends, fluorescence and other spectroscopic techniques, together with biochemical analyses and patch-clamp studies, are currently being undertaken. Correlations can be established between structural aspects of phospholipid packing in the immediate perimeter of AChR and other properties of these annular lipids revealed by dynamic spectroscopic and molecular modeling techniques. Lipid compositional analyses of the clonal muscle cell line BC3H-1 and chemical modification studies have been carried out by incubation of intact cells in culture and of membrane patches excised therefrom with liposomes of different lipid composition. These studies have been combined with electrophysiological measurements using the patch-clamp technique, with the aim of determining the possible effects of lipids on the channel properties of muscle-type AChR. A variety of experimental conditions, involving polar head and fatty acyl chain substitution of phospholipids and cholesterol incorporation, are being assayed in the BC3H-1 cells. Dedicated to the memory of the late E. De Robertis.     

Physical state of bulk and protein-associated lipid in nicotinic acetylcholine receptor-rich membrane studied by laurdan generalized polarization and fluorescence energy transfer.

The spectral properties of the fluorescent probe laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) were exploited to learn about the physical state of the lipids in the nicotinic acetylcholine receptor (AChR)-rich membrane and compare them with those in reconstituted liposomes prepared from lipids extracted from the native membrane and those formed with synthetic phosphatidylcholines. In all cases redshifts of 50 to 60 nm were observed as a function of temperature in the spectral emission maximum of laurdan embedded in these membranes. The so-called generalized polarization of laurdan exhibited high values (0.6 at 5 degrees C) in AChR-rich membranes, diminishing by approximately 85% as temperature increased, but no phase transitions with a clear Tm were observed. A still unexploited property of laurdan, namely its ability to act as a fluorescence energy transfer acceptor from tryptophan emission, has been used to measure properties of the protein-vicinal lipid. Energy transfer from the protein in the AChR-rich membrane to laurdan molecules could be observed upon excitation at 290 nm. The efficiency of this process was approximately 55% for 1 microM laurdan. A minimum donor-acceptor distance r of 14 +/- 1 A could be calculated considering a distance 0 < H < 10 A for the separation of the planes containing donor and acceptor molecules, respectively. This value of r corresponds closely to the diameter of the first-shell protein-associated lipid. A value of approximately 1 was calculated for Kr, the apparent dissociation constant of laurdan, indicating no preferential affinity for the protein-associated probe, i.e., random distribution in the membrane. From the spectral characteristics of laurdan in the native AChR-rich membrane, differences in the structural and dynamic properties of water penetration in the protein-vicinal and bulk bilayer lipid regions can be deduced. We conclude that 1) the physical state of the bulk lipid in the native AChR-rich membrane is similar to that of the total lipids reconstituted in liposomes, exhibiting a decreasing polarity and an increased solvent dipolar relaxation at the hydrophilic/hydrophobic interface upon increasing the temperature; 2) the wavelength dependence of laurdan generalized polarization spectra indicates the presence of a single, ordered (from the point of view of molecular axis rotation)-liquid (from the point of view of lateral diffusion) lipid phase in the native AChR membrane; 3) laurdan molecules within energy transfer distance of the protein sense protein-associated lipid, which differs structurally and dynamically from the bulk bilayer lipid in terms of polarity and molecular motion and is associated with a lower degree of water penetration.     

Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Laurdan is a fluorescent probe that detects changes in membrane phase properties through its sensitivity to the polarity of its environment in the bilayer. Variations in membrane water content cause shifts in the laurdan emission spectrum, which are quantified by calculating the generalized polarization (GP). We tested whether laurdan fluorescence could be used to distinguish differences in phospholipid order from changes in membrane fluidity by examining the temperature dependence of laurdan GP and fluorescence anisotropy in dipalmitoylphosphatidylcholine (DPPC) vesicles. The phase transition from the solid ordered phase to the liquid disordered phase was observed as a decrease in laurdan GP values from 0.7 to −0.14 and a reduction in anisotropy from 0.25 to 0.12. Inclusion of various amounts of cholesterol in the membranes to generate a liquid ordered phase caused an increase in the apparent melting temperature detected by laurdan GP. In contrast, cholesterol decreased the apparent melting temperature estimated from anisotropy measurements. Based on these results, it appeared that laurdan anisotropy detected changes in membrane fluidity while laurdan GP sensed changes in phospholipid order. Thus, the same fluorescent probe can be used to distinguish effects of perturbations on membrane order and fluidity by comparing the results of fluorescence emission and anisotropy measurements.     

Assembly of an adult type acetylcholine receptor in a mouse cell line transfected with rat muscle ε-subunit DNA

The mouse muscle cell line BC3H-1 expresses an acetylcholine receptor (AChR) composed of -,β-, and δ-subunits [1]. The functional characteristics of this AChR are comparable to the non-synaptic AChR subtype in mouse muscle [2,3]. To investigate the role of the ε-subunit, which is believed to replace the γ-subunit in forming the adult AChR subtype [4], BC3H-1 cells were stably transfected with cDNA encoding the rat muscle AChR ε-subunit. Expression of this cDNA was under the control of a heat shock promoter, and the plasmid carried the neomycin resistance gene for selection. Several clones were isolated that had integrated the plasmid DNA in a stable form and produced ε-subunit specific RNA after heat induction. Single-channel current recording from cells which contained abundant ε-subunit mRNA identified a novel AChR channel having a larger conductance than the native AChR in these cells. These results suggest that the rat muscle ε-subunit may assemble with mouse muscle -, β- and δ-subunits to form a mouse-rat hybrid AChR with properties similar to that of end-plate channels in the mature mammalian neuromuscular synapse. The novel AChR channel appears in the surface membrane within a few hours following the rise in ε-subunit mRNA. Thus, the notion that replacement of the γ-subunit by the ε-subunit during development is the result of the postnatal rise in the level of ε-subunit specific mRNA is further supported.     

Antibodies affect the ionic conductance of channels formed by amphotericin B in a lipid bilayer

For the first time poly- and monoclonal antibodies (class IgM) against the polyene antibiotic amphotericin B were obtained affecting the properties of a channel formed by the antibiotic and cholesterol in a lipid bilayer when amphotericin B was added to the solution at one (cis) side of the membrane. In the case of the symmetric distribution of cholesterol in the lipid bilayer, three molecules of monoclonal antibodies bind firmly to the channel at the trans-side of the membrane, thus strongly increasing the mean lifetime of the channel in the open state, and not changing practically the ion conductance of its open state. The antibodies did not alter the properties of these channels when added at the cis-side of the membrane as well as of the channels formed in the lipid bilayer when amphotericin B was added at both membrane sides. The antibodies obtained did not affect the conductance of channels in which amphotericin B and cholesterol were replaced with their analogs levorin and 5 alpha-androstan-3 beta-one, which points to a high specificity of the immunoglobulins isolated. When cholesterol was present only in the cis-monolayer of the lipid bilayer and was absent in the trans-monolayer, the same monoclonal antibodies when added at the trans-side of the membrane blocked the conductance of the channel formed by adding the antibiotic to the solution at the cis-side of the bilayer. The obtained evidence is of interest in elucidating the general features of interaction of antibodies with the ionic channels of cellular and model membranes.     

Time-resolved Fluorescence and Generalized Polarization: Innovative tools to assess bull sperm membrane dynamics during slow freezing

During slow freezing, spermatozoa undergo membrane alterations that compromise their ability of fertilizing. These alterations are cause either by cold shock or by the use of cryoprotectants known to be cytotoxic. However, little is known about the membrane changes that occurred during freezing. Here, we combined Generalized Polarization (GP), Time-resolved Fluorescence and laurdan fluorescence properties to investigate the changes in membrane fluidity and dynamics during slow freezing of bull sperm. We successfully demonstrated that laurdan may be distributed in three different local environments that correspond to different membrane lipid composition. These environments wont behave the same way when the cells will be subjected to either a chemical treatment (adding the cryoprotectants) or a physical treatment (freezing).     

Gangliosides asymmetrically alter the membrane order in cultured PC-12 cells

Exogenous gangliosides readily associate with the cell membranes and produce marked effects on cell growth and differentiation. We have studied the effect of bovine brain gangliosides (BBG) on the membrane dynamics of intact cells. The structural and dynamic changes in the cell membrane were monitored by the fluorescence probes DPH, TMA-DPH and laurdan. Incorporation of BBG into the cell membrane decreased the fluorescence intensity, lifetime and the steady state anisotropy of TMA-DPH. Analysis of the time resolved anisotropy decay by wobbling in the cone model revealed that BBG decreased the order parameter, and increased the cone angle without altering the rotational relaxation rate. The fluorescence intensity and lifetime of DPH were unaffected by BBG incorporation, however, a modest increase was observed in the steady state anisotropy. BBG incorporation reduced the total fluorescence intensity of laurdan with pronounced quenching of the 440-nm band. The wavelength sensitivity of generalized polarization of laurdan manifested an ordered liquid crystalline environment of the probe in the cell membrane. BBG incorporation reduced the GP values and augmented the liquid crystalline behavior of the cell membrane. BBG incorporation also influenced the permeability of cell membranes to cations. An influx of Na+ and Ca2+ and an efflux of K+ was observed. The data demonstrate that incorporation of gangliosides into the cell membrane substantially enhances the disorder and hydration of the lipid bilayer region near the exoplasmic surface. The inner core region near the center of the bilayer becomes slightly more ordered and remains highly hydrophobic. Such changes in the structure and dynamics of the membrane could play an important role in modulation of transmembrane signaling events by the gangliosides.     

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.     

Stable expression of the mouse nicotinic acetylcholine receptor in mouse fibroblasts. Comparison of receptors in native and transfected cells.

A stable cell line expressing mouse acetylcholine receptors (AChRs), named AM4, was established by cotransfecting into NIH 3T3 fibroblasts, alpha-, beta-, gamma-, and delta-subunit cDNAs plus the neor gene by calcium phosphate precipitation. Surface AChRs on AM4 cells contain all four subunits, sediment as a single approximately 9 S peak on sucrose gradients, and have the same ratio of alpha- to beta-subunits as surface AChRs from mouse BC3H-1 cells. The surface AChRs exhibit pharmacological properties identical to those obtained for BC3H-1 cells, including the association and dissociation rates of alpha-bungarotoxin, a low affinity and cooperative instantaneous dose-response curve, cooperative steady state agonist binding and desensitization, cooperative enhancement of agonist binding affinity by local anesthetics, and distinct affinities for curariform antagonists. Patch clamp measurements on AM4 cells reveal AChR single channel properties identical to those obtained from BC3H-1 cells, including a single class of channels with a conductance of 56 pS, short and long duration openings at low and high agonist concentrations, brief and intermediate closed duration components at low agonist concentrations, and six distinct closed duration components at high agonist concentrations. The biochemical, pharmacological, and single channel measurements indicate at least 95% of the surface AChRs on AM4 cells are alpha 2 beta gamma delta pentamers.     

pH modulation of large conductance potassium channel from adrenal chromaffin granules

We report here that large conductance K(+) selective channel in adrenal chromaffin granules is controlled by pH. We measured electrogenic influx of (86)Rb(+) into chromaffin granules prepared from bovine adrenal gland medulla. The (86)Rb(+) influx was inhibited by acidic pH. Purified chromaffin granule membranes were also fused with planar lipid bilayer. A potassium channel with conductance of 432+/-9 pS in symmetric 450 mM KCl was observed after reconstitution into lipid bilayer. The channel activity was unaffected by charybdotoxin, a blocker of the Ca(2+)-activated K(+) channel of large conductance. It was observed that acidification to pH 6.4 cis side of the membrane lowered the channel open probability and single channel conductance. Whereas only weak influence on the single channel current amplitude and open probability were observed upon lowering of the pH at the trans side. We conclude that a pH-sensitive large conductance potassium channel operates in the chromaffin granule membrane.     

The lipid environment of the nicotinic acetylcholine receptor in native and reconstituted membranes

Detailed knowledge of the membrane framework surrounding the nicotinic acetylcholine receptor (AChR) is key to an understanding of its structure, dynamics, and function. Recent theoretical models discuss the structural relationship between the AChR and the lipid bilayer. Independent experimental data on the composition, metabolism, and dynamics of the AChR lipid environment are analyzed in the first part of the review. The composition of the lipids in which the transmembrane AChR chains are inserted bears considerable resemblance among species, perhaps providing this evolutionarily conserved protein with an adequate milieu for its optimal functioning. The effects of lipids on the latter are discussed in the second part of the review. The third part focuses on the information gained on the dynamics of AChR and lipids in the membrane, a section that also covers the physical properties and interactions between the protein, its immediate annulus, and the bulk lipid bilayer.     

Vegetative insecticidal protein (Vip1Ac) of Bacillus thuringiensis HD201: evidence for oligomer and channel formation

The binding component (Vip1Ac) of the ADP-ribosylating vegetative insecticidal protein (Vip) of Bacillus thuringiensis HD201 was isolated from the supernatant of cell cultures. Vip1Ac protein solubilized at room temperature ran as oligomers on SDS-PAGE. These oligomers were not resistant to heating. Mass spectroscopic analysis of this high molecular mass band identified it as Vip1Ac. The protein formed in artificial lipid bilayer membranes channels with two conductance states of about 350 and 700 pS in 1 M KCl. The channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero-current membrane potential measurements showed that the Vip1Ac channel has a slightly higher permeability for chloride than for potassium ions. Asymmetric addition of Vip1Ac to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. The functional role of Vip1Ac and its relationship to other ADP-ribosylating toxins are discussed.     

The lipid bilayer of acetylcholine receptor clusters of cultured rat myotubes is organized into morphologically distinct domains

We have studied the composition and organization of the lipid bilayer at the large, substrate-associated clusters of acetylcholine receptors (AChR) that form in cultured rat myotubes. These clusters have a characteristic morphology consisting of alternating linear domains of AChR-rich and AChR-poor membrane, the latter involved in attaching the myotube to the substrate. We partially purified AChR clusters by extracting cultured rat myotubes with the cholesterol-specific detergent, saponin. The lipid bilayer of the cluster preparation was analyzed biochemically and the substructure of the bilayers was studied morphologically using the fluorescent probes, dansyl polymyxin B, and 3,3'-di(C12H25 and C18H37) indocarbocyanine iodide (C12- and C18-diI). Our results demonstrate that preparations of AChR clusters have a lipid composition biochemically similar to that of the surrounding plasma membrane. Morphologically, however, the lipid bilayer appears to be arranged into domains that resemble the interdigitating pattern seen for the AChR. This distinctive lipid organization is not due to the use of saponin to purify clusters, as we obtained similar results with clusters isolated by physically shearing myotube cultures. The domain-like organization of the bilayer at clusters is disrupted by treatments that disperse AChR clusters in intact myotubes or that remove peripheral membrane proteins from isolated clusters. This suggests that such proteins may contribute to the organization of the bilayer. Two additional factors may also contribute to the organization of the bilayer: physical constraints imposed by sites of substrate attachment and, to a lesser extent, "boundary" lipid associated with AChR.     

Amphiphiles capsaicin and triton X-100 regulate the chemotherapy drug colchicine’s membrane adsorption and ion pore formation potency

Chemotherapy drugs (CDs), e.g. colchicine derivative thiocolchicoside (TCC) and taxol, have been found to physically bind with lipid bilayer membrane and induce ion pores. Amphiphiles capsaicin (Cpsn) and triton X-100 (TX100) are known to regulate lipid bilayer physical properties by altering bilayer elasticity and lipid monolayer curvature. Both CDs and amphiphiles are predicted to physically accommodate alongside lipids in membrane to exert their membrane effects. The effects of their binary accommodation in the lipid membrane are yet to be known. Firstly, we have performed experimental studies to inspect whether membrane adsorption of CDs (colchicine or TCC) gets regulated due to any membrane effects of Cpsn or TX100. We find that the aqueous phase presence of these amphiphiles, known to reduce the membrane stiffness, works towards enhancing the membrane adsorption of CDs. Our recently patented technology ‘direct detection method’ helps address the membrane adsorption mechanisms. Secondly, in electrophysiology records, we measured the amphiphile effects on the potency of ion channel induction due to CDs. We find that amphiphiles increase the CD induced channel induction potency. Specifically, the membrane conductance, apparently due to the ion channel induction by the TCC, increases substantially due to the Cpsn or TX100 induced alterations of the bilayer physical properties. Thus we may conclude that the binary presence of CDs and amphiphiles in lipid membrane may influence considerably in CD’s membrane adsorption, as well as the membrane effects, such as ion pore formation.     

The prion peptide forms ion channels in planar lipid bilayers

One of the hypotheses concerning the pathogenic properties of the prion protein considers its influence on cellular ion homeostasis. Using the lipid bilayer technique, the influence of prion-derived peptides on the lipid bilayer conductance was characterized. To evaluate the physiological significance and possible pathological functions of the peptides, their effect on the membrane potential and respiration rate of hippocampal mitochondria was also studied. We used a peptide bearing the human prion protein sequence YSNQNNF (PrP [169-175]), and peptide SSQNNF (PrP [170-175]) bearing a naturally-occurring mutation in position 171 [N(r)S] linked to schizoaffective diseases in humans (Samaia, H.B., Mari, J.J., Vallada, H.P., Moura R.P., Simpson A.J.G., Brentani R.R. A prion-linked psychiatric disorder. Nature 390 (1997) 241). In this report, we show that PrP [170-175] N171S increases the conductance of planar lipid bilayers. Based on the conductance of single channel currents recorded in 500/500 mM KCl (cis/trans), we found a single channel conductance of 8 to 26 pS. The native prion peptide PrP [169-175] does not form ion channels in the lipid bilayer. Neither of the peptides significantly changed the membrane potential or respiration rate of isolated rat hippocampal mitochondria. We propose a possible mechanism for channel formation by aggregation of the prion-derived peptide.     

Lipid Bilayer Composition Influences the Activity of the Antimicrobial Peptide Dermcidin Channel

Antimicrobial peptides (AMPs) carry great potential as new antibiotics against “superbugs.” Dermcidin (DCD), a broad-spectrum AMP in human sweat, has been recently crystallized in its oligomeric state and showed channel-like properties. In this work, we performed multiscale molecular dynamics simulations to study how the membrane composition influences the behavior of a transmembrane pore formed by the DCD oligomer in the hope of revealing the origin of the membrane selectivity of this AMP toward bacteria. Our results indicate that bilayers composed of various lipids (DMPC, DPPC, and DSPC) with different thicknesses result in different orientations of the DCD oligomer when embedded in lipid bilayers. The thicker the bilayer, the less tilted the channel. Cholesterol makes the bilayers more rigid and thicker, which also affects the orientation of the channel. Furthermore, we observed that the predicted conductance of the channel from computational electrophysiology simulations is related to its orientation in the lipid bilayer: the larger the tilt, the larger the conductance. Our results indicate that the membrane composition has a significant influence on the activity of the DCD channel, with thicker, cholesterol-rich membranes showing lower conductance than that of thinner membranes.     

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     

Arachidonic acid and diacylglycerol act synergistically to activate protein kinase C in vitro and in vivo

Using a well-defined model membrane bilayer system, incorporation of both lipid second messengers, 1,2-diacylglycerol and arachidonic acid, at submaximal activating concentrations, resulted in a synergistic activation of protein kinase C in a Ca2+/phosphatidylserine-dependent manner as measured by monitoring phosphorylation of phosphoprotein substrates. The arachidonic acid appears to modulate membrane properties both at the hydrocarbon core and the membrane surface increasing the availability of the diacylglycerol which can bind to and subsequently activate the enzyme. Co-application of these two lipid activators to the Hermissenda photoreceptor reduced K+ channel conductance in a synergistic manner via a PKC-dependent pathway. Thus, these in vivo and in vitro studies suggest that the membrane bilayer properties of these PKC lipid activators interact to specifically regulate the cellular lipid microenvironment resulting in PKC activation.     

Ion-gated channel induced in planar bilayers by incorporation of (Na+,K+)-ATPase

An ion-gated channel was conferred on a planar lipid bilayer membrane upon incorporation of (Na+,K+)-ATPase. The channel exhibited two conductance states. The high conductance state was only observed when an ion gradient was present across the planar membrane. This state corresponded to an enzyme conformation which was ouabain and vanadate sensitive (i.e. conductance was inhibited by these compounds), while the low conductance state showed no sensitivity to either inhibitor. Single channel conductance behavior was observed when minimal amounts of enzyme were incorporated into the planar bilayer. The observed single channel conductance was 270 +/- 14 picosiemens. Similar transport behavior was observed for enzyme purified from ovine kidney using sodium dodecyl sulfate (anionic), eel electroplax using Lubrol-WX (nonionic), and kidney microsomes. In addition, the data strongly suggest that enzyme from the kidney microsomes was asymmetrically incorporated into the planar bilayer.     

Monazomycin-induced single channels. I. Characterization of the elementary conductance events

Monazomycin (a positively charged, polyene-like antibiotic) induces voltage-dependent conductance changes in lipid bilayer membranes when added to one of the bathing solutions. These conductance changes have generally been attributed to the existence of channels spanning the membrane. In this article we characterize the behavior of the individual conductance events observed when adding small amounts of monazomycin to one side of a lipid bilayer. We find that there are several apparent channel types with one or sometimes two amplitudes predominating. We find further that these fairly similar amplitudes represent two different states of the same fundamental channel entity, presumed to be the monazomycin channel. The current-voltage characteristics of these channels are weakly hyperbolic functions of applied potential. The average lifetimes are essentially voltage independent (between 50 and 400 mV). The average channel intervals, on the other hand, can be strongly voltage dependent, and we can show that the time-averaged conductance of a membrane is proportional to the average channel frequency.