Activation and conductance properties of ryanodine-sensitive calcium channels from brain microsomal membranes incorporated into planar lipid bilayers

Summary Rat brain microsomal membranes were found to contain high-affinity binding sites for the alkaloid ryanodine (k _d 3nm.B _max 0.6 pmol per mg protein). Exposure of planar lipid bilayers to microsomal membrane vesicles resulted in the incorporation, apparently by bilayer-vesicle fusion, of at least two types of ion channel. These were selective for Cl^– and Ca²⁺, respectively. The reconstituted Ca²⁺ channels were functionally modified by 1 m ryanodine, which induced a nearly permanently open subconductance state. Unmodified Ca²⁺ channels had a slope conductance of almost 100 pS in 54mm CaHEPES and a Ca²⁺/TRIS⁺ permeability ratio of 11.0. They also conducted other divalent cations (Ba²⁺>Ca²⁺>Sr²⁺>Mg²⁺) and were markedly activated by ATP and its nonhydrolysable derivative AMPPCP (1mm). Inositol 1,4,5-trisphosphate (1–10 m) partially activated the same channels by increasing their opening rate. Brain microsomes therefore contain ryanodine-sensitive Ca²⁺ channels, sharing some of the characteristics of Ca²⁺ channels from striated but not smooth muscle sarcoplasmic reticulum. Evidence is presented to suggest they were incorporated into bilayers following the fusion of endoplasmic reticulum membrane vesicles, and their sensitivity to inositol trisphosphate may be consistent with a role in Ca²⁺ release from internal membrane stores.     

Reversible electrical breakdown of lipid bilayers: formation and evolution of pores

The mechanism of reversible electric breakdown of lipid membranes is studied. The following stages of the process of pore development are substantiated. Hydrophobic pores are formed in the lipid bilayer by spontaneous fluctuations. If these water-filled defects extend to a radius of 0.3 to 0.5 nm, a hydrophilic pore is formed by reorientation of the lipid molecules. This process is favoured by a potential difference across the membrane. The conductivity of the pores depends on membrane voltage, and the type of this dependence changes with the radius of the pore. Hydrophilic pores of an effective radius of 0.6 up to more than 1 nm are formed, which account for the membrane conductivity increase observed. The characteristic times of changes in average radius and number of pores during the voltage pulse and after it are investigated.     

Single chloride-selective channel from cardiac sarcoplasmic reticulum studied in planar lipid bilayers

Summary The behavior of single Cl^– channel was studied by fusing isolated canine cardiac sarcoplasmic reticulum (SR) vesicles into planar lipid bilayers. The channel exhibited unitary conductance of 55 pS (in 260mm Cl^–) and steady-state activation. Subconductance states were observed. Open probability was dependent on holding potentials (–60 to +60 mV) and displayed a bell-shaped relationship, with probability values ranging from 0.2 to 0.8 with a maximum at –10 mV. Channel activity was irreversibly inhibited by DIDS, a stilbene derivative. Time analysis revealed the presence of one time constant for the full open state and three time constants for the closed states. The open and the longer closed time constants were found to be voltage dependent. The behavior of the channel was not affected by changing Ca²⁺ and Mg²⁺ concentrations in both chambers, nor by adding millimolar adenosine triphosphate, or by changing the pH from 7.4 to 6.8. The presence of sulfate anions decreased the unit current amplitude, but did not affect the open probability. These results reveal that at the unitary level the cardiac SR anion-selective channel has distinctive as well as similar electrical properties characteristic of other types of Cl^– channels.     

Formation of ion channels by Colicin B in planar lipid bilayers

Summary The gene for the antibacterial peptide colicin B was cloned and transformed into a host background where it was constitutively overexpressed. The purified gene product was biologically active and formed voltage-dependent, ion-conducting channels in planar phospholipid bilayers composed of asolectin. Colicin B channels exhibited two distinct unitary conductance levels, and a slight preference for Na⁺ over Cl^–. Kinetic analysis of the voltage-driven opening and closing of colicin channels revealed the existence of at least two conducting states and two nonconducting states of the protein. Both the ion selectivity and the kinetics of colicin B channels were highly dependent on pH. Excess colicin protein was readily removed from the system by perfusing the bilayer, but open channels could be washed out only after they were allowed to close. A monospecific polyclonal antiserum generated against electrophoretically purified colicin B eliminated both the biological and in vitro activity of the protein. Membrane-associated channels, whether open or closed, remained functionally unaffected by the presence of the antiserum. Taken together, our results suggest that the voltage-independent binding of colicin B to the membrane is the rate-limiting step for the formation of ion channels, and that this process is accompanied by a major conformational rearrangement of the protein.     

Anion channels from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Synaptic membranes from rat brain were incorporated into planar lipid bilayers, and the characteristics of two types of anion-selective channels (type I and type II) were investigated. In asymmetric BaCl₂ buffers (cis, 100mm/trans, 25mm), single channel conductances at –40 mV were 70 pS (type I) and 120 pS (type II). Permeability ratios (P _Na:P _Ba:P _Cl) calculated from the Goldman-Hodgkin-Katz current equation for type I and type II channels were 0.230.041 and 0.050.031, respectively. Both channels exhibited characteristic voltage-dependent bursting activities. Open probability for type I channels had a maximum of 0.7 at about 0 mV and decreased to zero at greater transmembrane potentials of either polarity. Type II channels were relatively voltage independent at negative voltages and were inactivated at highly positive voltages. Type I channels showed spontaneous irreversible inactivation often preceded by sudden transition to subconducting states. DIDS blocked type I channels only from thecis side, while it blocked type II channels from either side.     

Polypeptide neurotoxins modify gating and apparent single-channel conductance of veratridine-activated sodium channels in planar lipid bilayers

Summary The effects of scorpion and sea anemone polypeptide toxins on partially purified veratridine (VER)-activated Na channels from rat brain were studied at the single-channel level in planar lipid bilayers. The probability of the VER-activated channel being open (P_o) increased with depolarization;P_o was 0.5 at –40 to –50 mV. Saxitoxin (STX) blocked VER-activated channels with an apparent dissociation constant of about 1nm at –45 mV. The apparent single-channel conductance was approximately 9 pS, similar to that seen in VER-activated Na channels from skeletal muscle transverse tubules. Addition of sea anemone or scorpion polypeptide toxins to VER-activated Na channels resulted in a 19% increase in apparent single-channel conductance and a hyperpolarizing shift in theP_ovs. V_m relation such that the channels were more likely to be open at potentials <40 mV. These effects of the polypeptide toxins on the single-channel properties of VER-activated Na channels may account for the previously described potentiation of VER action by polypeptide toxins.     

Properties of an anion-selective channel from rat colonic enterocyte plasma membranes reconstituted into planar phospholipid bilayers

Summary Vesicles derived from epithelial cells of the colonic mucosa of the rat were fused to planar phospholipid bilayer membranes, revealing spontaneously switching anion-conducting channels of 50 pS conductance (at-30 mV with 200mm Cl^– each side). The equilibrium selectivity series was I^– (1.7)/Br^– (1.3)/Cl^– (1.0)/F^– (0.4)/HCO ₃ ^– (0.4)/Na (<0.11.). Only one dominant open-state conductance could be resolved, which responded linearly to Cl^– concentrations up to 600mm. The singlechannel current-voltage curve was weakly rectifying with symmetrical solutions. When 50 mV were exceeded at the highconductance branch of the curve, switching was arrested in the closed state. At more moderate voltages (±40 mV) kinetics were dominated by one open state of about 35-msec lifetime and two closed states of about 2 and 9-msec lifetime. Of these, the more stable closed state occurred less often. At these voltages one additional closed state of significantly longer lifetime (>0.5 sec) was observed.     

Wave-guide spectroscopy and ion transport in planar lipid bilayers

The refractive indices of the bilayer-electrolyte system allow the membrane to operate as a light-guide. This system is then able to monitor, optically, the flow of ions across the bilayer. The light is coupled into and decoupled from a spherically bulged bilayer by means of optical, single mode fibers. The light wave travels along the curved bilayer for several millimeters. This light transmission depends critically on the angle of incidence between the fiber axis and the tangent to the film. Three transmission peaks were observed when the angle of incidence was varied between 0° and 90°. The transmitted light intensity can be modulated by the application of an electric potential upon the bilayer. The center peak, with maximum light transmission, appears at an angle of incidence which is defined by the launching geometry. A quadratic field dependence (independent of the polarity) is observed, which originates from changes in the shape of the torus transition region. The transmission of the satellite peaks, which appear just before and after the central peak, can also be modulated by an external potential. This modulation signal reflects a linear dependence on the polarity of the external voltage. The phase of the modulation signal changes its sign at each satellite peak. It is shown that this modulation signal originates from the bimolecular area of the lipid film. We present evidence that this transmission modulation occurs as a result of ion transport through the lipid film. This provides the basis for the use of wave-guide spectroscopy to investigate membrane ionic fluxes.     

Evidence of pores and thinned lipid bilayers induced in oriented lipid membranes interacting with the antimicrobial peptides, magainin-2 and aurein-3.3

Dynamic structures of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers induced in oriented lipid membranes, which are interacting with membrane-acting antimicrobial peptides (AMPs), magainin-2 and aurein-3.3, were explored by ³¹P and ²H solid-state NMR (ssNMR) spectroscopy. Various types of phospholipid systems, such as POPC-d₃₁, POPC-d₃₁/POPG, and POPC-d₃₁/cholesterol, were investigated to understand the membrane disruption mechanisms of magainin-2 and aurein-3.3 peptides at various peptide-to-lipid (P:L) ratios. The experimental lineshapes of anisotropic ³¹P and ²H ssNMR spectra measured on these peptide-lipid systems were simulated reasonably well by assuming the presence of supramolecular lipid assemblies, such as toroidal pores and thinned bilayers, in membranes. Furthermore, the observed decrease in the anisotropic frequency span of either ³¹P or ²H ssNMR spectra of oriented lipid bilayers, particularly when anionic POPG lipids are interacting with AMPs at high P:L ratios, can directly be explained by a thinned membrane surface model with fast lateral diffusive motions of lipids. The spectral analysis protocol we developed enables extraction of the lateral diffusion coefficients of lipids distributed on the curved surfaces of pores and thinned bilayers on a few nanometers scale.     

Interaction of charged lipid vesicles with planar bilayer lipid membranes: Detection by antibiotic membrane probes

A technique has been developed for monitoring the interaction of charged phospholipid vesicles with planar bilayer lipid membranes (BLM) by use of the antibiotics Valinomycin, Nonactin, and Monazomycin as surface-charge probes. Anionic phosphatidylserine vesicles, when added to one aqueous compartment of a BLM, are shown to impart negative surface charge to zwitterionic phosphatidylocholine and phosphatidylethanolamine bilayers. The surface charge is distributed asymmertically, mainly on the vesicular side of the BLM, and is not removed by exchange of the vesicular aqueous solution. Possible mechanisms for the vesicle-BLM interactions are discussed.     

A chloride-permeable channel from Phaseolus vulgaris roots incorporated into planar lipid bilayers

Ion channels are key participants in physiological processes of plant cells. Here, we report the first characterization of a high conductance, Cl(-)-permeable channel, present in enriched fractions of plasma membranes of bean root cells. The Cl(-) channel was incorporated into planar lipid bilayers and its activity was recorded under voltage clamp conditions. The channel is voltage-dependent, excludes the passage of cations (K(+), Na(+), and Ca(2+)), and is inhibited by micromolar concentrations of Zn(2+). The Cl(-) conductance here characterized represents a previously undescribed channel of plant cells.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Pore formation by complement in the outer membrane of Gram-negative bacteria studied with asymmetric planar lipopolysaccharide/phospholipid bilayers

Summary The interaction of complement with an asymmetric planar lipopolysaccharide/phospholipid bilayer system as a model for the lipid matrix of the outer membrane of Gram-negative bacteria has been studied. The addition of whole human serum to the aqueous solution at the lipopolysaccharide side of the asymmetric membrane resulted in a rapid increase of the bilayer conductance in discrete steps, indicating the formation of transmembrane pores, which were not observed in the case of pure phospholipid membranes. The amplitudes of the discrete conductance steps varied over a range of more than one order of magnitude. The mean single step conductance was (0.39±0.24) nS for a subphase containing (inmm): 100 KCl, 5 MgCl₂ and 5 HEPES buffer. The steps were grouped into bursts of typically 9±3 events per burst and the conductance change within one burst was (8.25±4.00) nS.The pore-forming activity of serum at the asymmetric membrane system was independent of the presence of specific antibodies against the lipopolysaccharide but was dependent on calcium ions. Furthermore, the pore-forming activity required complement component C9.A model for the mode of pore formation by complement is proposed: The complement pore is generated in discrete steps by insertion of C9 monomers into the membrane and their irreversible aggregation to water-filled channels with a diameter of approximately 7 nm assuming a circular geometry.     

Construction of P-glycoprotein incorporated tethered lipid bilayer membranes

To investigate drug–membrane protein interactions, an artificial tethered lipid bilayer system was constructed for the functional integration of membrane proteins with large extra-membrane domains such as multi-drug resistance protein 1 (MDR1). In this study, a modified lipid (i.e., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino (polyethylene glycol)-2000] (DSPE-PEG)) was utilized as a spacer molecule to elevate lipid membrane from the sensor surface and generate a reservoir underneath. Concentration of DSPE-PEG molecule significantly affected the liposome binding/spreading and lipid bilayer formation, and 0.03 mg/mL of DSPE-PEG provided optimum conditions for membrane protein integration. Further, the incorporation of MDR1 increased the local rigidity on the platform. Antibody binding studies showed the functional integration of MDR1 protein into lipid bilayer platform. The platform allowed to follow MDR!-statin-based drug interactions in vitro. Each binding event and lipid bilayer formation was monitored in real-time using Surface Plasmon Resonance and Quartz Crystal Microbalance–Dissipation systems, and Atomic Force Microscopy was used for visualization experiments.     

Raman spectra of planar supported lipid bilayers

Raman scattering has been used to obtain high quality vibrational spectra of planar supported lipid bilayers (pslb's) at the silica/water interface without the use of resonance or surface enhancement. A total internal reflection geometry was used both to increase the bilayer signal and to suppress the water background. Polarization control permits the determination of four components of the Raman tensor, of which three are independent for a uniaxial film. Spectra are reported of the phospholipids DMPC, DPPC, and POPC, in the C-H stretching region and the fingerprint region. The temperature-dependent polarized spectra of POPC show only small changes over the range 14-41 °C. The corresponding spectra of DMPC and DPPC bilayers show large thermal changes consistent with a decreasing tilt angle from the surface normal and increasing chain ordering at lower temperatures. The thermal behavior of DMPC pslb's is similar to that of vesicles of the same lipid in bulk suspension. In contrast to calorimetry, which shows a sharp phase transition (L_α-L_β') with decreasing temperature, the changes in the Raman spectra occur over a temperature range of ca. 10 °C commencing at the calorimetric phase transition temperature.     

Toad bladder amiloride-sensitive channels reconstituted into planar lipid bilayers

In the present study we used established methods to obtain apical membrane vesicles from the toad urinary bladder and incorporated these membrane fragments to solvent-free planar lipid bilayer membranes. This resulted in the appearance of a macroscopic conductance highly sensitive to the diuretic amiloride added to the cis side. The blockage is voltage dependent and well described by a model which assumes that the drug binds to sites in the channel lumen. This binding site is localized at about 15% of the electric field across the membrane. The apparent inhibition constant (K(0)) is equal to 0.98 microM. Ca2+, in the micromolar range on the cis side, is a potent blocker of this conductance. The effect of the divalent has a complex voltage dependence and is modulated by pH. At the unitary level we have found two distinct amiloride-blockable channels with conductances of 160 pS (more frequent) and 120 pS. In the absence of the drug the mean open time is around 0.5 sec for both channels and is not dependent on voltage. The channels are cation selective (PNa/PCl = 15) and poorly discriminate between Na+ and K+ (PNa/PK = 2). Amiloride decreases the lifetime in the open state of both channels and also the conductance of the 160-pS channel.     

Effect of phospholipid surface charge on the conductance and gating of a Ca2+-activated K+ channel in planar lipid bilayers

Summary A Ca-activated, K-selective channel from plasma membrane of rat skeletal muscle was studied in artificial lipid bilayers formed from either phosphatidylethanolamine (PE) or phosphatidylserine (PS). In PE, the single-channel conductance exhibited a complex dependence on symmetrical K⁺ concentration that could not be described by simple Michaelis-Menten saturation. At low K⁺ concentrations the channel conductance was higher in PS membranes, but approached the same conductance observed in PE above 0.4m KCl. At the same Ca²⁺ concentration and voltage, the probability of channel opening was significantly greater in PS than PE. The differences in the conduction and gating, observed in the two lipids, can be explained by the negative surface charge of PS compared to the neutral PE membrane. Model calculations of the expected concentrations of K⁺ and Ca²⁺ at various distances from a PS membrane surface, using Gouy-Chapman-Stern theory, suggest that the K⁺-conduction and Ca²⁺-activation sites sense a similar fraction of the surface potential, equivalent to the local electrostatic potential at a distance of 9 Å from the surface.     

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).     

PEG blocking of single pores arising on phase transitions in unmodified lipid bilayers

Changes in ionic permeability of bilayer lipid membranes (BLM) from dipalmitoyl phosphatidylcholine at temperature of phase transition in 1 M LiCl solution in the presence of polyethyleneglycols (PEG) of various molecular masses are studied. The transition of ionic membrane channels from conducting to blocked nonconducting state using polymers makes it possible to calibrate lipid pores. It is shown that low-molecular weight glycerol and PEG with molecular weights of 300 and 600 decrease the amplitude of current fluctuations through the membrane, the decrease being proportional to the size of the polymer molecule incorporated. The addition of PEG with molecular masses of 1450, 2000, and 3350 decrease the current fluctuations to the basal noise level. The result is considered as a complete blockade of ion channel conductivity. In the presence of rather large polymers, such as PEG with molecular masses of 6000 and 20000, which are hardly incorporated in the pore, single current fluctuations occur again; however, their amplitudes are somewhat smaller than in the absence of PEG. It is assumed that a complete blockade of the conductivity of lipid ionic channels by PEG with molecular masses of 1450, 2000, and 3350 is due to dehydration of the pore gap and the conversion of the hydrophilic pore to a hydrophobic one.     

A cytolytic delta-endotoxin from Bacillus thuringiensis var. israelensis forms cation-selective channels in planar lipid bilayers

In order to determine the mechanism of action of the 27 kDa mosquitocidal delta-endotoxin of Bacillus thuringiensis var. israelensis we have studied its effects on the conductance of planar lipid bilayers. The toxin formed cation-selective channels in the bilayers, permeable to K+ and Na+ but not to N-methylglucamine or Cl-, showing very fast, cooperative opening and closing. Channel opening was greatly reduced in the presence of divalent cations (Ca2+, Mg2+) and the effect was reversed when these ions were removed. These results are consistent with our proposal that B. thuringiensis toxins act by a mechanism of colloid-osmotic lysis.