Svetlana S. Efimova

Novel Class of Spider Toxin: ACTIVE PRINCIPLE FROM THE YELLOW SAC SPIDER CHEIRACANTHIUM PUNCTORIUM VENOM IS A UNIQUE TWO-DOMAIN POLYPEPTIDE*

Venom of the yellow sac spider Cheiracanthium punctorium (Miturgidae) was found unique in terms of molecular composition. Its principal toxic component CpTx 1 (15.1 kDa) was purified, and its full amino acid sequence (134 residues) was established by protein chemistry and mass spectrometry techniques. CpTx 1 represents a novel class of spider toxin with modular architecture. It consists of two different yet homologous domains (modules) each containing a putative inhibitor cystine knot motif, characteristic of the widespread single domain spider neurotoxins. Venom gland cDNA sequencing provided precursor protein (prepropeptide) structures of three CpTx 1 isoforms (a–c) that differ by single residue substitutions. The toxin possesses potent insecticidal (paralytic and lethal), cytotoxic, and membrane-damaging activities. In both fly and frog neuromuscular preparations, it causes stable and irreversible depolarization of muscle fibers leading to contracture. This effect appears to be receptor-independent and is inhibited by high concentrations of divalent cations. CpTx 1 lyses cell membranes, as visualized by confocal microscopy, and destabilizes artificial membranes in a manner reminiscent of other membrane-active peptides by causing numerous defects of variable conductance and leading to bilayer rupture. The newly discovered class of modular polypeptides enhances our knowledge of the toxin universe.

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 dipole modifiers on the magnitude of the dipole potential of sterol-containing bilayers

The effects of various subclasses of flavonoids, Rose Bengal, and different styrylpyridinium dyes on the magnitude of the dipole potential of membranes composed of pure phospholipids and sterol-containing bilayers were investigated. Changes in the steady-state membrane conductance induced by cation-ionophore complexes were measured to examine the changes in the dipole potential of lipid bilayers. The characteristic parameters of the Langmuir adsorption isotherm for different flavonoids and Rose Bengal and the slope of the linear dependence of the dipole potential change on the aqueous concentrations of RH dyes were estimated. Chalcones (phloretin and phloridzin) and flavonols (quercetin and myricetin) strictly decrease the dipole potential of phospholipid- and sterol-containing membranes; the unsaturation of the C-ring and the hydrophobicity of the molecule contribute to the ability of the flavonoid to reduce the bilayer dipole potential. Rose Bengal decreases the magnitude of the bilayer dipole potential to a similar extent, but its affinity for membrane lipids is higher; the effects of RH dyes, chalcones, and phloroglucinol are determined by sterol concentration and type.

The Interaction of Dipole Modifiers with Polyene-Sterol Complexes

Recently, we showed that the effect of dipole modifiers (flavonoids and styrylpyridinium dyes) on the conductance of single amphotericin B (AmB) channels in sterol-containing lipid bilayers primarily resulted from changes in the membrane dipole potential. The present study examines the effect of dipole modifiers on the AmB multi-channel activity. The addition of phloretin to cholesterol-containing membranes leads to a significant increase in the steady-state AmB-induced transmembrane current. Quercetin significantly decreases and RH 421 increases the current through ergosterol-containing bilayers. Other tested flavonoids and styrylpyridinium dyes do not affect the channel-forming activity of AmB independently on the sterol composition of the bilayers. The effects obtained in these trials may instead be attributed to the direct interaction of dipole modifiers with AmB/sterol complexes and not to the effect of dipole potential changes. The presence of double bonds in the Δ7 and Δ22 positions of sterol molecules, the number of conjugated double bonds and amino sugar residues in polyene molecules, and the conformation and adsorption plane of dipole modifiers are important factors impacting this interaction.

Channel-forming activity of cecropins in lipid bilayers: effect of agents modifying the membrane dipole potential.

Cecropin A (CecA) and cecropin B (CecB) added to one side of a bilayer formed from equimolar mixtures of DOPS and DOPE, DPhPS and DPhPE, or DOPS, DOPE, and Chol leads to the formation of well-defined and well-reproducible ion channels of different conductance levels while cecropin P1 (CecP1) does not induce pore formation at micromolar concentrations. We found three populations of CecA channels: pores with weak cationic selectivity, pores with weak anionic selectivity, and pores that were nonselective. The dipole modifiers, flavonoids and styryl dyes, were used to modulate the channel-forming activity of CecA and CecB. The mean conductance of single CecA channels is affected by the influence of dipole modifiers on the lipid packing in the membrane. A decrease in the membrane dipole potential is accompanied by a decrease in the steady-state transmembrane current induced by CecA and CecB in cholesterol-free and cholesterol-containing bilayers. The observed changes in the channel-forming activity might be caused by an increase in the energy barrier for the interfacial accumulation of cecropin monomers. This finding indicates that the negative pole of the cecropin dipole is inserted into the membrane.

The interaction of dipole modifiers with amphotericin-ergosterol complexes. Effects of phospholipid and sphingolipid membrane composition

The influence of agents, known to affect the membrane dipole potential, phloretin and RH 421, on the multi channel activity of amphotericin B in lipid bilayers of various compositions, was studied. It was shown that the effects were dependent on the membrane’s phospholipid and sphingolipid type. Phloretin enhanced amphotericin B induced steady-state transmembrane current through bilayers made from binary mixtures of POPC (DOPC) and ergosterol and ternary mixture of DPhPC, ergosterol and stearoylphytosphingosine. RH 421 increased steady-state polyene induced transmembrane current through membranes made from binary mixtures of DPhPC (DPhPS) and ergosterol and ternary mixture of DPhPS, ergosterol and stearoylphytosphingosine. It was proposed that the observed effects reflect the fine balance of the interactions between the various components present: amphotericin B, ergosterol, phospholipid, sphingolipid and dipole modifier. The shape of lipid molecules seems to be an important factor impacting the responses of amphotericin B modified bilayers to dipole modifiers. The influence of different phospholipids and sphingolipids on the physical and structural properties of ordered lipid microdomains, enriched in AmB, was also discussed. It was also shown that RH 421 enhanced the antifungal activity of amphotericin B in vitro.

Phloretin-Induced Reduction in Dipole Potential of Sterol-Containing Bilayers

The phloretin-induced reduction in the dipole potential of planar lipid bilayers containing cholesterol, ergosterol, stigmasterol, 7-dehydrocholesterol and 5α-androstan-3β-ol was investigated. It is shown that effects depend on the type and concentration of membrane sterol. It is supposed that the effectiveness of phloretin in reducing the dipole potential of the bilayers that contain cholesterol, ergosterol and 7-dehydrocholesterol correlates with the ordering and condensing effects. The role of the concentration-dependent ability of different sterols to promote lateral heterogeneity in membranes is also discussed.

Direct visualization of solid ordered domains induced by polyene antibiotics in giant unilamellar vesicles.

Polyene antibiotics isolated from Streptomyces are frequently used in treatment of mycoses. Confocal fluorescence microscopy has been employed to investigate the influence of polyene macrolide antibiotics nystatin, amphotericin B, and filipin on the phase separation in giant unilamellar vesicles. It has been demonstrated that nystatin produced the solid ordered domains in vesicles made from DOPC/Chol, DOPC/Chol/SM, and POPC while DOPC vesicles remained homogenous in the presence of polyene antibiotics. The ability of various polyenes to produce the solid ordered phase in POPC membranes has been compared. It has been shown that amphotericin B produced phase separation at lower concentration as compared with nystatin and filipin. Filipin was less effective in promotion of gel domains. The observed efficiency of polyene antibiotics to induce phase separation in lipid bilayers correlates with their biological activity. Present findings probably indicate the limitations of using of polyenes as fluorescence membrane probes for determination of strerol-enriched domains in plasma membrane of live cells.

Local Anesthetics Affect Gramicidin A Channels via Membrane Electrostatic Potentials.

The effects of local anesthetics (LAs), including aminoamides and aminoesters, on the characteristics of single gramicidin A (GA) channels in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers were studied. Aminoamides, namely lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), and bupivacaine (BPV), reduced the conductance of GA channels. Aminoesters influenced the current fluctuations induced by GA differently; procaine (PC) did not affect the fluctuations, whereas tetracaine (TTC) distinctly reduced the conductance of single GA channels. Using electrophysiological technique, we estimated the changes in the membrane boundary potential at the adsorption of LAs; LDC, PLC, MPV, BPV, and TTC substantially increased, while PC did not affect it. To elucidate which component of the membrane boundary potential, the surface or dipole potential, is responsible for the observed effects of LAs, we employed a fluorescence assay. We found that TTC led to a significant increase in the membrane dipole potential, whereas the adsorption of LDC, PLC, MPV, BPV, and PC did not produce any changes in the membrane dipole potential. We concluded that aminoamides affected the surface potential of lipid bilayers. Together, these data suggest that the effects of LAs on the conductance of single GA channels are caused by their influence on membrane electrostatic potentials; the regulation of GA pores by aminoamides is associated with the surface potential of membranes, whereas TTC modulation of channel properties is predominantly due to changes in dipole potential of lipid bilayers. These data might provide some significant implications for voltage-gated ion channels of cell membranes.

Modifiers of membrane dipole potentials as tools for investigating ion channel formation and functioning.

Electrostatic fields generated on and within biological membranes play a fundamental role in key processes in cell functions. The role of the membrane dipole potential is of particular interest because of its powerful impact on membrane permeability and lipid-protein interactions, including protein insertion, oligomerization, and function. The membrane dipole potential is defined by the orientation of electric dipoles of lipid headgroups, fatty acid carbonyl groups, and membrane-adsorbed water. As a result, the membrane interior is several hundred millivolts more positive than the external aqueous phase. This potential decrease depends on the lipid, and especially sterol, composition of the membrane. The adsorption of certain electroneutral molecules known as dipole modifiers may also lead to significant changes in the magnitude of the potential decrease. These agents are widely used to study the effects of the dipole potential on membrane transport. This review presents a critical analysis of a variety of data from studies dedicated to ion channel formation and functioning in membranes with different dipole potentials. The types of ion channels found in cellular membranes and pores formed by antimicrobial agents and toxins in artificial lipid membranes are summarized. The mechanisms underlying the influence of the membrane dipole potential on ion channel activity, including dipole-dipole and charge-dipole interactions in the pores and in membranes, are discussed. A hypothesis, in which lipid rafts in both model and cellular membranes also modulate ion channel activity by virtue of an increased or decreased dipole potential, is also considered.