[Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films].

In studying of conductivity of single supernarrow pores (varying 1 to 15 nm in diameter), formed in thin membranes (10-12 microm in the thickness) from polyethylene terephthalate (PETP), there were revealed discrete changes of currents passing through such pores when applied from external source of potential difference from 200 to 1000 mV. By several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical the so-cold current of single ionic channels in the cell membranes. Supernarrow pores which properties are describes in the present work were obtained as a result of alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). Alkaline etching leads to formation of negative fixed charges on the walls of the pores compensated by positive counterions. When setting potential difference onto the PETP membrane, the latter cation layer is able to transfer the current and this transfer was called the surface conductance. In the case of nanometer pores, such surface conductance may be dominating. We have shown that these discrete changes of currents passing through nanometer pores are associated with metastability of the surface conductance. In the case of highly cation-selective channels in the cell membranes it is inevitable, that at least a part of these channels should have dominating cation surface conductance and mentioned above conductance metastability as well. Our findings allow us to propose a new explanation of the origin of the characteristic discreteness of the currents of cation-selective ionic channels in the cell membranes.

Cholesterol-dependent gramicidin A channel inactivation in red blood cell membranes and lipid bilayer membranes.

The exchange diffusions of tracer cations (22Na+, 86Rb+) are studied on gramicidin-A-treated red blood cell (RBC) membranes. A time-dependent decrease in cation permeability has been observed and has been considered to be the result of a channel inactivation process. The channel inactivation appears at 20 and 30 degrees C but not at a temperature as low as 6 degrees C. The gramicidin A channel inactivation can be monitored by a conductivity decay of molecular lipid membranes (BLM) prepared either from cholesterol or from a mixture of cholesterol and phospholipids but not of pure phosphatidylethanolamine. The role of cholesterol in the channel inactivation is discussed.

Sterol specific inactivation of gramicidin A induced membrane cation permeability.

Channel inactivation, a time-dependent decrease of the high-cationic permeability induced by gramicidin A, has been found both in cholesterol containing red blood cell membranes and lipid bilayers (Schagina et al., (1989) Biochim. Biophys. Acta 978, 145-150). The rate of channel inactivation strongly depends on the phospholipid to cholesterol molar ratio of the membrane. The channel inactivation is suggested to be the result of an interaction between gramicidin and cholesterol in a stoichiometry of 1:5. Cholesterol dependent inactivation is shown also for gramicidin A analogs: tryptophan-N-formylated gramicidin A, o-pyromellitilgramicidin and malonylbisdesformylgramicidin. When cholesterol in the membrane is substituted by sitosterol, the inactivation of gramicidin-induced cation permeability is preserved, while in the presence of either ergosterol or 7-dehydrocholesterol no indication of the channel inactivation is observed. Thus, the structure of the 'B', ring, not the apolar tail of the sterol molecule, appears to be important in the inactivation process.

Defective dystrophin in Duchenne and Becker dystrophy myotubes in cell culture.

We examined normal and dystrophic human myotubes in cell culture for expression of dystrophin, the protein product of the Duchenne muscular dystrophy locus. Dystrophin levels in developing myotubes detected by Western blotting increased after 24 hours and reached maximum levels after 10 days in fusion medium. We did not detect dystrophin in myotubes cultured from Duchenne myoblasts (7 cases). Myotubes from a Becker muscular dystrophy patient's biopsy produced a lower molecular weight (approximately 408 kd) dystrophin, which was the same size in a whole muscle preparation from the same biopsy. This 408-kd dystrophin was the expected size for this Becker patient whose DNA was deleted for exons 45-48 of the Duchenne gene. This cell culture system will allow a detailed analysis of the effects of potential pharmacologic agents on steady-state dystrophin levels.

Rapid switching of ion current in narrow pores: implications for biological ion channels.

Ions flowing through purely synthetic filters made of polyethylene terephthalate which have been etched to produce narrow pores show: (i) rapid transitions between a high-conducting and a low-conducting state; (ii) selectivity of ion flow; and (iii) inhibition by divalent cations and protons. These features resemble those displayed by many biological ion channels. We interpret our results in terms of the special properties of ion conductance at an interface that may be observed whenever the contribution of bulk conductance is minimal.

The mode of action of some antibiotics on red blood cell membranes.

Data are presented on the interaction of gramicidin, primycin and valinomycin with red blood cell membranes and compared with those obtained for artificial lipid bilayer membranes. The channel forming antibiotics gramicidin and primycin show specific kinetic behaviour in living cell membranes. It could be shown that the penetration of these antibiotics into the red blood cell membrane is a cooperative process resulting in the occurrence of aggregates in the lipid lattice of the membrane.

The influence of cytotoxins from Central Asian cobra venom and melittin from bee venom on the thermodynamic properties of phospholipid bilayer.

The interaction of cytotoxin Vc1 and Vc5 from Central Asian cobra and melittin from the bee venom with multilayer liposomes prepared from dimiristoylphosphatidylcholine with an addition of phosphatidic acid have been studied by the method of differential scanning calorimetry. Incorporation of Vc1, Vc5 and melittin into the lipid resulted in pronounced changes in the thermodynamic properties of the lipid. Polypeptides studied induced lateral phase separation in the lipid. Interaction between molecules of the toxins and the lipid resulted in the formation of a new lipid phase characterized by a higher melting temperature and lower phase transition enthalpy.

[Change in the state of a lecithin molecular layer at the heptane-aqueous KCl solutions interface on the introduction of the ionophore, valinomycin]. / Izmenenie sostoianiia sloia molekul letsitina na granitse razdela geptan-vodyne rastvory KCl pri vvedenii inofora--valinomitsina.

The influence of ionophore--valinomycin on the organization of the lipid layer located between a heptane solution of lecithin and KCl water solutions of different concentrations has been shown by measurements of the interfacial tension. Specific changes in the interfacial tension found at high KCl concentrations are related to the transition of valinomycin into a form of its complex with K+-ion. A conclusion on the difference in the lipid layer organisation in the presence of the neutral ionophore and its charged complex has been made.

[Comparison of the electrical and isotope characteristics of ionic transport through bilateral phospholipid membranes]. / Sopostavlenie élektricheskikh i izotopnykh kharakteristik ionnogo transporta cherez fosfolipidnye bisloinye membrany

Simultaneous measurements of electrochemical characteristics and tracer fluxes have been made using large spherical bilayer lipid membranes (1-2 cm2 area). The results obtained show the predominant contribution of electrically silent chloride molecules to the fluxes through unmodified membranes. In cases of valinomycin modified (valinomycin concentration=10(-7)M) membranes (in 0.1 M solutions of KCl, RbCl and CsCl) the fluxes are determined exclusively by cations. The membrane permeability for 22Na, in 0.1 NaCl solution, does not depend on the valinomycin concentration. The selectivity sequences obtained by electrochemical and tracer methods are compared and discussed.

[Study of interaction between cation fluxes in gramicidin A-modified bilayer phospholipid membranes. Determination of the number of ion-binding sites and their position in the gramicidin channel]. / Issledovanie vzaimodeistviia potokov kationov v bisloinykh fosfolipidnykh membranakh, modifitsirovannykh gramitsidinom A. Opredelenie chisla ion-sviazyvaiushchikh mest i ikh polozheniia v gramitsidinovom kanale.

Simultaneous studies were carried out of isotope and electric parameters of spheric bilayer membranes modified with gramicidin A and its analog O-pyromellithylgramicidin (PG) having three negative charges on the N-end of the molecule. The relationship between the electric coefficients of permeability and the isotope ones PG/P* = n was determined by two independent methods. It has been found that for the membranes modified with gramicidin A in RbCl concentrations from 2.2 x 10(-3) to 10(-1) M the value n is constant and approximates 2 and with RbCl concentration 1 M, n = 1.6. For the membranes modified with PG in 0.1 M solutions of PbCl n = 2. The results obtained in terms of the model of unilinear ion diffusion in a narrow pore indicate that in a gramicidin channel there are two sites of cation binding which are located near the channel mouth.

[Phase separation in dipalmitoyl phosphatidylcholine bilayers induced by ionophores and binary electrolytes]. / Razdelenie faz v dipal'mitoilfosfatidilkholine v prisutstvii ionoforov i binarnykh élektrolitov.

Thermotropic behavior of unsonicated aqueous dispersion of dipalmitoyl phosphatidylcholine (DPPC) has been studied by scanning microcalorimetry and fluorescent probe method. Phase separation in the lipid bilayers was observed for systems containing ionophores (valinomycin, dinactin) and 1 : 1 electrolytes (NaCl, KCl, RbCl, CsCl). The ratio of lipid phases coexisting in the systems appeared to be dependent on the concentration of the electrolytes. Changes in the thermotropic properties of the lipid phase induced by valinomycin were observed when K+ and Rb+ ions-forming complexes with the ionophore were present in the systems. The latter phenomenon was not found for the systems containing dinactin possessing a lower ability for complex formation with the cations.

[Acute angle x-ray scattering by phospholipid membranes after treatment with valinomycin]. / Rasseianie pod malymi uglami rentgenovskikh luchei fosfolipidnymi membranami pri vvedenii valinomitsina.

It has been found by means of small-angle X-ray analysis of oriented bilayers of synthetic dimyristoyl phosphatidyl choline that the introduction of valinomycin modifies their structure and these modifications depend on the phase state of lipid.