[Interaction of melittin with ion channels of excitable membranes].

The effect of the neurotoxin melittin on the activation of ion channels of excitable membrane, the plasmalemma of Characeae algae cells, isolated membrane patches of neurons of mollusc L. stagnalis and Vero cells was studied by the method of intracellular perfusion and the patch-clamp technique in inside-out configuration. It was shown that melittin disturbs the conductivity of plasmalemma and modifieds Ca(2+)-channels of plant membrane. The leakage current that appears by the action of melittin can be restored by substituting calmodulin for melittin. Melittin modifies K(+)-channels of animal cell membrane by disrupting the phospholipid matrix and forms conductive structures in the membrane by interacting with channel proteins, which is evidenced by the appearance of additional ion channels.

[The mechanism of antiarrhythmic action of a new ammonium derivative of lidocaine (LKhT-12-02)].

The results of electrophysiological investigation of the effects of LKhT-12-02 (a quaternary ammonium derivative of lidocaine) on the intact cat heart and the isolated ion channels of Lymnaea stagnalis snail showed that this compound belongs to class 1B antiarrhythmic agents (Vaughan - Williams classification). The drug does not suppress the automatic nonmonotonic rhythm driver, does not influence the conductance in ventricles, auricles, and atrioventricular node in the sinus rhythm, and does not elongate the effective refractory period of the auricles and atrioventricular node. LKhT-12-02 decreases the rate of fast depolarization of the action potential, while not reducing its duration. The compound does not influence the conduction of sodium ion channels.

The potential-dependent K+ channel in molluscan neurones is organized in a cluster of elementary channels.

Single potential-dependent K+ channels were studied using the patch-voltage-clamp method. Two types of channel with identical, but oppositely directed, potential dependences were found. The channels of the first type (slow channels) are assumed to be responsible for the outward rectification. The properties of the channels of the second type (fast channels) are similar to those of the K+ channels in neurone soma which create the fast transient currents. The kinetic characteristics of both types of channel are presented. The conductances of slow and fast K+ channels are approx. 30 and 40 pS, respectively, at zero membrane potential and a K+ concentration of 50 mmol/l at the inner side of the membrane. The following sequence of channel selectivity with respect to monovalent cations was found: T1+ greater than K+ greater than Rb+ much greater than Cs+ approximately equal to Li+ approximately equal to Na+. The probability of the channel open state monotonically decreases with free Ca2+ concentration at the inside membrane surface for both types of channel. It was found that the channels have discrete and multiple conductance substates . It is supposed that a unitary K+ channel consists of approx. 16 elementary ones with conductances of approx. 2 pS (slow channels) and approx. 2.5 pS (fast channels) at zero potential. At +100 mV the elementary conductances are equal to approx. 4 and 5.5. pS, respectively. Thus, according to this assumption, the unitary channel is a cluster of elementary channels.

Single potential-dependent K+ channels and their oligomers in molluscan glial cells.

Single K+ channels were studied using the patch-clamp method. A potential-dependent K+ channel of large conductance (about 100 pS at 100 mM of KCl on both membrane sides) was detected. Some properties of the channel (current-voltage relations, kinetic parameters, etc.) are presented. The channel was found to have about 16 resolvable quantized conductance substates. The data are confirmed by spontaneous channel degradation, i.e., spontaneous splitting of the channel conductance into independent conductance oligomers. Some properties of the conductance oligomers of different order are described. The degree of potential dependency of the conductance oligomer parameters is a function of potential dependency. The data obtained are in agreement with a hypothesis that the channels studied are clusters (aggregates) of elementary channel subunits.

Preliminary microwave irradiation of water solutions changes their channel-modifying activity.

Earlier we have shown that millimetre microwaves (42.25 GHz) of non-thermal power, upon direct admittance into an experiment bath, greatly influence activation characteristics of single Ca(2+)-dependent K+ channels (in particular, the channel open state probability, Po). Here we present new data showing that similar changes in Po arise due to the substitution of a control bath solution for a preliminary microwave irradiated one of the same composition (100 mmol/l KCl with Ca2+ added), with irradiation time being 20-30 min. Therefore, due to the exposure to the field the solution acquires some new properties that are important for the channel activity. The irradiation terminated, the solution retains a new state for at least 10-20 min (solution memory). The data suggest that the effects of the field on the channels are mediated, at least partially, by changes in the solution properties.

Dual effects of microwaves on single Ca(2+)-activated K+ channels in cultured kidney cells Vero.

Using the patch voltage-clamp method, possible effects of millimetre microwaves (42.25 GHz) on single Ca(2+)-activated K+ channels in cultured kidney cells (Vero) were investigated. It was found that exposure to the field of non-thermal power (about 100 microW/cm2) for 20-30 min greatly modifies both the Hill coefficient and an apparent affinity of the channels for Ca2+(i). The data suggest that the field alters both cooperativity and binding characteristics of the channel activation by internal Ca2+. The effects depend on initial sensitivity of the channels to Ca2+ and the Ca2+ concentration applied.

[Analysis of long-lived substates of the conductivity of single ion channels]. / Analiz dolgozhivushchikh podsostoianii provodimosti odinochnykh ionnykh kanalov.

Using the patch-voltage-clamp method kinetic parameters of single ionic channels were studied. It was found that the channels have long-lasting conductance substates along with the short-living ones. The conductance of a long-lasting substate fluctuates near an average sublevel in the boundaries of a restrict number (k) of elementary conductance steps. There is a direct relationship: the greater k, the longer is average duration (tau k) of the substate. For a given k the falling phase of tau k value distribution is approximately one-exponential. The substates of k-th order result in the multi-exponentiality of the ion current kinetics.

[Discrete parameters of current oscillation in single ion channels]. / Diskretnost' parametrov ostsiliiatsii toka v odinochnykh ionnykh kanalakh.

Using the patch-voltage-clamp method it was shown that oscillations of an open channel are fast current transitions between 64 multiple sublevels. Average values of elementary conductance step (gamma) and substate lifetime (tau el) were determined for different kinds of ionic channels. The values of gamma lie in the range from 1.5 to 6 pS, and tau el--in the range from 0.15 to 0.5 ms. The channel transitions between the substates are highly cooperative processes. The data are regarded in terms of the hypothesis about clustery organization of ionic channels.

[Mechanism of blocking K+-channels with tetraethylammonium]. / Mekhanizm blokirovaniia K+-kanalov tetraétilammoniem.

Using the patch-voltage-clamp method action of tetraethylammonium on the fast (30 pS) and slow K+ channels was investigated. The slow K+ channels were presented by two types: with whole (30 pS) and decreased (20 pS) conductance. In all cases tetraethylammonium decreased the current magnitude and modified the channel kinetic parameters. Apparent blocking constants determined from the current decreasing are as 8-50 and 4-12 mM for the slow K+ channels with whole and decreased conductance, respectively, and 0.05-0.08 mM--for the fast K+ channel. The potential dependency of the blocking constants correlates with that of the channel conductance. Probability of the channel open state for the slow K+ channels decreases, and that for the fast K+ channel increases under application of tetraethylammonium. It is concluded that there are two sites of tetraethylammonium binding: the first site is into the channel pore, and the second one--into the regulatory centre responsible for the channel kinetic behaviour. Blocking of general conductance of the slow channels is accompanied by proportional decrease of the channel substate conductances without change of their number and cooperatively. Block of the fast K+ channel occurs without change of the channel elementary conductance but with decrease of the number of the channel substates and reversible violation of the channel transition cooperativity. The data are discussed from the point of the hypothesis on the channel clustery organization.

[Synchronization of calcium channel activity in mollusk neurons after treatment with ferricyanide and barium ions]. / Sinkhronizatsiia aktivnosti kalievykh kanalov neironov molliuska pod deistviem ferritsianida i ionov bariia.

Using the patch-voltage-clamp method on the isolated membrane patches from molluscan neurons, effects of ferricyanide and barium on fast potassium channels with a priori destroyed synchronism in the transitions between conductance sublevels were studied. Ferricyanide (0.1-0.5 mM) applied at the inner membrane side produced irreversible transformation of occasional transitions of the channel conductance between intermediate states into highly cooperative and potential-dependent process. Barium ions completely or partly reversibly restore synchronism.

[Inactivation of choline receptors induced by membrane depolarization]. / Inaktivatsiia kholkinoretseptorov, vyzyvaemaia membrannoi depoliarizatsiei.

It was found that membrane depolarization of Lymnaea stagnalis neurons inhibited the conductance induced by acetylcholine (ACh). This phenomenon (cholinoreceptor inactivation) was shown to be connected with a potential influence on none of the following characteristics: reversal potential (Er) of the ACh-induced currents, affinity of ACh to the receptors, ACh concentration near the receptive membrane surface, and desensitization rate.

[A discrete type of potassium channel conductance in snail neurons]. / Diskretnyi kharakter provodimosti kalievogo kanala v neironakh prudovika.

The results of further investigations on a single potential dependent K+ channel are described. It was shown that ionic selectivity of the channel for monovalent ions is too high: Li+, Na+, and Cs+ are practically impermeant ions. Permeability of the channel for Rb+ is approximately 10 times less, and for Tl+ it is 2 times more than permeability for K+. Besides, it was found that open K+ channel has 16 multiple conductance levels.

[Cooperative phenomena in the activity of single ion channels]. / Kooperativnye iavleniia v aktivnosti odinochnykh ionnykh kanalov.

Using the patch-voltage-clamp method kinetics of the fast potential-dependent K+-channels in molluscan neurones was investigated. It was found that under given experimental conditions the amplitudes of single current impulses have a wide spectrum. The amplitudes are proportional to a number of the current substates involved. Averaged fronts of the current impulses are S-shaped, and have duration greater than 1 ms. Averaged duration of the current impulses increases (from 0.25 to 30-40 ms) with the impulse amplitude (or with the number of the substates involved). There is a sharp bend of the dependence at the impulse amplitude 0.6-0.7 of maximal value. The phenomena investigated reflect, probably, cooperativity of the channel transitions between the substates. The degree of the cooperativity depends on the membrane potential value.

[Temperature dependence of the conductivity of individual potential-dependent K+-channels in mollusk neurons]. / Temperaturnye zavisimosti provodimosti odinochnykh potentsialozavisimykh K+-kanalov v neironakh molliuska.

Using the patch-clamp method temperature dependences of the chord conductance of single potential--dependent slow and fast K+ channels in mollusk neurons were studied. Under control conditions (20 degrees C, 0 mV, [K+]o = 1.5 mM and [K+]i = 100 mM) the conductances of the fast and slow K+ channels were equal to 20-25 pS and 30-40 pS, respectively. Besides, the temperature dependences of the currents through the K+ channels of lesser conductance (5-20 pS) were studied. Some of these channels may be regarded as subtypes of the fast and slow K+ channels named above. It was found that for the channels of all types single channel currents arise with temperature. However, in the range of 10-20 degrees C an anomalous conductance decrease at temperature elevation was observed. For all channels except for the fast one at temperatures above 20 degrees C activation energy (delta Ea) calculated from the Arrhenius plots of the currents was about 4 kcal/mol. At the temperatures below 10 degrees C delta Ea was equal to about 12 kcal/mol. In this temperature range delta Ea had a pronounced potential dependency. Temperature dependences of the fast K+ channel conductance were opposite to those of the slow K+ channel to some extent.

[Intracellular calcium-initiator of inactivation of choline receptors]. / Vnutrikletochnyi kal'tsii - initsiator inaktivatsii kholinoretseptorov.

It has been shown that inactivation of cholinoreceptors observed in the region of neuron depolarization is conditioned by the supply of Ca ions into the cell through the electro--excitable Ca2+-channels. It is proved by three means: 1) variation of outer concentration of Ca2+(Ca2+h); 2) application of the blockers of electroexcitable Ca2+ channels; 3) variation of intracellular concentration Ca2+(Ca2+b).

[Calculation of local Hurst exponents in the Ca(2+)-activated K(+)-channel dwell time]. / Raschet lokal'nykh pokazatelei Khersta v posledovatel'nostiakh vremen zhizni Ca(2+)-aktiviruemykh K(+)-kanalov.

A novel method based on the maximum overlap wavelet transform of dwell time series is proposed. Information on local multifractal properties of the series, namely local Hurst exponents or Holder exponents, was obtained. The results confirm the presence of multifractality and intrinsic correlations in the Ca(2+)-activated K+ channel dwell time series. The data on the local multifractal structure of the series can be interpreted in terms of processes having self-organized criticality. The proposed approach allows one to widen the store of methods for the analysis of single ion channel activity.

[Single Ca2+-activated K+-channels in cultured kidney cells Vero]. / Odinochnye Ca2+-aktiviruemye K+-kanaly v kul'tiviruemykh pochechnykh kletkakh Vero.

Using the patch voltage-clamp method ("inside-out" mode) properties of single Ca(2+)-activated K+ channels in cultured kidney cell Vero were studied. Two types of kinetically distinct channels (fast and slow) were found. The fast and slow channels differ by single channel conductance, kinetic parameters, characteristics of interaction with Ca2+, and voltage dependency degree of the kinetics parameters, but are similar in ionic selectivity in respect to monovalent cations. It was found that the channel conductance varies from patch to patch between 140 and 230 pS. Average values of single channel open duration and an apparent dissociation constant were correlated with the single channel conductance. The relationship was found: the more the conductance the lesser the open time duration and more the dissociation constant. The data suggest that there is almost continuous spectrum of subtypes of the Ca(2+)-activated K+ channels, the fast and slow channels being extreme groups of this spectrum. The data are regarded from the viewpoint of the clustery hypothesis.

[Fractal properies of gating in potential-dependent K+-channels in Lymnaea stagnalis neurons]. / Fraktal'nye svoistva vorotnogo mekhanizma potentsialozavisimykh K+ - kanalov v neironakh Lymnaea stagnalis.

Sets of the channel open times, [tau(o)], and closed times, [tau(c)], and the full set of the channel open and closed times, [tau(o), tau(c)], in the activity of single voltage-dependent K+-channels in mollusc L. stagnalis neurons were analyzed using the rescaled range analysis (Hurst method), fast Fourier and wavelet transforms. It was found that the Hurst dependence for each time series could be approximated by a polygonal line with at least two slopes: H1 and H2 (Hurst exponents). The averaged values of H1 and H2 for the sets [tau(o), tau(c)] were equal to 0.61 +/- 0.03 and 0.83 +/- 0.11, respectively; for the [tau(o)] sets H1 = 0.66 +/- 0.03 and H2 = 0.95 +/- 0.10; for the [tau(c)] sets, H1 = 0.62 +/- 0.05 and H2 = 0.85 +/- 0.10. In some cases, a third slope appeared on the Hurst dependences. It was very variable and ranged between 0.5 and 1. The Hurst exponents H1, H2, and H3 characterized short, intermediate, and long time ranges, respectively. The ranges greatly varied from experiment to experiment. The data obtained show that the channel openings and closings (gating process) represent a persistent process correlated in time. The randomization of the time sets resulted in a single slope, H, of 0.52 +/- 0.02 characteristic of random processes. The results were confirmed by the fast Fourier and wavelet transforms. In addition, possible voltage dependences of Hurst exponents and their correlation with tau(o) and tau(c) were investigated. As a whole, single channel activity may be characterized as a multifractal process with a slight voltage dependence of the Hurst exponents.

[Temperature dependence of kinetic parameters of single potential dependent K+ channels in mollusc neurons. Similarity in the action of potential and temperature]. / Temperaturnye zavisimosti kineticheskikh parametrov odnochnykh potentsialozavisimykh K+-kanalov v neironakh molliuska. Skhodstvo v deistvo v deistvii potentsiala i temperatury.

Using the patch-voltage-clamp method on excised membrane fragments from molluscan neurones temperature dependences of kinetic parameters of the fast and slow K(+)-channels were investigated in the temperature range 1 to 40 degrees C. Temperature dependences of probability of the channel open state (P0) for the slow and fast K(+)-channels are, generally, opposite, that is P0 increases for the slow channel and decreases for the fast channel with temperature. Similar dependences characterize durations of single channel open intervals (tau 0) and burst durations (t(p)). Durations of interburst and interpulse intervals (respectively, t(i) and tau) decrease for the slow channel and increase, in contrast, for the fast channel with temperature. For the channels of both types temperature dependences of P0 (as for other parameters) are essentially nonmonotonous. There are two local extrema, at least: for the slow K(+)-channel-maximum at 15 degrees C (minimum for the fast channel) and minimum at 20-25 degrees C (maximum for the fast channel). In some cases the number of local extrema may be greater than two. Some similarity in the action of temperature and membrane potential on the kinetic parameters was observed. For the slow K(+)-channel P0, tau 0 and t p increase with temperature and membrane potential. For the fast channel these parameters decrease at the same conditions. Moreover, for the channels of both types temperature dependences of the kinetic parameters are slightly pronounced at the potentials where potential dependences of the parameters are least. As a whole, temperature measurements showed that there are, possibly, several points of structural transitions (similar to phase transitions) in the temperature range 0 to 40 degrees C. Primarily, the kinetic parameters are determined by these transitions.

[Study of fractal properties of single ionic channel gating mechanism by the fast Fourier transform]. / Issledovanie fraktal'nykh svoistv "vorotnogo" mekhanizma odinochnykh ionnykh kanalov metodom bystrogo Fur'e-preobrazovaniia.

Sets of the channel open times (tau o), closed times (tau c) and the full set of the channel open and closed times (tau o, tau c) in the activity of single Ca(2+)-activated K+ channels in cultured kidney cells Vero were analyzed using the fast Fourier transform. It was found that in the low-frequency range (about 0.01-10 Hz), power density can be described by the equation S(f) approximately f-alpha (as a rule, 0 < alpha < 1), and this part of the Fourier spectrum usually consists of narrow peaks at almost multiple frequencies. It was shown that the upper frequency boundary of this spectrum is determined by the kinetic parameters tau o [symbol: see text] tau c. The data obtained show that ion channel gating is a fractal process (correlated in time) and can be regarded as a random signal modulated by some periodical functions (sinuses). The data obtained by the Fourier method are in agreement with the earlier results obtained using the rescaled-range analysis.