V.I. Geletyuk’s research while affiliated with Institute of Cell Biophysics, Russian Academy of Sciences and other places

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.

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.

The patch method in isolated membrane fragments of mollusc neurones in the range from 1 to 40 °C has been used to investigate the temperature dependences of the kinetic parameters of the activity of the fast and slow K ⁺ channels. It was established that the temperature dependences of the probability of finding the channel in the open state (P 0 ) for the fast and slow K ⁺ channels are, as a whole, opposed: for the slow K ⁺ channel the P 0 value increases with rise in temperature and for the fast channel it decreases. It is shown that similar dependences characterize the durations of the single current pulses (τ 0 ) and packets of current pulses (t p ); the durations of the interpulse and interpacket intervals (respectively, τ i and t i ) diminish with rise in temperature for the slow and increase for the fast K ⁺ channels. It was found that for the channels of both types the temperature dependences of 0 (like the other parameters) are profoundly non-monotonic: there are at least two local extremes-maximum at 15 °C for the slow K ⁺ channel (minimum for the fast channel) and a minimum at 20-25 °C (maximum for the fast K ⁺ channel); in some cases the number of local extremes may be far greater. Some similarity was established in the effect on the kinetic parameters of changes in temperature and membrane potential: for the slow channel the P 0 , t p and τ 0 values equally increase with rise both in temperature and membrane potential; for the fast channel the same parameters diminish with rise in temperature or membrane potential; for the channels of both types the temperature dependence of the kinetic parameters is weakly marked for those potentials at which their potential dependence is minimal. From analysis of the results of the temperature measurements of the activity of the ion channels it is concluded that in the 0-40 °C range there may be several structural changes in the membrane (similar to phase transitions) which primarily also determine the kinetic parameters.

The patch method ("inside-out" configuration) has been used to investigate the temperature dependences of the chord conductivity of single potential dependent channels fast and slow K ⁺ channels in the neurones of the fresh water mollusc Lymnaea stagnalis. It was established that in control conditions (20 °C, 0 mV, [K ⁺ ] o ] = 1·5 mM, [K ⁺ ] i = 100 mM) the conductivity of the fast and slow K ⁺ channels is, respectively, 20-25 and 30-40 pCm. In addition, the temperature dependences of currents in the K ⁺ channels of lower conductivity (from 5 to 20 pCm) have been investigated. It is shown that some of these channels may be regarded as subtypes of fast and slow K ⁺ channels. It has been established that for all the channels the currents (and conductivity) increase with rise in temperature although in the 10-20 °C interval anomalous behaviour of the currents is observed (they either do not change or even decrease with rise in temperature), possibly due to phase transitions in the membrane. It is shown that outside the anomalous zone the temperature dependences of currents in Arrhenius coordinates may be approximated by straight lines, the slope of which are determined by the apparent activation energy values of the conductivity of the channels (ΔE a ). It was found that for all channels except the fast at temperatures above 20 °C the ΔE a value is ~16·7 kJ/mole and corresponds to the free diffusion of ions in aqueous solution. For these temperatures the magnitude ΔE a weakly depends on the membrane potential: at temperatures below 10 °C the potential dependence of ΔE a for the slow K ⁺ channels is significant and is observed, in the main, in the range -20 to +20 mV (the magnitude ΔE a is equal, on average, to ~50·2 kJ/mole); the potential dependence of ΔE a for channels of lower conductivity is more weakly marked. The temperature dependences of currents in the fast K ⁺ channels are to some degree opposite to the corresponding dependences for the slow K ⁺ channels.

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.

The patch method has been used on isolated membrane fragments of mollusc neurones to study the effect of ferricyanide and the Ba2+ ion on the K+ channels in which the synchronization of the transitions between substates of conductivity was disturbed. Ferricyanide (0·1-0·5 mm) and Ba2+ (1 μm) were applied to the inner surface of the membrane. It is shown that in several tens of minutes ferricyanide causes irreversible transformation of the random transitions between substates to a highly cooperative process; the Ba2+ ion in some cases reversibly and completely restores the synchrony of the transition of the channel between the sublevels of conductivity and in others partially restores it.

The patch method on isolated membrane fragments has shown that the oscillations of the open ionic channel represent fast transitions of current between 64 sublevels. The mean values of the elementary jump of conductivity (γ = 1·5-6 pCm) and the lifetimes of the substates (τel = 0·15-0·5 msec) have been determined for different types of ionic channel. It was established that the transitions of the channel between substates represent a highly cooperative process. The findings are considered on the basis of the notions of the hypothesis of the cluster organization of the ionic channels.

Properties of the single Cl channels were studied in excised patches of surface membrane from molluscan neurones using single-channel recording technique. These channels are controlled by Ca2+ and K+ acting on cytoplasmic and outer membrane surfaces, respectively, and by the membrane potential. The channels display about 16 intermediate conductance sublevels, each of them being multiples of approximately 12.5 pS. The upper level of the channel conductance is about 200 pS. The channel behavior is consistent with an aggregation of channel-forming subunits into a cluster.

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.