Functional expression of alpha-latrotoxin in baculovirus system.

To facilitate the study of the mechanism of alpha-latrotoxin action, it is necessary to create a biologically active recombinant toxin. Mature alpha-latrotoxin is naturally produced by post-translational cleavage, probably at two furin sites located at the N- and C-termini of the precursor. A recombinant baculovirus has now been constructed, which encodes the melittin signal peptide fused to the 130-kDa mature toxin between the furin sites. Insect cells, infected with this baculovirus, secreted recombinant alpha-latrotoxin. This was partially purified and proved indistinguishable from the natural toxin with respect to its molecular mass, immunostaining, toxicity to mice, binding to alpha-latrotoxin receptors (latrophilin or neurexin Ialpha) and electrophysiological recording in the mouse diaphragm. The successful expression of recombinant alpha-latrotoxin permits mutational analysis of the toxin.

Some properties of sodium channels in neuroblastoma cells modified with scorpion toxin and chloramine-T. Single channel measurements.

Currents through sodium channels of neuroblastoma cells were measured using patch technique in outside-out configuration. Scorpion toxin (ScTX) produced 3 to 4 fold prolongation of mean open time and increased number of reopenings. The mean open times showed slow fluctuations around some average values. The distribution of channel open times for ScTX-modified channels required more than one exponential to be fitted. Chloramine-T (ChT) produced qualitatively similar, though weaker, prolongation of open times.

[Effect of several m-cholinoblockaders on the functioning of sodium potential-dependent channels of neuroblastoma cells]. / Vliianie nekotorykh m-kholinoblokatorov na funktsionirovanie natrievykh potentsialzavisimykh kanalov kletok neiroblastomy.

Effects of two central antimuscarinic drugs, namely N-methyl-4-piperidinol benzylate and tropine phenylcyclopentylglycolate on Na currents in internally dialyzed mouse neuroblastoma cells were studied using the suction-pipette voltage-clamp technique. Like amine local anesthetics, the both compounds produce two phenomenologically different types of sodium current inhibition: a steady block without conditioning and a cumulative (use-dependent) block which develops during repetitive membrane pulsing. The ability of these m-cholinoblockers to modulate voltage-dependent ionic permeability of the neuronal membranes is supposed to be one of possible components in the mechanism of their both main and accessory (non-synaptic) effects.

[Effect of blood plasma and serum and its fractions on the activity of the sodium channels in neuroblastoma cells]. / Vliianie plazmy, syvorotki krovi i ee fraktsii na aktivnost' natrievykh kanalov neiroblastomy.

Blood plasma, serum and its fractions containing components of different molecular masses obtained by dialysis and gradual ultrafiltration were tested for their action on the sodium currents of voltage clamped internally perfused neuroblastoma cells. Blood plasma free of platelet factor 4, serum and its fractions containing components of molecular masses more than 50 kD produced an increase in sodium channel currents and shifts in activation and inactivation curves along the voltage axis towards more negative or positive potentials, respectively. Moreover, the serum deficient in components with the molecular masses less than 50 kD produced a more prominent effect on all the parameters tested. Thus, the low-molecular fractions were suggested to exert an inhibitory activity on the stimulatory effects produced by the high-molecular components of the serum.

[Effect of ruthenium red on the sodium channel inactivation of neuroblastoma cells]. / Vliianie rutenievogo krasnogo na inaktivatsiiu natrievykh kanalov kletok neiroblastomy.

Currents through sodium channels in the neuroblastoma cell membrane were measured after internal and external application of the ruthenium red. The treatment of the membrane by the dye resulted in a slowdown of inactivation kinetics of sodium currents and in the changes of activation and inactivation stationary parameters, which were more pronounced after internal application of the ruthenium red.

[Action of chloramine-T on the processes of sodium channel activation and inactivation in neuroblastoma cells]. / Issledovanie deistviia khloramina-T na protsessy aktivatsii i inaktivatsii natrievykh kanalov kletok neiroblastomy.

Effect of chloramine-T, the specific reagent on methionine residues, on gating of sodium channels was studied in neuroblastoma cell membrane. After the chloramine treatment the inactivation became slower, incomplete and the steepness of its voltage dependence considerably decreased; the inactivation curve was shifted towards depolarization. Time course of activation did not change. The activation curve shifted towards negative potentials, its slope being insignificantly decreased. Effective charge of activation as determined from the limiting logarithmic slope of the activation decreased by a factor of 1.17. Possible explanations of the phenomena observed are discussed.

Modification of Na channels by synthetic dihydrobatrachotoxinin A-20 alpha-benzoate.

Sodium channels in nodal membrane modified by a synthetic analog of batrachotoxin, 7,8-dihydrobatrachotoxinin A-20 alpha-benzoate, were studied under voltage clamp conditions. The voltage dependence of channel activation was shifted by 70-80 mV towards more negative potentials. Selectivity sequence determined from peak current reversal potential was as follows: Na:NH4:K = 1:0.46:0.23. Our data suggest that 7,8-dihydrobatrachotoxinin A-20 alpha-benzoate has qualitatively similar effects on the properties of sodium channels as does natural batrachotoxin.

[Effect of glutaraldehyde on the activation and inactivation of sodium channels in frog nerve fibers]. / Vliianie glutaral'degida na aktivatsiiu i inaktivatsiiu natrievykh kanalov nervnogo volokna liagushki.

Effect of glutaraldehyde on gating of sodium channels was studied in the nodal membrane of the frog nerve fibre. After treatment the inactivation became slower, incomplete and the steepness of its voltage-dependence considerably decreased. The activation became slower and the steepness of its voltage-dependence decreased though both effects for the activation were less pronounced than for the inactivation. Effective charge of the activation as determined from the limiting logarithmic slope of the activation curve did not change significantly. Possible explanations of the phenomena observed are discussed.

Voltage dependent modification of sodium channel gating with water-soluble carbodiimide.

Currents through sodium channels in frog myelinated fibre were measured before and after the treatment of the membrane with water-soluble carbodiimide (WSC). The WSC treatment produces dramatic changes both in activation and inactivation when membrane potential during the treatment is held at levels from -80 to -100 mV. Both gating processes are slowed and their voltage dependence is reduced. The effective charge of activation as measured by limiting logarithmic potential sensitivity is reduced after 10 min of the WSC treatment by the factor of 1.66. The same WSC treatment applied at zero potential induced no change in the effective charge of activation. Other parameters of gating are changed after such treatment but to a lesser degree and in a somewhat different fashion than after the treatment at high negative potential. The results are consistent with the hypothesis that part of a mobile gating charge is presented by carboxyl groups migrating from the external surface to the interior of the channel molecule when the channel opens or inactivates.

A study on the potential-dependence of proton block of sodium channels.

Ionic currents through sodium channels in nodal membranes were measured under voltage clamp conditions both at normal and at low (4.8-4.9) external solution pH. The measurements of so-called 'instantaneous' currents were used to distinguish between the proton blockage in open channels and the influence of low pH on channel gating processes. It is shown that the amount of the proton blockage in open channels decreases as membrane potential becomes more positive. This result suggests that at least one of the acid groups accessible from the outside is located within the conducting pore. The influence of the other group(s) on the degree of potential-dependence of proton blockage is discussed.

[Potential-dependent interaction of chemical reagents with the sodium channel gating mechanism in a nerve fiber]. / Potentsialozavisimoe vzaimodeistvie khimicheskikh reagentov s vorotnym mekhanizmom natrievogo kanala v nervnom volokne.

Effect of two water-soluble carbodiimides (WRC) and Woodward's reagent K (RK) on gating in sodium channels of nodal membrane was examined. The WRC treatment (pH 4.8-5.2) at holding potential from -80 to -100 mV resulted in a considerable slowing down of activation and inactivation processes, and rendered the gating machinery less sensitive to membrane potential changes. Effective charge of activation (Zef) determined from limiting logarithmic slope of activation curve at potentials where channels just begin to open was reduced by the factor of 1.7. The same treatment but at zero holding potential did not reduce Zef, and changed other parameters of gating function to a less degree. RK at high negative holding potentials induced changes in properties of gating machinery similar to those induced by WRC at zero potential. RK effects were less potential-dependent than those of WRC. The results suggest that gating machinery of sodium channel incorporates at least two types of carboxyl groups. The first type: "mobile", play the role of gating particles; during depolarization they move from the surface into the membrane causing opening or inactivation of the channel. The second type: "immobile", is always exposed to the external solution regardless of the channel state.

Potential-dependent interaction of toxin from venom of the scorpion Buthus eupeus with sodium channels in myelinated fibre: voltage clamp experiments.

1. The steady-state characteristics of the sodium channel gating in the nodal membrane were determined under voltage clamp conditions before and after treatment with toxins from the venom of scorpion, Buthus eupeus. 2. The apparent binding constant (KA) of the toxin was determined for different levels of the membrane potential. At potentials more negative than -120 mV, KA tends to a constant level. KA is maximum at about -80 mV, and it decreases as the potential is teduced to 0 mV. 3. A model assuming that the voltage dependency of KA is mainly due to the difference in electrical energy between inactivated states of normal and poisoned channels is proposed. An additional decrease in overall binding of toxin results from the transition of a fraction of the sodium channels into the state of slow inactivation.

[Features of the kinetic and stationary characteristics of aconitine-modified sodium channels]. / Nekotorye osobennosti kineticheskikh i statsionarnykh kharakteristik natrievykh kanalov, modifitsirovannykh akonitinom.

Kinetic and steady-state characteristics of aconitine-modified sodium channels were studied in the Ranvier node membrane. Aconitine-modified sodium channels are shown to be inactivated only partially. The voltage dependence of the fraction of noninactivated channels (h infinity) may be described by a three-state model of the channel with closed, open and inactivated states. A reasonable agreement with the data was obtained when parameters of the inactivated state were supposed to be not changed after aconitine modification of the channels. The membrane repolarization to -70 divided by -110 mV, after long (10 ms) depolarizing shift induces firstly fast current decay ("tail") and then its rather slow increase to a steady-state level. Kinetics of this current requires two or more open states to be postulated.

[Kinetics of sodium current decay during normal axon membrane repolarization and in the presence of scorpion toxin]. / Kinetika spada natrievogo toka pri repoliarizatsii aksonnoi membrany v norme i v prisuststvii toksina skorpiona.

Decay of sodium currents in repolarization ("tail current") was studied in from axonal membrane. The decay in the membrane repolarization to -40 divided by -60 mV has two exponential components: fast and slow. The fraction of the slow component in the total "tail current" (theta M) decreases as the repolarization potential (Vp) becomes more negative; at Vp more negative than -80 mV "tail" follows practically one-exponential time course. When lengthening the test pulse (at the given Vp) the fraction of the fast component in the "tail" decreases quicker than that of the slow component, following approximately the kinetics of inactivation during the tests pulse. Scorpion toxin treatment results in slowing down "tail" kinetics mainly at the expense of increasing the fraction of the slow component. A kinetic diagram assuming two open state for the channel is suggested. A hypothesis is advanced that scorpion toxin, DDT and trinitrophenol have a common "site" to interact with the gating mechanism of the sodium channel.

[Comparative study of the action of procaine and benzocaine on normal and aconitine-modified sodium channels]. / Sravnitel'noe izuchenie deistviia prokaina i benzokaina na normal'nye i modifitsirovannye akonitinom natrievye kanaly.

Ionic currents of normal and aconitine modified sodium channels of the Ranvier node membrane were measured under voltage clamp conditions. The experiments with local anesthetics in the external Ringer solution have showed that dissociation constant (Kdis) of normal channel-anesthetic complex for procaine is 0.27 + 0.03 mM, and for benzocaine is 0.68 +/- 0.04 mM. With aconitine modified channels, Kdis increases and becomes 1.32 +/- 0.5 mM and 1.52 +/- 0.3 mM for procaine and benzocaine, respectively. It is ascertained that the development of aconitine effect is inhibited by neutral benzocaine to a lesser extent than by procaine. It is shown that the aconitine effect cannot be reversed by a high concentration of anesthetic. Hence, it appears that aconitine and anesthetic receptors do not coincide.

[Change in the selectivity of the sodium channels of a nerve fiber membrane under the action of veratrine]. / Izmenenie selektivnosti natrievykh kanalov membrany nervnogo volokna pri deistvii veratrina.

The ionic currents of the nodal membrane were measured under voltage clamp conditions. The membrane being +40 mv. The replacing of the external Na+-ions to K+- and NH4+-ions have showed that the relative pearmeabilities of the veratrine-modified channels calculated from the constant field theory are arranged in the following row: PNa:PK:PNH4 = 1:0.29:0.61, which differs from the same row for the normal channels. The decreasing of the slope of current-voltage relations of the modified channels with the replacing of Na+-ions to K+- and NH4+-ions is the evidence of a more strong binding of these ions to external mouth of the modified channel compared to the binding of Na+-ions.

The permeability of aconitine-modified sodium channels to univalent cations in myelinated nerve.

1. Ionic currents through the sodium system of nodes of Ranvier treated with aconitine were measured under voltage clamp conditions in a Ringer solution containing Na+ or an equimolar amount of various test cations. 2. Average shifts in reversal potentials in nodes of Ranvier treated with aconitine with NH4+, Li+, K+, Rb+, Cs+ in place of Na+ in the Ringer solution are 7.6, --6.8, --25.0, --41.0 and --51.5 mV at 13--14degrees C. At 20--22degrees C the sequence of shifts is 7.5, --5.5, --13.5, --29.0 and --41.0 mV. For Tl+ the the average reversal potential shift is +3 mV at 20--22degrees C. 3. The slope of the instantaneous current-voltage relation at the reversal potential in nodes treated with aconitine changed with the various cations tested. The ratios are NH4+/Na+/K+/Rb+/Cs+/Li+ = 1.14 : 1.0 : 0.80 :0.67 :0.53 : 0.53. 4. Using a three energy barrier model some of the parameters for the aconitine-modified Na+ channels were estimated (Chizmadgev, Yu. A., Khodorov, B.I. and Aityan, S.Kh. (1974) Bioelectrochem. Bioenerg. 1, 301--312).