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I V Chizhmakov

Publications and source records attributed to I V Chizhmakov.

14 recordsLinked to original sources
PubMed2003

Differences in conductance of M2 proton channels of two influenza viruses at low and high pH.

The M2 protein of influenza A viruses forms a proton channel involved in modifying virion and trans Golgi pH during infection. Previous studies of the proton current using whole-cell patch clamp of mouse erythroleukaemia (MEL) cells expressing the M2 protein of the "Weybridge" strain provided evidence for two protonation sites, one involved in permeation, the other in activation by acid pH. The present report compares the M2 channels of two different strains of influenza virus, "Weybridge" (WM2) and "Rostock" (RM2). Whereas with external acid pH the current-voltage relations showed similar small degrees of inward rectification, a similar apparent K(d) of approximately 10 microM for proton permeation and a high selectivity for protons over Na(+), the two M2 proteins differed in whole-cell conductance at low and high pH. The proton conductance of unit membrane area was on average 7-fold greater in RM2- than WM2-expressing MEL cells. At high external pH WM2 was shown previously to have small conductance for outward current at positive driving potential. In contrast, RM2 shows high conductance for outward current with high external pH, but shows small conductance for inward current with high internal pH, conditions in which WM2 shows high conductance for inward current. The different properties of the conductances due to the two channels at high pH were determined by three amino acids in their transmembrane domains. All intermediate mutants possessed one or other property and transformation of the WM2 phenotype into that of RM2 required substitution in all three residues V27I, F38L and D44N; single substitutions in RM2 effected the opposite phenotypic change. The significance of this difference for virus replication is not clear and it may be that the higher proton flux associated with RM2 is the main factor determining its increased ability to dissipate pH gradients. It is apparent, however, from the specific differences in the sidedness of the pH-induced changes in voltage dependence of the whole-cell current that this is an intrinsic property of the virus proton channel which may have parallels with regulation of other proton channels.

Animals↗
PubMed1996

Selective proton permeability and pH regulation of the influenza virus M2 channel expressed in mouse erythroleukaemia cells.

1. The M2 protein of influenza A virus is implicated in transmembrane pH regulation during infection. Whole-cell patch clamp of mouse erythroleukaemia cells expressing the M2 protein in the surface membrane showed a conductance due to M2 which was specifically blocked by the anti-influenza drug rimantadine. 2. The ion selectivity of the rimantadine-sensitive current through M2 was determined. Reversal potentials were close to equilibrium potentials for transmembrane pH gradients and not to those for Na+, K+ or Cl- concentration gradients. M2 permeability to Na+ relative to H+ was estimated to be less than 6 x 10(-7). 3. The M2 conductance increased as external pH decreased below 8.5 and approached saturation at an external pH of 4, effects attributable to increased permeability due to increased driving potential and to activation by low external pH. Both activation and permeation could be described by interaction of protons with sites on M2, with apparent dissociation constants of approximately 0.1 microM and 1 microM, respectively, under physiological conditions. 4. The M2 protein can transfer protons selectively across membranes with the H+ electrochemical gradient, properties consistent with its role in modifying virion and trans-Golgi pH during virus infection.

Animals↗
PubMed1994

The action of a phorbol ester on voltage-dependent parameters of the sodium current in isolated hippocampal neurons.

The action of a phorbol ester (phorbol-12,13-diacetate) on the voltage-activated sodium current has been investigated by the voltage-clamp method in acutely isolated pyramidal neurons from rat hippocampus. The intracellular perfusion of isolated pyramidal neurons for 30-40 min induced a gradual 10-15 mV shift in both the current-voltage relationship and voltage-dependent steady-state inactivation to more negative potentials. The application of phorbol ester (1-10 microM) to isolated neurons for the same time increased the amplitude of sodium current by 15-20%, shifted the above-mentioned voltage-dependent parameters for an additional 10-15 mV in the same direction and changed the slope of the steady-state inactivation curve. In contrast, after prolonged incubation of slices in the phorbol ester-containing solution (1-10 microM) for 0.5-3 h, subsequent application of phorbol ester at the same concentration caused neither the addition shift of the voltage-dependent characteristics of sodium channels nor the change of the slope of the steady-state inactivation curve. However, in this case an increase in the amplitude of sodium current by 15-20% during 30-40 min intracellular perfusion was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

R56865 and flunarizine as Na(+)-channel blockers in isolated Purkinje neurons of rat cerebellum.

Dose-related blocking effects of R56865, flunarizine and nimodipine on voltage-activated Na+ currents recorded in the whole-cell voltage clamp mode were studied in acutely isolated Purkinje neurons of rat cerebellum. The dose-dependences of blocking action were obtained for all drugs at a holding potential of -110 mV and rare stimulation. At stimulation frequencies 5 and 15 Hz the block produced by R56865 was increased showing a shift of dose-dependence to lower concentrations of antagonist. This shift was less pronounced for flunarizine, practically absent for nimodipine, and increased for all drugs with an increase in the amplitude of stimulating voltage pulse. With the change in holding potential to -80 mV the block produced by R56865 and flunarizine increased showing a dose-dependence shift to lower concentrations of antagonists. All the drugs tested induced parallel shifts of the steady-state voltage-dependence of inactivation of Na+ channels to more negative membrane potentials. R56865, and to a lesser extent flunarizine, slowed down the recovery of Na+ channels from steady-state inactivation increasing the relative number of channels which showed slow recovery. In the absence of Na+ current inactivation (treatment by intracellular pronase) R56865 at a concentration of 1 microM blocked modified channels preferentially in the open state, while the block produced by flunarizine showed no dependence on voltage pulse protocol. R56865 was shown to decrease the cell leakage while other drugs produced little or no effect. It is concluded that R56865 and flunarizine block Na+ currents predominantly by interacting with inactivated Na+ channels. The higher ability of R56865 to block open channels and to increase slow inactivation underlies its higher frequency-dependence. These characteristics suggest the use of R56865 and flunarizine in the treatment of cerebral ischemia.

Animals↗
PubMed1992

A highly potent and selective N-methyl-D-aspartate receptor antagonist from the venom of the Agelenopsis aperta spider.

Agatoxin-489, extracted from the venom of the Agelenopsis aperta spider, was studied on acutely isolated perfused hippocampal neurons of rat using the concentration clamp technique. Agatoxin-489 proved to be a selective N-methyl-D-aspartate antagonist; responses to applications of N-methyl-D-aspartate or L-aspartate were blocked by concentrations of agatoxin-489 ranging between 0.1 nM and 1 microM, while responses to kainate were not affected by agatoxin-489 at concentrations up to 10 microM. The actions of agatoxin-489 against responses to N-methyl-D-aspartate or L-aspartate were use- and voltage-dependent, being less pronounced with an increase in the holding potential from -100 to -30 mV. The action of agatoxin-489 could be completely or partially reversed only after washout in the presence of an N-methyl-D-aspartate agonist. The washout was more effective at positive membrane potentials ranging from 0 to +20 mV. These results imply that the spider toxin agatoxin-489, like dizocilpine, is a potent and selective N-methyl-D-aspartate antagonist which preferentially interacts with activated N-methyl-D-aspartate receptors and/or open N-methyl-D-aspartate-activated ionic channels.

Agatoxins↗
PubMed1992

Two types of steady-state desensitization of N-methyl-D-aspartate receptor in isolated hippocampal neurones of rat.

1. Whole-cell voltage-clamp recordings were made from rat isolated hippocampal neurones. Aspartate (Asp) and/or glycine (Gly) were applied by a method in which the external solution could be changed within 30 ms and thereafter held constant. 2. Asp and Gly applied together at maximal concentrations (5 mM and 10 microM, respectively) evoked an inward current due to activation of N-methyl-D-aspartate (NMDA) receptors. The current peaked and then declined to a steady state during the application. The time constant of desensitization (tau) was about 1 s when the agonists were applied soon after the onset of whole-cell recording. The desensitization became more rapid (tau = 0.3 s) and more complete during the first 15 min of recording, and thereafter remained stable; the amplitude of the peak response did not change throughout. In solutions containing 10 microM-Gly, Asp had an apparent Kd of 51 microM at the peak of response and 20 microM measured at the steady state. The steady-state current was 14% of the peak current. 3. Asp was applied after a conditioning exposure of the cell of Gly (from 1 to 50 microM), together with the same Gly concentration. The maximum current evoked by the application of Asp was increased while increasing Gly in the conditioning solution, with no change in the apparent Kd for Asp at the peak of Asp-activated response. 4. Various concentrations of Asp (plus 10 microM-Gly) were applied after a conditioning exposure to Asp (which alone was without effect). The maximum current induced by Asp applications was only 28% of that observed without conditioning Asp application, but the apparent Kd was unchanged (about 57 microM). 5. Test solution containing maximal concentrations of Asp and Gly was applied after conditioning exposure to both Asp (varying concentrations) and Gly (10 microM). Complete desensitization was caused by 200 microM-Asp. The apparent Kd for Asp to induce desensitization (8.7 microM) was less than the Kd as an agonist (51 microM). 6. Test solution containing maximal concentrations of Asp and Gly was applied after conditioning exposure to both Gly (varying concentrations) and Asp (5 mM). Complete desensitization was caused by 1 microM-Gly. The apparent Kd for Gly to induce desensitization (120 nM) was less than the Kd as a co-agonist (about 1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Desensitization of NMDA receptors does not proceed in the presence of kynurenate.

The agonist-induced steady-state desensitization of the N-methyl-D-aspartate (NMDA) receptor was investigated by means of whole cell patch and concentration clamp in isolated pyramidal neurons from rat hippocampus. When administered against a background of previously applied agonist, glycine (Gly) produced a response that was smaller than the response elicited by simultaneous application of agonist and Gly. This feature could be explained by an agonist-induced suppression of NMDA receptor sensitivity to the facilitatory action of Gly. This type of 'steady-state desensitization' did not develop when the preincubating solutions contained kynurenate (250 microM). It is proposed that NMDA receptors at all concentrations of agonist may acquire two distinct and interconverting conformations that are sensitive or insensitive to the facilitatory action of Gly. Kynurenate shifts the equilibrium between these states keeping the receptor in a Gly-sensitive conformation.

Animals↗
PubMed1989

Glycine action on N-methyl-D-aspartate receptors in rat hippocampal neurons.

The modulatory effect of glycine and other amino acids on the activity of N-methyl-D-aspartate (NMDA) receptors has been investigated using a concentration clamp method in the acutely isolated pyramidal neurons from rat hippocampus. The dose-response curves for L-aspartate, measured on the background of several glycine concentrations, were close to Langmuir isotherms with Kd values practically independent of the concentration of glycine. The facilitatory action of glycine appeared at concentrations below 0.1 microM and saturated between 10 and 100 microM. This effect demonstrated marked densensitization, at least at concentrations higher than 5 microM. Facilitation of the responses was shared by amino acids with the potencies diminishing as follows: glycine, D-serine, D-alanine greater than L-proline, D-proline, hydroxy-L-proline, taurine greater than L-alanine, L-serine. This sequence did not correlate with the ability of these amino acids to activate strychnine-sensitive glycine receptors.

Amino Acids↗
PubMed1987

[New findings on the mechanism of action of blocking toxins on electrically excitable sodium channels].

The paper summarizes the results obtained from the 10-12 years studies on the mechanism of action of blocking toxins--tetrodotoxin (TTX) and saxitoxin (STX)--on voltage-operated sodium channels. Experimental data can be interpreted on the basis of two models of blocking action of toxins: channel blockade, when guanidine group of toxins penetrates into the channel and causes its blockade and allosteric action on sodium conductance. Special attention is devoted to peculiarities of cooperative interaction between the blockers and channels. The analysis of experimental findings permits a better understanding of functional organization of sodium channels, in particular, the interrelation between the receptor of blocking toxins and its other structural elements.

Animals↗
PubMed1986

[Temperature-independence of the effect of osmotic pressure on the inward sodium current in the membrane of isolated spinal ganglion neurons of the rat].

The temperature dependence of the effect of transmembrane osmotic pressure on sodium TTX-sensitive inward current was studied on isolated neurons of rat dorsal root ganglia using intracellular perfusion under voltage clamp conditions. It was found that the effect of transmembrane osmotic pressure on the kinetic parameters of sodium current does not depend on temperature in a wide range (from 8 to 40 degrees C). The apparent values of activation energies for the activation and inactivation processes do not depend on osmolality. The overall results indicate that the most satisfactory way to account for the present observations is to postulate that the effect of transmembrane osmotic pressure is determined by the water flux crossing the membrane. It is supposed that this flux takes place within the protein molecule which forms the sodium channel. The molecular mechanisms of interaction between water pathways and gating are discussed.

Animals↗