Displacement currents in sodium channels of the ranvier node membrane

The displacement current was recorded in the Ranvier node membrane ofRana ridibunda. This current was shown to be due to conversion of charges from the initial state in which they were when a high negative voltage was present on the membrane into the final state. The dependence of the displacement charge on the membrane potential and state of activation of the sodium channels suggests that the displacement current is connected with activation of the m gates of the sodium channels. Considering the density of the displaced charges, the density of the sodium channels can be estimated to be 5000 channels/µ².A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 410–417, July–August, 1976.     

Ionic current through batrachotoxin-modified sodium channels of the ranvier node membrane at high positive and negative potentials

Ionic current through batrachotoxin (BTX)-modified sodium channels within a wide range of membrane potentials were measured by the voltage clamp method on the membrane of a myelinated frog nerve fiber. At high positive voltages (above +80 mV) the current decreased with time; with an increase in voltage the steady-state level of the currents fell. The results of measurement of "instant" currents showed that this phenomenon is connected with a decrease in overall conductivity of the modified channels. Scorpion toxin had no significant effect on the kinetics of decline of the currents. This indicates that they are due to processes which differ from ordinary inactivation. In the presence of procaine, at high positive voltages slow (tens of milliseconds) potential-dependent blocking of BTX-modified channels was observed. An increase in negative potentials above ?100 mV caused a decrease in "instant" currents, connected with rapid potential-dependent blocking of BTX-modified sodium channels by calcium ions.     

Activation and inactivation of batrachotoxin-modified sodium channels in the frog nerve fiber membrane

Currents through batrachotoxin (BTX)-modified sodium channels were measured under voltage clamp conditions on the Ranvier node membrane. Potential-dependence of the fraction of activated BTX-modified channels was determined on the basis of data showing nonlinearity of the momentary current-voltage characteristic curve in the region of high negative potentials. BTX induces a shift of the sodium channel activation curve toward negative potentials on average by 67 mV, but does not, under these circumstances, alter the potential-sensitivity of their activation mechanism. The results of experiments with preliminary depolarization, of varied amplitude and duration, showed that BTX-modified sodium channels are capable of partial inactivation. The high level of steady-state conduction of the modified channels is evidently due to the fact that as a result of modification by BTX the open state of the channel becomes energetically more advantageous than the inactivated state. It is concluded that the action of BTX on inactivation differs in principle from the action of pronase.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 16, No. 1, pp. 18–26. January–February, 1984.     

Effects of glutaraldehyde on sodium channel activation and inactivation in frog nerve fiber

The effects of glutaraldehyde on sodium channel gating were investigated in the membrane of the node of Ranvier in frog nerve fiber. It was found that treating the membrane with glutaraldehyde slows the rate of inactivation, renders the inactivation curve considerably less steep, and leads to the appearance of a steady-state current component. It also decelerated the activation rate and reduced the slope of the central portion of the activation curve, which was shifted over to depolarization at the membrane. This produced no significant change in the effective charge in the effective charge of activation as determined from the limiting logarithmic slope of the activation curve. The mechanisms possibly underlying these changes in sodium channel gating are discussed.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 18, No. 5, pp. 579–586, September–October, 1986.     

Inhibition of sodium currents in frog ranvier node treated with local anesthetics Role of slow sodium inactivation

Effects of different local anesthetics of sodium permeability were studied in single nerve fibres of frog by the method of voltage clamp. Inhibition of sodium current by externally applied tertiary anesthetics, procaine and trimecaine, was the sum of a potentially independent block (reduced $\text{P}{}_{\mathrm{rmNa}}$) and slow sodium inactivation with time constants ranging from tens to hundreds of ms depending on membrane potential (at room temperature). Externally applied uncharged benzocaine produced a potentially independent block only. According to dose-response curves both processes are one-to-one reactions. In the case of trimecaine equilibrium constant the reaction responsible for reduction of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ is about 0.3 mM, while that for slow inactivation is more than ten times less (0.02 mM). Increasing pH from 5.6 to 8.5 markedly accelerated the slow inactivation process at all potential values. Divalent cations Ca²⁺ and Ni²⁺ shifted the steady-state slow inactivation curve along the potential axis and simultaneously reduced slow inactivation at the saturation level. Permanently charged quaternary trimecaine was ineffective when applied externally. Internally applied tertiary anesthetics and quaternary trimecaine as well as externally applied quaternary derivative of lidocaine QX-572 produced a progressively irreversible block enhanced by depolarization and inhibition reversibly increased by repetitive short-term depolarization (frequency-dependent inhibition). Inhibition of sodium currents by repetitive stimulation observed also in the case of externally applied tertiary anesthetics is due mainly to slow inactivation. The data suggests the existence of several types of receptor sites through which local anesthetics exert their blocking action on sodium permeability.     

Hydrogen ion current through sodium channels in myelinated nerve fiber membrane

Ionic currents through the frog Ranvier node membrane were measured by the voltage clamp method on the membrane of a single myelinated frog's nerve fiber under conditions when Na⁺ in the external solution was replaced by nonpenetrating cations. When pH fell below 4.0, small (under 0.1 nA) inward currents were found and on the basis of various features (kinetics, region of activation, and blocking by the local anesthetic benzocaine — 1.0 mM) were identified as currents through sodium channels. The results of control experiments with variation of the concentrations of cations in the external solution led to the conclusion that the H⁺ (or H₃O⁺) ion is the main carrier of the measured inward current. According to the results of measurement of the reversal potential of these currents, the relative permeability of sodium channels for hydrogen ions (P_H/P_Na) averages 205 ± 14. The results are discussed in terms of a model of the water pore with saturation. It is concluded that the energy barriers for H⁺ in the sodium channel are low. It was also shown that the velocity of passage of protons through the channel is limited by binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 499–507, September–October, 1982.     

Effect of aconitine on some properties of the sodium channels of the ranvier node membrane

Aconitine causes the appearance of two types of modified channels in the Ranvier node membrane. Channels of the first type are activated at high negative potentials and are inactivated only partly or not at all; their selectivity is sharply reduced: The mean ratio of potassium to sodium permeability is 0.72. The properties of these channels are stable with time. The second type of modified channels lose their conductivity during the action of aconitine, and their kinetic characteristics and region of activation are similar to those of normal sodium channels. They are less selective than normally: the ratio of potassium to sodium permeability is 0.22. The sensitivity of the modified channels to tetrodotoxin is at the same level as that of normal sodium channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 152–160, March–April, 1976.     

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

Change in the selectivity of the sodium channels of a nerve fiber membrane under the action of veratrine]

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.     

Kinetic model of the gating system of the sodium channel in a Ranvier node membrane

A kinetic model of the sodium channel gating system consisting of four subunits with three states--closed (X), open (Y) and inactivated (Z)--is proposed. For the channel to conduct, all the four subunits must be in the open state. The transitions between states X and Y are independent, while those between states X and Z are coupled, so that for the particle considered transition of one of two neighbouring particles into state Z increases the activation energy of the step by kT. The model fits rather well to the experimental data.     

The role of the putative inactivation lid in sodium channel gating current immobilization

We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.     

Reconstitution of the sodium channel with partially solubilized lobster nerve membrane

Reconstitution experiments were carried out with particles obtained from lobster nerve plasma membrane preparations by detergent treatment, differential centrifugation and ammonium sulfate fractionation. The NA channel activity of the three fractions obtained, which have different amounts of the same peptides present in the original membrane, appears related to their content in a large component which does not enter the 9% polyacrylamide gel and in peptides with 220,000 and 110,000 apparent molecular weight. Other reconstitution experiments made with two fractions obtained by detergent treatment, differential centrifugation and gel exclusion chromatography, revealed that the Na channel active fraction contains the material which does not enter the gel in addition to the 220,000 and 110,000 molecular weight peptides. The other fraction was inactive and does not contain those components. The 220,000 dalton peptide has a molecular weight similar to those determined for the tetrodotoxin-saxitoxin receptor and the scorpion toxin receptor of the Na channel. Whether any of the other peptides is a Na channel constituent is unknown at present.     

Ion selectivity of the nerve membrane sodium channel incorporated into liposomes

Tetrodotoxin-sensitive sodium channels of lobster nerve membranes were incorporated into soybean liposomes by the freeze-thaw-sonication procedure and their ionic selectivity was studied. Veratridine and grayanotoxin-I were used to activate the sodium channels and the increment of the ionic flux through them was specifically abolished by tetrodotoxin. The drug-sensitive 22Na+, 42K+, 86Rb+ and 137Cs+ influxes were measured. The permeability ratios calculated directly from ion fluxes showed that the channels preferably allow the passage of Na+. No anion influx ([32P]phosphate, [35S]sulfate, 36Cl) sensitive to the drugs was observed. The data reveal that the sodium channels incorporated into liposomes remain cation-selective and discriminate among different cations.     

Comparison of ion current noise predicted from different models of the sodium channel gating mechanism in nerve membrane

Summary The theoretical power density spectrumS(f) of ion current noise is calculated from several models of the sodium channel gating mechanism in nerve membrane. Sodium ion noise experimental data from the frog node of Ranvier [Conti, F.,et al. (1976),J. Physiol. (London) 262:699] is used as a test of the theoretical results. The motivation for recent modeling has been evidence for a coupling between sodium activation and inactivation from voltage clamp data. The two processes are independent of one another in the Hodgkin and Huxley (HH) model [Hodgkin, A.L., Huxley, A.F. (1952),J. Physiol. (London) 117:500] The noise data is consistent with HH, as noted by Contiet al. (1976). The theoretical results given here appear to indicate that only one case of coupling models is also consistent with the noise data.     

Hydrogen currents through aconitine-modified sodium channels in the nerve fiber membrane

Ionic currents through aconitine-modified sodium channels of the Ranvier node membrane were measured by a voltage clamp method in an external medium free from sodium ions. A shift of pH of the solution below 4.6 led to the appearance of inward ionic currents, whose kinetics and activation region were characteristic of aconitine-modified sodium channels at low pH. These currents were blocked by the local anesthetic benzocaine in a concentration of 2 mM. Experiments with variation of the concentration of Ca⁺⁺, Tris⁺, TEA⁺, and choline⁺ in acid sodium-free solutions showed that these cations make no appreciable contribution to the inward current. It is concluded that the inward currents observed under these conditions are carried by H⁺ (or H₃O⁺) through aconitine-modified sodium channels. From the shifts of reversal potentials of the ionic currents the relative permeability (P_H/P_Na) for H⁺ was determined: 1059 ± 88. The results agree with the view that the aconitine-modified sodium channel is a relatively wide water pore, and that movement of H⁺ through it is limited by its binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 508–516, September–October, 1982.     

Sodium channel opening as a precursor to inactivation

The time constant of the process producing the delay in Na inactivation development as determined by the two pulse method (_delay) was extracted and compared to that of the slowest Na activation process ₃ for the I _Na during the conditioning pulse of that same determination. _delay and two pulse inactivation _c values were computer generated using a nonlinear least squares algorithm. _h and single pulse inactivation _h values were independently generated for each determination also with the aid of the computer using the same non-linear least squares algorithm. In one determination at 2 mV, _c was 4.68 and _delay 0.494 ms while _h was 4.70 and ₃ 0.491 ms for a _c/_h of 0.996 and a _delay/₃ of 1.006. Mean _delay/₃ from five determinations in four axons, both Cs and K perfused, and spanning a potential range of-27 to 2mV was 1.068. The precursor process to inactivation is channel opening. Some fraction of channels presumably inactivate via another route where prior channel opening is not required.     

Modelled temperature-dependent excitability behaviour of a single ranvier node for a human peripheral sensory nerve fibre

The objective of this study was to determine whether the Hodgkin–Huxley model for unmyelinated nerve fibres could be modified to predict excitability behaviour at Ranvier nodes. Only the model parameters were modified to those of human, with the equations left unaltered. A model of a single Ranvier node has been developed as part of a larger model to describe excitation behaviour in a generalised human peripheral sensory nerve fibre. Parameter values describing the ionic and leakage conductances, corresponding equilibrium potentials, resting membrane potential and membrane capacitance of the original Hodgkin–Huxley model were modified to reflect the corresponding parameter values for human. Parameter temperature dependence was included. The fast activating potassium current kinetics were slowed down to represent those of a slow activating and deactivating potassium current, which do not inactivate. All calculations were performed in MATLAB^TM. Action potential shape and amplitude were satisfactorily predicted at 20, 25 and 37°C, and were not influenced by activation or deactivation of the slow potassium current. The calculated chronaxie time constant was 65.5 μs at 37°C. However, chronaxie times were overestimated at temperatures lower than body temperature.