Aconitine-induced modification of single sodium channels in neuroblastoma cell membrane

Aconitine-modified sodium channels in the neuroblastoma cell membrane were investigated with patch-clamp technique in outside-out configuration. When aconitine (0.1 mmol/l) was present in the pipette solution two types of modified single sodium channels were observed. The first type showed openings with normal amplitude (slope conductance 15.5 pS) and bursting behaviour. The second type of modified channel openings was characterized with low amplitude (slope conductance 2.8 pS) and longer open time as comparing to unmodified channels. The low-amplitude channels were shown to have altered ion selectivity: they were permeable to NH4+. Both populations of aconitine-modified channels could be blocked by tetrodotoxin. In contrast to macroscopic current experiments (Mozhayeva et al. 1977) the development of aconitine modification was not affected by repetitive stimulation and external application of the agent had no effect on single sodium channels in outside-out membrane patch.     

Interaction between sodium channels in mouse neuroblastoma cells

Single sodium channels in mouse neuroblastoma cells (N1E 115) were studied in cell-attached patches. During a series of consecutive responses to depolarizing pulses, records with and without channel opening were seen to form clusters rather than appearing randomly. The probability of finding open channels on a record seemed to increase with increasing number of channel openings. The open times of channels became shorter with increasing closed time interval measured between consecutive channel openings. Overlapping openings showed a voltage-dependent open time, in contrast to single openings which had voltage-independent open time. On the basis of these observations interaction between neighbouring sodium channels is suggested.Abbreviations RP resting potential - OT channel open time     

Properties of the sodium channels of the membrane of neuroblastoma clone N18 A-1 cultured cells

Ionic currents through sodium channels of dialyzed mouse neuroblastoma N18 A-1 cells were measured under voltage clamp conditions. The PNa/PK ratio evaluated by reversal potential shifts was 10.4 +/- 0.7. Parameters of steady-state fast inactivation curves (h--V) and peak sodium conductance curves (gNa--V) were determined. The inactivation kinetics had usually a two-exponential time course. The internal perfusion of cells by trypsin and pronase caused a slowing-down of the sodium current falling phase, pronase being more specific in this respect. An external application of the purified scorpion toxin in concentration of 1.42 X 10(-7) M leads to a fast and sharp slowing-down of sodium inactivation. The same toxin in concentration of 5 X 10(-6) M, applied internally was quite unaffective. Experimental results demonstrate similarities in the main features between the sodium channels of neuroblastoma cells and those of other excitable cell membranes.     

Hydrogen ion block of single sodium channels in neuroblastoma cells

The effects of pH of the external medium on amplitude of currents through single sodium channels at the membrane of cultured neuroblastoma cells were investigated in mice belonging to strain C 1300, clone N18A-1. Currents through single sodium channels in isolated membrane segments (outside-out configuration) were registered with normal (7.2) and reduced (5.4) pH levels in the external medium. Reducing the pH to 5.4 was found to decrease current amplitude reversibly by about twofold (–10 to –30 mV for test potentials). Findings would confirm that the depression of macroscopic sodium currents produced by reducing the pH of the extracellular solution is due to a decline in ionic flow through single open sodium channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 101–105, January–February, 1989.     

Modulation of nerve membrane sodium channels by chemicals

1. Modulations of sodium channel kinetics by grayanotoxins and pyrethroids have been studied using voltage clamped, internally perfused giant axons from crayfish and squid. 2. Grayanotoxin I and alpha-dihydrograyanotoxin II greatly depolarize the nerve membrane through an increase in resting sodium channel permeability to sodium ions. 3. Grayanotoxins modify a fraction of sodium channel population to give rise to a slow conductance increase with little or no inactivation, and the slow conductance-membrane potential curve is shifted toward hyperpolarization. This accounts for the depolarization. 4. The tail current associated with step repolarization during the slow current in grayanotoxins decays with a dual exponential time course. 5. (+)-trans tetramethrin and (+)-trans allethrin also modify a fraction of sodium channel population in generating a slow current, which attains a maximum slowly and decays very slowly during a maintained depolarizing step. The membrane is depolarized only slightly. 6. The tail current associated with step repolarization during the slow current in the pyrethroids is very large in initial amplitude and decays very slowly. 7. The rate at which the sodium channel arrives at the modified open state in the presence of pyrethroids is expressed by a dual exponential function, and the slow phase disappears following removal of the sodium inactivation mechanism by internal perfusion of pronase. 8. A kinetic model is proposed to account for the actions of both grayanotoxins and pyrethroids on sodium channels. Both chemicals interact with the channel at both open and closed states to yield a modified open state which results in a slow sodium current.     

Evidence for multiple open states of sodium channels in neuroblastoma cells

Summary Open times of voltage-gated sodium channels in neuroblastoma cells were measured during repolarization (following a short depolarizing conditioning pulse) and during moderate depolarization. Conditional and unconditional channel open-time histograms were best fitted by the sum of two exponentials. (The conditional open time was measured from the end of the conditioning pulse until an open channel shuts provided it was open att=0). Time constants of both histograms depended on the postpulse and were shifted to more positive potentials with increasing conditioning pulse potential. This shift could be explained by assuming more than two time constants in the histograms, which could not be separated. Channel open-time histograms from single-pulse experiments showed a maximum att>0. These histograms could be best fitted by an exponential function with three time constants. One term of this function included the difference of two exponentials resulting in a maximum att>0. Open-time histograms showed a definite time dependence. At 2 to 6.5 msec after the beginning of the depolarization the best fit could be obtained by the difference of two exponentials. To these components another term had to be added at 0 to 2 msec. Between 6.5 and 14.0 msec the sum of two exponentials, and after 14.0 msec a single exponential resulted in a good fit. The results support the hypothesis that sodium channels in neuroblastoma cells may have multiple open states. Two of these states are irreversibly coupled.     

Modulation of voltage-gated sodium channels by recombinant interferon-alpha2b in the human neuroblastoma cell line IMR-32

Clonal human neuroblastoma cells imr-32 are a suitable model system for studies of neuronal excitability modulation. The ability interferon-alpha 2b "laferon" to modulate the mechanisms of electrical activity was studied in whole-cell patch-clamped undifferentiated human neuroblastoma cells IMR-32. It was shown that 1 h incubation of IMR-32 cells at 37 degrees C in medium with laferon (600 U/ml) exerted changes in voltage-dependent properties of Na(+)-channels. The results of the present study demonstrate that laferon decreased of Na(+)-channels sensitivity to changes of membrane potential leading of IMR-32 cells electrical excitability decrease.     

Effects of chloramine-T on activation and inactivation of sodium channels in neuroblastoma cells

The effects of chloramine-T, a reagent specific to methionine residues, on sodium channel gating mechanisms was investigated in neuroblastoma cell membrane. Treating the membrane with chloramine was found to retard inactivation kinetics and considerably reduce the slope of the inactivation curve, while pushing the activation curve toward hyperpolarization ranges without changing the slope of the central portion perceptibly. Effective activation charge, as determined from the limiting logarithmic slope of activation, was reduced by a factor of 1.17. Possible reasons for the changes observed in sodium channel gating mechanisms are discussed.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 19, No. 6, pp. 789–795, November–December, 1987.     

Effect of gamma radiation on sodium channels in different conformations in neuroblastoma cells

We studied the dose-response relationship between gamma radiation and batrachotoxin-stimulated sodium influx in neuroblastoma cells in tissue culture. We also tested the hypothesis that changes in sodium channel conformation may alter the radiosensitivity of the channel. We found that gamma radiation inhibited toxin-stimulated 22Na uptake at doses beyond a threshold of 200-300 Gy. No effects were seen following doses below 100 Gy. This inhibition of sodium permeability was seen when the cells were irradiated with sodium channels in the closed or inactivated, nonconducting states. However, when the channels were in the toxin-opened, conducting state, gamma radiation had no effect at doses up to 2000 Gy. Our results support earlier electrophysiological studies that showed that high doses of ionizing radiation are required to produce a measurable decrease in sodium permeability. In addition, our data suggest that by changing the sodium channel conformation, batrachotoxin appears to alter radiosensitive chemical bonds in the gating or ion-conducting portion of the channel.     

Gating kinetics of batrachotoxin-modified sodium channels in neuroblastoma cells determined from single-channel measurements

We have observed the opening and closing of single batrachotoxin (BTX)-modified sodium channels in neuroblastoma cells using the patch-clamp method. The conductance of a single BTX-modified channel is approximately 10 pS. At a given membrane potential, the channels are open longer than are normal sodium channels. As is the case for normal sodium channels, the open dwell times become longer as the membrane is depolarized. For membrane potentials more negative than about -70 mV, histograms of both open-state dwell times and closed-state dwell times could be fit by single exponentials. For more depolarized potentials, although the open-state histograms could still be fit by single exponentials, the closed-state histograms required two exponentials. This data together with macroscopic voltage clamp data on the same system could be accounted for by a three-state closed-closed-open model with transition rates between these states that are exponential functions of membrane potential. One of the implications of this model, in agreement with experiment, is that there are always some closed BTX-modified sodium channels, regardless of membrane potential.     

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.     

Amiloride-sensitive apical membrane sodium channels of everted Ambystoma collecting tubule

Patch clamp methods were used to characterize sodium channels on the apical membrane of Ambystoma distal nephron. The apical membranes were exposed by everting and perfusing initial collecting tubules in vitro. In cell-attached patches, we observed channels whose mean inward unitary current averaged 0.39±0.05 pA (9 patches). The conductance of these channels was 4.3±0.2 pS. The unitary current approached zero at a pipette voltage of –92 mV. When clamped at the membrane potential the channel expressed a relatively high open probability (0.46). These characteristics, together with observation that doses of 0.5 to 2 m amiloride reversibly inhibited the channel activity, are consistent with the presence of the high amiloride affinity, high sodium selectivity channel reported for rat cortical collecting tubule and cultured epithelial cell lines.We used antisodium channel antibodies to identify biochemically the epithelial sodium channels in the distal nephron of Ambystoma. Polyclonal antisodium channel antibodies generated against purified bovine renal, high amiloride affinity epithelial sodium channel specifically recognized 110, 57, and 55 kDa polypeptides in Ambystoma and localized the channels to the apical membrane of the distal nephron. A polyclonal antibody generated against a synthetic peptide corresponding to the C-terminus of Apx, a protein associated with the high amiloride affinity epithelial sodium channel expressed in A6 cells, specifically recognized a 170 kDa polypeptide. These data corroborate that the apically restricted sodium channels in Ambystoma are similar to the high amiloride affinity, sodium selective channels expressed in both A6 cells and the mammalian kidney.This work was supported by American Heart Association, New York Affiliate Grant 91007G (LCS) and National Institute of Diabetes and Digestive and Kidney Disease Grants DK-37206 (DJB) and DK46705 (PRS).     

Modification of nerve membrane sodium channels by the insecticide pyrethroids

1. The synthetic pyrethroids exert potent and selective actions on nerve membrane sodium channels. (+)-trans tetramethrin and (+)-trans allethrin cause repetitive discharges to be produced in the isolated crayfish and squid giant axons in response to a single stimulus as a result of an increase in depolarizing after-potential. 2. The latter effect is due to slowing of the sodium channel kinetics which causes a prolonged sodium current following the normal peak sodium current. 3. A kinetic model is proposed to account for the action of the pyrethroids in which the pyrethroid molecule binds to the sodium channels at both closed and open states to produce a modified open state. 4. (-)-trans and (-)-cis isomers of tetramethrin are ineffective in causing the effects, but prevent the active (+)-trans and (+)-cis isomers from exerting the effects. This stereospecificity provides us with an excellent opportunity for the study of binding sites of pyrethroids and other sodium channel modulators.     

Current-dependent block of nerve membrane sodium channels by paragracine.

Paragracine, isolated from the coelenterate species Parazoanthus gracilis, selectively blocks sodium channels of squid axon membranes in a frequency-dependent manner. The blocking action depends on the direction and magnitude of the sodium current rather than on the absolute value of the membrane potential. Paragracine blocks the channels only from the axoplasmic side and does so only when the current is in the outward direction. This block may be reversed by generating inward sodium currents. In axons in which sodium inactivation has been removed by pronase, the frequency-dependent block persists, and a slow time-dependent block is observed. A slow interaction with its binding site in the channel may account for the frequency-dependent block.     

Molecular mechanism of allosteric modification of voltage-dependent sodium channels by local anesthetics

The hallmark of many intracellular pore blockers such as tetra-alkylammonium compounds and local anesthetics is their ability to allosterically modify the movement of the voltage sensors in voltage-dependent ion channels. For instance, the voltage sensor of domain III is specifically stabilized in the activated state when sodium currents are blocked by local anesthetics. The molecular mechanism underlying this long-range interaction between the blocker-binding site in the pore and voltage sensors remains poorly understood. Here, using scanning mutagenesis in combination with voltage clamp fluorimetry, we systematically evaluate the role of the internal gating interface of domain III of the sodium channel. We find that several mutations in the S4-S5 linker and S5 and S6 helices dramatically reduce the stabilizing effect of lidocaine on the activation of domain III voltage sensor without significantly altering use-dependent block at saturating drug concentrations. In the wild-type skeletal muscle sodium channel, local anesthetic block is accompanied by a 21% reduction in the total gating charge. In contrast, point mutations in this critical intracellular region reduce this charge modification by local anesthetics. Our analysis of a simple model suggests that these mutations in the gating interface are likely to disrupt the various coupling interactions between the voltage sensor and the pore of the sodium channel. These findings provide a molecular framework for understanding the mechanisms underlying allosteric interactions between a drug-binding site and voltage sensors.     

Sodium and calcium channels in the somatic membrane of artificially differentiated neuroblastoma cells

Using an intracellular dialysis technique a study was made on calcium and sodium inward currents at the neuroblastoma somatic cell membrane in suspension and during the course of artificial morphological differentiation produced by raising the pH of the culture medium to 8.0–8.2. The density of sodium currents in the somata of cells cultured in the suspension averaged 7.3±0.8 µA/µF, while this value varied from 37±5.2 to 54.7±3.6 µA/µF at various stages of culture. These values equalled 1.4±0.2 and 1.1±0.2 to 2.8±0.4 µA/µF in the case of calcium currents. Reciprocal changes were produced in the density of sodium and calcium channels by altering the culture medium.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 2, pp. 207–214, March–April, 1986.     

Further evidence that membrane thickness influences voltage-gated sodium channels.

The short-chain phospholipid, diheptanoyl phosphatidylcholine, at 520 microM, reduced the maximum inward sodium current in voltage-clamped squid giant axons by greater than 50%. Analysis of these currents by means of the Hodgkin-Huxley equations showed this reduction to be mainly the result of a large depolarizing shift in the voltage dependence of the steady state activation parameter, m infinity. The voltage dependence of the steady state inactivation parameter, h infinity, was also moved in the depolarizing direction and the axonal membrane capacitance per unit area measured at 100 kHz was increased. A longer chain length derivative, didecanoyl phosphatidylcholine, had no significant effect on the axonal sodium current at concentrations of 3.7 and 18.5 microM. Dioctanoyl phosphatidylcholine was intermediate in its effects, 200 microM producing approximately the same current suppression as 520 microM diheptanoyl phosphatidylcholine, together with depolarizing shifts in m infinity and h infinity. These effects may be contrasted with those of the normal and cyclic alkanes (1-3), which tend to move both m infinity and h infinity in the hyperpolarizing direction and to reduce the capacitance per unit area at 100 kHz. The above results are all consistent with the hypothesis that small hydrocarbons thicken, while short-chain phospholipids thin, the axonal membrane. Thus membrane thickness changes may be of considerable importance in determining the behavior of the voltage-gated sodium channel.     

Interaction of n-alkylguanidines with the sodium channels of squid axon membrane

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.