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.     

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.     

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.     

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.     

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.     

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.     

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).     

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.     

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.     

Electrically operated sodium channels in the somatic membrane of sympathetic neurons

Electrically operated sodium channels in the somatic membrane of isolated neurons from the rat superior cervical ganglion were investigated using an intracellular dialysis technique and voltage clamping. It was found that sodium currents can be conveyed along two independent systems of sodium channels in these neurons. A mathematical analysis was made of voltage-dependent tetrodotoxin-sensitive fast sodium currents within the framework of the Hodgkin-Huxley model and their kinetic properties were compared with those described in other subjects. It was also shown that the tetrodotoxin-sensitive sodium channels in the somatic membrane of sympathetic neurons have a high affinity for sodium ions. The kinetic and voltage-dependent characteristics of slow tetrodotoxin-sensitive inward sodium current are described. It is also noted that this component of the sodium current was observed in only a limited number of neurons (not more than 2%).A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 18, No. 1, pp. 108–117, January–February, 1986.     

Mechanisms of sodium/calcium selectivity in sodium channels probed by cysteine mutagenesis and sulfhydryl modification.

A conserved lysine residue in the "P loop" of domain III renders sodium channels highly selective. Conversion of this residue to glutamate, to mimic the homologous position in calcium channels, enables Ca2+ to permeate sodium channels. Because the lysine-to-glutamate mutation converts a positively charged side chain to a negative one, it has been proposed that a positive charge at this position suffices for Na+ selectivity. We tested this idea by converting the critical lysine to cysteine (K1237C) in mu 1 rat skeletal sodium channels expressed in Xenopus oocytes. Selectivity of the mutant channels was then characterized before and after chemical modification to alter side-chain charge. Wild-type channels are highly selective for Na+ over Ca2+ (PCa/PNa < 0.01). The K1237C mutation significantly increases permeability to Ca2+ (PCa/PNa = 0.6) and Sr2+. Analogous mutations in domains I (D400C), II (E755C), and IV (A1529C) did not alter the selectivity for Na+ over Ca2+, nor did any of the domain IV mutations (G1530C, W1531C, and D1532C) that are known to affect monovalent selectivity. Interestingly, the increase in permeability to Ca2+ in K1237C cannot be reversed by simply restoring the positive charge to the side chain by using the sulfhydryl modifying reagent methanethiosulfonate ethylammonium. Single-channel studies confirmed that modified K1237C channels, which exhibit a reduced unitary conductance, remain permeable to Ca2+, with a PCa/PNa of 0.6. We conclude that the chemical identity of the residue at position 1237 is crucial for channel selectivity. Simply rendering the 1237 side chain positive does not suffice to restore selectivity to the channel.     

Electrofusion: a biophysical modification of cell membrane and a mechanism in exocytosis

The molecular bases of the exocytosis process remain poorly known. Many proteins have been recognized to play key roles in the machinery. Their functions are well characterized in the specificity of the docking processes. Forces involved in the merging of the two partners must take into account the physics of membrane interfaces. The target membrane and the vesicle are both electrically charged interfaces. Strong electrostatic fields are triggered when they are brought in close neighborhood. These fields are high enough to induce an electropermeabilisation process. It is now well known that when applied on a cell, an external field induces a modulation of the transmembrane potential difference. When high enough the transmembrane potential may induce a membrane destabilisation. This results in a free exchange of polar molecules across well defined parts of the cell surface. Furthermore, when permeabilization is present on two cells, if those parts of the cell surfaces are brought in close contact, membrane merging occurs spontaneously. Cell fusion results from this membrane coalescence. The similarity with what is taking place in exocytosis is striking. The present review describes the state-of-the-art in the knowledge on electrofusion. It is emphasized that it results from electropermeabilisation and not from a direct effect of the external field. A local destabilisation of the vesicle membrane results from electrostatic interactions while keeping unaffected its viability. Such processes appear relevant for what takes place during exocytosis.     

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.     

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.