Single Sodium Channels Research Articles

The actions of a red tide toxin from Ptychodiscus brevis on single sodium channels in mammalian neuroblastoma cells

The actions of brevetoxin (PbTX-3) were studied on single, voltage-dependent sodium channels and whole-cell currents from the neuroblastoma × glioma cell line NG108-15. Purified PbTX-3 shifted the activation of sodium channels to membrane potentials negative to normal. PbTX-3 did not alter the single-channel mean open lifetime, suggesting that the toxin does not change the rate of sodium channel inactivation from the open state. There was also no change in single-channel conductance. These results indicate that brevetoxin increases sodium current at rest by shifting the voltage dependence of channel activation and that the resulting depolarization is limited by channel inactivation.

Temperature-dependent subconducting states and kinetics of deltamethrin-modified sodium channels of neuroblastoma cells

The effects of temperature on the properties of sodium channels from mouse neuroblastoma cells modified by the pyrethroid insecticide deltamethrin were investigated using the patch-clamp technique. The study was aimed at determining various states of modified channels which were expected to be revealed by raising the temperature as a result of an increase in channel activity. After exposure to 10 microM deltamethrin, the decay of whole cell sodium current at -30 mV was drastically slowed. It is expressed by two exponential functions at 11 degrees C and by three exponential functions at room temperature (22 +/- 1 degree C). Thus, raising the temperature reveals a new process. Whole cell sodium tail currents associated with step repolarization from -30 mV to -100 mV were best fit by the sum of two exponential functions both at 11 degrees C and at room temperature. The decay of the summed modified single sodium channel currents at -30 mV was expressed by a single exponential function at 11 degrees C, and by two exponential functions at room temperature. In keeping with these results, the open time histograms show the single (11 degrees C) and double (room temperature) exponential distributions. Thus, raising the temperature allows a new single channel process to be revealed. Other modified open states observed previously at 11 degrees C were also found at room temperature including a flickering state and a subconducting state. In addition, several new subconducting states were found at room temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Hydrogen ion block of single sodium channels in neuroblastoma cells

The effect of external pH on the amplitude of currents through single sodium channels in cultured mouse neuroblastoma cells C 1300, clone N18A-1 was studied. Currents through single sodium channels in outside-out membrane patches were measured at normal (7.2) and low (5.4) pH of the external solution. With a decrease of the external pH to 5.4, about two-fold reversible reduction of the amplitude of single sodium channel currents (at testing potentials of -10-30 mV) was observed. The data obtained confirm the suggestion that the inhibition of macroscopic sodium currents with lowering of pH of the extracellular solution is due to the decrease in the ionic current flowing through single open channels.

Kinetic properties of single sodium channels modified by fenvalerate in mouse neuroblastoma cells

(1) The kinetic properties of single sodium channels modified by the pyrethroid fenvalerate have been analyzed by patch clamp techniques using the cultured mouse neuroblastoma cells. (2) Fenvalerate drastically prolonged the open time of single sodium channels from the normal value of 5 ms to several hundred milliseconds during a depolarizing pulse. The channels remained open after termination of a depolarizing pulse for as long as several seconds. (3) The channel lifetime varied with the membrane potential, attained a maximum at -70 mV, and decreased with hyperpolarization and depolarization from -70 mV. (4) Prolonged openings of the modified channels allowed a current-voltage curve for a single channel to be plotted by sweeping a ramp pulse. The single channel conductance had a value of 11 pS and was linear over potentials ranging from 0 to -100 mV. (5) Power density spectral analysis of the open channel current noise indicated a single Lorentzian curve with a cut-off frequency at 90 Hz, indicating that the increase in noise during channel opening resulted from a relatively slow kinetic process. (6) The probability of the channel being modified by fenvalerate was independent of the length of time during which the channel was opened. This observation suggests that channel modification had taken place before the channel opened. This study of the prolonged opening at the single channel level provides a new insight into open channel properties and the kinetics of channel modification.

Kinetic properties of single sodium channels in rat heart and rat brain.

Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics.

Flickering of Subconductance States of Single Sodium Channels in Neuroblastoma Cells

Flickering of Subconductance States of Single Sodium Channels in Neuroblastoma Cells

Two open states or two different Na channels in skeletal muscle fibers: Markov models of the decay of Na currents in frog skeletal muscle

The rate of sodium current decay at −140 mV was studied as a function of the duration and amplitude of the activating voltage pulse. These sodium current decays or tails of current showed a biexponential decline in amplitude which depended upon the duration of the activating pulse. At 12°C, the two exponential components of the Na tail currents exhibited time constants of 72 and 534 μs. As the duration of an activating pulse was lengthened, the relative amplitude of the slow component of the decay increased compared to the fast component, without any changes in the fast and slow time constants. This slowing of the decay of current as a function of the duration of the activating pulse is found only in fibers with inactivation intact. A number of Markov models were tested for their ability to predict the biexponential decays found in muscle fibers with inactivation intact and removed. A homogeneous population of channels having only a single open state fails to predict the behavior. A homogeneous population of channels having two open states predicts the behavior. The behavior can also be predicted by two different types of single open-state sodium channels, with one ensemble of channels carrying a minority of the current and exhibiting a much slower closing rate. If a homogeneous population of channels is present, the simulations show that the observed changes in decay rates are driven by inactivation.

Mechanism of inactivation of single sodium channels after modification by chloramine-T, sea anemone toxin and scorpion toxin.

Single sodium-channel currents were measured in neuroblastoma cells after inhibition of inactivation by chloramine-T (CHL-T), sea anemone toxin II (ATX-II) and scorpion toxin (SCT). The decaying phase of the averaged single-channel currents recorded with 90-msec pulses in cell-attached patches was clearly slower than that of the ummodified channels, suggesting inhibition of macroscopic inactivation. Each substance caused repetitive openings and a moderate increase in the channel open time. At Vm = RP + 20 mV and T = 12 degrees C, the mean channel open times were 1.4, 1.6 and 1.8 msec for CHL-T, ATX-II and SCT, respectively, as opposed to 1.07 msec for native channels. Open-time histograms could be best fitted by the sum of two exponentials. The time constants of the fits were similar for histograms constructed from single openings and from openings during bursts. This suggests that the population of channels is homogeneous and that in bursts the same open conformations of channels occur as in single openings. Mean burst durations for bursts consisting of more than one opening at Vm = RP + 20 mV were 4.9, 5.8 and 6.1 msec for CHL-T, ATX-II and SCT, respectively. Burst open-time histograms constructed from two or three openings were fitted by the gamma function. The different time constants of the fits obtained for ATX-II and SCT suggested multiple open conformations of channels for openings of bursts. However, significantly different open-time histograms constructed from the first, second and third openings of bursts could not be obtained systematically. A positive correlation was found for the dwell time of the first and the second, as well as for the second and the third opening of bursts with each substance, but a negative one for the dwell time of an opening and the neighboring closing of bursts with ATX-II. The results suggest a model with multiple open and inactivated states. In this model the inactivated states are weakly absorbing.

Conditional open and delay time histograms of sodium channels

Currents through single sodium channels were recorded in neuroblastoma cells. Open time histograms were constructed from openings which appeared between 2.0 and 5.0 ms after the onset of the depolarization. Histograms constructed from openings which were not preceded by other openings showed a maximum at t > 0 in contrast to those, which were preceded by other openings. Time constants of delay time histograms fitted by the sum of two exponentials were different for the first, second and third records of runs. The results support the view that sodium channels have multiple open and closed states and the transition probabilities among the states depends on local conditions of the membrane.

Single sodium channels from the squid giant axon

Since the work of A. L. Hodgkin and A. F. Huxley (1952. J. Physiol. [Lond.].117:500-544) the squid giant axon has been considered the classical preparation for the study of voltage-dependent sodium and potassium channels. In this preparation much data have been gathered on macroscopic and gating currents but no single sodium channel data have been available. This paper reports patch clamp recording of single sodium channel events from the cut-open squid axon. It is shown that the single channel conductance in the absence of external divalent ions is approximately 14 pS, similar to sodium channels recorded from other preparations, and that their kinetic properties are consistent with previous results on gating and macroscopic currents obtained from the perfused squid axon preparation.

Subconductance states of single sodium channels modified by chloramine-T and sea anemone toxin in neuroblastoma cells

Single channel currents of chloramine-T (Chl-T) and sea anemone toxin (ATX-II) modified sodium channels were studied in neuroblastoma cells. With both substances similar subconductance states have been observed. The conductances of the sublevels were multiples of the "unit" step which was about one-forth of the most frequently occurring main conductance. Thus, the current levels observed were one fourth, half and five-fourths of the main current size. Both substances caused a slower decay of the averaged current compared to the current of the native channels. The main single-channel conductance was 15.2 pS (T = 16 degrees C) for the Chl-T and 10.8 pS (T = 12 degrees C) for the ATX-II modified channels. The channel open time was doubled by ATX-II, but was not increased significantly by Chl-T. The existence of the subconductance states suggests that the native channels may also have multiple open conformations.

Effects of lidocaine on single cardiac sodium channels

Lidocaine block of single cardiac sodium channels was studied in cell free inside-out patches of ventricular cells isolated from guinea-pig hearts. When applied to the inner surface of the membrane lidocaine depressed Na channel currents by decreasing the probability P of the channels to open measured from the peaks of the averaged currents. In parallel to the decrease in P the relative number of empty sweeps (nulls) was increased. Half maximum block of the activity of single Na channels was observed at 2.9 microM. Lidocaine affected the gating behaviour of Na channels by shortening of the mean open time tau 0 from 0.44 +/- 0.17 (control) to 0.19 +/- 0.13 (5 microM lidocaine, holding potential-120 mV, test potential -60 mV). Five micromolar lidocaine completely suppressed burst-like openings of Na channels and abolished the slow decaying phase of the averaged currents. A shift from - 120 towards - 160 mV exerted relief from the effect of both P and tau 0.

Effects of veratridine on single neuronal sodium channels expressed inXenopus oocytes

(1) Chick neuronal Na+ channels were expressed in Xenopus laevis oocytes after injection with total messenger ribonucleic acid (mRNA) isolated from chick brain. The currents were investigated with the whole cell voltage clamp and with the patch clamp technique. Activation and inactivation of the induced current, and its sensitivity towards tetrodotoxin (TTX) and veratridine were reminiscent of vertebrate neuronal Na+ channels. (2) In the presence of veratridine normal single channel openings often converted into small amplitude openings of long duration. These small amplitude openings persisted for hundreds of milliseconds after return to the holding potential. (3) The slope conductance of the veratridine modified open channel state was 5-6 pS as compared to the normal state with 21-25 pS in the voltage range between -35 and +5 mV. (4) The modified channel showed saturation behaviour towards Na+ ions. Half saturation of the single channel amplitude was observed at 330 mM Na+ at a membrane potential of -100 mV. (5) Final closure of the modified channel after return to the holding potential followed an exponential time course. Its potential dependence was similar to that of the time course of the veratridine induced tail currents in the whole cell configuration. (6) The properties of the Na+ channel derived from chick forebrain are compared with the properties of the same channel derived from chick skeletal muscle. Both were expressed in the same membrane environment, the Xenopus oocyte plasma membrane. While earlier results with Na+ channels of muscle origin showed two channel populations, one with short and another with long mean open times, Na+ channels of neuronal origin were homogeneous and characterized by short open times.

Acetylcholine activates single sodium channels in smooth muscle cells

Using the whole-cell clamp technique with a patch electrode, single channel activities were recorded in dispersed single smooth muscle cells of the guinea-pig ileum in response to application of greater than 1 microM acetylcholine (ACh) or carbachol (CCh). Under physiological conditions (bath:Krebs, pipette:High K), these channels have a single channel conductance of 20-25 pS at the membrane potentials ranging between -100 and -40 mV and are activated in the membrane potential dependent manner. The amplitude of the channel current showed a strong dependence on the extracellular Na concentration but the reversal potential obtained by the extrapolation of the I-V relationship was not consistent with the equilibrium potential of Na ion. An approximate estimation of permeability ratio based on the independent principle described by Hodgkin et al. gave a value of Na:K = 1.0:0.3-0.4 to this channel. From features of the macroscopic and single channel currents, it is concluded that these muscarinic ACh(CCh)-activated channels mainly pass Na ion and play a major part in the membrane depolarization produced by ACh or CCh in mammalian intestinal smooth muscles.

Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid.

1. The properties of fast transient Na channels induced in the Xenopus laevis oocyte plasma membrane after injection of the oocyte with foreign messenger ribonucleic acid (mRNA) were investigated with the whole-cell voltage clamp and with the patch-clamp technique. 2. The time course of expression and the effect of the metabolic inhibitors actinomycin D and tunicamycin were studied. The rate of channel insertion reached a maximum only about 3 days after injection with mRNA and corresponded to the incorporation of 20 active channels/s, into a single oocyte. When applied intracellularly tunicamycin blocked the appearance of active channels nearly completely while actinomycin D added to the medium had no effect. 3. The whole-cell currents showed activation and inactivation properties reminiscent of skeletal muscle Na+ currents. The maximal peak current amplitude was 6 microA. Tetrodotoxin blocked the observed transient inward current. 50% inhibition was observed at 10 nM concentration. Veratridine depressed inactivation of the current and led to prolonged tail currents. 4. After removal of the surrounding layers of the oocyte tight seals were obtained with a patch-clamp electrode pushed on the surface membrane. Single-channel currents endogenous to the oocyte and Na+-channel currents induced by injected mRNA could be recorded. The single-channel slope conductance of the latter was 12-15 pS. Two different types of kinetic behaviour were evident from an analysis of single-channel currents and ensemble average currents. One type showed fast inactivation (tau less than 1 ms) and brief channel openings (less than 1 ms) whereas the second type was characterized by slower inactivation and a bursting behaviour. 5. When veratridine (75 microM) was present in the pipette solution the single-channel behaviour was modified in a complex manner. In addition to the channel openings with normal conductance a second open state was observed with a slope conductance of 3.5 pS. This second type of channel opening could still be recorded after return to the holding potential. Its final closure followed an exponential time course with a constant time constant of 0.5 s at -100 mV. These events probably underlie the tail currents in the whole-cell configuration. 6. The Xenopus oocyte represents a useful system for the study of the expression of channels induced by foreign mRNA, for the characterization of their single-channel behaviour and for the investigation of the action of pharmacologically active substances on these channels. This system may prove useful for the study of channels that are not accessible to patch-clamp experiments 'in situ'.

Voltage-dependent gating of single sodium channels from mammalian neuroblastoma cells

Single sodium channel currents have been studied in cell-attached patches from the mouse neuroblastoma cell line N1E115. Distributions of open duration, latency until first opening, and the average probability of a channel being open after a voltage step, p(t), were analyzed and compared to predicted distributions from various kinetic models for voltage-dependent gating. It was found that, over most of the voltage range under which channel gating occurs, the slow steps in gating are opening transitions and that inactivation of open channels is significantly faster than the decline in p(t) (tau h). This view of gating is confirmed by comparison of the kinetics of ensemble averages of single-channel currents obtained from step- and tail-current records at the same voltage. The probability of a channel reopening after having closed was calculated by comparing p(t) with the convolution of the first-latency probability density and the conditional probability of remaining open t milliseconds after opening. This reopening probability is small but slightly voltage dependent over the voltage range where the mean open duration remains constant and tau h changes considerably. The voltage dependence of open channel inactivation and deactivation were calculated from the probability of reopening and the mean open duration. The equivalent gating charge for the inactivation rate is a few tenths of an electronic charge, whereas the equivalent charge for the closing rate is 2.5-3.5 electronic charges.

Modified gating behaviour of aconitine treated single sodium channels from adult cardiac myocytes

Currents through single Na channels of ventricular cells from adult mouse and guinea pig hearts were studied using the patch clamp technique. Under control conditions the majority of openings is brief and a clustering at the beginning of the depolarizing pulse can be observed. Only between 1 and 8% of the sweeps show long lasting bursts. The bursting may account for a second slow phase of decay of macroscopic currents. In the presence of aconitine in the pipette up to 80% of the sweeps showed bursts underlying the slow inactivation of aconitine modified macroscopic currents. The mean open times are unchanged but the number of openings per sweep is dramatically increased due to aconitine. It is discussed that Na channels may function in different "modes". One mode is characterized by a fast transition into an absorbing state the other one by frequent reopenings ("bursts"). aconitine favours the "bursting" mode.

Toxins That Modulate the Sodium Channel Gating Mechanisma

A variety of toxins and chemicals has been shown to modulate the gating kinetics of the sodium channel. Studies of batrachotoxin, grayanotoxins and pyrethroids are summarized here as examples. Batrachotoxin and grayanotoxins eliminate the sodium channel inactivation thereby causing a prolonged, steady-state sodium current to flow during a depolarizing step. The sodium channel activation kinetics are not affected markedly. Batrachotoxin appears to bind to a site in the sodium channel to which the inactivation gate normally binds, thus causing an inhibition of sodium inactivation. Single channel recording experiments have shown that the mean open time of individual sodium channels is greatly prolonged by batrachotoxin. It appears that individual sodium channels are modified by batrachotoxin in an all-or-none manner. Pyrethroids which are synthetic derivatives of pyrethrins also modify the kinetics of sodium channels in a very drastic manner. In the presence of type I pyrethroids which lack a cyano group at the alpha position (e.g., allethrin and tetramethrin), a large steady-state sodium current appears during a step depolarization and a large slowly decaying sodium tail current appears upon repolarization. Thus both the activation and inactivation kinetics are slowed. Type II pyrethroids which contain an alpha-cyano group (e.g., deltamethrin, cyphenothrin, and fenvalerate) exert effects on sodium channels qualitatively similar to those of type I pyrethroids. However, the amplitudes of the steady-state sodium current and sodium tail current are smaller and the time constant of tail current decay is much longer. The mean open time of single sodium channels is greatly prolonged by the pyrethroids, and the effect is much more pronounced in type II than in type I pyrethroids. A high degree of stereospecificity has been found among four isomers of tetramethrin, (+)-trans and (+)-cis isomers being highly active and (-)-trans and (-)-cis isomers almost totally inactive. The inactive isomers bind to the sodium channel sites, thus preventing the action of the active isomers. Because of the unique action of pyrethroids in modulating the sodium channels, they are becoming useful tools for channel physiology and pharmacology.

Stabilization of sodium channel states by deltamethrin in mouse neuroblastoma cells.

The effect of the pyrethroid insecticide deltamethrin on sodium channels of mouse neuroblastoma cells was investigated using the patch-clamp technique. The study was aimed at determining how the effects of deltamethrin at the whole cell level would be reflected in the modified properties of single sodium channel currents. Whole cell recording showed that deltamethrin prolonged sodium currents in neuroblastoma cells by several orders of magnitude. Single channel recordings showed that a variety of channel states were prolonged by deltamethrin. Not only was the open state prolonged by several orders of magnitude but a closed or inactivated state was also prolonged, leading to less frequent channel openings. A subconducting state and a flickering state were observed in the presence of deltamethrin as well as a state in which channels opened with some delay after the termination of a depolarizing pulse. The results are compatible with the hypothesis that deltamethrin stabilizes a variety of channel states by reducing the transition rates between them. This allows states that are normally very brief to be detected more easily.

Developmental Appearance of Sodium Channel Subtypes in Rat Skeletal Muscle Cultures

22Na influx was measured in the established muscle cell line L-6 and in primary rat skeletal muscle cultures following activation of sodium channels by veratridine and sea anemone toxin II. Inhibition of the activated channels by tetrodotoxin (TTX) was analyzed with computer-assisted fits to one- or two-site binding models. In L-6 cultures, two inhibitable sodium channel populations were resolved at all ages in culture: a TTX-sensitive (K = 0.6-5.0 X 10(-8) M) and an insensitive population (Ki = 3.3-4.9 X 10(-6) M). In primary rat muscle cultures, the sensitivity of the toxin-stimulated channels to TTX changed with time in culture. In 4-day-old cultures, a single sodium channel population was detected using TTX (Ki = 2.4 X 10(-7)M). A single population was also found in 6-day-old cultures (Ki = 5.3 X 10(-7) M). By day 7 in culture, the inhibition of 22Na influx by TTX could be resolved into two components with high- and low-affinity sites for the toxin (Ki = 1.3 X 10(-9) M and 9.6 X 10(-7) M). We conclude that a single, toxin-activated sodium channel population with low affinity for TTX exists at early stages, whereas a second, high-affinity population evolves with time in primary rat muscle cultures. The expression of a high-affinity site apparently does not require ongoing neuronal involvement and may reflect an intrinsic property of the muscle cells.