Tetrodotoxin-sensitive Sodium Channels Research Articles

Sodium Current-Induced Release of Calcium from Cardiac Sarcoplasmic Reticulum

The role of sodium-calcium exchange at the sarcolemma in the release of calcium from cardiac sarcoplasmic reticulum was investigated in voltage-clamped, isolated cardiac myocytes. In the absence of calcium entry through voltage-dependent calcium channels, membrane depolarization elicited release of calcium from ryanodine-sensitive internal stores. This process was dependent on sodium entry through tetrodotoxin-sensitive sodium channels. Calcium release under these conditions was also dependent on extracellular calcium concentration, suggesting a calcium-induced trigger release mechanism that involves calcium entry into the cell by sodium-calcium exchange. This sodium current-induced calcium release mechanism may explain, in part, the positive inotropic effects of cardiac glycosides and the negative inotropic effects of a variety of antiarrhythmic drugs that interact with cardiac sodium channels. In response to a transient rise of intracellular sodium, sodium-calcium exchange may promote calcium entry into cardiac cells and trigger sarcoplasmic calcium release during physiologic action potentials.

Hypothalamic warm-sensitive neurons possess a tetrodotoxin-sensitive sodium channel with a high Q 10

We have investigated the ionic current responses to temperature of dissociated cells from the preoptic and anterior hypothalamus (PO/AH) of rat, using the ‘whole-cell’ configuration. The majority of the recorded neurons showed a linear increase in a non-inactivating inward current during warming (30–40°C), and the Q 10 was about 2. However, about 24% of PO/AH neurons were markedly sensitive to warming and the increase in non-inactivating inward current to a rise in temperature in the hyperthermic range (35–40°C) had a high Q 10 (4.3–7.0). This increase in current in the hyperthermic range was reversibly blocked by tetrodotoxin (TTX). The inward current in neurons with a Q 10 of 2 was not affected by TTX. The results show that some neurons in the PO/AH possess a non-inactivating sodium channel that is highly temperature-sensitive in the hyperthermic range. These neurons are presumably the ‘primary’ warm-sensitive neurons.

Optical recording of electrical activity from axons and glia of frog optic nerve: potentiometric dye responses and morphometrics.

Voltage-sensitive dyes were used to study the changes in membrane potential in axons and glial cells of the frog optic nerve following electrical stimulation. The lack of a signal in the unstained nerve and the multiphasic action spectra after staining indicated that the optical responses were from the extrinsic dyes. Changes in dye absorption and fluorescence had rapid and slow phases. The rapid phases resulted from action potentials in myelinated and unmyelinated axons. The kinetics of the slow phase of the optical response were similar to the depolarization recorded from the glial cells with intracellular electrodes. The ratio of the amplitudes of the fast and slow phases was characteristic for each type of dye. Pharmacological analysis of the action potential of the unmyelinated axons revealed tetrodotoxin-sensitive sodium channels and 4-aminopyridine-sensitive potassium channels. Repeated exposure of the stained preparation to light led to photodynamic damage as shown by a block of recovery of the glial depolarization. An electron microscopic morphometric study of the nerve was carried out in an effort to understand the contribution of the various anatomical elements to the compound optical response. The ratio of unmyelinated axon membrane to glial membrane was much greater than was the ratio of the fast and slow components of the signal, suggesting that the dyes either had a higher affinity for glial membrane or did not penetrate the nerve uniformly.

Tetrodotoxin-sensitive sodium channels in a continuously cultured cell line derived from the adult rat cerebellum

A method to establish continuously cultured cell lines from adult cerebellar cortex is reported. Clones (prepared by this procedure) were isolated from cerebellar established cultures at the 25th passage and after 15 months in vitro. One clone (UCHCC1) was maintained in culture and studied while the others were frozen. The cerebellar cell line UCHCC1 retained a neuronal-like morphology; the addition of dimethylsulfoxide (DMSO) to the culture medium elicited a reproducible morphological ‘differentiation’ event, characterized mainly by process extension. In ‘differentiated’ cells, veratridine significantly increased the uptake of 22Na. Such enhanced uptake was blocked by tetrodotoxin (TTX) with a half-maximal inhibitory concentration of 0.9 nM. Binding of an [ 3H]ethylenediamine derivative of TTX ([ 3H]en-TTX) to the microsomal fraction prepared from same DMSO-treated cells, showed a single class of receptors with a maximal binding (B max) of 173 fmol/mg protein and aK d of 1.1 nM. Thyroid UCHT1 cells and ‘undifferentiated’ (cultured without DMSO) cerebellar cells, did not show significant effects of veratridine on 22Na-uptake, or [ 3H]en-TTX binding. The ‘differentiated’ nerve-like properties, induced by appropriate environmental manipulation, demonstrate the usefulness of cerebellar UCHCC1 cells as a model system for the developing central neuron. On the other hand, the novel transforming procedure opens new possibilities for obtaining permanent cell lines from other regions of the adult CNS.

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%).

Cyclic adenosine 3':5'-monophosphate and cytosolic calcium exert opposing effects on biosynthesis of tetrodotoxin-sensitive sodium channels in rat muscle cells

We have previously presented evidence that electrical activity and increased cytosolic calcium reduce the density of sarcolemmal tetrodotoxin (TTX)-sensitive sodium channels in cultured rat muscle cells (Sherman, S. J., and W. A. Catterall (1984) Proc. Natl. Acad. Sci. U. S. A. 81: 262-266). We show here that growth of cells in ryanodine has a biphasic effect on sodium channel number. At low concentrations (0.3 to 10 microM) where this drug releases calcium from the sarcoplasmic reticulum into the cytoplasm, sodium channel number is reduced 62%; whereas, at higher concentrations where total cellular calcium is depleted, the density of sodium channels is increased 40% above control. These results provide further evidence for modulation of sodium channel number by cytosolic calcium. Growth of muscle cells in the presence of agents that mimic cyclic AMP (cAMP) action or increase intracellular cAMP levels including 8-bromo-cyclic AMP (8-BrcAMP), cyclic nucleotide phosphodiesterase inhibitors, and forskolin increased sodium channel density up to 125%. This action did not involve changes in spontaneous electrical activity. Dibutyryl cGMP had no effect. Measurement of the turnover rate of sodium channels after block of channel accumulation by tunicamycin (1.5 micrograms/ml) gave a half-time of 18 hr for exponential decay of TTX-sensitive sodium channels in cultured rat muscle cells after an initial 6-hr lag period. Treatments which modulate sodium channel number through changes in cytosolic calcium or cAMP had no effect on the rate of channel turnover. The increase of sodium channel number after inhibiton of electrical activity or treatment with 8-BrcAMP was half-maximal at 17 hr, consistent with an increase in the rate of sodium channel biosynthesis and/or incorporation into the sarcolemma without a change in channel turnover time. We conclude that cytosolic calcium decreases and cAMP increases sodium channel number by modulating the rate of biosynthesis and/or processing of channel components. The biochemical mechanisms of these regulatory effects are considered.

Electrical activity and cytosolic calcium regulate levels of tetrodotoxin-sensitive sodium channels in cultured rat muscle cells.

Pharmacological blockade of the spontaneous electrical activity present in primary cultures of rat myotubes by growth in bupivacaine, tetrodotoxin, or KCl was found to increase the number of voltage-sensitive Na+ channels 38-83% as measured by the specific binding of [3H]saxitoxin. The inhibition of spontaneous electrical activity and increase in channel density by bupivacaine displayed an identical dose response, with a half-maximal effect at 3.0 microM. Growth of myotubes in the presence of 1 microM A23187, a Ca2+-specific ionophore, resulted in a 30-60% decrease in the number of tetrodotoxin-sensitive channels with no change in affinity for [3H]saxitoxin. A23187 was able to overcome the increase in channel density produced by bupivacaine. These results suggest the presence of a Ca2+-mediated negative feedback system in which electrical excitability may be regulated by altering the number of tetrodotoxin-sensitive Na+ channels.

Tetrodotoxin-sensitive sodium channels in rat muscle cells developing in vitro.

Tetrodotoxin-sensitive sodium channels in rat muscle cells developing in vitro.

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.

Inhibition by α-amanitin of development of tetrodotoxin-sensitive spike induced by brain extract in cultured chick skeletal muscle cells

The enhancing effect of brain extract on the development of tetrodotoxin-sensitive sodium channels in cultured chick skeletal muscle cells is blocked by treatment of culture with α-amanitin, an RNA polymerase inhibitor. The result is interpreted as indicating that the trophic substance in the brain extract exerts its effect on the development of sodium channels through regulation of gene transcription.

Ionic currents in the somatic membrane of rat dorsal root ganglion neurons—I. Sodium currents

Measurements of sodium transmembrane ionic currents evoked by depolarizing shifts in membrane potential have been performed on isolated dorsal root ganglion neurons of 5–10-day-old rats. Potassium currents were eliminated by dialysing the neurons with potassium-free solutions. In 10–15% of investigated neurons a tetrodotoxin-resistant component has been revealed in the sodium inward current which differs in its potential-dependent and kinetic characteristics from the main tetrodotoxinsensitive one. The activation kinetics of the tetrodotoxin-sensitive sodium current could be described by the Hodgkin-Huxley model using the cubic power of the m-variable, whereas the activation kinetics of the tetrodotoxin-resistant one can be described using only the square power of m. The time constants of activation and inactivation of the tetrodotoxin-resistant current were about ten times longer than those of the tetrodotoxin-sensitive current. The tetrodotoxin-resistant current was highly sensitive to all extracellular agents which are known as effective blockers of calcium channels (Co 2+, Mn 2+, Cd 2+, D-600 and its derivatives). At the same time, the selectivity of the corresponding channels did not differ significantly from the selectivity of the tetrodotoxin-sensitive sodium channels. The sequence of relative permeabilities for univalent cations was P Na: P Li: P hydrazinium:P NH 4 :P hydroxylammonium:P K = 1.0:0.79:0.43:0.33:0.25:0.18 for the tetrodotoxin-sensitive channels and 1.0:0.98:0.47:0.42:0.26:0.26 for the tetrodotoxin-resistant ones. Thus, the tetrodotoxin-resistant sodium channels combine some features of sodium (the selective filter) and calcium (gating mechanism and binding properties) channels.

Tetrodotoxin-sensitive sodium channels in normal human fibroblasts and normal human glia-like cells.

Tetrodotoxin-sensitive sodium channels are detectable in normal human fibroblasts and in "glia-like" cells at appreciable levels when compared to what is observed in established neuronal cell lines in culture. Two- to 3-fold stimulations of sodium influx are observed in the presence of 0.2 mM veratridine and scorpion venom at 0.1 mg/ml. Tetrodotoxin (2 microM) inhibits the observed stimulation of sodium influx. Previous work has indicated that these neurotoxins act on the voltage-sensitive sodium ionophore of excitable cells, and the presence of such channels in cells generally considered nonexcitable raises questions regarding both the uniqueness of this ionophore as a property of excitable cells and the origin of the cells generally described as fibroblasts.

Dopamine release from the rat substantia nigra in vitro. Effect of raphe lesions and veratridine stimulation

In order to eliminate the 5-hydroxytryptaminergic input to the substantia nigra lesions were placed in the dorsal and medial raphe nuclei in a number of rats. The release of exogenously applied [ 3H]dopamine from the partially denervated substantia nigra was determined in vitro and found to be very similar to the release observed from slices of control substantia nigra. These results lend further support to the theory that the release of exogenously applied [ 3H]dopamine at the level of the substantia nigra occurs mainly from dopaminergic dendrites, rather than from terminals of 5-hydroxytryptamine-containing neurons. A veratridine-induced release of [ 3H]dopamine from the pars reticulata of the substantia nigra is also described. An almost complete blockade of veratridine (3.0 μM) stimulation was observed with 100 nM tetrodotoxin. Similar effects of veratridine and tetrodotoxin were also observed on [ 3H]dopamine release from slices of corpus striatum. These results suggest that dendrites of the dopaminergic neurones in the substantia nigra contain fast, tetrodotoxin-sensitive sodium channels.