A Pharmacological Approach to the Structure of Sodium Channels in Myelinated Axons
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Abstract:
Figure 6 summarizes the present state of our knowledge on the sodium channel in myelinated nerve fibers. Two sites have been discussed in detail: a metal cation binding site accessible by tetrodotoxin and saxitoxin from the outside surface only; and a second site accessible from the inside surface with which local anesthetics combine. Hydrogen ions gain access to this region of the sodium channel (and hence determine the relative local concentration of protonated drug) more readily from the extracellular fluid than from the axoplasm (Schwarz et al 1977). In addition, a variety of other sites have been mentioned, binding of drugs to which alters selectively the kinetics of opening and closing of the h and m gates. In myelinated nerve fibers these channels are packed tightly on the nodal membrane. The highest estimate for the sodium channel density in the mammalian node is 10,000 micron2. A re-evaluation of the effective nodal area, however, might reduce this value to 3000-5000/micron 2. This would still leave the nodal membrane rather crowded with sodium channels. Furthermore, the channel density would still be greater than the density of particles, sometimes believed to be sodium channels seen in freeze fracture studies (Rosenbluth 1976). One possibility for resolving this problem is that the units detected by X-ray inactivation (Levinson & Ellory 1973), and those seen in freeze-fracture studies (Rosenbluth 1976) represent not single sodium channels but groups of three. Catterall & Morrow (1978) in a comparison of the binding of saxitoxin and Leiurus sculpturatus scorpion toxin venom have concluded that there are three saxitoxin binding sites for each scorpion toxin binding site. On this basis, three saxitoxin molecules might act to block independently each of the three openings of the channels; while the the conformational change produced by the scorpion venom molecule would affect the inactivation process of all three channels.Keywords:
Saxitoxin
Tetrodotoxin
Scorpion toxin
Batrachotoxin
Axoplasm
Abstract: We have previously shown that the [ 3 H]saxitoxin binding site of the sodium channel is expressed independently of the [ 125 I]scorpion toxin binding site in chick muscle cultures and in rat brain. In the present work, we studied the development of the sodium channel protein during chemically induced differentiation of N1E‐115 neuroblastoma cells, using [ 3 H]saxitoxin binding, [ 125 I]scorpion toxin binding, and 22 Na uptake techniques. When grown in their normal culture medium, these cells are mostly undifferen‐tiated, bind 90 ± 10 fmol of [ 3 H]saxitoxin/mg of protein and 112 ± 14 fmol of [ 125 I]scorpion toxin/mg protein, and, when stimulated with scorpion toxin and batrachotoxin, take up 70 ± 5 nmol of 22 Na/min/mg of protein. Cells treated with dimethyl sulfoxide (DMSO) or hexamethy‐lene‐bis‐acetamide (HMBA) differentiate morphologically within 3 days. At this time, the [ 3 H]saxitoxin binding, the [ 125 I]scorpion toxin binding, and the 22 Na uptake values are not very different from those of undifferentiated cells. With subsequent time in DMSO or HMBA, these values continue to increase, a result indicating that the main period of sodium channel expression occurs well after the cells have assumed the morphologically differentiated state. The data indicate that the expression of sodium channels and morphological differentiation are independently regulated neuronal properties, that the attainment of morphological differentiation is necessary but not in itself sufficient for full expression of the sodium channel proteins, and that, in contrast to the chick muscle cultures and rat brain, the [ 3 H]saxitoxin site and [ 125 I]scorpion toxin site appear to be coregulated in N1E‐115 cells.
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Batrachotoxin
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Voltage clamp measurements on myelinated nerve fibers show that tetrodotoxin, saxitoxin, and DDT specifically affect the sodium channels of the membrane. Tetrodotoxin and saxitoxin render the sodium channels impermeable to Na ions and to Li ions and probably prevent the opening of individual sodium channels when one toxin molecule binds to a channel. The apparent dissociation constant of the inhibitory complex is about 1 nM for the cationic forms of both toxins. The zwitter ionic forms are much less potent. On the other hand, DDT causes a fraction of the sodium channels that open during a depolarization to remain open for a longer time than is normal. The effect cannot be described as a specific change in sodium inactivation or as a specific change in sodium activation, for both processes continue to govern the opening of the sodium channels and neither process is able to close the channels. The effects of DDT are very similar to those of veratrine.
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Tetrodotoxin
Neurotoxin
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Sodium channel blocker
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Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.
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Abstract Skeletal analogues of saxitoxin (STX) that possess a fused‐type tricyclic ring system, designated FD‐STX, were synthesized as candidate sodium ion channel modulators. Three kinds of FD‐STX derivatives 4 a – c with different substitution at C13 were synthesized, and their inhibitory activity on sodium ion channels was examined by means of cell‐based assay. (−)‐FD‐STX ( 4 a ) and (−)‐FD‐dcSTX ( 4 b ), which showed moderate inhibitory activity, were further evaluated by the use of the patch‐clamp method in cells that expressed Na V 1.4 (a tetrodotoxin‐sensitive sodium channel subtype) and Na V 1.5 (a tetrodotoxin‐resistant sodium channel subtype). These compounds showed moderate inhibitory activity towards Na V 1.4, and weaker inhibitory activity towards Na V 1.5. Uniquely, however, the inhibition of Na V 1.5 by (−)‐FD‐dcSTX ( 4 b ) was “irreversible”.
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Figure 6 summarizes the present state of our knowledge on the sodium channel in myelinated nerve fibers. Two sites have been discussed in detail: a metal cation binding site accessible by tetrodotoxin and saxitoxin from the outside surface only; and a second site accessible from the inside surface with which local anesthetics combine. Hydrogen ions gain access to this region of the sodium channel (and hence determine the relative local concentration of protonated drug) more readily from the extracellular fluid than from the axoplasm (Schwarz et al 1977). In addition, a variety of other sites have been mentioned, binding of drugs to which alters selectively the kinetics of opening and closing of the h and m gates. In myelinated nerve fibers these channels are packed tightly on the nodal membrane. The highest estimate for the sodium channel density in the mammalian node is 10,000 micron2. A re-evaluation of the effective nodal area, however, might reduce this value to 3000-5000/micron 2. This would still leave the nodal membrane rather crowded with sodium channels. Furthermore, the channel density would still be greater than the density of particles, sometimes believed to be sodium channels seen in freeze fracture studies (Rosenbluth 1976). One possibility for resolving this problem is that the units detected by X-ray inactivation (Levinson & Ellory 1973), and those seen in freeze-fracture studies (Rosenbluth 1976) represent not single sodium channels but groups of three. Catterall & Morrow (1978) in a comparison of the binding of saxitoxin and Leiurus sculpturatus scorpion toxin venom have concluded that there are three saxitoxin binding sites for each scorpion toxin binding site. On this basis, three saxitoxin molecules might act to block independently each of the three openings of the channels; while the the conformational change produced by the scorpion venom molecule would affect the inactivation process of all three channels.
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Tetrodotoxin
Scorpion toxin
Batrachotoxin
Axoplasm
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Tetrodotoxin
Sodium channel blocker
Saxitoxin
Batrachotoxin
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Veratridine
Batrachotoxin
Tetrodotoxin
Aconitine
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Scorpion toxin
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The inward movement of sodium ions during the propagation of action potential in most nerve membranes occurs through voltage-sensitive sodium channels. The molecular properties of sodium channels are defined predominantly by the interaction of specific neurotoxins with the discrete receptor sites of the voltage-sensitive sodium channels, such as sodium influx inhibitors (tetrodotoxin, saxitoxin) sodium channel activators (batrachotoxin, veratridine, aconitine), and inactivation inhibitors (sea anemone toxin, scorpion toxin) (Lazdunski and Renaud, 1982; Catterall, 1984). Because the neurotoxins bind to their receptor sites with a high affinity and specificity, they have been used as pharmacological probes to study the structural and functional properties of voltage-sensitive sodium channels in variety of excitable membranes.
Veratridine
Batrachotoxin
Tetrodotoxin
Scorpion toxin
Neurotoxin
Saxitoxin
Aconitine
Electrophorus
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Scorpion toxin
Batrachotoxin
Tetrodotoxin
Neurotoxin
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