Sodium currents in voltage clamped nerve fiber of frog under the combined action of batrachotoxin and procaine

Bi, Khodorov; Peganov, É. M.; Revenko, S. V.; Shishkova, L. D.

doi:10.1016/0006-8993(75)90771-4

Cited by 76 publications

(33 citation statements)

References 8 publications

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“…The combination of 1 gN batrachotoxin and 0.1 gM scorpion toxin maximally activates Na + channels and, in the presence of normal (145 raM) Na +, depolarizes the synaptosomes . In electrophysiological studies, both of these toxins also inhibit Na § channel inactivation (Koppenh6fer & Schmidt, 1968 ;Khodorov et al, 1975 ;Okamoto, Takahashi & Yamashita, 1977). As shown in Fig.…”

Section: Binding Of 3h-stx To Synaptosomesmentioning

confidence: 95%

Saxitoxin binding to synaptosomes, membranes, and solubilized binding sites from rat brain

et al. 1979

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Binding of 3H-saxitoxin to Na+ channels was studied in subcellular fractions prepared from rat brain homogenates. Saxitoxin binding to synaptosomes was saturable with an apparent dissociation constant of about 1 nM; about 1 pmol/mg protein was bound at saturating saxitoxin concentrations. A linear, nonsaturable component of saxitoxin binding accounted for less than 3% of the total binding at 30 nM. Saxitoxin binding to synaptosomes was unaffected by depolarization with elevated K+ concentrations, or by activation of the Na+ channels with batrachotoxin plus a purified polypeptide toxin from the scorpion Leiurus quinquestriatus. A procedure is described for preparing a membrane fraction that contains 70--80% of the total saxitoxin binding activity of the crude homogenate. The specific activity of this fraction was about 4 to 6 pmol/mg protein. About 60--70% of the saxitoxin binding sites were solubilized by incubating these membranes with the nonionic detergent Triton X-100; the detergent-solubilized binding sites eluted at a position corresponding to a mol wt of about 700,000 on gel filtration chromatography. Both membrane-bound and solubilized saxitoxin binding were assayed by a new cation exchange column method. The binding of saxitoxin to both membrane-bound and detergent-solubilized binding sites was saturable with an apparent dissociation constant of about 2 nM. Dissociation of the saxitoxin-receptor complex followed a single exponential decay with a rate constant at 0 degrees of 0.1 min-1 for membrane bound and 0.2 min-1 for detergent-solubilized binding sites. The measured association rate constant was 6 X 10(8) M-1 min-1 at 0 degrees for membrane-bound saxitoxin binding sites.

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Section: Binding Of 3h-stx To Synaptosomesmentioning

confidence: 95%

Saxitoxin binding to synaptosomes, membranes, and solubilized binding sites from rat brain

et al. 1979

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“…BTX is known to penetrate slowly to its site of action in a variety of nerve and muscle cells (2,3,13), and the requirement for repetitive stimulation for BTX-elicited depolarizations in the electroplax again indicates a slow penetration. The observations on the electroplax can be interpreted most simply in terms of three pools for BTX ( (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…A report that BTX is more effective when applied internally to the squid giant axon (12) suggests that BTXint could simply be the interior cell cytoplasm. An attempt to pharmacologically isolate BTX-modified sodium channels in frog nerve fibers with local anesthetics during depolarizing voltage clamping (13) suggests that BTX is specifically associated with inactive sodium channels; thus BTXint could be highly specific complexes of this type. A third possibility is simply to extend Scheme I by dividing BTXint into BTXint', an interior cell phase, and BTXint", a specific complex with inactive sodium channels.…”

Section: Discussionmentioning

confidence: 99%

Effect of batrachotoxin on the electroplax of electric eel: evidence for voltage-dependent interaction with sodium channels.

Bartels-Bernal¹,

Rosenberry²,

Daly³

1977

Proc. Natl. Acad. Sci. U.S.A.

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Batrachotoxin under certain conditions has a strong depolarizing effect on the innervated membrane of the monocellular electroplax preparation from the electric eel, Electrophorus electricus. No effect is observed when the toxin (50-200 nM) is applied to the resting membrane for periods up to 1 hr. However, if the membrane is exposed to batrachotoxin and the cell is subjected to stimulation at a stimulus voltage slightly above the threshold for action potential firing, a progressive prolongation of the action potential and concomitant progressive depolarization of the innervated membrane is observed. When the membrane is depolarized by 15-20 mV, a further abrupt all-or-none depolarization occurs, and the potential attains a steady-state value between 0 and -10 mV. Brief stimulation of a cell in the presence of batrachotoxin is sufficient to define a batrachotoxin-treated cell, even though negligible depolarization occurs. If depolarizing agents such as carbamoylcholine or potassium chloride are introduced to such a cell in concentrations that normally produce a 20-30 mV depolarization, the abrupt all-or-none depolarization immediately occurs. All-or-none depolarizations arising from either electrical stimulation or depolarizing agents are unaffected by d-tubocurarine but are completely reversed by tetrodotoxin. Batrachotoxin thus appears to activate only the action potential sodium channels. In the batrachotoxin-treated membrane, these channels can attain stable steady states in either a closed configuration at the normal resting potential or in an open configuration after complete depolarization. A striking hysteresis cycle thus can be generated, which is strongly indicative of a voltage-dependent interaction of the toxin with the action potential sodium channels.

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“…It modifies the voltage-gated Na+ channel in excitable membranes and has been widely utilized to probe the properties ofthis class of channel (2,3). Khodorov et aL have made extensive electrophysiological observations ofthe action ofBTX on Na+ channels in the node of Ranvier (4)(5)(6). In these studies, BTX shifted Na+ channel activation to more negative potentials, and the kinetics of activation of the toxin-modified channel were slowed and inactivation was eliminated.…”

mentioning

confidence: 99%

Modification of single Na+ channels by batrachotoxin.

Quandt¹,

Narahashi²

1982

Proc. Natl. Acad. Sci. U.S.A.

123

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The modifications in the properties of voltage-gated Na' channels caused by batrachotoxin were studied by using the patch clamp method for measuring single channel currents from excised membranes of Modified and normal open states of Na' channels coexist in batrachotoxin-exposed membrane patches. Unlike the normal condition, Na' channels exposed to batrachotoxin open spontaneously at large negative potentials. These spontaneous openings apparently cause the toxin-induced increase in Na' permeability which, in turn, causes membrane depolarization.Batrachotoxin (BTX) is an extremely toxic steroidal alkaloid isolated from the skin of the Colombian arrow poison frog (1). It modifies the voltage-gated Na+ channel in excitable membranes and has been widely utilized to probe the properties ofthis class of channel (2,3). Khodorov et aL have made extensive electrophysiological observations ofthe action ofBTX on Na+ channels in the node of Ranvier (4-6). In these studies, BTX shifted Na+ channel activation to more negative potentials, and the kinetics of activation of the toxin-modified channel were slowed and inactivation was eliminated. Similar observations recently have been made for the actions of BTX on Na+ currents in neuroblastoma cells (7) and in squid axons (unpublished observations). BTX-modified channels were also less selectively permeable to Na+ over K+, Rb+, or Cs+ than normal channels. Further, the maximal Na+ permeability, if all voltage-gated channels were open, was substantially decreased after exposure to BTX. BTX also causes nerve and muscle membranes to depolarize due to an increase in resting Na+ permeability (8, 9).These observations on the actions of BTX were primarily based on measurements of membrane currents recorded from the whole preparation by using conventional voltage clamp techniques. However, such currents represent the average behavior of a large population of channels of unknown number. Many questions remain regarding the action of BTX. Either reductions in the rate ofchannel closing or changes in the probability of channel opening could underlie the apparent lack of inactivation. The observed decrease in the maximal permeability for voltage-gated Na+ channels could be caused either by a block of Na+ channels or a reduction in the Na+ permeability for each modified channel. Modification ofNa+ channels might be either in a graded or in an all-or-none manner. Further, changes in gating of the channel could be coupled to the changes in the permeation properties ofthe channel, or the two effects could be independent. Recently it has become possible to measure the current through individual voltage-gated Na+ channels (10-12). Changes in the fundamental properties ofthe Na' channel that are modified by the toxin can be measured directly by using this approach.NIE-115 neuroblastoma cells were utilized in this study because this preparation has a number of advantages for single channel studies. (i) There is free access to the plasma membrane, permitting a good seal between the membra...

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Sodium currents in voltage clamped nerve fiber of frog under the combined action of batrachotoxin and procaine

Cited by 76 publications

References 8 publications

Saxitoxin binding to synaptosomes, membranes, and solubilized binding sites from rat brain

Saxitoxin binding to synaptosomes, membranes, and solubilized binding sites from rat brain

Effect of batrachotoxin on the electroplax of electric eel: evidence for voltage-dependent interaction with sodium channels.

Modification of single Na+ channels by batrachotoxin.

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