The sodium channel in non-impulsive cells Interaction with specific neurotoxins

The cell line C₉ used in this paper has a resting potential of ?50 mV (±10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na⁺ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstrated by electrophysiological techniques and by ²²Na⁺ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. ²²Na⁺ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of ²²Na⁺ influx into the C₉ cell. The stimulation of ²²Na⁺ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of ²²Na⁺ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.     

Interaction of nonylguanidine with the sodium channel.

Alkyl and aromatic guanidines interact strongly with the tetrodotoxin (TTX)- receptor site in eel electroplaque membranes, showing competition with TTX. That these guanidines could be useful as highly reversible small molecular weight blockers of Na+ currents is therefore suggested. We have investigated the mechanisms of interaction of one of these derivatives, nonylguanidine, by studying its effects on Na+ currents in squid giant axons using voltage clamp techniques. Although nonylguanidine competed with TTX for binding to eel electroplaque membrane fragments (Ki = 1.8 X 10(-5) M), it reversibly blocked both inward and outward Na+ currents in intact axons only if applied to the interior. In axons with the Na+ inactivation removed by papain nonylguanidine produced a time-dependent block very similar to that reported for strychnine and pancuronium. The reduction of steady-state currents in these axons was also voltage-dependent, with increasing block observed with increasing step depolarization. These results suggest that nonylguanidine binds to a site accessible from the axoplasmic side of the channel, simulating Na+ inactivation in papain-treated axons and competing with the normal inactivation process in untreated axons. The competition between internal nonylguanidine and external TTX may result from perturbation by the positively charged nonylguanidine of the TTX-binding site from within the channel itself.     

Interaction of syntaxins with the amiloride-sensitive epithelial sodium channel.

Amiloride-sensitive sodium channels mediate sodium entry across the apical membrane of epithelial cells in variety of tissues. The rate of Na(+) entry is controlled by the regulation of the epithelial sodium channel (ENaC) complex. Insertion/retrieval of the ENaC complex into the apical membrane as well as direct kinetic effects at the single channel level are recognized mechanisms of regulation. Recent data suggest that the syntaxin family of targeting proteins interact with and functionally regulate a number of ion channels and pumps. To evaluate the role of these proteins in regulating ENaC activity, we co-expressed rat ENaC cRNA (alpha, beta, gamma subunits) with syntaxin 1A or 3 cRNAs in Xenopus oocytes. Basal ENaC currents were inhibited by syntaxin 1A and stimulated by syntaxin 3. Both syntaxin 1A and syntaxin 3 could be co-immunoprecipitated with ENaC subunit proteins, suggesting physical interaction. Interestingly, immunofluorescence data suggest that with either syntaxin isoform the ENaC-associated epifluorescence on the oocyte surface is enhanced. These data indicate that (i) both syntaxin isoforms increase the net externalization of the ENaC channel complex, (ii) that the functional regulation is isoform specific, and (iii) suggest that ENaC may be regulated through mechanisms involving protein-protein interactions.     

Interaction of scorpion toxins with the sodium channel

Scorpion toxins are miniproteins that have been characterized with regard to their molecular properties including their pharmacological action. On these bases, toxins specific to mammals or to insects have been described and within the first category alpha- and beta-toxins identified. These toxins were chemically modified and used as molecular probes of the sodium channel. Thus, the interaction of the toxins with the pharmacological target could be followed and a better definition of this target could be obtained.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Interaction of deoxycholate with the sodium channel of squid axon membranes

Deoxycholate can react with sodium channels with a high potency. The apparent dissociation constant for the saturable binding reaction is 2 microM at 8 degrees C, and the heat of reaction is approximately -7 kcal/mol. Four independent test with Na-free media, K-free media, tetrodotoxin, and pancuronium unequivocally indicate that it is the sodium channel that is affected by deoxycholate. Upon depolarization of the membrane, the drug modified channel exhibits a slowly activating and noninactivating sodium conductance. The kinetic pattern of the modified channel was studied by increasing deoxycholate concentration, lowering the temperature, chemical elimination of sodium inactivation, or conditioning depolarization. The slow activation of the modified channel can be represented by a single exponential function with the time constant of 1--5 ms. The modified channel is inactivated only partially with a time constant of 1 S. The reversal potential is unchanged by the drug. Observations in tail currents and the voltage dependence of activation suggest that the activation gate is actually unaffected. The apparently slow activation may reflect an interaction betweem deoxycholate and the sodium channel in resting state.     

The presence of Na+ channels in myometrial smooth muscle cells is revealed by specific neurotoxins

This paper shows the presence, in rat myometrial smooth muscles, of low affinity binding sites for tetrodotoxin with a K0.5 value of 2 microM. Electrophysiological experiments using both intact strips and single isolated myometrial cells in culture have shown that veratridine and sea anemone toxins reveal functional Na+ channels. The activity of these channels was blocked by tetrodotoxin (10 microM) or by removal of Na+ ions. Results presented here are the first direct demonstration of the existence in rat myometrium of Na+ channels of the tetrodotoxin-resistant type.     

The sodium channel of human excitable cells is a target for gambierol.

BACKGROUND: Gambierol is a polycyclic ether toxin with the same biogenetic origin as ciguatoxins. Gambierol has been associated with neurological symptoms in humans even though its mechanism of action has not been fully characterized. METHODS: We studied the effect of gambierol in human neuroblastoma cells by using bis-oxonol to measure membrane potential and FURA-2 to monitor intracellular calcium. RESULTS: We found that this toxin: i) produced a membrane depolarization, ii) potentiated the effect of veratridine on membrane potential iii) decreased ciguatoxin-induced depolarization and iv) increased cytosolic calcium in neuroblastoma cells. CONCLUSION: These results indicate that gambierol modulate ion fluxes by acting as a partial agonist of sodium channels.     

Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 mum) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to + 80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

Interaction of spin-labeled local anesthetics with the sodium channel of squid axon membranes

Summary The effects of spin-labeled local anesthetics on sodium currents of internally perfused squid axons were studied using the voltage-clamp technique. Internal application (10 m) of the most potent spin-labeled local anesthetic used in this study produced a small initial block of sodium currents. However, after sixty repetitive pulses (to +80 mV) given at 1 Hz, the sodium currents were drastically reduced. In addition to this frequency-dependent phenomenon, the anesthetic effect on the sodium currents was also sensitive to the voltage of the pulses. Both the frequency- and voltage-dependent properties remained intact after removal of sodium inactivation with pronase. The recovery of sodium currents from this frequency-dependent anesthetic effect followed a single exponential curve with a surprisingly long time constant of about 10 min. Such a long recovery time, which is longer than any known sodium inactivation process, led us to suggest that the recovery process represents the dissociation of drug molecules from their binding sites. We have also found that increasing hydrophobic character of the homologues series of spin-labeled local anesthetics enhances the frequency- and voltage-dependent block of sodium currents. This effect strongly suggests that hydrophobic interaction is an integral component of the binding site. These probes with their selective effects on the sodium currents, are expected to be highly useful in studying the molecular structure of the sodium channels.     

Interaction between TRPC channel subunits in endothelial cells

Transient Receptor Potential Canonical (TRPC) proteins have been identified in mammals as a family of plasma membrane calcium-permeable channels activated by different kinds of stimuli in several cell types. We have studied TRPC subunit expression in bovine aortic endothelial (BAE-1) cells, where stimulation with basic fibroblast growth factor (bFGF), a potent angiogenetic factor, induces calcium entry carried at least partially by TRPC1 channels. By means of a RT-PCR approach, we have found that, in addition to TRPC1, only TRPC4 is expressed, both at the mRNA and protein level, as confirmed by immunoblotting and immunocytochemical analysis. Because functional TRPC channels are formed by assembly of four subunits in either homo- or heterotetrameric structures, we have carried out immunoprecipitation experiments and showed that TRPC1 and TRPC4 interact to form heteromers in these cells, independently from culture conditions (high or low percent of fetal calf serum, stimulation with bFGF). Moreover, the data show that TRPC subunits are not tyrosine-phosphorylated after bFGF stimulation and they do not co-immunoprecipitate with the type 1 FGF receptor. These results suggest that BAE-1 cells are a suitable model to study function and regulation of endogenous TRPC1/TRPC4 heteromers.     

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     

Tumour calcitonin. Interaction with specific calcitonin receptors.

The human epidermoid bronchial carcinoma (BEN) cell line has been shown to have specific membrane binding sites for calcitonin and to secrete high-molecular-weight forms (ranging from 40000 to 10000) of immunoreactive calcitonin. Synthetic salmon and human calcitonins and a thyroid extract of porcine calcitonin have been shown to displace 125I-labelled salmon calcitonin from the receptors in a dose-related fashion. The binding to these receptors of calcitonins derived from the BEN cell line and a medullary thyroid carcinoma with molecular weights ranging from 28000 to 3500 (both separated by gel-filtration chromatography) has been investigated. Neither major peaks of BEN-cell-line calcitonin showed receptor binding activity. Only one form of medullary thyroid carcinoma calcitonin, that which co-eluted with synthetic calcitonin monomer on gel-filtration chromatography, caused any significant displacement of labelled hormone from the receptors.     

Open sodium channel properties of single canine cardiac Purkinje cells.

Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).