5-HT(3) receptors and neurotransmitter release in the CNS: a nerve ending story?

Serotonin (5-HT) 5-HT(3) receptors are ligand-gated ion channels, which are generally thought to be involved in the presynaptic modulation of neurotransmitter release. However, analysis of published data reveals that most of the evidence for the alleged presynaptic role of 5-HT(3) receptors in modulating CNS neurotransmitter release is not compelling. Nevertheless, 5-HT(3) receptors are present in nerve terminals from some brain regions. The increased basic knowledge of the cellular physiology of central 5-HT(3) receptor ligand-gated ion channels provides opportunities for a detailed characterization of the specific presynaptic effects of 5-HT(3) receptors. Such reconsideration is required for the full appreciation of the functional role of 5-HT(3) receptors in the CNS.

Single Ca(2+)-activated K+ channels in human erythrocytes: Ca2+ dependence of opening frequency but not of open lifetimes.

Using the patch-clamp technique single-channel properties of Ca(2+)-activated K+ (CaK) channels were investigated in inside-out membrane patches of human erythrocytes. In a physiological K+ gradient (5 mM K+ externally: 150 mM K+ internally) the single CaK channel conductance is 15 pS in the membrane potential range of -40 to +40 mV. The channel open probability, opening frequency and open and closed time distributions are voltage-independent. The open probability and the opening frequency of the CaK channel depend on [Ca2+]i and increase between 0.5 and 60 microM Ca2+ from approx. 10% to 90% of the maximum value obtained at 115 microM. The relation between open probability and [Ca2+]i can be described by a sigmoid concentration-effect curve with an EC50 of 4.7 microM and a slope factor of 1. Independent of [Ca2+]i open time distributions yield two time constants of 5.3 and 22 ms. The relative amplitudes of the fast and slow components of the open time histogram as well as the maximum open probability and the maximum opening frequency of CaK channels vary considerably. In addition, CaK channels in multiple channel patches are highly interdependent. It is concluded that the Ca(2+)-dependence of CaK channels in human erythrocytes is due to the modulation of opening frequency by internal Ca2+. The results are consistent with a classical receptor-agonist model in which ligand interaction kinetics are much faster than channel gating.

Distinct metal ion binding sites on Ca(2+)-activated K+ channels in inside-out patches of human erythrocytes.

Effects of Cd2+, Co2+, Pb2+, Fe2+ and Mg2+ (1-100 microM) on single-channel properties of the intermediate conductance Ca(2+)-activated K+ (CaK) channels were investigated in inside-out patches of human erythrocytes in a physiological K+ gradient. Cd2+, Co2+ and Pb2+, but not Fe2+ and Mg2+, were able to induce CaK channel openings. The potency of the metals to open CaK channels in human erythrocytes follows the sequence Pb2+, Cd2+ > Ca2+ > or = Co2+ >> Mg2+, Fe2+. At higher concentrations Pb2+, Cd2+ and Co2+ block the CaK channel by reducing the opening frequency and the single-channel current amplitude. The potency of the metals to reduce CaK channel opening frequency follows the sequence Pb2+ > Cd2+, Co2+ >> Ca2+, which differs from the potency sequence Cd2+ > Pb2+, Co2+ >> Ca2+ to reduce the unitary single-channel current amplitude. Fe2+ reduced the channel opening frequency and enhanced the two open times of CaK channels activated by Ca2+, whereas up to 100 microM Mg2+ had no effect on any of the measured single-channel parameters. It is concluded that the activation of CaK channels of human erythrocytes by various metal ions occurs through an interaction with the same regulatory site at which Ca2+ activates these channels. The different potency orders for the activating and blocking effects suggest the presence of at least one activation and two blocking sites. A modulatory binding site for Fe2+ exists as well. In addition, the CaK channels in human erythrocytes are distinct from other subtypes of Ca(2+)-activated K+ channels in their sensitivity to the metal ions.

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier.

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.

Voltage-gated and Ca(2+)-activated K+ channels in intact human T lymphocytes. Noninvasive measurements of membrane currents, membrane potential, and intracellular calcium.

Voltage-gated n-type K(V) and Ca(2+)-activated K+ [K(Ca)] channels were studied in cell-attached patches of activated human T lymphocytes. The single-channel conductance of the K(V) channel near the resting membrane potential (Vm) was 10 pS with low K+ solution in the pipette, and 33 pS with high K+ solution in the pipette. With high K+ pipette solution, the channel showed inward rectification at positive potentials. K(V) channels in cell-attached patches of T lymphocytes inactivated more slowly than K(V) channels in the whole-cell configuration. In intact cells, steady state inactivation at the resting membrane potential was incomplete, and the threshold for activation was close to Vm. This indicates that the K(V) channel is active in the physiological Vm range. An accurate, quantitative measure for Vm was obtained from the reversal potential of the K(V) current evoked by ramp stimulation in cell-attached patches, with high K+ solution in the pipette. This method yielded an average resting Vm for activated human T lymphocytes of -59 mV. Fluctuations in Vm were detected from changes in the reversal potential. Ionomycin activates K(Ca) channels and hyperpolarizes Vm to the Nernst potential for K+. Elevating intracellular Ca2+ concentration ([Ca2+]i) by ionomycin opened a 33-50-pS channel, identified kinetically as the CTX-sensitive IK-type K(Ca) channel. The Ca2+ sensitivity of the K(Ca) channel in intact cells was determined by measuring [Ca2+]i and the activity of single K(Ca) channels simultaneously. The threshold for activation was between 100 and 200 nM; half-maximal activation occurred at 450 nM. At concentrations > 1 microM, channel activity decreased. Stimulation of the T-cell receptor/CD3 complex using the mitogenic lectin, PHA, increased [Ca2+]i, and increased channel activity and current amplitude resulting from membrane hyperpolarization.

Intracellular Ca2+ oscillations and membrane potential fluctuations in intact human T lymphocytes: role of K+ channels in Ca2+ signaling.

In intact human T lymphocytes, voltage-gated K+ [K(V)] channels and Ca(2+)-activated K+ [K(Ca)] channels have been recorded using the patch clamp technique in the cell-attached configuration. The reversal potential of the voltage-gated current with high K+ solution in the pipette gives a measure for the cell membrane potential (VM). The open probability of the K(Ca) channels gives a measure for intracellular Ca2+ concentration ([Ca2+]i). By simultaneous recording of both types of K+ channels, the interaction of VM and [Ca2+]i in T lymphocytes was investigated. It was demonstrated that VM fluctuates under resting conditions in a 20 mV range around an average value of -60 mV. In response to T cell receptor stimulation by PHA, rises in [Ca2+]i occur, which vary between cells from transient or sustained elevations to Ca2+ oscillations, in parallel with amplification of the hyperpolarizing deflections of VM. The correlation between VM and [Ca2+]i suggests that Ca2+ oscillations are modulated by positive feedback between Ca2+ influx, [Ca2+]i and VM mediated by K(Ca) channels and by intrinsic VM fluctuations caused by negative feedback between VM and the K(V) channel. Differences in the ratio between K(Ca) and K(V) channel numbers can account for the variability in Ca2+ responses between cells. The results predict periodic K(V) channel activity at rest and alternating K(V) and K(Ca) channel activity during Ca2+ signaling, which was consistent with subsequent observations.

Agonist and antagonist effects of apomorphine enantiomers on 5-HT3 receptors.

Direct effects of the enantiomers of the classical dopamine receptor ligand apomorphine on 5-HT3 receptors were examined in NIE-115 neuroblastoma cells in whole-cell voltage clamp mode. R(-)-apomorphine (R(-)APO; 3-300 microM) evokes a small, transient inward ion current. At 30 microM, R(-)APO induces its maximum inward current, which is approximately 3% of the amplitude of the inward current induced by 10 microM 5-HT. The R(-)APO-induced current is completely and reversibly inhibited after superfusion of 50 nM of the selective 5-HT3 receptor antagonist MDL 72222 and after desensitization of the 5-HT3 receptors by 10 microM 5-HT. The results indicate that R(-)APO is a partial agonist at the 5-HT3 receptor. R(-)APO (30 microM) evokes a depolarization of the membrane potential with an amplitude which is 26% of the 10 microM 5-HT-induced depolarization. In addition, the 5-HT-induced depolarization is reduced (from 29 to 15 mV) after prolonged exposure of the cell to R(-)APO. S(+)-apomorphine (S(+)APO; 3-300 microM) does not evoke an ion current. Instead, S(+)APO antagonizes the 5-HT3 receptor with an IC50 of 32 microM. The combined results indicate that enantiomers of apomorphine act directly on 5-HT3 receptors, and suggest that the in vivo effects of apomorphine are partially attributable to a direct interaction with 5-HT3 receptors.

Allosteric potentiation of the 5-HT3 receptor-mediated ion current in N1E-115 neuroblastoma cells by 5-hydroxyindole and analogues.

Potentiation of the 5-HT3 receptor-mediated ion current in mouse N1E-115 neuroblastoma cells by 5-hydroxyindole (5-OHi) and three analogues (5-aminoindole, catechol and indole) was examined using whole-cell voltage clamp and single channel patch clamp techniques. The substances tested enhanced the amplitude of the maximum 5-HT-evoked ion current by 12-30%. The rank order (at 1 mM) to potentiate the 5-HT-induced current was: 5-OHi approximately 5-aminoindole approximately catechol > indole. The concentration-effect curve of 5-HT was shifted leftwards by 1 mM 5-OHi, resulting in a two-fold increase of the apparent affinity of 5-HT from 1.4 microM to 0.7 microM, without affecting the Hill coefficient. The time constant of reversal of activation of the 5-HT-induced ion current upon washout of the agonist was delayed by 1 mM 5-OHi from 4.0 sec to 12.8 sec. 5-HT3 receptor-gated single channel events in cell-attached patches in the presence and absence of 1 mM 5-OHi were indistinguishable, apart from a slight increase in the event frequency. The results suggest that 5-OHi and analogues potentiate the 5-HT3 receptor-mediated ion current by delaying agonist dissociation and thereby increase the probability of channel opening. From the increased apparent affinity of 5-HT and the non-surmountability of the potentiating effect, it is concluded that 5-OHi and analogues are allosteric modulators of 5-HT3 receptors.

Pharmacological characterization of serotonin 5-HT3 receptor-mediated electrical response in cultured mouse neuroblastoma cells.

The aim of this study was to investigate the pharmacological characteristics of the 5-hydroxytryptamine-(5-HT)-induced electrical response in cultured neuroblastoma N1E-115 cells of the mouse. In these cells 5-HT induces a transient membrane depolarization, which is associated with a transient inward current, that has been recorded in voltage clamp experiments on whole cells. The peak amplitude of the inward current depends on the concentration of 5-HT applied. Maximum peak inward current was evoked by 10 microM 5-HT and half maximum effect by 2 microM. Responses to 5-HT were blocked by nanomolar concentrations of selective 5-HT3-receptor antagonists, whereas the selective agonist 2-methyl-5-HT mimicked the membrane depolarization induced by 5-HT. A number of agonists and antagonists, which are known to act on 5-HT1-like, 5-HT2, dopaminergic and adrenergic receptors failed to affect the response to 5-HT in neuroblastoma cells. Observed antagonistic effects of SCH 23390 [(R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepi n-7-ol hemimaleate] and haloperidol are discussed. The inhibitory effect of the 5-HT3 receptor antagonist, ICS 205-930 [(3 alpha-tropanyl)-1H-indole-3-carboxylic acid ester] has been demonstrated. When cells were exposed to 0.1 nM ICS 205-930 the maximum evoked response was reduced by about 50%, but a surmountable shift of the concentration-response curve of 5-HT was not observed. The kinetics of the 5-HT-induced inward current remained unchanged in the presence of ICS 205-930. Recovery from the block by ICS 205-930 was very slow.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction between enantiomers of mianserin and ORG3770 at 5-HT3 receptors in cultured mouse neuroblastoma cells.

Stereoselective effects of mianserin and ORG3770 on serotonin 5-HT3 receptors in mouse neuroblastoma N1E-115 cells have been investigated in radioligand binding and in whole-cell voltage clamp experiments. The specific binding of [3H]GR65630 to 5-HT3 recognition sites in N1E-115 cell homogenates is reduced by mianserin and ORG3770 and their enantiomers. The pKi values of the more potent (R)enantiomers of mianserin and ORG3770 are 8.44 and 8.62, respectively. The (R)enantiomers of mianserin and ORG3770 are 15 and 37 times more potent than their respective (S)enantiomers. The racemates are only 1.9 and 3.3 times less potent than the corresponding (R)enantiomers. In voltage clamp experiments the (R)enantiomers block the 5-hydroxytryptamine(5-HT)-induced ion current with pIC50 values of 8.52 for (R)mianserin and 8.26 for the (R)enantiomer of ORG3770. The (R)enantiomers of mianserin and ORG3770 are 24 and 145 times more potent in blocking the 5-HT-induced ion current than their respective (S)enantiomers. The racemates are 6 and 13 times less potent than the corresponding (R)enantiomers. In addition, the block of 5-HT-induced ion current by the (R)enantiomer of ORG3770 is partially reversed by a low concentration of its (S)enantiomer. The results indicate that the two enantiomers block the 5-HT3 receptor-mediated ion current in a mutually dependent manner.

Properties of neuronal type acetylcholine receptors in voltage clamped mouse neuroblastoma cells.

The pharmacological and physiological characteristics of nicotinic acetylcholine receptor-mediated ion current in mouse neuroblastoma N1E-115 cells have been investigated by superfusion of voltage clamped cells with known concentrations of agonists and antagonists for short periods. The acetylcholine-induced inward current is blocked by d-tubocurarine and by kappa-bungarotoxin with IC50 values of 0.5 microM and 30 nM, respectively. The inward current is resistant to alpha-bungarotoxin up to a concentration of 0.5 microM. This allows classification of the acetylcholine receptors of N1E-115 cells as neuronal type nicotinic receptors. The amplitude of the inward current increases with increasing concentration of the agonists acetylcholine and carbamylcholine, resulting in concentration-effect curves with EC50 values of 53 and 240 microM and slope factors slightly below unity. Conversely, at the highest concentrations of the agonists the amplitude of the inward current is reduced and a transient increase of the current appears when the agonist is removed. The characteristics of this transient tail current indicate that the agonists cause rapid ion channel block by interacting with a low affinity site. In the continued presence of acetylcholine the peak inward current is reduced by desensitization. The IC50 value and the slope factor of the steady-state desensitization curve are 1.1 microM and 0.58, respectively. At a low concentration of acetylcholine both the onset of desensitization and the inward current decay are described by similar dual exponential kinetics, but the steady-state inward current is smaller than expected from the degree of desensitization. Neuronal nicotinic receptors in N1E-115 cells and end-plate nicotinic receptors have several characteristics in common. However, the present results indicate that these receptors are distinct, not only in their sensitivity to snake toxins, but also with respect to functional properties.

Selection of distinct conformational states of the 5-HT3 receptor by full and partial agonists.

1. 5-Hydroxytryptamine 5-HT3 receptor-mediated ion currents evoked by 5-HT, quaternary 5-HT (5-HTQ), meta-chlorophenylbiguanide (mCPBG), dopamine and tryptamine in N1E-115 mouse neuroblastoma cells have been investigated in whole-cell voltage clamp and single channel patch clamp experiments. 2. The concentration-dependent activation and desensitization of the ion currents evoked by the agonists yield the potency order: mCPBG > 5-HTQ approximately 5-HT >> tryptamine > dopamine, and the efficacy order: 5-HT approximately mCPBG approximately 5-HTQ >> dopamine approximately tryptamine. Thus, 5-HT, 5-HTQ and mCPBG are full agonists, whereas dopamine and tryptamine are partial agonists at the 5-HT3 receptor. 3. Full and partial agonists cause complete cross-desensitization and activate single channels with similar conductances and open lifetimes. This shows that full and partial agonists act on the same population of 5-HT3 receptors. 4. The time course of recovery from desensitization depends on the agonist used. Recovery from partial agonist-induced desensitization is single exponential, whereas the desensitization induced by full agonists recovers with sigmoid kinetics, suggesting at least 3 steps between 4 states. 5. During the process of recovery from cross-desensitization, the full agonists activate a larger fraction of the 5-HT3 receptors than the partial agonists, irrespective of the agonist used to induce desensitization. 6. It is concluded that full and partial agonists induce distinct desensitized states and, during recovery from desensitization, recognize distinct conformations of unoccupied 5-HT3 receptors. This conformational selection is likely to account for the different efficacies of full and partial 5-HT3, receptor agonists.

5-Hydroxyindole slows desensitization of the 5-HT3 receptor-mediated ion current in N1E-115 neuroblastoma cells.

Effects of 5-hydroxyindole (5-OHi) on 5-HT3 receptor-operated ion current were investigated in voltage-clamped N1E-115 neuroblastoma cells. In the presence of 1 mM 5-OHi, the amplitudes of inward currents induced by the agonists 5-hydroxytryptamine (5-HT), 2-methyl-5-HT and dopamine were enhanced and desensitization of the responses was markedly slowed down. The results indicate that 5-OHi selectively modifies the desensitization of the 5-HT3 receptor-mediated ion current.

Competitive and non-competitive effects of 5-hydroxyindole on 5-HT3 receptors in N1E-115 neuroblastoma cells.

1. Effects of 5-hydroxyindole (5-OH-indole) (the aromatic moiety of 5-hydroxytryptamine (5-HT)) on 5-HT-evoked ion current and the nature of these effects on 5-HT3 receptors have been investigated in whole-cell voltage clamp and radioligand binding experiments on cultured N1E-115 mouse neuroblastoma cells. 2. The amplitude of 10 microM 5-HT-evoked ion currents was enhanced up to 150% of the control value by increasing concentrations up to 10 mM 5-OH-indole with half maximum effect of 0.8 mM. At concentrations between 10 mM and 50 mM, 5-OH-indole blocked the 5-HT-evoked ion current. Both the enhancement and the block by 5-OH-indole were accompanied by a marked slowing of the kinetics of decay of the 5-HT-evoked inward currents. 3. The blocking effect was surmounted when the 5-HT concentration was raised from 10 microM to 100 microM. Conversely, the increase in amplitude and the slowing of the decay of the 5-HT-evoked ion current induced by 1 mM 5-OH-indole were not reversed by the same increase of 5-HT concentration. 4. The binding of the selective antagonist [3H]-GR65630 to 5-HT3 receptors was displaced by 5-OH-indole in a concentration-dependent manner with a pKi of 1.96. In saturation binding experiments 10 mM 5-OH-indole reduced the affinity of [3H]-GR65630, whereas the total number of binding sites remained unaffected. 5. It is concluded that the blocking effect of high concentrations of 5-OH-indole is due to a competitive interaction with the antagonist recognition sites of 5-HT3 receptors, whereas the potentiating effect of lower concentrations of 5-OH-indole appears to be mediated by a distinct non-competitive interaction.

Metal interactions with voltage- and receptor-activated ion channels.

Effects of Pb and several other metal ions on various distinct types of voltage-, receptor- and Ca-activated ion channels have been investigated in cultured N1E-115 mouse neuroblastoma cells. Experiments were performed using the whole-cell voltage clamp and single-channel patch clamp techniques. External superfusion of nanomolar to submillimolar concentrations of Pb causes multiple effects on ion channels. Barium current through voltage-activated Ca channels is blocked by micromolar concentrations of Pb, whereas voltage-activated Na current appears insensitive. Neuronal type nicotinic acetylcholine receptor-activated ion current is blocked by nanomolar concentrations of Pb and this block is reversed at micromolar concentrations. Serotonin 5-HT3 receptor-activated ion current is much less sensitive to Pb. In addition, external superfusion with micromolar concentrations of Pb as well as of Cd and aluminum induces inward current, associated with the direct activation of nonselective cation channels by these metal ions. In excised inside-out membrane patches of neuroblastoma cells, micromolar concentrations of Ca activate small (SK) and big (BK) Ca-activated K channels. Internally applied Pb activates SK and BK channels more potently than Ca, whereas Cd is approximately equipotent to Pb with respect to SK channel activation, but fails to activate BK channels. The results show that metal ions cause distinct, selective effects on the various types of ion channels and that metal ion interaction sites of ion channels may be highly selective for particular metal ions.

L- and T-type calcium channels in cultured neuronal cell lines are insensitive to tetanus toxin.

Possible effects of tetanus toxin (TeTox) on voltage-activated Ca2+ channels of the mouse neuroblastoma cell line N1E-115 and of the neuroblastoma x glioma hybrid cell line NG108-15 have been investigated using the whole-cell voltage clamp technique. Similar to N1E-115 cells, differentiated NG108-15 cells express transient (T-type) as well as long-lasting (L-type) Ca2+ channels. Using various treatment protocols N1E-115 and NG108-15 cells were exposed to TeTox externally and by internal dialysis. In the cells treated with TeTox normal Ca2+ channel activity was present, as measured by the voltage-activated Ba2+ currents under voltage clamp conditions. In addition, intracellular microelectrode recordings showed that TeTox did not block the Ca2+ action potential in N1E-115 cells. It is concluded that TeTox, in contrast to previously reported results, does not affect voltage-activated T- and L-type Ca2+ channels in cultured neuronal cell lines. The results also indicate that Ca2+ channel block is unlikely to be an explanation for the block of neurotransmitter release by TeTox in vivo.

alpha 4 beta 2 subunit combination specific pharmacology of neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

Pharmacological characteristics of native neuronal nicotinic acetylcholine receptor-mediated ion currents in mouse N1E-115 neuroblastoma cells have been investigated by superfusion of voltage clamped cells with known concentrations of the agonists acetylcholine, nicotine and cytisine, and the antagonists alpha-bungarotoxin and neuronal bungarotoxin. The sensitivity of the nicotinic acetylcholine receptor for agonists followed the agonist potency rank-order: nicotine approximately acetylcholine >> cytisine. The EC50 values of acetylcholine and nicotine are 78 microM and 76 microM, respectively. Equal concentrations of acetylcholine and nicotine induce inward currents with approximately the same peak amplitude whereas cytisine induces much smaller inward currents. Acetylcholine-induced currents are unaffected by high concentrations of alpha-bungarotoxin. Conversely, at 10 and 90 nM neuronal bungarotoxin reduces the amplitude of the 1 mM acetylcholine-induced inward current to 47% and 11% of control values, respectively. Both the agonist potency rank-order and the differential sensitivity to snake toxins of nicotinic receptors in N1E-115 cells are consistent with the known pharmacological profile of alpha 4 beta 2 nicotinic receptors expressed in Xenopus oocytes and distinct from those of all other nicotinic acetylcholine receptors of known functional subunit compositions. All data indicate that the native nicotinic acetylcholine receptor in N1E-115 cells is an assembly of alpha 4 and beta 2 subunits, the putative major subtype of nicotinic acetylcholine receptor in the brain.

Sodium depletion in the periaxonal space of the squid axon treated with pyrethroids.

Pyrethroids are known to increase the steady-state sodium current during a step depolarization and to increase and prolong the tail sodium current associated with a step repolarization of the membrane. The pyrethroid-induced tail sodium current of squid axons developed as a function of the duration of the conditioning depolarizing pulse. However, with further lengthening the conditioning pulse duration, it decreased, further increased, or remained constant depending on the direction of sodium current during the depolarization, irrespective of the membrane potential per se. The depletion or accumulation of sodium in the periaxonal space during a 200-ms conditioning depolarizing pulse in the axon internally treated with pronase, pyrethroids, or both, was demonstrated by measurements of the changes in sodium reversal potential. Thus the observed changes in tail current amplitude as a function of the conditioning pulse duration are explicable in terms of changes in sodium concentration in the periaxonal space without assuming inactivation of the pyrethroid-modified channel.

Dual, non-competitive interaction of lead with neuronal nicotinic acetylcholine receptors in N1E-115 neuroblastoma cells.

In cultured mouse neuroblastoma N1E-115 cells, inorganic lead (Pb2+) affects inward currents induced by activation of neuronal type nicotinic acetylcholine receptors (nAChR) in a biphasic manner. At nanomolar concentrations a blocking action is observed, while at submillimolar concentrations this blocking effect is reversed, resulting in a U-shaped concentration-effect curve. Maximal block by 90% is observed at 1-3 microM Pb2+. The interactions of Pb2+ with nAChR were examined at the blocking concentration of 10 nM Pb2+ and at 10 microM Pb2+, presenting the reversal of block. The fitted Emax for nAChR receptor activation by ACh was attenuated at both high and low Pb2+ concentrations by 24% and 54%, respectively. The EC50 values of the activation curves were not significantly altered; amounting to 53 microM, 64 microM and 86 microM ACh in the control situation and in the presence of 10 nM and 10 microM Pb2+, respectively. Further, receptor desensitization and ion channel block by ACh were also not affected by Pb2+. The results indicate that Pb2+ affects nAChR in a dual manner that involves inhibition and potentiation, both by non-competitive interactions. Neuronal nAChR expressed in N1E-115 cells resemble a combination of alpha 4 and beta 2 subunits, that constitute the predominant subunits in the central nervous system. The differential inhibition and potentiation of nAChR, together with the high sensitivity, are of interest with respect to Pb2+ neurotoxicity.

Interactions of pyrethroids and octylguanidine with sodium channels of squid giant axons.

Kinetics of pyrethroid-modified sodium channels and the interaction of N-octylguanidine with the modified channels have been studied with internally perfused and voltage-clamped squid giant axons. The pyrethroids used were 1R-cis-phenothrin; 1R-cis-permethrin; 1R-cis-cyphenothrin; and 1R-cis-deltamethrin. Modification of sodium channels by pyrethroids resulted in marked slowing of opening and closing kinetics. The rate at which sodium channels arrived at the open pyrethroid-modified state during a depolarizing step was independent of the concentration of pyrethroids applied. The time of exposure to pyrethroids required for the pyrethroid-induced sodium tail current following a step depolarization to reach a steady-state amplitude was independent of the frequency of short (5 ms) depolarizing pulses, and in the pronase-treated axons was independent of the membrane potential (0 mV or -90 mV). We conclude that sodium channels are modified by pyrethroids primarily in the closed resting state. A small fraction of sodium channels is modified in the open state. The dose-response curve for N-octylguanidine block of sodium channels was not shifted by pyrethroids. The rate at which the pyrethroid-modified sodium channels were blocked by octylguanidine during a depolarizing step depended neither on the concentration of pyrethroids nor on the depolarizing potential, but depended on the concentration of octylguanidine. The time course of the pyrethroid-induced slow sodium tail current was not altered by octylguanidine. We conclude that the actions of pyrethroids and N-octylguanidine on sodium channels are independent of each other.