Jay Z. Yeh

Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels

The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance.

Differential actions of insecticides on target sites: basis for selective toxicity.

Whereas the selective toxicity of insecticides between insects and mammals has a long history of studies, it is now becoming abundantly clear that, in many cases, the differential action of insecticides on insects and mammalian target receptor sites is an important factor. In this paper, we first introduce the mechanism of action and the selective toxicity of pyrethroids as a prototype of study. Then, a more detailed account is given for fipronil, based primarily on our recent studies. Pyrethroids keep the sodium channels open for a prolonged period of time, causing elevation of the depolarizing after-potential. Once the after-potential reaches the threshold for excitation, repetitive after-discharges are produced, resulting in hyperexcitation of intoxicated animals. Only about 1% of sodium channels needs to be modified to produce hyperexcitation, indicating a high degree of toxicity amplification from sodium channels to animals. Pyrethroids were >1000-fold more potent on cockroach sodium channels than rat sodium channels, and this forms the most significant factor to explain the selective toxicity of pyrethroids in insects over mammals. Fipronil, a phenylpyrazole, is known to act on the γ-aminobutyric acid receptor to block the chloride channel. It is effective against certain species of insects that have become resistant to most insecticides, including those acting on the γ-aminobutyric acid receptor, and is much more toxic to insects than to mammals. Recently, fipronil has been found to block glutamate-activated chloride channels in cockroach neurons in a potent manner. Since mammals are devoid of this type of chloride channel, fipronil block of the glutamate-activated chloride channel is deemed responsible, at least partially, for the higher selective toxicity to insects over mammals and for the lack of cross-resistance. Human & Experimental Toxicology (2007) 26, 361-366

Effects of Ethanol on Tonic GABA Currents in Cerebellar Granule Cells and Mammalian Cells Recombinantly Expressing GABAA Receptors

The effects of ethanol on the GABAA receptors, which are regarded as one of the most important target sites of ethanol, are very controversial, ranging from potentiation to no effect. The δ subunit-containing GABAA receptors expressed in Xenopus oocytes were recently reported to be potently augmented by ethanol. We performed patch-clamp experiments using the cerebellar granule cells and mammalian cells expressing recombinant GABAA receptors. In granule cells, the sensitivity to GABA increased from 7 to 11 days in vitro. Furosemide, an antagonist of α6-containing GABAA receptors, inhibited GABA-induced currents more potently at 11 to 14 days than at 7 days. Ethanol at 30 mM had either no effect or an inhibitory effect on currents induced by low concentrations of GABA in granule cells. On α4β2δ, α6β2δ, or α6β3δGABAA receptors expressed in Chinese hamster ovary cells, ethanol at 10, 30, and 100 mM had either no effect or an inhibitory effect on GABA currents. Ethanol inhibition of GABAA receptor was observed in all of the subunit combinations examined. In contrast, the perforated patch-clamp method to record the GABA currents revealed ethanol effects on the α6β2δ subunits ranging from slight potentiation to slight inhibition. Ethanol seems to exert a dual action on the GABAA receptors and the potentiating action may depend on intracellular milieu. Thus, the differences between the GABAA receptors expressed in mammalian host cells and those in Xenopus oocytes in the response to ethanol might be due to changes in intracellular components under patch-clamp conditions.

Potent modulation of neuronal nicotinic acetylcholine receptor-channel by ethanol.

Controversies remain over which ion channels are the most sensitive to ethanol. We have found that ethanol potently modulates the neuronal nicotinic acetylcholine receptor-channel at micromolar concentrations with an EC50 of 88.5 microM, which is significantly lower than most values previously reported for other ion channels. Prolonged application of ethanol accelerated the decay phase of acetylcholine-induced currents, caused single-channels to open in bursts, and shortened the mean open time, all of which reflect increased receptor desensitization. However, ethanol slowed the decay phase of the current induced by a brief application of acetylcholine, which may indicate that ethanol manifests its action by causing an increase in the affinity of the receptor for acetylcholine. These results suggest that neuronal nicotinic acetylcholine receptors may be important target sites of ethanol, particularly in the early stages of ethanol intoxication.

Fipronil modulation of glutamate-induced chloride currents in cockroach thoracic ganglion neurons.

Fipronil is the first phenylpyrazole insecticide introduced for pest control. It is effective against some insects that have become resistant to other insecticides, and exhibits low mammalian toxicity. Although fipronil is known to block GABA receptors, the mechanisms of its selective toxicity and efficacy against insects with dieldrin-resistant GABA receptors are not fully understood. We studied the effects of fipronil on the inhibitory glutamate receptor-chloride channel complex, which is found only in invertebrates. Glutamate-activated chloride currents were recorded from neurons isolated from cockroach thoracic ganglia using the whole-cell patch clamp technique. When glutamate was applied to a neuron, it evoked inward currents with an EC50 of 36.8 +/- 3.0 microM and a Hill coefficient of 1.56 +/- 0.17. The similarity between the reversal potential and the calculated chloride equilibrium potential indicated that glutamate-induced currents were carried by chloride ions. Fipronil suppressed the glutamate-induced peak currents in a dose-dependent manner with an IC50 of 0.73 +/- 0.27 microM and a Hill coefficient of 0.68 +/- 0.15. The current decay phases were greatly prolonged after fipronil application in a concentration-dependent manner. Picrotoxinin (PTX) at 100 microM slightly suppressed glutamate-induced currents to 87.8 +/- 3.7% of the control, and dieldrin at 100 microM had no effect (96.7 +/- 3.1%). AP5 and CNQX, mammalian glutamate receptor antagonists, were without effect on glutamate-induced Cl- currents. It is concluded that the potent blocking action of fipronil against glutamate-gated chloride channels may contribute to the higher toxicity against insects than mammals, as well as the efficacy against insects resistant to other insecticides.

Use‐ and Voltage‐Dependent Block of the Sodium Channel by Saxitoxin

Saxitoxin and tetrodotoxin have long fascinated electrophysiologists because of their remarkable specificity and potency of action on sodium channels in various excitable membranes.’.’ Much effort has been made to elucidate the mechanism of sodium channel blocking action by these toxins. Kao and Nishiyama’ first proposed that the tetrodotoxin and saxitoxin molecules, which are too large to pass through the sodium channel, physically blocked the passage of other ions by binding in the channel, acting on the channel like a cork in a bottle, with the guanidinium group of the toxin being the cork. This seemed to be a reasonable hypothesis then since free guanidinium ions can pass through the channel, and since the TTX and STX molecules are thought to be too bulky to pass through the Na channel. This notion was further elaborated by Hille! By comparing the structure of TTX with STX, and by incorporating the theory of ion permeation through the channel, Hille proposed a hypothetical structural basis to account for the Na channel blocking action of these two toxins. Hille proposed that the progress of an ion through the open sodium channel can be simulated by the passage of the ion over a series of energy barriers according to Eyring rate theory.’ The highest energy barrier in the channel is viewed as being the basis for the channel’s selectivity among different permeating ions. This barrier is viewed mechanistically as being the narrowest part of the channel, and would require ions to shed at least some of their water of hydration. Hille termed this constriction the “selectivity filter,” proposing that it consists of a 3 x 5A oxygen-lined constriction that allows close interaction of the partially dehydrated ion with a highly negative field strength site, presumably an ionized carboxylic acid. This selectivity filter is also the binding site for both H+ and CaZ+ ions6. Since the site is located within the membrane field, the blocking action is expected to vary with voltage according to a Boltzmann distribution. This is indeed the case for the block in the presence of H+ and Ca2+ ions.6.’ In Hille’s model for toxin block, the guanidinium group was pictured as being bound to a carboxylic acid group in the selectivity filter, with five hydrogen bonds forming between the toxin molecule and the lining of the sodium channel. This TTX-carboxylic acid binding hypothesis was supported by experiments with trimethyloxonium tetrafluoroborate (TMO), a carboxyl group alkylating reagent. After the treatment of nerve or muscle membranes with TMO, TTX could no longer bind to the sodium channel’ nor block the sodium currents? However, the TTX-resistant channels had normal selectivity and were still sensitive to hydrogen ion block, suggesting that the carboxyl group necessary for TTX binding is distinct from that in the selectivity filter. Two other lines of evidence further argue against STX and TTX binding to the

Isoflurane modulation of neuronal nicotinic acetylcholine receptors expressed in human embryonic kidney cells.

Background: It is well established that neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to inhalational anesthetics. The authors previously reported that halothane potently blocked &agr;4&bgr;2-type nAChRs of rat cortical neurons. However, the effect of isoflurane, which is widely used clinically, on nAChRs largely remains to be seen. The authors studied the effects of isoflurane as compared with sevoflurane and halothane on the human &agr;4&bgr;2 nAChRs expressed in human embryonic kidney cells. Methods: The whole-cell and single-channel patch clamp techniques were used to record currents induced by acetylcholine. Results: Isoflurane, sevoflurane, and halothane suppressed the acetylcholine-induced currents in a concentration-dependent manner with 50% inhibitory concentrations of 67.1, 183.3, and 39.8 &mgr;m, respectively, which correspond to 0.5 minimum alveolar concentration or less. When anesthetics were coapplied with acetylcholine, isoflurane and sevoflurane decreased the apparent affinity of receptor for acetylcholine, but halothane, in addition, decreased the maximum acetylcholine current. When isoflurane was preapplied and coapplied, its inhibitory action was independent of acetylcholine concentration. Isoflurane blocked the nAChR in both resting and activated states. Single-channel analyses revealed that isoflurane at 84 &mgr;m decreased the mean open time and burst duration without inducing “flickering” during channel openings. Isoflurane increased the mean closed time. As a result, the open probability of single channels was greatly reduced by isoflurane. Conclusions: Isoflurane, sevoflurane, and halothane potently blocked the &agr;4&bgr;2 nAChR. Isoflurane suppression of whole-cell acetylcholine currents was a result of decreases in the open time, burst duration, and open probability and an increase in the closed time of single channels. The high sensitivity of neuronal nAChRs to inhalational anesthetics is expected to play an important role in several stages of anesthesia.

Voltage-dependent block of sodium channels in mammalian neurons by the oxadiazine insecticide indoxacarb and its metabolite DCJW

Indoxacarb is a newly developed insecticide with high insecticidal activity and low toxicity to non-target organisms. Its metabolite, DCJW, is known to block compound action potentials in insect nerves and to inhibit sodium currents in cultured insect neurons. However, little is known about the effects of these compounds on the sodium channels of mammalian neurons. We compared the effects of indoxacarb and DCJW on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion neurons by using the whole-cell patch clamp technique. Indoxacarb and DCJW at 1-10 microM slowly and irreversibly blocked both TTX-S and TTX-R sodium channels in a voltage-dependent manner. The sodium channel activation kinetics were not significantly modified by 1 microM indoxacarb or 1 microM DCJW. The steady-state fast and slow inactivation curves were shifted in the hyperpolarization direction by 1 microM indoxacarb or 1 microM DCJW indicating a higher affinity of the inactivated sodium channels for these insecticides. These shifts resulted in an enhanced block at more depolarized potentials, thus explaining voltage-dependent block, and an apparent difference in the sensitivity of TTX-R and TTX-S channels to indoxacarb and DCJW near the resting potential. Indoxacarb and its metabolite DCJW cause toxicity through their action on the sodium channels.

Halothane and Propofol Modulation of γ -aminobutyric Acida Receptor Single-channel Currents

Halothane and propofol enhance the activity of the γ-aminobutyric acid (GABA) system, which is one of the most important systems in the mechanism of anesthesia. To determine whether halothane and propofol enhance GABAergic responses by the same mechanism, we performed single-channel patch-clamp experiments with rat cortical neurons in primary culture. Each of the open-time and closed-time distributions of GABAA receptor single channels was expressed by a sum of fast and slow time constants. Neither halothane nor propofol changed the single-channel conductance. Halothane increased the probability of the channel being open via a prolongation of the slow phase of open time, whereas propofol increased the channel open probability via a shortening of the slow phase of closed time. Thus, although both halothane and propofol augmented the channel open probability, thereby causing an increase in charge transfer during inhibitory transmitter action, they acted by different mechanisms.

Single-Channel Analyses of Ethanol Modulation of Neuronal Nicotinic Acetylcholine Receptors

BACKGROUND We have previously reported that ethanol potentiates the acetylcholine-induced currents of the alpha4beta2 neuronal nicotinic acetylcholine receptors in rat cortical neurons and of those that are stably expressed in human embryonic kidney cells. The potentiation of the maximal currents evoked by high concentrations of acetylcholine suggests that ethanol affects the channel gating. METHODS We performed single-channel patch-clamp experiments to elucidate the detailed mechanism of ethanol modulation of the alpha4beta2 receptor that is stably expressed in human embryonic kidney cells. RESULTS At least two conductance states, 40.5 pS and 21.9 pS, were activated by acetylcholine. Acetylcholine at 30 nM predominantly induced the high conductance state currents (85% of total). Ethanol did not affect the single-channel conductance but selectively modulated the high-conductance state currents. The high-conductance state currents exhibited two open time constants. Both time constants were increased by 100 mM ethanol, from 1.9 msec to 2.8 msec and from 9.0 msec to 15.5 msec, respectively. Ethanol also prolonged the burst duration and the open time within burst and increased the probability of channel opening. CONCLUSIONS These changes in single-channel parameters indicate that ethanol stabilizes the alpha4beta2 receptor-channel in the opening state, explaining how the maximum acetylcholine-induced whole-cell currents are further potentiated by ethanol.