William Marszalec

Neuronal nicotinic acetylcholine receptors: a new target site of ethanol.

Whereas a variety of neuroreceptors and ion channels have been demonstrated to be affected by ethanol including GABAA receptors, NMDA receptors, non-NMDA glutamate receptors, 5-HT3 receptors and voltage-gated calcium channels, neuronal nicotinic acetylcholine receptors (nnAChRs) have recently emerged as a new target site of ethanol. The nnAChRs are different from the muscle type nicotinic AChRs with respect to their molecular architecture and pharmacology. This article briefly reviews the structure, distribution and function of nnAChRs for which a considerable amount of information has been rapidly accumulated during the past 5-10 years. The potent and unique action of ethanol on nnAChRs has been unveiled only during the past few years. Most recent developments along this line of ethanol action are discussed in this paper.

Use-dependent pentobarbital block of kainate and quisqualate currents.

The effects of pentobarbital on whole-cell excitatory amino acid-induced currents were studies in cultured rat cortical neurons. Currents evoked by 40 microM kainate were reversibly inhibited by pentobarbital with an IC50 value of 50 microM. The block of the kainate response by pentobarbital was use dependent, requiring kainate stimulation. In the absence of kainate activation, 10 min perfusions of 100 microM pentobarbital inhibited kainate-induced currents less than 10%. Recovery from pentobarbital block also exhibited use dependence, reversing in 5-10 s with kainate stimulation, while persisting 10 min or more in the absence of agonist. Pentobarbital inhibition of the kainate response was not voltage dependent. Responses evoked by 10 microM quisqualate consisted of a peak current desensitizing to a smaller steady-state current. The co-application of 100 microM pentobarbital reduced the steady-state current by 49 +/- 5%. The peak current before desensitization, however, was inhibited less than 10%. Currents evoked by 25 microM N-methyl-D-aspartate were not significantly inhibited by co-application of 100 microM pentobarbital. The results suggest that the pentobarbital-induced inhibition of kainate responses involves open channel block and that the block of quisqualate currents primarily involve non-desensitizing receptor channels that generate steady-state currents.

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.

Mechanisms of alcohol-nicotine interactions: alcoholics versus smokers.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Toshio Narahashi and Bo Söderpalm. The presentations were (1) Nicotinic mechanisms and ethanol reinforcement: Behavioral and neurochemical studies, by Bo Söderpalm, M. Ericson, P. Olausson, and J. A. Engel; (2) Chronic nicotine and ethanol: Differential regulation in gene expression of nicotinic acetylcholine receptor subunits, by X. Zhang and A. Nordberg; (3) Nicotine-ethanol interactions at neuronal nicotinic acetylcholine receptors, by Toshio Narahashi, William Marszalec, and Gary L. Aistrup; (4) Relapse prevention in alcoholics by cigarette smoking? Treatment outcome in an observational study with acamprosate, by L.G. Schmidt, U. Kalouti, M. Smolka, and M. Soyka; and (5) Effect of nicotine on voluntary ethanol intake and development of alcohol dependence in male rats, by L. Hedlund and G. Wahlström.

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.

Alcohol modulation of cloned GABAA receptor-channel complex expressed in human kidney cell lines.

The effects of n-octanol on GABA-induced currents were examined on the alpha 1 beta 2 gamma 2s and alpha 1 beta 2 combinations of GABAA receptor subunits expressed in a human kidney cell line (HEK 293), using the whole-cell variation of the patch clamp technique. The EC50 of the GABA dose-response curve for the alpha 1 beta 2 combination was lower than that for the alpha 1 beta 2 gamma 2s combination. n-Octanol at 100 microM augmented the GABA-induced currents in a dose-dependent manner, decreasing the EC50 of the GABA dose-response curve without affecting the maximal response. The magnitude of n-octanol potentiation was nearly the same in both combinations. In contrast, a benzodiazepine agonist, chlordiazepoxide, augmented the currents of the alpha 1 beta 2 gamma 2s combination only. We conclude that the potentiation of GABAA receptor-mediated currents by a long carbon chain n-alcohol does not require the gamma 2 subunit.

Critical Roles for the M3–S2 Transduction Linker Domain in Kainate Receptor Assembly and Postassembly Trafficking

Kainate receptors (KARs) are neuronal proteins that exhibit a highly polarized distribution in the mammalian CNS. Assembly, intracellular trafficking, and synaptic targeting of KARs and other ionotropic glutamate receptors are processes controlled, in part, by various determinants within the constituent subunit proteins themselves. Here, we demonstrate that the linker region between the M3 and S2 domains, which in current structural models is thought to transduce ligand-binding energy into channel opening, additionally has an essential role in receptor biogenesis. Our results show that this gating-associated domain is engaged at two distinct critical stages of KAR biogenesis: first, during the transition from dimeric to tetrameric assembly states and, second, at a postassembly trafficking checkpoint within the endoplasmic reticulum. Alteration of a basic residue, arginine 663, altered the desensitization properties of the GluR6 kainate receptor in response to glutamate application, and these changes were weakly correlated with intracellular retention of the mutant receptors. Elimination of the positive charge also significantly attenuated oligomerization and stability of the intracellular subunit protein. Furthermore, charge swapping with an adjacent residue, glutamate 662, normalized the receptor physiological behavior and reversed the deficits in assembly and degradation, but only partially restored plasma membrane expression of the receptors. These results reveal a new role for this linker domain in glutamate receptor biogenesis and contribute to understanding the cellular controls of receptor assembly and trafficking, which will be important for relating receptor stoichiometry to their neuronal targeting and function.

Ion channel modulation as the basis for general anesthesia.

(1) Modulation of the function of the GABA(A) and neuronal nicotinic acetylcholine receptor channels caused by general anesthetics and modulation of the GABA(A) receptor-channel by halothane, enflurane, isoflurane, and n-octanol was channel state-dependent. (3) Halothane modulation of the GABA(A) receptor was independent of subunits, but n-octanol modulation was subunit-dependent. (4) Ethanol at 30-100 microM was very potent in accelerating the desensitization of currents induced by acetylcholine. (5) The ethanol modulation was subunit- and state-dependent, occurring in the alpha3beta4 combination but only weakly in the alpha3beta2 combination. (6) In contrast, halothane at 430 microM (approximately 1 MAC) potently suppressed ACh-induced currents in the alpha3beta2 subunit combination.

Effects of the Nootropic Drug Nefiracetam on the GABAA Receptor-channel Complex in Dorsal Root Ganglion Neurons

The effects of nefiracetam on GABA-induced chloride currents were studied with rat dorsal root ganglion neurons in primary culture using the whole-cell patch-clamp technique. The dose-response curve for GABA-induced currents was shifted by 16 microM to lower concentrations by 10 microM nefiracetam while the maximal response was reduced by 22.84 +/- 0.68%. Thus at a low concentration (10 microM) of GABA, the chloride currents were potentiated by nefiracetam in a concentration-dependent manner. With 10 microM nefiracetam, the potentiation occurred slowly and the recovery after washout was also slow. The desensitization of the GABAA receptor at high concentration (100 microM) of GABA was accelerated by nefiracetam. The recovery process of chloride currents from desensitization was not affected by nefiracetam. KT 5720 (0.56 microm), a specific protein kinase A (PKA) inhibitor, blocked the transient potentiation of GABA-activated currents by nefiracetam, but did not affect the acceleration of desensitization. Nefiracetam suppression of GABA-induced currents was also abolished by KT 5720 or the pertussis toxin. Thus, nefiracetam may inhibit Gi/G(o) proteins leading to a cascade of events that increase the intracellular cAMP level, activate the PKA system, and suppress GABA-induced currents. Nefiracetam-induced transient potentiation and acceleration of desensitization of GABA-induced currents may involve other pathways. The nefiracetam modulation of the GABAA receptor function will result in a nootropic effect on the central nervous system through modification of synaptic transmission.