The potentiation of glycine-induced responses by ethanol (EtOH) was studied in neurons freshly dissociated from the ventral tegmental area (VTA) of 5- to 14-day-old postnatal rats using whole-cell and gramicidin-perforated patch-clamp techniques. Under current-clamp conditions, EtOH increased glycine-induced membrane depolarization and action potential firing. Under voltage-clamp conditions, EtOH (0.1-40mM) alone did not elicit a current. When coapplied with glycine, EtOH enhanced the glycine-induced current in 35% (180 of 474) of the neurons. The EtOH-induced enhancement of glycine current was independent of membrane potential (between 260 and 160 mV); the reversal potential was not changed. Concentration-response analysis showed that in the presence of EtOH (10 mM), the EC50 for glycine decreased from 25 6 4 to 14 6 3 mM; the Hill coefficient increased from 1.5 6 0.2 to 1.9 6 0.3. Kinetic analysis of glycine currents indicated that EtOH decreased the time constant of activation and increased the time constant of deactivation of glycine-gated chloride channels. EtOH may accelerate glycine association with its receptor at the agonist binding site and increase the apparent agonist affinity. Our observations suggest that, at pharmacologically relevant concentrations, EtOH alters the function of glycine receptors and thus the excitability of neonatal VTA neurons. This action of EtOH may contribute to the neurobehavioral disturbances associated with fetal alcohol syndrome.

Previous work has shown that protonated taurine and aminosulfonate pH buffers, including HEPES, can directly and reversibly inhibit connexin channels that contain connexin26 (Cx26) (1). The structural requirements for this inhibition were explored by studies of the effects of structural analogs of taurine on the activity of Cx26-containing reconstituted hemichannels from native tissue. Several analogs inhibited the channels, with a range of relative affinities and efficacies. Each active compound contains a protonated amine separated from an ionized sulfonate or sulfinate moiety by several methylene groups. The inhibition is eliminated if the sulfonate/sulfinate moiety or the amine is not present. Compounds that contain a protonated amine but lack a sulfonate/sulfinate moiety do not inhibit, but competitively block the effect of the active compounds. Compounds that lack the protonated amine do not significantly inhibit or antagonize inhibition. The results suggest involvement of the protonated amine in binding and of the ionized sulfur-containing moiety in effecting the inhibition. Maximal effect of the inhibitory compounds is enhanced when a carboxyl group is linked to the α-carbon. Inhibition but not binding is stereospecific, with L-isomers being inhibitory, and the corresponding D-isomers being inactive, but able to antagonize inhibition by the L-isomers. While not all connexins are sensitive to aminosulfonates, the well-defined structural requirements described here argue strongly for a highly specific regulatory interaction with some connexins. The finding that cytoplasmic aminosulfonates inhibit connexin channels while other cytoplasmic compounds antagonize the inhibition suggests that gap junction channels are regulated by a complex interplay of cytoplasmic ligands.

We demonstrated previously that ethanol depresses glycine-induced currents in 45% of neurons freshly isolated from the ventral tegmental area (VTA) of rats (Ye et al., 2001b), and that protein kinase C (PKC) modulates this action of ethanol (Tao and Ye, 2002). In the present study, we investigated the time course of this effect of ethanol on VTA neurons from young rats. For 70% of the neurons in which ethanol reduced glycine-evoked currents, this depressant effect gradually diminished during continuous superfusion with ethanol. Its action decayed faster when ethanol was applied in several brief pulses than by continuous superfusion. On the other hand, the decay was especially slower when ethanol was applied in pulses at longer intervals or by preincubation. Phorbol ester 12,13-dibutyrate (PDBu, 1 μM), an activator of PKC, also depressed glycine-induced currents. In ～40% (6/15) of the neurons, the effect of PDBu diminished with time and was antagonized by the specific PKC inhibitor, chelerythrine (7μM). Chelerythrine also attenuated the ethanol-induced depression of glycine-induced currents and its time-dependent decay, thus confirming our previous evidence that PKC mediates, at least in part, the decay of the depressant effect of ethanol on glycine-induced currents of VTA neurons.

Tao, Liang, Yu Huang, and Jean-Pierre Bourreau. Control of the mode of excitation-contraction coupling by Ca2+ stores in bovine trachealis muscle. Am J Physiol Lung Cell Mol Physiol 279: L722–L732, 2000.—Full muscarinic stimulation in bovine tracheal smooth muscle caused a sustained contraction and increase in intracellular Ca2+ concentration ([Ca2+]i) that was largely resistant to inhibition by nifedipine. Depletion of internal Ca2+ stores with cyclopiazonic acid resulted in an increased efficacy of nifedipine to inhibit this contraction and the associated increase in [Ca2+]i. Thus internal Ca2+ store depletion promoted electromechanical coupling between full muscarinic stimulation and muscle contraction to the detriment of pharmacomechanical coupling. A similar change in coupling mode was induced by ryanodine even when it did not significantly modify the initial transient increase in [Ca2+]i induced by this stimulation, indicating that depletion of internal stores was not necessary to induce the change in excitation-contraction coupling mode. Blockade of the Ca2+-activated K1 channel by tetraethylammonium, charybdotoxin, and iberiotoxin all induced the change in excitation-contraction coupling mode. These results suggest that in this preparation, Ca2+ released from the ryanodine-sensitive Ca2+ store, by activating Ca2+-activated K1 channels, plays a central role in determining the expression of the pharmacomechanical coupling mode between muscarinic excitation and the Ca2+ influx necessary for the maintenance of tone.

1 The inhibitory effects of n-alcohols (methanol to dodecanol) on glycine-activated currents were studied in neurons freshly dissociated from the ventral tegmental area of neonatal rats using wholecell patch-clamp recording technique. 2 Ethanol enhanced and depressed glycine-activated currents in 35% and 45%, respectively, of neurons of ventral tegmental area of neonatal rats. In this report, we extended our focus of ethanolinduced inhibition of glycine currents to other straight-chain alcohols. 3 Aliphatic n-alcohols, which have carbon numbers less than nine, suppressed glycine currents in 45% (71/158) of the neurons. All results from this study are obtained from the 45% of cells displaying inhibition; the other 55% of the neurons were not studied. 4 Alcohol potency increased as the number of carbon atoms increased from one to five, and was at a maximal plateau from five to nine; alcohols with 10 or more carbons did not inhibit glycineactivated currents. Thus, a cutoff' point in their potency for inhibition of glycine receptor function occurred at about decanol. 5 A coapplication of dodecanol with ethanol eliminated the inhibition resulting from ethanol. Thus, dodecanol may bind to the receptor silently and compete with ethanol. 6 These observations indicate that straight-chain n-alcohols exhibit a cutoff' point in their potency for inhibition of the glycine receptor function between nine and 10 carbon atoms. The inability of longer alcohols to change the activation properties of the receptors may contribute to the cutoff effect.