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.

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.

The brain is particularly sensitive to alcohol during its growth spurt period. To better understand the mechanism(s) involved, we studied the effects of ethanol on neurons freshly dissociated from the ventral tegmental area (VTA) in neonatal rats. Ethanol enhanced (35%) or depressed (45%) glycine-induced responses in VTA neurons (Ye et al., 2001a, 2001b). In this report, we investigated the role of protein kinase C (PKC) and protein kinase A (PKA) in ethanol-induced inhibition of glycine-activated current, using whole-cell patch-clamp technique. Ethanol inhibited glycine-activated current when it was coapplied with the agonist. This inhibition was enhanced when neurons were pretreated with ethanol before the subsequent coapplication of ethanol and glycine. Ethanol's inhibition of glycine-activated currents increased with the length of ethanol pretreatment time (ranging from 1 to 30 s), and reached the maximum at 30 s. However, this enhanced inhibition was not seen in the absence of internal ATP. In addition, phorbol-12-myristate-13-acetate (PMA, 100 nM), a PKC activator, markedly inhibited glycineactivated current. Blockade of PKC by chelerythrine or by PKC inhibitor peptide significantly attenuated ethanol-induced inhibition. Although partial increase of PKC activity by 1 nM PMA enhanced ethanol inhibition, pretreatment of ethanol did not increase ethanol inhibition after the neurons were treated with 100 nM PMA. These data suggest that ethanol and PKC share the same pathway to suppress glycine receptors. H-89 (1

The brain is particularly sensitive to alcohol during the period of its rapid growth. To better understand the mechanism(s) involved, we studied ethanol effects on glycine-activated responses of ventral tegmental area (VTA) neurons isolated from the newborn rat, using whole cell and gramicidin perforated patch-clamp techniques. Previously we reported that 0.1-40m M ethanol enhances glycineinduced responses of 35% of VTA neurons (Ye et al. 2001). We now direct our attention to the inhibitory effects of ethanol observed in 45% (312 of 694) of neonatal VTA neurons. Under current-clamp conditions, 1 mM ethanol had no effect on the membrane potential of these cells, but it decreased glycine-induced membrane depolarization and the frequency of spontaneous action potentials. Under voltageclamp conditions, 0.1-10 mM ethanol did not elicit a current but depressed the glycine-induced currents. The ethanol-induced inhibition of glycine current was independent of membrane potential (between 260 and 160 mV). Likewise, ethanol did not alter the reversal potential of the glycine-activated currents. Ethanol-mediated inhibition of glycine current depended on the glycine concentration. While ethanol strongly depressed currents activated by 30μM glycine, it had no appreciable effect on maximal currents activated by 1 mM glycine. In the presence of ethanol (1 mM), the EC50 for glycine increased from 32±5 to 60±3μM. Thus ethanol may decrease the agonist affinity of glycine receptors. A kinetic analysis indicated that ethanol shortens the time constant of glycine current deactivation but has no effect on activation. In conclusion, by altering VTA neuronal function, ethanol-induced changes in glycine receptors may contribute to neurobehavioral manifestations of the fetal alcohol syndrome.