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.

Interleukin (IL)-2 is a brain-derived cytokine that influences mesocorticolimbic dopamine release, and is associated with pathological outcomes that are mediated, at least in part, by aberrations in mesolimbic neurotransmission. The mechanisms by which IL-2 modulates mesolimbic transmission, however, are not known. The NMDA receptor/channel (NMDAR) plays an essential role in neuronal excitability of mesolimbic neurons; we thus examined in neonatal rats the effects of IL-2 on NMDA-activated current (I) in NMDA voltage-clamped neurons freshly isolated from the ventral tegmental area (VTA), the site of origin of the mesolimbic system. IL-2 (0.01-500ng/ml) alone had no effect on membrane conductance. When co-applied with NMDA, IL-2 (50-500ng/ml) significantly potentiated I. In contrast, doses as low as 0.01ng/ml markedly decreased the NMDA response. Dose–response analysis showed that NMDA IL-2 (.50 ng/ml) increased the maximal I, without changing the EC, indicating that IL-2 potentiates I by increasing the NMDA 50 NMDA efficacy of the NMDAR. Moreover, current-voltage analysis revealed that IL-2 potentiation of I was voltage-dependent, being NMDA greater at negative potentials. In contrast, IL-2 inhibition of I was voltage-independent, and IL-2 did not alter the reversal potential. NMDA Additionally, IL-2 (1ng/ml) shifted the NMDA concentration-response curve to the right, significantly increasing the EC for NMDA 50 without changing the maximal I, suggesting that IL-2 inhibits the NMDAR by a competitive mechanism. IL-2 thus acts as a potent NMDA modulator of the NMDAR. IL-2-induced alterations of responses to NMDAR activation may contribute to synaptic plasticity in the mesolimbic system and to pathological outcomes associated with this system.

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.

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.