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The ion current induced by the glutamate receptor agonist N-methyl-D-aspartate (NMDA) in voltage-clamped hippocampal neurons was inhibited by ethanol (EtOH). Inhibition increased in a concentration-dependent manner over the range 5 to 50 mM, a range that also produces intoxication. The amplitude of the NMDA-activated current was reduced 61 percent by 50 mM EtOH; in contrast, this concentration of EtOH reduced the amplitude of current activated by the glutamate receptor agonists kainate and quisqualate by only 18 and 15 percent, respectively. The potency for inhibition of the NMDA-activated current by several alcohols is linearly related to their intoxicating potency, suggesting that alcohol-induced inhibition of responses to NMDA receptor activation may contribute to the neural and cognitive impairments associated with intoxication.

The effect of ethanol on the function of P2X4 receptors expressed in Xenopus oocytes was studied using two‐electrode voltage‐clamp recording. The amplitude of current activated by 1 μM ATP was decreased by ethanol in a concentration‐dependent manner over the concentration range 1–500 mM. The concentration of ethanol that produced 50% inhibition (IC50) of current activated by 1 μM ATP was 58 mM. Ethanol inhibition of ATP‐activated current was not dependent on membrane potential from −60 to +20 mV, and ethanol did not change the reversal potential of ATP‐activated current. Ethanol, 50 mM, shifted the ATP concentration‐response curve to the right, increasing the EC50 for ATP from 9.1 to 16.0 μM, but did not reduce the maximal response to ATP. The results suggest that ethanol may inhibit P2X4 receptors by decreasing the apparent affinity of the binding site for ATP. Since the P2X4 receptor is the most abundant P2X subunit in the brain, these receptors could be important effectors of ethanol action in the central nervous system. British Journal of Pharmacology (2000) 130, 1394–1398; doi:10.1038/sj.bjp.0703439

1 We investigated ethanol inhibition of the rat P2X4 receptor and the contribution of the three histidine residues in the extracellular loop of this receptor to ethanol inhibition of receptor function, using site‐directed mutagenesis and electrophysiological characterization of recombinant receptors. 2 In the wild‐type receptor, 50, 200 and 500 mM ethanol increasingly shifted the ATP concentration–response curve to the right in a parallel manner, increasing the EC50 value without affecting Emax. However, 750 or 900 mM ethanol did not produce a further increase in the EC50 value of the ATP concentration–response curve, suggesting that this inhibition is not competitive. 3 The P2X4 receptor mutations H140A and H286A did not significantly alter ethanol inhibition of ATP‐activated current. By contrast, the mutation H241A changed the mechanism by which ethanol inhibits receptor function; viz., ethanol inhibition was not associated with an increased EC50 value of the ATP concentration–response curve, instead, ethanol decreased the maximal response to ATP without affecting the EC50 value of the ATP concentration–response curve. 4 Ethanol inhibition of the H241A mutant was voltage independent between −60 and +20 mV and ethanol did not alter the reversal potential of ATP‐activated current. In addition, ethanol decreased the desensitization rate of the H241A‐mediated current. 5 The purinoceptor antagonists, suramin and pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), did not alter the magnitude of ethanol inhibition of ATP‐activated current in the H241A mutant. 6 The results suggest that ethanol inhibits the wild‐type rat P2X4 receptor by an allosteric action to increase the EC50 value of the ATP concentration–response curve, the P2X4 receptor mutation H241A alters the mechanism by which ethanol inhibits P2X4 receptor function, and ethanol and PPADS or suramin appear to inhibit H241A‐mutated receptors at independent sites. British Journal of Pharmacology (2005) 145, 576–586. doi:10.1038/sj.bjp.0706192

The role of protein kinase C in regulating Ca2 + channel activity was investigated using the whole-cell patch-clamp technique in the mouse pituitary tumor cell line AtT-20. The Ca2 + current was activated by depolarizing voltage steps from a holding potential of –80 mV. Extracellular application of the protein kinase C activator 1-oleoyl-2-acetylglycerol (OAG) reduced voltage-dependent Ca2 + current. This effect was reversible and dose dependent (10–100 µM). Pertussis toxin did not block the effect of OAG on Ca2 + current, suggesting that OAG does not affect Ca2 + channels via a pertussis toxin sensitive guanosine triphosphate binding protein. Na + -free solutions did not block the effect of OAG on Ca2 + channels, suggesting that this effect of OAG does not involve the Na+/H+ antiporter. The phorbol esters 12-deoxyphorbol-13-isobutyrate (10 µM) and phorbol-12,13-diacetate (100 µM) also reduced Ca2 + current. The results suggest that protein kinase C may be an inhibitory regulator of voltage-dependent Ca2 + channels.

As the number of carbon atoms in an aliphatic n-alcohol is increased from one to five, intoxicating potency, lipid solubility, and membrane lipid disordering potency all increase in a similar exponential manner. However, the potency of aliphatic n-alcohols for producing intoxication reaches a maximum at six to eight carbon atoms and then decreases. The molecular basis of this \"cutoff\" effect is not understood, as it is not correlated with either the lipid solubility or the membrane disordering potency of the alcohols, which continue to increase exponentially. Since it has been suggested that inhibition of N-methyl-D-aspartate (NMDA) receptors by alcohols may play a role in alcohol intoxication, we investigated whether a series of aliphatic n-alcohols would exhibit a cutoff in potency for inhibition of NMDA receptors. We found that although potency for inhibition of NMDA receptors increased exponentially for alcohols with one to five carbon atoms, potency for inhibition of NMDA receptors reached a maximum at six to eight carbon atoms and then abruptly disappeared. This cutoff for alcohol inhibition of NMDA receptors is consistent with an interaction of the alcohols with a hydrophobic pocket on the receptor protein. In addition, the similarity of the cutoffs for alcohol inhibition of NMDA receptors and alcohol intoxication suggests that the cutoff for NMDA receptor inhibition may contribute to the cutoff for alcohol intoxication, which is consistent with an important role of NMDA receptors in alcohol intoxication.

The whole-cell patch-clamp technique was used in a study of the modulation of the ATP-gated cation channel by zinc2positive in mammalian neurons.

For almost a century, alcohols have been thought to produce their effects by actions on the membrane lipids of central nervous system neurons-the well known \"lipid theory\" of alcohol action. The rationale for this theory is the correlation of potency with oil/water or membrane/buffer partition coefficient. Although a number of recent studies have shown that alcohols can affect the function of certain neuronal neurotransmitter receptors, there is no evidence that the alcohols interact directly with these membrane proteins. In the present study, we report that inhibition of a neuronal neurotransmitter receptor, an ATP-gated ion channel, by a series of alcohols exhibits a distinct cutoff effect. For alcohols with a molecular volume of ≤≤42.2 ml/mol, potency for inhibiting ATP-activated current was correlated with lipid solubility (order of potency: 1-propanol = trifluoroethanol > monochloroethanol > ethanol > methanol). However, despite increased lipid solubility, alcohols with a molecular volume of ≥46.1 ml/mol (1-butanol, 1-pentanol, trichloroethanol, and dichloroethanol) were without effect on the ATP-activated current. The results suggest that alcohols inhibit the function of this neurotransmitter receptor by interacting with a small hydrophobic pocket on the receptor protein.

The slow excitatory postsynaptic potential (EPSP) was investigated in frog sympathetic ganglion cells. In contrast to the increased conductance associated with other known EPSP's, during the slow EPSP resting membrane conductance was decreased. Electrical depolarization of the membrane potentiated the slow EPSP, whereas progressive hyperpolarization decreased its size and then reversed it to a hyperpolarizing potential (the opposite of the effect of membrane polarization on other EPSP's). The reversal potential of the slow EPSP was close to the potassium equilibrium potential. We propose that the slow EPSP, in contrast to classical EPSP's, is generated by an inactivation of resting potassium conductance.

Despite the increasing recognition that ATP is an important extracellular excitatory mediator in the nervous system, the regulation of ATP receptors is poorly understood. Because the extracellular Zn2+concentration is regulated in a variety of biological tissues, we studied modulation of the ATP-gated cation channel by Zn2+in mammalian neurons using the whole-cell patch-clamp technique. In ≈73% of cells tested, the amplitude of ATP-activated membrane ion current increased up to 5-fold in the presence of micromolar concentrations of Zn2+. The characteristics of this action suggest that Zn2+increases the apparent affinity of the receptor for ATP. In addition, Zn2+increased membrane depolarization and action potential firing elicited by ATP. These observations suggest that Zn2+may play a physiological role in regulating the excitatory action of ATP on mammalian neurons.

Brief stimulation of cholinergic preganglionic nerve fibers resulted in an increase in guanosine 3′, 5′ monophosphate (cyclic GMP) in the bullfrog sympathetic ganglion. When the release of synaptic transmitter was prevented by a high-magnesium, low-calcium Ringer solution, stimulation of preganglionic nerve fibers did not increase cyclic GMP in the ganglion. The increase in cyclic GMP caused by preganglionic stimulation was also blocked by the muscarinic antagonist, atropine. The data indicate that the increase in cyclic GMP is associated with synaptic transmission and support the possibility that cyclic GMP may mediate the postsynaptic action of acetylcholine at muscarinic cholinergic synapses.