Robert W. Peoples

Single-channel and whole-cell analysis of ethanol inhibition of NMDA-activated currents in cultured mouse cortical and hippocampal neurons

The effects of 0.1 to 500 mM ethanol on NMDA-activated currents were studied in primary cultures of mouse cortical and hippocampal neurons. In whole-cell recordings the IC50S for inhibition of NMDA-activated currents by ethanol were 129 mM +/- 20 mM in hippocampal neurons and 126 +/- 18 mM in cortical neurons. In single-channel recordings from excised outside-out patches of cortical neurons, ethanol inhibited total charge per minute with an IC50 of 174 +/- 23 mM, which was not significantly different from the IC50S for inhibition of whole-cell current. The reduction in mean open channel lifetime by ethanol was fit by the logistic equation with an apparent IC50 of 340 +/- 28 mM. Analysis of single-channel data indicated that ethanol inhibition of NMDA currents did not involve substantial changes in fast closed state kinetics, changes in open channel conductance, or block of the open channel. At the whole-cell IC50 of ethanol, mean open channel lifetime would decrease by 28% and frequency of opening would decline by 31% to account for the reduction in current. Single-channel data were consistent with ethanol being an allosteric modulator of gating which reduces agonist efficacy.

Ethanol inhibition of N‐methyl‐D‐aspartate‐activated current in mouse hippocampal neurones: whole‐cell patch‐clamp analysis

1 The action of ethanol on N‐methyl‐D‐aspartate (NMDA)‐activated ion current was studied in mouse hippocampal neurones in culture using whole‐cell patch‐clamp recording. 2 Ethanol inhibited NMDA‐activated current in a voltage‐independent manner, and did not alter the reversal potential of NMDA‐activated current. 3 Concentration–response analysis of NMDA‐ and glycine‐activated current revealed that ethanol decreased the maximal response to both agonists without affecting their EC50 values. 4 The polyamine spermine (1 μM) increased amplitude of NMDA‐activated current but did not alter the percentage inhibition of ethanol. 5 Compared to an extracellular pH of 7.0, pH 6.0 decreased and pH 8.0 increased the amplitude of NMDA‐activated current, but these changes in pH did not significantly alter the percentage inhibition by ethanol. 6 The sulphydryl reducing agent dithiothreitol (2 mM) increased the amplitude of NMDA‐activated current, but did not affect the percentage inhibition by ethanol. 7 Mg2+ (10, 100, 500 μM), Zn2+ (5, 20 μM) or ketamine (2, 10 μM) decreased the amplitude of NMDA‐activated current, but did not affect the percentage inhibition by ethanol. 8 The observations are consistent with ethanol inhibiting the function of NMDA receptors by a non‐competitive mechanism that does not involve several modulatory sites on the NMDA receptor–ionophore complex.

Ethanol inhibition of N-methyl-d-aspartate-activated ion current in rat hippocampal neurons is not competitive with glycine

The interaction of ethanol with glycine at the N-methyl-D-aspartate (NMDA)-activated ion channel was investigated in voltage-clamped rat cultured hippocampal neurons. As shown previously, glycine increased, and ethanol inhibited, the NMDA-activated current in these cells. Concentration-response data for glycine (0.1-100 microM) indicate that the inhibition of NMDA-activated current by ethanol does not involve a competitive interaction with glycine. Thus, ethanol appears to inhibit NMDA-activated current at a locus different from the glycine modulatory site.

Modulation of glycine-activated ion channel function by G-protein |[beta]||[gamma]| subunits

Glycine receptors (GlyRs), together with GABAA and nicotinic acetylcholine (ACh) receptors, form part of the ligand-activated ion channel superfamily and regulate the excitability of the mammalian brain stem and spinal cord. Here we report that the ability of the neurotransmitter glycine to gate recombinant and native ionotropic GlyRs is modulated by the G protein βγ dimer (Gβγ). We found that the amplitude of the glycine-activated Cl− current was enhanced after application of purified Gβγ or after activation of a G protein–coupled receptor. Overexpression of three distinct G protein α subunits (Gα), as well as the Gβγ scavenger peptide ct-GRK2, significantly blunted the effect of G protein activation. Single-channel recordings from isolated membrane patches showed that Gβγ increased the GlyR open probability (nPo). Our results indicate that this interaction of Gβγ with GlyRs regulates both motor and sensory functions in the central nervous system.

Alcohols inhibit N-methyl-d-aspartate receptors via a site exposed to the extracellular environment

N-Methyl-D-aspartate (NMDA) receptors are important CNS target sites of alcohols, but the site and mechanism of action of alcohols on NMDA receptors remains unclear. In CHO-K1 cells transfected with NR1/NR2B NMDA receptor subunits, ethanol inhibited NMDA-activated current with an IC(50) of 138 mM. Truncation of the intracellular C-terminal domain of the NR1 subunit (NR1T) did not alter ethanol sensitivity when combined with the NR2B subunit, but a similar truncation of the NR2B subunit (NR2BT) slightly enhanced ethanol sensitivity of receptors formed from coexpression with either NR1 or NR1T subunits. 1-Pentanol applied externally inhibited NMDA receptors with an IC(50) of 9.9 mM, but intracellular application of 1-pentanol (25 mM) did not alter NMDA receptor inhibition by externally applied ethanol or 1-pentanol. In addition, the amplitude of NMDA-activated current did not decrease during the time required for 1-pentanol (25 mM) to diffuse throughout the cytoplasm. Ethanol did not inhibit NMDA receptors when bath-applied in cell-attached patches or when applied to the cytoplasmic face of inside-out membrane patches. These results appear to be best explained by an action of alcohols on the NMDA receptor-channel protein, at a site located in a domain exposed to, or only accessible from, the extracellular environment.

Alcohol and Anesthetic Actions on Excitatory Amino Acid—Activated Ion Channels

The actions of alcohol and anesthetics have been studied on excitatory amino acid activated ion channels in mammalian neurons. Ethanol inhibits NMDA-activated current over a concentration range that produces intoxication, and the potency of several alcohols for inhibiting the NMDA-activated current is correlated with 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. Studies on the mechanism of ethanol inhibition of NMDA-activated current indicate that ethanol does not appear to block the ion channel, alter the ion selectivity of the channel, or interact with previously described binding sites on the NMDA receptor/ionophore complex. The linear relation between the potency of several alcohols for inhibiting the NMDA-activated current and the hydrophobicity of the alcohols suggests that ethanol may inhibit the NMDA-activated ion current by a novel type of interaction with a hydrophobic site associated with the NMDA channel. In addition, different types of general anesthetic agents exhibit different inhibitory actions on NMDA-, kainate-, and quisqualate-activated currents, suggesting that differences in the profile of inhibition of excitatory amino acid neurotransmission in the CNS among different classes of general anesthetics may contribute to the differences in their behavioral and physiological effects.

Volatile general anaesthetic actions on recombinant nAChα7, 5-HT3 and chimeric nAChα7-5-HT3 receptors expressed in Xenopus oocytes

The effect of halothane and isoflurane was studied on the function of recombinant neurotransmitter receptors expressed in Xenopus oocytes. Both anaesthetics inhibited nicotinic acetylcholine type α7 (nAChα7) receptor‐mediated responses, potentiated 5‐hydroxytryptamine type 3 (5‐HT3) receptor‐mediated responses at low agonist concentrations, and inhibited the function of a chimeric receptor (with the N‐terminal domain from the nAChα7 receptor and the transmembrane and C‐terminal domains from the 5‐HT3 receptor) in a manner similar to that of the nAChα7 receptor. Since the N‐terminal domain of the chimeric receptor was from the nAChα7 receptor, the observations suggest that the inhibition involves the N‐terminal domain of the receptor.

Ethanol-induced inhibition of a neuronal P2X purinoceptor by an allosteric mechanism

Ethanol inhibits a neuronal P2X purinoceptor by shifting the ATP concentration–response curve to the right in an apparently competitive manner. However, the underlying mechanism has not been determined. We investigated the effects of ethanol on the activation and deactivation time constants for ATP‐activated current in bullfrog dorsal root ganglion neurones. Ethanol decreased the time constant of deactivation of ATP‐gated ion channels without affecting the time constant of activation. The observations are not consistent with a competitive mechanism of inhibition by ethanol, but may be explained by an allosteric action of ethanol to decrease apparent agonist affinity. This represents a novel mechanism of action of ethanol on a neurotransmitter‐gated ion channel.

Trichloroethanol potentiation of γ-aminobutyric acid-activated chloride current in mouse hippocampal neurones

1 The action of 2,2,2‐trichloroethanol on γ‐aminobutyric acid (GABA)‐activated Cl− current was studied in mouse hippocampal neurones in tissue culture by use of whole‐cell patch‐clamp recording. 2 Trichloroethanol increased the amplitude of currents activated by 1 μm GABA or 0.1 μm muscimol. Trichloroethanol, 1–25 mm, potentiated current activated by 1 μm GABA in a concentration‐dependent manner with an EC50 of 3.0 ± 1.4 mm and a maximal response (Emax) of 576 ± 72% of control. 3 Trichloroethanol potentiated currents activated by GABA concentrations < 10 μm, but did not increase the amplitude of currents activated by concentrations of GABA ≥ 10 μm. Despite marked potentiation of currents activated by low concentrations of GABA, trichloroethanol did not significantly alter the EC50, slope, or Emax of the GABA concentration‐response curve. 4 Trichloroethanol, 5mM, potentiated GABA‐activated current in neurones in which ethanol, 10–500 mm, did not. The effect of trichloroethanol was not altered by the putative ethanol antagonist, Ro 15–4513. Trichloroethanol did not potentiate currents activated by pentobarbitone. 5 In the absence of exogenous GABA, trichloroethanol at concentrations ≥ 2.5 mm activated a current that appeared to be carried by Cl− as its reversal potential changed with changes in the Cl− gradient and as it was inhibited by the GABAA antagonists, bicuculline methiodide and picrotoxin. 6 Since trichloroethanol is thought to be the active metabolite of chloral hydrate and other chloral derivative anaesthetics, potentiation of the GABA‐activated current in central nervous system neurones by trichloroethanol may contribute to the sedative/hypnotic effects of these agents.

Distinct ATP-activated currents in different types of neurons dissociated from rat dorsal root ganglion

Rat dorsal root ganglion neurons can be classified into at least three distinct groups based on cell size, afferent fiber diameter, electrophysiological properties, sensitivity to vanilloid agonists such as capsaicin, and function. In the present study, ATP-activated current in these neurons was characterized using whole-cell patch-clamp recording. Small diameter (<30 microm) cells had high capsaicin sensitivity, high affinity for ATP, and rapidly desensitizing ATP-activated current. Medium diameter (30-50 microm) cells had no capsaicin sensitivity, lower affinity for ATP and slowly desensitizing ATP-activated current. Large diameter (>50 microm) cells were insensitive to both capsaicin and ATP. These findings suggest that distinct types of ATP receptor-ion channels are expressed in different types of dorsal root ganglion neurons, and may contribute to the functional differences among these types of neurons.