No abstract

Benzodiazepines are positive allosteric modulators of the GABA receptor (GABAR), acting at the α-γ subunit interface to enhance GABAR function. GABA or benzodiazepine binding induces distinct conformational changes in the GABAR. The molecular rearrangements in the GABAR following benzodiazepine binding remain to be fully elucidated. Using two molecular models of the GABAR, we identified electrostatic interactions between specific amino acids at the α-γ subunit interface that were broken by, or formed after, benzodiazepine binding. Using two-electrode voltage clamp electrophysiology in oocytes, we investigated these interactions by substituting one or both amino acids of each potential pair. We found that Lys in the α subunit forms an electrostatic bond with Asp of the γ subunit after benzodiazepine binding and that this bond stabilizes the positively modified state of the receptor. Substitution of these two residues to cysteine and subsequent covalent linkage between them increased the receptor's sensitivity to low GABA concentrations and decreased its response to benzodiazepines, producing a GABAR that resembles a benzodiazepine-bound WT GABAR. Breaking this bond restored sensitivity to GABA to WT levels and increased the receptor's response to benzodiazepines. The α Lys and γ Asp interaction did not play a role in ethanol or neurosteroid modulation of GABAR, suggesting that different modulators induce different conformational changes in the receptor. These findings may help explain the additive or synergistic effects of modulators acting at the GABAR.

The glycine receptor is a pentameric ligand-gated ion channel that is involved in fast inhibitory neurotransmission in the central nervous system. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low-micromolar concentrations and inhibiting its effects at higher concentrations. Low-nanomolar concentrations of contaminating zinc in electrophysiological buffers are capable of synergistically enhancing receptor modulation by other compounds, such as ethanol. This suggests that, unless accounted for, previous studies of glycine receptor modulation were measuring the effects of modulator plus comodulation by zinc on receptor function. Since zinc is present in vivo at a variety of concentrations, it will influence glycine receptor modulation by other pharmacologic agents. We investigated the utility of previously described "zinc-enhancement-insensitive" 1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. We found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using1 W170S receptors, we established that, in addition to ethanol, zinc interacts with inhalants, but not volatile anesthetics, to synergistically enhance channel function. Additionally, we determined that this interaction is abolished at higher zinc concentrations when receptor-enhancing binding sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc, in addition to enhancing channel function on their own.

Ethanol and psychostimulant use disorders exhibit comorbidity in humans and cross‐sensitization in animal models, but the neurobiological underpinnings of this are not well understood. Ethanol acutely increases dopamine neuron excitability, and psychostimulants such as cocaine or methamphetamine increase extracellular dopamine through inhibition of uptake through the dopamine transporter (DAT) and/or vesicular monoamine transporter 2 (VMAT2). Psychostimulants also depress dopamine neuron activity by enhancing dendritic dopamine neurotransmission. Here, we show that mice with a previous history of ethanol drinking are more sensitive to the locomotor‐stimulating effects of a high dose (5 mg/kg), but not lower doses (1 and 3 mg/kg) of methamphetamine or any tested dose of cocaine (3, 10, and 18 mg/kg), compared with water‐drinking controls. We next investigated the impact of a history of ethanol drinking, in a separate group of mice, on methamphetamine‐ or cocaine‐induced enhancement of dendritic dopamine transmission using whole‐cell voltage clamp electrophysiology in mouse brain slices. Methamphetamine, applied at a concentration (10 μM) that affects both DAT and VMAT2, enhanced D2 receptor‐mediated inhibitory postsynaptic currents (D2‐IPSCs) in both groups, but this effect was blunted in mice with a history of ethanol drinking. As methamphetamine action at VMAT2 disrupts dopamine neurotransmission, these results may suggest enhanced action of methamphetamine at VMAT2. Furthermore, there were no differences in low‐dose methamphetamine or cocaine‐induced enhancement of D2‐IPSCs, suggesting intact DAT function. Disruption of methamphetamine‐induced enhancement of dendritic dopamine transmission would result in decreased inhibition of dopamine neurons, ultimately increasing downstream release and the behavioral effects of methamphetamine.