Alcohol modulation of GABAA receptors : subunit requirements and structure-activity relationships

Whitten, Rhonda Joyce (1995) Alcohol modulation of GABAA receptors : subunit requirements and structure-activity relationships. Masters thesis, Memorial University of Newfoundland.

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Abstract

Alcohols are believed to exert their behavioural effects at least in part by a selective enhancement of γ-aminobutyric acid type A (GABAA) receptor activity. However, the mechanism(s) by which ethanol and other alcohols facilitate GABAA receptor function have remained elusive. Historically, alcohols have been thought to act by partitioning into membrane lipids to cause changes in the function of membrane proteins. However, more recent evidence suggests that alcohols interact selectively with hydrophobic regions of specific membrane proteins or phospholipids to alter their function. If a drug-protein interaction is involved in the effects of alcohols on GABAA receptor function, it was hypothesized that stereoisomers of a simple straight-chain alcohol might show differential effects on receptor function. Alternatively, there may be differences in the effects of closely related alcohols that are not predicted by differences in lipid solubility, and/or a significant influence of the subunit composition of the GABAA receptor itself. To test these hypotheses concerning the mechanisms of action of alcohols, cloned subunits of human GABAA receptors were expressed in Xenopus laevis oocytes, and the two-electrode voltage-clamp technique was used to quantify the membrane current response to GABA in the presence and absence of different alcohols. -- 1-butanol and 2-butanol differentially potentiated GABA responses in both α₁β₂γ₂L and α₂β₂γ₂L receptor constructs, with 2-butanol being significantly more potent than 1-butanol as a modulator of GABAA receptor function. However, 2-butanol has a lower membrane/buffer partition coefficient, and is therefore less lipid soluble than 1-butanol. Thus, the structure of the alcohol, rather than simple lipid solubility, was more important as a predictor of modulation of GABAA receptor function. In contrast, the stereoisomers (R)-2-butanol and (S)-2-butanol did not differ in their modulation of receptor function. 1-butanol and 2-butanol were also significantly more potent as modulators at α₂β₂γ₂L compared to α₁β₂γ₂L GABAA receptor constructs. Thus, the subunit composition of the receptor protein also influences modulation of receptor function by alcohols. The γ₂L subunit, which is reported to be necessary for ethanol to potentiate GABAA receptor activity, was not required for 1-butanol or 2-butanol to increase GABA-mediated responses. Low concentrations (20 mM) of ethanol potentiated GABAA receptor function at α₁β₂γ₂L receptors. Ethanol potentiation of GABAA receptor function was blocked by 0.5 μM Ro15-4513, a benzodiazepine receptor partial inverse agonist. However, Ro15-4513 at concentrations up to 5 μM did not block potentiation of GABAA receptor activity by butanol, heptanol, or the non-volatile anesthetic propofol. -- Differential effects of alcohols on GABAA receptor function in response to changes in subunit composition or structure of the alcohol suggest specific interactions of these agents with the receptor complex. In addition, it appears that longer-chain (≥ 4 carbons) alcohols act at a different site and/or induce a different conformational change in the receptor compared to ethanol. In conclusion, these data support the idea that alcohols have specific interactions with the GABAA receptor, rather than simply disordering membrane lipids. In addition, there may be at least two distinct mechanisms and/or sites by which alcohols can act to modulate activity at the GABAA receptor.

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