Multiple sites of ethanol action in α1 and α2 glycine receptors suggested by sensitivity to pressure antagonism

Abstract

The current study used an ethanol antagonist, increased atmospheric pressure, to test the hypothesis that ethanol acts on multiple sites in glycine receptors (GlyRs). The effects of 12 times normal atmospheric pressure of helium-oxygen gas (pressure) on ethanol-induced potentiation of GlyR function in Xenopus oocytes expressing human a1, a2 or the mutant a1(A52S) GlyRs were measured using two-electrode voltage clamp. Pressure reversibly antagonized potentiation of glycine in a1 GlyR by 40-200 mM ethanol, but did not antagonize 10 and 25 mM ethanol in the same oocytes. In contrast, pressure did not significantly affect potentiation of glycine by 25-100 mM ethanol in a2 GlyRs, nor did pressure alter ethanol response in the A52S mutant. Pressure did not affect baseline receptor function or response to glycine in the absence of ethanol. These findings provide the first direct evidence for multiple sites of ethanol action in GlyRs. The sites can be differentiated on the basis of ethanol concentration, subunit and structural composition and sensitivities to pressure antagonism of ethanol. Parallel studies with butanol support this conclusion. The mutant a1(A52S) GlyR findings suggest that increased attention should be focused on the amino terminus as a potential target for ethanol action.

Charge and Geometry of Residues in the Loop 2 β Hairpin Differentially Affect Agonist and Ethanol Sensitivity in Glycine Receptors

“…Previous work found that ethanol potentiation of GlyR function is more robust and reliable when tested in the presence of low concentrations of glycine (typically EC 2-10 ) (Davies et al, 2004;Perkins et al, 2008). This is because the largest effects of potentiation are seen at low agonist concentrations and are essentially masked at I max .…”

“…Oocytes expressing WT or mutant ␣1GlyRs were exposed to glycine for 30 s, using 5-to 15-min washouts between applications to ensure complete resensitization (Mascia et al, 1996a,b;Davies et al, 2004;Crawford et al, 2007). Pilot experiments found that WT and mutant GlyR agonist responses, using a 1-min glycine application, reached a steady-state equilibrium with results that did not differ appreciably from results using 30-s applications.…”

“…Based on these studies, we used a concentration of glycine producing 2 Ϯ 0.3% of the maximal effect (EC 2 ). When testing ethanol potentiation, the oocytes were preincubated with ethanol for 60 s before coapplication of ethanol and glycine (Davies et al, 2004). Washout periods (5-15 min) were allowed between drug applications to ensure complete resensitization of receptors.…”

“…Experiments using molecular strategies combined with a novel ethanol antagonist, increased atmospheric pressure, pointed to extracellular domain loop 2 of GlyRs as an important target for ethanol action (Davies et al, 2004;Perkins et al, , 2010. Other studies, using the substituted cysteine accessibility method (Karlin and Akabas, 1998) with the alcohol-like reagent propyl methanethiosulfonate (MTS), identified position 52 (Ala52) in loop 2 of the extracellular domain (Crawford et al, 2007) and position 267 (Ser267) in the transmembrane (TM) domain (Mihic et al, 1997) as sites of ethanol action in GlyRs.…”

Glycine and GABAAUltra-Sensitive Ethanol Receptors as Novel Tools for Alcohol and Brain Research

“…Prior studies identified extracellular loop 2 of GlyRs and GABA A Rs as a site of ethanol action, and found that structural modifications in this region profoundly influence receptor sensitivity to ethanol (Mascia et al, 1996b;Davies et al, 2003Davies et al, , 2004Crawford et al, 2007;Perkins et al, 2008). This initial work found that replacing loop 2 of a1 GlyR and g2 GABA A R subunits with loop 2 of the more ethanol-sensitive d GABA A Rs significantly increased ethanol sensitivity of the resultant receptor (Perkins et al, 2009), and identified important physical-chemical properties of loop 2 that alter receptor sensitivity to ethanol and agonist Perkins et al, 2012).…”