Evidence of inter- and intra-subunit alcohol and anesthetic binding cavities in the glycine receptor

Alcohol is abundantly consumed by society and general anesthetics are used everyday in operating suites throughout the world, yet the sites and mechanisms of action for these drugs are not completely understood. Glycine receptors (GlyRs) are pentameric ion channels expressed throughout the brain and spinal cord and have become increasingly popular targets in the study of alcohol action. Each GlyR subunit is composed of four alpha helical transmembrane segments (TM1-4), and although amino acids involved with alcohol action have been previously identified in TM1-4, the orientation of each of these residues with respect to a putative alcohol/anesthetic binding cavity remains controversial. In order to better characterize this binding cavity within the GlyR, we conducted a series of experiments using cysteine mutagenesis and biochemical cross-linking. In Aim 1, the participation of TM1 with TM3 in a common alcohol/anesthetic binding cavity was further investigated. We used two-electrode voltage clamp electrophysiology in Xenopus oocytes to demonstrate the ability of A288 in TM3 to form cross-links with I229 in TM1, which reduced the ability of both alcohol and anesthetics to modulate GlyR function. Aim 2 investigated whether TM3 could also participate in a binding cavity with TM4. We have shown that residues in TM4 are able to form cross-links with A288 in TM3, and found that cross-linking between TM3 and those residues in TM4 also reduced the ability of alcohol and anesthetics to enhance GlyR function. Aim 3 determined whether these cross-links are formed between residues within the same subunit (intra-subunit) or between subunits (inter-subunit), and ultimately whether these residues participate in a common alcohol/anesthetic binding cavity within or between GlyR subunits. GlyR protein, which measures about 50 kDa, was extracted from oocytes injected with the cysteine mutants, and immunoblotting was used with a GlyR-specific antibody to subsequently help quantify band ratios between cross-linked and uncross-linked conditions. We found an increase in the 100:50 kDa band ratio for the TM1-3 mutant only, but not TM3-4 mutant or the wild-type, which suggests TM1-3 may participate in an alcohol binding cavity between GlyR subunits while TM3-4 may contribute to a binding cavity within a subunit.