Mutation of sites in alpha subunits alters pharmacology and function of glycine and GABA[subscript A] receptors
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Glycine receptors (GlyR) are ligand-gated ion channels that conduct chloride in response to glycine and inhibit neurotransmission in the mammalian spinal cord and brainstem, and mutations in GlyR have been shown to cause human hyperekplexia (startle disease). GABAA receptors are also ligand-gated ion channels that conduct chloride and inhibit neurotransmission, and GABAA receptors share significant homology with GlyR. Point mutations at the putative alcohol and anesthetic binding site at the extracellular edge of transmembrane region 2 have previously been shown to eliminate the action of alcohol and anesthetics at GABAA receptors and GlyR. Here we demonstrate that mutation at the putative alcohol binding site in GABAA receptors can also nonspecifically alter allosteric modulation. Additionally, to determine if GlyR may be involved in the acute action of alcohol in vivo, we created transgenic mice expressing a mutant GlyR that possesses a mutation (S267Q) at the putative alcohol binding site that eliminates the effects of ethanol in vitro. Using several behavioral tests, these transgenic mice were less sensitive to the acute effects of ethanol, supporting the idea that ethanol action at GlyR is involved with some of alcohol’s acutely intoxicating effects in vivo. We also characterized a novel knock-in mouse harboring the alcohol-insensitive S267Q GlyR mutation. Mutant S267Q GlyR demonstrated altered function compared to wild-type GlyR in vitro, and mice homozygous for this mutation die within three weeks postnatally. Mice heterozygous for the S267Q mutation exhibit a more dramatic (dominant negative) hyperekplexia-like phenotype than mice heterozygous for the null mutation. These results demonstrate that the putative alcohol binding site in GlyR is also critically important for channel function and allosteric modulation, and this new mouse model of GlyR dysfunction will likely be useful for additional investigations regarding GlyR function in vivo.