title: Charge and geometry of residues in the loop 2 β hairpin differentially affect agonist and ethanol sensitivity in glycine receptors.
abstract: Recent studies highlighted the importance of loop 2 of α1 glycine receptors (GlyRs) in the propagation of ligand-binding energy to the channel gate. Mutations that changed polarity at position 52 in the β hairpin of loop 2 significantly affected sensitivity to ethanol. The present study extends the investigation to charged residues. We found that substituting alanine with the negative glutamate at position 52 (A52E) significantly left-shifted the glycine concentration response curve and increased sensitivity to ethanol, whereas the negative aspartate substitution (A52D) significantly right-shifted the glycine EC₅₀ but did not affect ethanol sensitivity. It is noteworthy that the uncharged glutamine at position 52 (A52Q) caused only a small right shift of the glycine EC₅₀ while increasing ethanol sensitivity as much as A52E. In contrast, the shorter uncharged asparagine (A52N) caused the greatest right shift of glycine EC₅₀ and reduced ethanol sensitivity to half of wild type. Collectively, these findings suggest that charge interactions determined by the specific geometry of the amino acid at position 52 (e.g., the 1-Å chain length difference between aspartate and glutamate) play differential roles in receptor sensitivity to agonist and ethanol. We interpret these results in terms of a new homology model of GlyR based on a prokaryotic ion channel and propose that these mutations form salt bridges to residues across the β hairpin (A52E-R59 and A52N-D57). We hypothesize that these electrostatic interactions distort loop 2, thereby changing agonist activation and ethanol modulation. This knowledge will help to define the key physical-chemical parameters that cause the actions of ethanol in GlyRs.
authors.affiliation: Alcohol and Brain Research Laboratories, Departments of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA, USA.
authors.forename: Daya I
authors.initials: DI
authors.lastname: Perkins
authors.forename: James R
authors.initials: JR
authors.lastname: Trudell
authors.forename: Liana
authors.initials: L
authors.lastname: Asatryan
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Davies
authors.forename: Ronald L
authors.initials: RL
authors.lastname: Alkana
mesh_terms: D000409:Alanine / Q000737:chemistry; D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D000431:Ethanol / Q000737:chemistry*; D005260:Female; D018698:Glutamic Acid / Q000737:chemistry; D005998:Glycine / Q000378:metabolism; D006801:Humans; D015640:Ion Channel Gating; D007473:Ion Channels / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D009865:Oocytes; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018009:Receptors, Glycine / Q000819:agonists* / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D014982:Xenopus laevis
chemical_list: D007473:Ion Channels; D018009:Receptors, Glycine; D001224:Aspartic Acid; D000431:Ethanol; D018698:Glutamic Acid; D000409:Alanine; D005998:Glycine