A single point mutation of the GABA(A) receptor alpha5-subunit confers fluoxetine sensitivity.
Fluoxetine has been reported to be a novel allosteric modulator of GABA(A) receptors with the notable exception of receptors that contain the alpha5-subunit isoform [Robinson, R.T., Drafts, B.C., Fisher, J.L., 2003. Fluoxetine increases GABA(A) receptor activity through a novel modulatory site. J. Pharmacol. Exp. Ther. 304, 978-984]. A mutagenic strategy has been used to investigate the structural basis for the insensitivity of this subunit. An alpha1/alpha5-subunit chimeragenesis approach first demonstrated the importance of the alpha1-subunit N-terminal sequence E165-D183 (corresponding to alpha5 E169-D187) in fluoxetine modulation. Specific amino acid substitutions in this domain subsequently revealed that a single mutation in the alpha5-subunit to the equivalent residue in alpha1 (T179A) was sufficient to confer fluoxetine sensitivity to the alpha5-containing receptor. However, the reciprocal mutation in the alpha1-subunit (A175T) did not result in a loss in sensitivity, suggesting the involvement of additional determinants for fluoxetine modulation. A comparative modeling approach was used to probe amino acids that may lie in close proximity to alpha1A175. This led serendipitously to the identification of a specific residue, alpha1F45, which, when mutated to an alanine, resulted in a significant decrease in potency for activation of the receptor by GABA and also reduced the efficacies of the partial agonists, THIP and P4S.
Derry
Jason M C
JM
Department of Pharmacology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. jasonder@cancerboard.ab.ca
Paulsen
Isabelle M
IM
Davies
Martin
M
Dunn
Susan M J
SM
D019943:Amino Acid Substitution; D000818:Animals; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004558:Electric Stimulation; D005473:Fluoxetine / Q000494:pharmacology*; D018755:GABA Agonists / Q000494:pharmacology; D007555:Isoxazoles / Q000494:pharmacology; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008958:Models, Molecular; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation / Q000502:physiology*; D009865:Oocytes; D018408:Patch-Clamp Techniques / Q000379:methods; D021122:Protein Subunits; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology*; D013329:Structure-Activity Relationship; D014982:Xenopus laevis; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology
D005473:Fluoxetine; D018755:GABA Agonists; D007555:Isoxazoles; D021122:Protein Subunits; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D005680:gamma-Aminobutyric Acid; C015542:gaboxadol