title: Methylene blue inhibits GABAAreceptors by interaction with GABA binding site.
abstract: Methylene blue (MB) is commonly used in diagnostic procedures and is also used to treat various medical conditions. Neurological effects of MB have been reported in clinical observations and experimental studies. Thus the modulation of GABAAreceptor function by MB was investigated. Whole-cell GABA-activated currents were recorded from HEK293 cells expressing various GABAAreceptor subunit configurations. MB inhibition of GABA currents was apparent at 3 μM, and it had an IC50of 31 μM in human α1β2γ2 receptors. The MB action was rapid and reversible. MB inhibition was not mediated via the picrotoxin site, as a mutation (T6'F of the β2 subunit) known to confer resistance to picrotoxin had no effect on MB-induced inhibition. Blockade of GABAAreceptors by MB was demonstrated across a range of receptors expressing varying subunits, including those expressed at extrasynaptic sites. The sensitivity of α1β2 receptors to MB was similar to that observed in α1β2γ2 receptors, indicating that MB's action via the benzodiazepine or Zn2+site is unlikely. MB-induced inhibition of GABA response was competitive with respect to GABA. Furthermore, mutation of α1 F64 to A and β2 Y205 to F in the extracellular N-terminus, both residues which are known to comprise GABA binding pocket, remarkably diminished MB inhibition of GABA currents. These data suggest that MB inhibits GABAAreceptor function by direct or allosteric interaction with the GABA binding site. Finally, in mouse hippocampal CA1 pyramidal neurons, MB inhibited GABA-activated currents as well as GABAergic IPSCs. We demonstrate that MB directly inhibits GABAAreceptor function, which may underlie some of the effects of MB on the CNS.
authors.affiliation: Center for Neuroscience Discovery, Institute of Healthy Aging, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, United States.
authors.forename: Zhenglan
authors.initials: Z
authors.lastname: Chen
authors.affiliation: Center for Neuroscience Discovery, Institute of Healthy Aging, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, United States.
authors.forename: Ran
authors.initials: R
authors.lastname: Liu
authors.affiliation: Center for Neuroscience Discovery, Institute of Healthy Aging, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, United States.
authors.forename: Shao-Hua
authors.initials: SH
authors.lastname: Yang
authors.affiliation: Center for Neuroscience Discovery, Institute of Healthy Aging, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, United States.
authors.forename: Glenn H
authors.initials: GH
authors.lastname: Dillon
authors.affiliation: Center for Neuroscience Discovery, Institute of Healthy Aging, University of North Texas Health Science Center at Fort Worth, Fort Worth, TX 76107, United States. Electronic address: ren-qi.huang@unthsc.edu.
authors.forename: Renqi
authors.initials: R
authors.lastname: Huang
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites / Q000187:drug effects* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D066298:In Vitro Techniques; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects / Q000235:genetics; D008297:Male; D008751:Methylene Blue / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000235:genetics / Q000378:metabolism*; D026941:Sodium Channel Blockers / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000378:metabolism / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D011963:Receptors, GABA-A; D026941:Sodium Channel Blockers; D013779:Tetrodotoxin; D005680:gamma-Aminobutyric Acid; D008751:Methylene Blue
keywords: 2-amino-5-phosphonopentanoic acid (AP-5) (PubChem CID: 135342); 3-dione (CNQX) (PubChem CID: 3721046); 6-cyano-7-nitroquinoxaline-2; GABA (PubChem CID:119); Hippocampus; IPSC; Memory enhancing; Methylene blue (PubChem CID: 6099); Muscimol (PubChem CID:4266); Picrotoxin (PubChem CID: 518601); Recombinant receptors; Tetrodotoxin (TTX) (PubChem CID: 11174599)