title: Targeting AMPA receptor gating processes with allosteric modulators and mutations.
abstract: Allosteric modulators and mutations that slow AMPAR desensitization have additional effects on deactivation and agonist potency. We investigated whether these are independent actions or the natural consequence of slowing desensitization. Effects of cyclothiazide (CTZ), trichlormethiazide (TCM), and CX614 were compared at wild-type GluR1 and "nondesensitizing" GluR1-L497Y mutant receptors by patch-clamp recording with ultrafast perfusion. CTZ, TCM, or L/Y mutation all essentially blocked GluR1 desensitization; however, the effects of L/Y mutation on deactivation and glutamate EC50 were three to five times greater than for modulators. CTZ and TCM further slowed desensitization of L/Y mutant receptors but paradoxically accelerated deactivation and increased agonist EC50. Results indicate that CTZ and TCM target deactivation and agonist potency independently of desensitization, most likely by modifying agonist dissociation (koff). Conversely, CX614 slowed desensitization and deactivation without affecting EC50 in both wild-type and L/Y receptors. The S750Q or combined L497Y-S750Q mutations abolished all CTZ and TCM actions without disrupting CX614 activity. Notably, the S/Q mutation also restored L/Y deactivation and EC50 to wild-type levels without restoring desensitization, further demonstrating that desensitization can be modulated independently of deactivation and EC50 by mutagenesis and possibly by allosteric modulators.
authors.affiliation: Center for Neuropharmacology & Neuroscience, Albany Medical College, Albany, New York 12208, USA.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Mitchell
authors.forename: Mark W
authors.initials: MW
authors.lastname: Fleck
mesh_terms: D019943:Amino Acid Substitution; D001581:Benzothiadiazines / Q000008:administration & dosage*; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems / Q000379:methods*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D007668:Kidney / Q000187:drug effects / Q000502:physiology*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010078:Oxazines / Q000008:administration & dosage*; D018091:Receptors, AMPA / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014237:Trichlormethiazide / Q000008:administration & dosage*
chemical_list: D001581:Benzothiadiazines; D010078:Oxazines; D018091:Receptors, AMPA; D014237:Trichlormethiazide; C414782:2H,3H,6aH-pyrrolidino(2'',1''-3',2')1,3-oxazino(6',5'-5,4)benzo(e)1, 4-dioxan-10-one; C004639:cyclothiazide