Browsing by Subject "GABAA receptors"
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Item Anionic Ligand-Gated Ion Channels: The Convulsive Site and Mechanism of Action(2001-08-01) Dibas, Mohammed I.; Hriday Das; Thomas Yorio; Neeraj AgarwalDibas, Mohammed, Anionic Ligand-Gated Ion channels: The Convulsive Site And Mechanism of Action. Doctor of Philosophy (Biomedical Sciences), August 2001, pp153, 1 table, 24 illustrations, 76 titles. Picrotoxin, a CNS convulsant inhibits all anionic ligand gated ion channels. The mechanism and the binding site for picrotoxin and its related ligands are still undefined. The second transmembrane (TMII) domain of these ligand gated ion channels is found to play a key role in the mechanism of block by picrotoxin. It has been shown that the incorporation of a phenylalanine residue in place of threonine at position 6’ within the TMII domain of B2 subunit conferred high resistance toward picrotoxin in GABAA a3B2(T6’F)y2 receptors. Mediating their blocking effect through the PTX-site, PTZ, TBPS, and U-93631 lost their inhibitory effects due to the same mutation B2(T6’F). Interestingly, this mutation uncovered a low affinity, highly efficacious stimulatory site for PTZ. PTZ seems to mediate its stimulatory effect through a novel distinct site different from that for benzodiazepine. The effect of varying subunit configuration of GABAA receptors dramatically affected the ability of the mutation B2(T6’F) to abolish the inhibitory effect of picrotoxin. While picrotoxin failed to block the current induced by GABA in a3B2(T6’F)y2 receptors, picrotoxin partially blocked the current in a3B2(T6’F)y2 receptors. In B2(T6’F)y2 receptors, picrotoxin restores its full efficacy. When phenylalanine was incorporated at position 6’ in the a1 subunit, picrotoxin completely blocked the current induced by GABA in a1(T6’F)B2y2 receptors. The combined results showed that the ability of (T6’F) mutation to regulate the inhibitory mechanism of picrotoxin as dependent on the subunit configurations and at which subunit is mutated. In addition, picrotoxin is known to inhibit GABAA receptors in use-facilitated mechanism, while it inhibits the glycine receptor in a non-use facilitated fashion. The molecular determinant behind the use-facilitated mechanism was modulated by the nature of the amino acid at position 15’ within the second transmembrane domain. The mutation of serine 15’ to either glutamine or asparagine in the glycine a1 receptors converted picrotoxin from a non-use facilitated blocker to a use-facilitated one. The latter finding suggested that this residue might residue within the PTX binding site or play a key role in the transduction pathway for picrotoxin mechanism. The overall results further support the fact that TMII domain plays a key role in the picrotoxin mechanism.Item Elucidation of Mechanism and Molecular Determinants Important in Picrotoxin Action in the 5-Hydroxytryptamine Type 3 Receptor(2003-09-01) Das, Paromita; Basu, Alakananda; Forster, Michael J.; Luedtke, Robert R.Das, Paromita, Elucidation of mechanism and molecular determinants important in picrotoxin action in the 5-hydroxytryptamin type 3 receptor. Doctor of Philosophy (Pharmacology and Neuroscience), September 2003, pp. 192, 3 tables, 26 illustrations, 67 titles. The 5-HT3 receptor belongs to the superfamily of ligand-gated ion channels (LGIC), which mediate fast neurotransmission. Till date, only two subtypes of the receptor i.e. 5-HT3A and 5-HT3B have been investigated. The GABAA receptor antagonist picrotoxin inhibits other anion-selective members of the LGIC. Whether PTX inhibits the cation-selective 5-HT3 receptors was previously unknown. Thus, the primary goal of this study was to elucidate the mechanism of action of PTX and identify the amino acids involved in the action of PTX in 5-HT3 receptors. The overall hypothesis tested was that PTX inhibits the 5-HT3 receptor by interacting in the ion channel. PTX-mediated blockade of the 5-HT3A receptors was non-competitive and use-facilitated similar to GABAA receptors suggesting a conserved site of action of these ligands. The inhibitory effect of PTX was reduced drastically in heteromeric 5-HT3A/3B receptors, compared to homomeric 5-HT3A receptors. Picrotoxin should prove to be a useful probe for determining the presence of homomeric vs. heteromeric 5-HT3 receptors in native tissue and recombinant receptor preparations. In anion-selective ion channels, the 2’, 3’ and 6’resides in cytoplasmic aspect of TM2 are known to modulate PTX sensitivity. While mutation of 2’ and 3’ residues in 5-dramatic loss of sensitivity to PTX in 5-HT3A receptors. A converse mutation at 6’ residue in the 5-HT3B subunit caused gain of sensitivity to PTX, suggesting that 6’ is a key determinant of PTX sensitivity. A novel finding was the involvement of 7’ residue in increasing PTX sensitivity in 5-HT3A but not the 5-HT3B subunit. The lack of specific binding by radioligand [3H]EBOB in 5-HT3A receptors suggested that the site of action of convulsants may be different from that anion-selective receptors. The overall results suggest that PTX interacts from that in the anion-selective receptors. The overall results suggest that PTX interact in the ion channel in the 5-HT3 receptors but also underscores the complexity of its interaction with LGICs.Item The 6' and 7' Residue of the Second Transmembrane Domain of Ligand-Gated Ion Channels Influence Gating and Picrotoxin Sensitivity(2005-07-01) Gonzales, Eric B.; Dillon, Glenn; Luedtke, Robert R.; Martin, MichaelGonzales, Eric B., The 6’ and 7’ residue of the second transmembrane domain of ligand-gated ion channels influence gating and picrotoxin sensitivity. Doctor of Philosophy (Pharmacology and Neuroscience), July 2005, pp213, 4 tables, 33 illustrations, 89 titles. The GABAA and glycine receptor are members of the Cys-loop family of ion channels. These receptors mediate rapid neurotransmission in the nervous system. Picrotoxin (PTX) interacts within the channel near the TM2 2’-6’ position, with the most critical interaction at the 6’ position. The present studied addressed the stoichiometric dependence and molecular requirements of the TM2 6’ position on PTX sensitivity. I hypothesized that there is not a stoichiometric dependence and that residues with a hydroxyl group remain sensitive to PTX. Further, work previously completed in the laboratory demonstrated that the TM2 7’ position influences channel kinetics in the serotonin type-3 receptor. However, similar work has not been performed in other members of the Cys-loop family of receptors. I hypothesize that the TM2 7’ position influences both gating in the glycine α1 receptor similarly to that in the serotonin type-3 receptor. Additionally, the TM2 7’ position could influence the PTX in the glycine α1 receptor. Picrotoxin sensitivity was determined to not be stoichiometrically dependent on the subunit location of the T6’F mutation in α1β2 or α1β2γ2 GABAA receptors, a single T6’F mutation was sufficient to eliminate PTX sensitivity. The α1(T6’F) β2 receptor showed PTX concentration-dependent stimulation. Picrotoxin sensitivity had a rank order of potency in α1β2(mutant) as follows: Serine [greater than] Threonine = Alanine [greater than] Cysteine [greater than] Tryptophan. In several cases, the kinetics of the T6’F mutant receptors exhibited rapid desensitization during prolonged application of agonist. In combinations of subunits with the T6’F mutation, two or fewer mutant receptors appeared to have normal phenotypes. Three or four T6’F mutations exhibited rapid desensitization, and strongly suggests that the stoichiometery of the α1β2 GABAA receptor is two α subunits and three β subunits in the receptor. The Gly α1(T7’L) mutation exhibited enhanced glycine sensitivity with slower gating kinetics than the wild type (approximately 3-fold slower); the T7’A mutation had significantly reduced glycine affinity. The glycine EC50 kinetics of the α1(T7’A) mutant receptor was more complex than the wild type. There appears to be a complex interaction between agonist binding and gating of the channel that is disrupted by the 7’ position mutations. Picrotoxin sensitivity in the mutation were enhanced with either a T7’A or T7’L substitution. The data suggest that the 7’ residue may play an accessory role in shaping the PTX site. Finally, these residues are also critical in gating of the receptor. Residues critical for gating and PTX sensitivity may be coupled in the normal function of the Cys-loop family of receptors.