Stoichiometric analysis of the TM2 6′ phenylalanine mutation on desensitization in α1β2 and α1β2γ2 GABAA receptors

Gonzales, Eric B.; Bell-Horner, Cathy L.; Dibas, Mohammed; Huang, Ren-Qi; Dillon, Glenn H.

doi:10.1016/j.neulet.2007.11.039

Cited by 14 publications

(9 citation statements)

References 14 publications

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“…BB, GB, DTZ, and PXN inhibition is subunit dependent at the 5-HT 3 receptor, similar to the actions of these compounds at other Cys-loop receptors (Das and Dillon, 2003;Erkkila et al, 2008;Gonzales et al, 2008;Thompson et al, 2011). 5-HT 3 AB receptors were less sensitive to PXN than 5-HT 3 A receptors, and mutation of the 5-HT 3 A 6Ј residue to its B subunit counterpart (T6ЈS) completely abolished inhibition by BB, GB, and PXN.…”

Section: Discussionsupporting

confidence: 52%

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor

2011

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Bilobalide (BB), ginkgolide B (GB), diltiazem (DTZ), and picrotoxinin (PXN) are 5-hydroxytryptamine type 3 (5-HT 3 ) receptor antagonists in which the principal sites of action are in the channel. To probe their exact binding locations, 5-HT 3 receptors with substitutions in their pore lining residues were constructed (NϪ4ЈQ, EϪ1ЈD, S2ЈA, T6ЈS, L7ЈT, L9ЈV, S12ЈA, I16ЈV, D20ЈE), expressed in Xenopus laevis oocytes, and the effects of the compounds on 5-HT-induced currents were examined. EC 50 values at mutant receptors were less than 6-fold different from those of wild type, indicating that the mutations were well tolerated. BB, GB, DTZ, and PXN had pIC 50 values of 3.33, 3.14, 4.67, and 4.97, respectively. Inhibition by BB and GB was abolished in mutant receptors containing T6ЈS and S12ЈA substitutions, but their potencies were enhanced (42-and 125-fold, respectively) in S2ЈA mutant receptors. S2ЈA substitution also caused GB ligand trap. PXN potency was modestly enhanced (5-fold) in S2ЈA, abolished in T6ЈS, and reduced in L9ЈV (40-fold) and S12ЈA (7-fold) receptors. DTZ potency was reduced in L7ЈT and S12ЈA receptors (5-fold), and DTZ also displaced [3 H]granisetron binding, indicating mixed competitive/noncompetitive inhibition. We conclude that regions close to the hydrophobic gate of M2 are important for the inhibitory effects of BB, GB, DTZ, and PXN at the 5-HT 3 receptor; for BB, GB, and PXN, the data show that the 6Ј channel lining residue is their major site of action, with minor roles for 2Ј, 9Ј, and 12Ј residues, whereas for DTZ, the 7Ј and 12Ј sites are important.

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Section: Discussionsupporting

confidence: 52%

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor

2011

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“…For example, in the ␣1␤ Gly receptor activation by agonists is markedly affected by mutations in the ␣1 subunit, but not in the homologous ␤ mutant subunits (30). In the GABA A receptor, asymmetric contribution of subunits has been reported for permeability (31), effect of gain-of-function mutations (L9Ј and F6Ј) (32)(33)(34) and mutations that ablate pH modulation (35).…”

Section: Discussionmentioning

confidence: 99%

In Glycine and GABAA Channels, Different Subunits Contribute Asymmetrically to Channel Conductance via Residues in the Extracellular Domain

Moroni

Meyer

Lahmann

et al. 2011

Journal of Biological Chemistry

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Single-channel conductance in Cys-loop channels is controlled by the nature of the amino acids in the narrowest parts of the ion conduction pathway, namely the second transmembrane domain (M2) and the intracellular helix. In cationic channels, such as Torpedo ACh nicotinic receptors, conductance is increased by negatively charged residues exposed to the extracellular vestibule. We now show that positively charged residues at the same loop 5 position boost also the conductance of anionic Cys-loop channels, such as glycine (␣1 and ␣1␤) and GABA A (␣1␤2␥2) receptors. Charge reversal mutations here produce a greater decrease on outward conductance, but their effect strongly depends on which subunit carries the mutation. In the glycine ␣1␤ receptor, replacing Lys with Glu in ␣1 reduces single-channel conductance by 41%, but has no effect in the ␤ subunit. By expressing concatameric receptors with constrained stoichiometry, we show that this asymmetry is not explained by the subunit copy number. A similar pattern is observed in the ␣1␤2␥2 GABA A receptor, where only mutations in ␣1 or ␤2 decreased conductance (to different extents). In both glycine and GABA receptors, the effect of mutations in different subunits does not sum linearly: mutations that had no detectable effect in isolation did enhance the effect of mutations carried by other subunits. As in the nicotinic receptor, charged residues in the extracellular vestibule of anionic Cys-loop channels influence elementary conductance. The size of this effect strongly depends on the direction of the ion flow and, unexpectedly, on the nature of the subunit that carries the residue.Channels in the nicotinic superfamily are formed by five subunits arranged quasi-symmetrically around the central pore. Structural information from the Torpedo acetylcholine nicotinic receptor and related prokaryotic channels, such as GLIC and ELIC (all cation-permeable), shows that the conduction path for ions starts with a wide vestibule formed by the extracellular domains (1-3). This tapers to a narrower transmembrane region, which is lined mainly by the second transmembrane domains (M2) of each of the five subunits. In eukaryotic channels, this is connected to the cytoplasm by openings in the subunits intracellular domains, which are formed by the M3-M4 loop. The rate of ion flow along this pathway (and therefore the single-channel conductance) is strongly affected by the nature of the residues that line it, especially those that line the narrowest parts. In particular, there are rings of charged amino acids at the extracellular and intracellular ends of the transmembrane part of the pore (positions Ϫ4Ј and 20Ј in the M2 domain (4) and around the fenestrations of the cytoplasmic domain (5). It was recently found that single-channel conductance can also be influenced by residues in the extracellular domain (ECD). The first clue came from the structure of a member of the family of ACh-binding proteins (AChBP), molluscan homopentameric soluble proteins, which are homologous to the ECD part...

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“…The stoichiometry of recombinant ␣ 4 ␤ 2 GABA A Rs was 2␣ 4 :3␤ 2 , whereas the ␣ 1 ␤ 2 GABA A R stoichiometry was 3␣ 1 :2␤ 2 (Table 1). Our previous work, using tandem subunit constructs, also indicated that ␣ 1 ␤ 2 GABA A Rs stoichiometry was 3␣ 1 :2␤ 2 (6), but others have reported a 2␣ 1 :3␤ 2 stoichiometry (38,39), suggesting that both stoichiometries are possible. Studies examining ␣ 6 ␤ 2 GABA A R subtypes report that the subunit stoichiomety is 3␣ 6 :2␤ 2 (40), whereas for ␣ 1 ␤ 3 GABA A Rs the stoichiometry is 2␣ 1 :3␤ 3 (3).…”

Section: Discussionmentioning

confidence: 91%

Stoichiometry of Expressed α4β2δ γ-Aminobutyric Acid Type A Receptors Depends on the Ratio of Subunit cDNA Transfected

Wagoner

Czajkowski

2010

Journal of Biological Chemistry

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The ␥-aminobutyric acid type A receptor (GABA A R) is the target of many depressants, including benzodiazepines, anesthetics, and alcohol. Although the highly prevalent ␣␤␥ GABA A R subtype mediates the majority of fast synaptic inhibition in the brain, receptors containing ␦ subunits also play a key role, mediating tonic inhibition and the actions of endogenous neurosteroids and alcohol. However, the fundamental properties of ␦-containing GABA A Rs, such as subunit stoichiometry, are not well established. To determine subunit stoichiometry of expressed ␦-containing GABA A Rs, we inserted the ␣-bungarotoxin binding site tag in the ␣ 4 , ␤ 2 , and ␦ subunit N termini. An enhanced green fluorescent protein tag was also inserted into the ␤ 2 subunit to shift its molecular weight, allowing us to separate subunits using SDS-PAGE. Tagged ␣ 4 ␤ 2 ␦ GABA A Rs were expressed in HEK293T cells using various ratios of subunit cDNA, and receptor subunit stoichiometry was determined by quantitating fluorescent ␣-bungarotoxin bound to each subunit on Western blots of surface immunopurified tagged GABA A Rs. The results demonstrate that the subunit stoichiometry of ␣ 4 ␤ 2 ␦ GABA A Rs is regulated by the ratio of subunit cDNAs transfected. Increasing the ratio of ␦ subunit cDNA transfected increased ␦ subunit incorporation into surface receptors with a concomitant decrease in ␤ 2 subunit incorporation. Because receptor subunit stoichiometry can directly influence GABA A R pharmacological and functional properties, considering how the transfection protocols used affect subunit stoichiometry is essential when studying heterologously expressed ␣ 4 ␤ 2 ␦ GABA A Rs. Successful bungarotoxin binding site tagging of GABA A R subunits is a novel tool with which to accurately quantitate subunit stoichiometry and will be useful for monitoring GABA A R trafficking in live cells.The ␥-aminobutyric acid type A receptor (GABA A R) 2 is the main inhibitory ligand-gated ion channel in the brain and is the target for a wide range of drugs, including benzodiazepines, anesthetics, neurosteroids, and barbiturates. The actions of these drugs are dependent on the GABA A R subunit isoforms present, 16 of which have been identified, including ␣1-6, ␤1-3, ␥1-3, ␦, ⑀, , and . Although the highly prevalent GABA A R subtype comprised of ␣, ␤, and ␥ subunits mediates the majority of fast synaptic inhibition in the brain, ␣␤␦ GABA A Rs also play a key role, mediating tonic inhibition and the molecular actions of endogenous neuroactive steroids, alcohol, and several anesthetic agents (1).Heterologous expression of GABA A R subtypes in cell lines (e.g. HEK293 and Chinese hamster ovary cells) and Xenopus laevis oocytes has been used extensively to study their structural, electrophysiological, and pharmacological properties. Although the subunit stoichiometry of ␣␤␥ GABA A Rs has been established as 2␣:2␤:1␥ using a variety of methods, including using a reporter mutation (2), quantifying antibody bound per subunit (3), fluorescence resonance energy transfer (4),...

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Stoichiometric analysis of the TM2 6′ phenylalanine mutation on desensitization in α1β2 and α1β2γ2 GABAA receptors

Cited by 14 publications

References 14 publications

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor

In Glycine and GABAA Channels, Different Subunits Contribute Asymmetrically to Channel Conductance via Residues in the Extracellular Domain

Stoichiometry of Expressed α4β2δ γ-Aminobutyric Acid Type A Receptors Depends on the Ratio of Subunit cDNA Transfected

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Stoichiometric analysis of the TM2 6′ phenylalanine mutation on desensitization in α1β2 and α1β2γ2 GABAA receptors

Cited by 14 publications

References 14 publications

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT3 Receptor

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT3 Receptor

In Glycine and GABAA Channels, Different Subunits Contribute Asymmetrically to Channel Conductance via Residues in the Extracellular Domain

Stoichiometry of Expressed α4β2δ γ-Aminobutyric Acid Type A Receptors Depends on the Ratio of Subunit cDNA Transfected

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Product

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Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor

Binding Sites for Bilobalide, Diltiazem, Ginkgolide, and Picrotoxinin at the 5-HT₃ Receptor