Allosteric modulation in spontaneously active mutant γ-aminobutyric acidA receptors

Findlay, Geoffrey S.; Ueno, S.; Harrison, Neil L.; Harris, R. Adron

doi:10.1016/s0304-3940(01)01646-9

Cited by 27 publications

(29 citation statements)

References 12 publications

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“…This suggests that a neurosteroid binding site is likely located on the 1 subunit and that the modulatory effects of allopregnanolone are maintained when combined with 1 and 2L subunits. Substitution of the serine at position 265 on the 1 subunit dramatically decreases the enhancement of GABA-induced currents by allopregnanolone, which also supports the hypothesis that the allopregnanolone binding site is on 1 (Findlay et al 2000). Bullfrogs possess this critical serine at the equivalent position on the 1 subunit.…”

Section: Discussionsupporting

confidence: 75%

“…Similarly, subunit composition also determines if and how neurosteroids modulate receptor function (Paul & Purdy 1992, Hauser et al 1997, Akk et al 2001, Findlay et al 2000. Molecular cloning of the subunits, coupled with photoaffinity labeling and point mutation studies, has led to the identification of specific binding domains and structural characteristics of receptor subtypes (Smith & Olsen 1995).…”

Section: Introductionmentioning

confidence: 99%

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Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

Hollis

Goetz²,

Roberts³

et al. 2004

Journal of Molecular Endocrinology

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The inhibitory neurotransmitter -aminobutyric acid (GABA) has multiple receptors. In mammals, the GABA A receptor subtype is modulated by neurosteroids. However, whether steroid interaction with the GABA A receptor is unique to mammals or a conserved feature in vertebrates is unknown. Thus, neurosteroid modulation of the GABA A receptor was investigated in the brain of the bullfrog (Rana catesbeiana) using the mammalian GABA A receptor agonist [ 3 H]muscimol. Two neurosteroids, allopregnanolone and pregnenolone sulfate, affected [ 3 H]muscimol specific binding in bullfrog brain membrane preparations. Allopregnanolone significantly increased [ 3 H]muscimol specific binding in a dose-and time-dependent manner. The pattern of allopregnanolone modulation supports the hypothesis that the bullfrog brain possesses both high-affinity and low-affinity [ 3 H]muscimol binding sites. Unlike allopregnanolone, pregnenolone sulfate showed biphasic modulation with increased [ 3 H]muscimol specific binding at low nanomolar concentrations and decreased specific binding at micromolar concentrations. Additionally, three cDNA fragments with significant homology to mammalian GABA A receptor subunits were isolated from the bullfrog brain. These fragments belong to the 1, 1, and 2 subunit families. In mammals, GABA A receptors composed of these specific subunit isoforms are effectively modulated by neurosteroids, including allopregnanolone. Neurosteroid modulation of the amphibian brain GABA A receptor is therefore supported by both [ 3 H]muscimol binding studies and subunit sequences. Allopregnanolone and pregnenolone sulfate modulation of this receptor may thus represent a significant mechanism for steroid influence on amphibian brain and behavior.

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Section: Discussionsupporting

confidence: 75%

Section: Introductionmentioning

confidence: 99%

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

Hollis

Goetz²,

Roberts³

et al. 2004

Journal of Molecular Endocrinology

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“…Changes in desensitization are demonstrated in the present study, where the Ile294Thr 5-HT 3A receptor shows a marked reduction in extent and rate of desensitization. Other mutation-induced changes that have been reported are changes in affinity, shift from antagonism to agonism (Bertrand et al, 1992), loss of allosteric modulation (Findlay et al, 2001), and production of tonically open channels (Findlay et al, 2001). Collectively, these data suggest that mutations in TM2 alter the statedependence of these channels by shifting the probability of closed 3 open 3 desensitized transitions.…”

Section: Discussionmentioning

confidence: 67%

“…Mutations in either TM2 or in the N terminus have been described that produce tonically open channels, an extreme case of increased probability of channel opening wherein a certain fraction of receptors are constantly open. These receptors have very reduced or no sensitivity to allosteric modulation by alcohols and anesthetics (Thompson et al, 1999;Findlay et al, 2001;Zhang et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

A Mutation in Transmembrane Domain II of the 5-Hydroxytryptamine_3A Receptor Stabilizes Channel Opening and Alters Alcohol Modulatory Actions

Sessoms-Sikes

Hamilton

Liu

et al. 2003

J Pharmacol Exp Ther

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Mutant 5-hydroxytryptamine (5-HT) 3A receptors, in which changes were made at Ile294, position 16Ј, of the second transmembrane domain, were assessed for alterations in macroscopic response kinetics and modulation by alcohols. Function of heterologously expressed receptors was measured in Xenopus oocytes in the two-electrode voltage clamp configuration and in human embryonic kidney 293 cells using whole cell patch-clamp electrophysiological recordings with rapid drug application. Compared with the wild-type receptor, a decrease in the 5-HT EC 50 value in the Ile294Thr mutant was observed, whereas an increase in the 5-HT EC 50 value in the Ile294Leu mutant was measured. Ile294Thr receptors showed a marked reduction in the extent of desensitization. Ethanol and 2,2,2-trichloroethanol (TCEt) enhanced 5-HT-mediated currents in wild-type and Ile294Leu receptors, but inhibited or had little stimulatory effect in the Ile294Thr mutant. Kinetic analysis revealed that in the presence of TCEt, the slope of activation was unchanged in the Ile294Thr mutant and increased in the wild-type receptor. Alcohol cutoff was altered with wild-type ϭ heptanol and Ile294Leu ϭ hexanol. Kinetic changes in the Ile294Thr mutant that favor the open channel state, as well as reduction in the rate of channel activation in the presence of TCEt, likely underlie this mutant's altered response to n-chain alcohols.The 5-hydroxytryptamine 3 (5-HT 3 ) receptor is a member of the superfamily of ligand-gated ion channels, of which the nicotinic acetylcholine (nACh) receptor is the prototype (Derkach et al., 1989;Peters et al., 1994). To date, two subunits of the 5-HT 3 receptor, 5-HT 3A (Maricq et al., 1991;Belelli et al., 1995;Miyake et al., 1995;Lankiewicz et al., 1998) and 5-HT 3B (Davies et al., 1999;Dubin et al., 1999) have been cloned. Recent reverse transcriptase and immunohistochemical studies demonstrated that the B subunit is largely expressed in the peripheral nervous system, suggesting that the predominant form in the brain is the A homomer (Morales and Wang, 2002). The most well documented actions of 5-HT 3 receptors are to alter gastrointestinal motility and to regulate the vomiting reflex (Aapro, 1991). Moreover, the 5-HT 3 receptor has been implicated in altering the voluntary intake of ethanol in humans (Johnson et al., 1993) and rodents (Knapp and Pohorecky, 1992;Hodge et al., 1993).The 5-HT 3 receptor is modulated by pharmacologically relevant concentrations of n-chain alcohols and anesthetics. Alcohols and volatile anesthetics, in the presence of low 5-HT concentrations, potentiate native and heterologously expressed 5-HT 3 receptors (Lovinger and White, 1991;Machu and Harris, 1994). The mechanism for enhancement of receptor function by alcohols has been assessed in NCB-20 cells, which express the 5-HT 3 receptor endogenously. Ethanol and 2,2,2-trichloroethanol (TCEt) enhanced peak currents evoked by a maximally effective concentration of dopamine, which is a weak partial agonist at the 5-HT 3 receptor (Lovinger et al., 200...

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“…2A. Residue L293 is in the same 15′ position as a residue in the GABA A receptor that has important roles in channel gating (Findlay et al, 2001;Scheller & Forman, 2002) and modulation by alcohols and general anesthetics (Mihic et al, 1997;Wick et al, 1998). This amino acid residue is thought to reside on the non-porefacing side of TM2 domain (Panicker et al, 2002;Reeves et al, 2001).…”

Section: Role Of L293 In the Modulatory Action Of 5-himentioning

confidence: 99%

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

Lovinger

2008

Neuropharmacology

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Allosteric modulation of ligand-gated ion channels can play important roles in shaping synaptic transmission. The function of the 5-hydroxytryptamine (serotonin) type 3 (5-HT 3 ) receptor, a member of the Cys-loop ligand-gated ion channel superfamily, is modulated by a variety of compounds such as alcohols, anesthetics and 5-hydroxyindole (5-HI). In the present study, the molecular determinants of allosteric modulation by 5-HI were explored in N1E-115 neuroblastoma cells expressing the native 5-HT 3 receptor and HEK 293 cells transfected with the recombinant 5-HT 3A receptor using molecular biology and whole-cell patch-clamp techniques. 5-HI potentiated 5-HT-activated currents in both N1E-115 cells and HEK 293 cells, and significantly decreased current desensitization and deactivation. Substitution of Leu293 (L293, L15′) in the second transmembrane domain (TM2) with cysteine (L293C) or serine (L293S) abolished 5-HI modulation. Other mutations in the TM2 domain, such as D298A and T284F, failed to alter 5-HI modulation. The L293S mutation enhanced dopamine efficacy and converted 5-HI into a partial agonist at the mutant receptor. These data suggest that 5-HI stabilizes the 5-HT 3A receptor in the open state by decreasing both desensitization and 5-HT unbinding/channel closing; and L293 is a common site for both channel gating and allosteric modulation by 5-HI. Our observations also indicate existence of a second 5-HI recognition site on the 5-HT 3A receptor which may overlap with the 5-HT binding site and is not involved in the positive modulation by 5-HI. These findings support the idea that there are two discrete sites for 5-HI allosteric modulation and direct activation in the 5-HT 3A receptor.

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Allosteric modulation in spontaneously active mutant γ-aminobutyric acidA receptors

Cited by 27 publications

References 12 publications

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

A Mutation in Transmembrane Domain II of the 5-Hydroxytryptamine_3A Receptor Stabilizes Channel Opening and Alters Alcohol Modulatory Actions

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

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Allosteric modulation in spontaneously active mutant γ-aminobutyric acidA receptors

Cited by 27 publications

References 12 publications

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

A Mutation in Transmembrane Domain II of the 5-Hydroxytryptamine3A Receptor Stabilizes Channel Opening and Alters Alcohol Modulatory Actions

The L293 residue in transmembrane domain 2 of the 5-HT3A receptor is a molecular determinant of allosteric modulation by 5-hydroxyindole

Contact Info

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A Mutation in Transmembrane Domain II of the 5-Hydroxytryptamine_3A Receptor Stabilizes Channel Opening and Alters Alcohol Modulatory Actions