Threonine-for-leucine mutation within domain M2 of the neuronal α 7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist

E. Palma, A. M. Mileo, F. Eusebi, R. Miledi

Research output: Contribution to journalArticle

Abstract

A study was made of the effects of 5-hydroxy-tryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken α 7 subunit. Acetylcholine (AcCho) elicited large currents (I(AcChu)) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC 50) of 56 μM and a Hill coefficient (nH) of 1.2. The inhibition of IAacCHO BY %HT by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild- type α 7 subunit cDNA. In contrast, 5HT elicited membrane currents (I5(HT)) in oocytes injected with cDNA encoding an α 7 mutant subunit with a threonine-for-leucine-247 substitution (L247T α 7). I(5HT) was inhibited by the potent nicotinic receptor blockers α-bungarotoxin (100 nM) and methyllycaconitine (1 μM). Furthermore, the characteristics of I(5HT), including its voltage dependence, were similar to those of I(AcCho). The 5HT dose-I(5HT) response gave an apparent dissociation constant EC 50 of 23.5 μM and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T α 7 for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal α 7 nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

Original languageEnglish
Pages (from-to)11231-11235
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume93
Issue number20
DOIs
Publication statusPublished - Oct 1 1996

Fingerprint

Serotonin Antagonists
Nicotinic Receptors
Threonine
Leucine
Mutation
Acetylcholine
Oocytes
Complementary DNA
tryptamine
Bungarotoxins
Membranes
Xenopus
Inhibitory Concentration 50
Chickens
Serotonin

Keywords

  • acetylcholine receptors
  • neurotransmitter interactions
  • Xenopus oocytes

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

@article{c9c59f915d6d476086f1dcbe9791398d,
title = "Threonine-for-leucine mutation within domain M2 of the neuronal α 7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist",
abstract = "A study was made of the effects of 5-hydroxy-tryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken α 7 subunit. Acetylcholine (AcCho) elicited large currents (I(AcChu)) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC 50) of 56 μM and a Hill coefficient (nH) of 1.2. The inhibition of IAacCHO BY {\%}HT by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild- type α 7 subunit cDNA. In contrast, 5HT elicited membrane currents (I5(HT)) in oocytes injected with cDNA encoding an α 7 mutant subunit with a threonine-for-leucine-247 substitution (L247T α 7). I(5HT) was inhibited by the potent nicotinic receptor blockers α-bungarotoxin (100 nM) and methyllycaconitine (1 μM). Furthermore, the characteristics of I(5HT), including its voltage dependence, were similar to those of I(AcCho). The 5HT dose-I(5HT) response gave an apparent dissociation constant EC 50 of 23.5 μM and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T α 7 for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal α 7 nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.",
keywords = "acetylcholine receptors, neurotransmitter interactions, Xenopus oocytes",
author = "E. Palma and Mileo, {A. M.} and F. Eusebi and R. Miledi",
year = "1996",
month = "10",
day = "1",
doi = "10.1073/pnas.93.20.11231",
language = "English",
volume = "93",
pages = "11231--11235",
journal = "Proceedings of the National Academy of Sciences of the United States of America",
issn = "0027-8424",
number = "20",

}

TY - JOUR

T1 - Threonine-for-leucine mutation within domain M2 of the neuronal α 7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist

AU - Palma, E.

AU - Mileo, A. M.

AU - Eusebi, F.

AU - Miledi, R.

PY - 1996/10/1

Y1 - 1996/10/1

N2 - A study was made of the effects of 5-hydroxy-tryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken α 7 subunit. Acetylcholine (AcCho) elicited large currents (I(AcChu)) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC 50) of 56 μM and a Hill coefficient (nH) of 1.2. The inhibition of IAacCHO BY %HT by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild- type α 7 subunit cDNA. In contrast, 5HT elicited membrane currents (I5(HT)) in oocytes injected with cDNA encoding an α 7 mutant subunit with a threonine-for-leucine-247 substitution (L247T α 7). I(5HT) was inhibited by the potent nicotinic receptor blockers α-bungarotoxin (100 nM) and methyllycaconitine (1 μM). Furthermore, the characteristics of I(5HT), including its voltage dependence, were similar to those of I(AcCho). The 5HT dose-I(5HT) response gave an apparent dissociation constant EC 50 of 23.5 μM and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T α 7 for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal α 7 nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

AB - A study was made of the effects of 5-hydroxy-tryptamine (5HT) on homomeric neuronal nicotinic receptors (nAcChoR) expressed in Xenopus oocytes after injection of cDNA encoding the wild-type chicken α 7 subunit. Acetylcholine (AcCho) elicited large currents (I(AcChu)) that were reduced by 5HT in a reversible and dose-dependent manner, with a half-inhibitory concentration (IC 50) of 56 μM and a Hill coefficient (nH) of 1.2. The inhibition of IAacCHO BY %HT by 5HT was noncompetitive and voltage independent, a behavior incompatible with a channel blockade mechanism. 5HT alone did not elicit membrane currents in oocytes injected with the wild- type α 7 subunit cDNA. In contrast, 5HT elicited membrane currents (I5(HT)) in oocytes injected with cDNA encoding an α 7 mutant subunit with a threonine-for-leucine-247 substitution (L247T α 7). I(5HT) was inhibited by the potent nicotinic receptor blockers α-bungarotoxin (100 nM) and methyllycaconitine (1 μM). Furthermore, the characteristics of I(5HT), including its voltage dependence, were similar to those of I(AcCho). The 5HT dose-I(5HT) response gave an apparent dissociation constant EC 50 of 23.5 μM and a Hill coefficient n(H) of 1.7, which were not modified by the presence of AcCho. Similarly, the apparent affinity of L247T α 7 for AcCho as well as its cooperativity were not influenced by 5HT, indicating a lack of mutual interactions between 5HT and AcCho. These results show that 5HT is a potent noncompetitive antagonist of neuronal α 7 nAcChoR, but it becomes a noncompetitive agonist following mutation of the highly conserved leucine residue 247 located in the channel domain M2.

KW - acetylcholine receptors

KW - neurotransmitter interactions

KW - Xenopus oocytes

UR - http://www.scopus.com/inward/record.url?scp=0029763194&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0029763194&partnerID=8YFLogxK

U2 - 10.1073/pnas.93.20.11231

DO - 10.1073/pnas.93.20.11231

M3 - Article

C2 - 8855338

AN - SCOPUS:0029763194

VL - 93

SP - 11231

EP - 11235

JO - Proceedings of the National Academy of Sciences of the United States of America

JF - Proceedings of the National Academy of Sciences of the United States of America

SN - 0027-8424

IS - 20

ER -