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 language | English |
|---|---|
| Pages (from-to) | 11231-11235 |
| Number of pages | 5 |
| Journal | Proceedings of the National Academy of Sciences of the United States of America |
| Volume | 93 |
| Issue number | 20 |
| DOIs | |
| Publication status | Published - Oct 1 1996 |
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Keywords
- acetylcholine receptors
- neurotransmitter interactions
- Xenopus oocytes
ASJC Scopus subject areas
- Genetics
- General
Cite this
Threonine-for-leucine mutation within domain M2 of the neuronal α 7 nicotinic receptor converts 5-hydroxytryptamine from antagonist to agonist. / Palma, E.; Mileo, A. M.; Eusebi, F.; Miledi, R.
In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 93, No. 20, 01.10.1996, p. 11231-11235.Research output: Contribution to journal › Article
}
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 -