Protein kinase C modulates exogenous acetylcholine current in <i>Xenopus</i> oocytes

Mileo, Anna Maria; Palma, Eleonora; Polenzani, Lorenzo; Limatola, Cristina; Grassi, Francesca; Eusebi, Fabrizio

doi:10.1002/jnr.490410403

Cited by 8 publications

(6 citation statements)

References 34 publications

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“…The inhibition was obtained by incubation with increasing concentrations of MG624 for 30 s before the application of ACh at about its EC 50 value. The IC 50 and n H values were 3.2 μ M and 1.2 ( n =7) for the α4β2 subtype (ACh, 2 μ M ; Fucile et al ., 1997 , and 2.9 μ M and 0.9 (ACh, 10 μ M ; Mileo et al ., 1995 ) for the α1β1γδ subtype, thus indicating that the half inhibitory concentrations of MG624 for α4β2 or α1β1γδ nicotinic AChRs were much higher than those of α7 nicotinic AChRs.…”

Section: Resultsmentioning

confidence: 99%

Selective effects of a 4‐oxystilbene derivative on wild and mutant neuronal chick α7 nicotinic receptor

Maggi

Palma

Eusebi

et al. 1999

British J Pharmacology

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1 We assessed the pharmacological activity of triethyl-(b-4-stilbenoxy-ethyl) ammonium (MG624), a drug that is active on neuronal nicotinic receptors (nicotinic AChR). Experiments on the major nicotinic AChR subtypes present in chick brain, showed that it inhibits the binding of [ 125 I]-aBungarotoxin (aBgtx) to the a7 subtype, and that of [ 3 H]-epibatidine (Epi) to the a4b2 subtype, with K i values of respectively 106 nM and 84 mM. 2 MG624 also inhibited ACh elicited currents (I ACh ) in the oocyte-expressed a7 and a4b2 chick subtypes with half-inhibitory concentrations (IC 50 ) of respectively 109 nM and 3.2 mM. 3 When tested on muscle-type AChR, it inhibited [ 125 I]-aBgtx binding with a K i of 32 mM and ACh elicited currents (I ACh ) in the oocyte-expressed a1b1gd chick subtype with an IC 50 of 2.9 mM. 4 The interaction of MG624 with the a7 subtype was investigated using an a7 homomeric mutant receptor with a threonine-for-leucine 247 substitution (L247T a7). MG624 did not induce any current in oocytes expressing the wild type a7 receptor, but did induce large currents in the oocyteexpressed L247T a7 receptor. The MG624 elicited current (I MG624 ) has an EC 50 of 0.2 nM and a Hill coe cient nH of 1.9, and is blocked by the nicotinic receptor antagonist methyllycaconitine (MLA). 5 These binding and electrophysiological studies show that MG624 is a potent antagonist of neuronal chick a7 nicotinic AChR, and becomes a competitive agonist following the mutation of the highly conserved leucine residue 247 located in the M2 channel domain.

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Section: Resultsmentioning

confidence: 99%

Selective effects of a 4‐oxystilbene derivative on wild and mutant neuronal chick α7 nicotinic receptor

Maggi

Palma

Eusebi

et al. 1999

British J Pharmacology

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“…Drugs were dissolved in oocyte's Ringer solution and bath applied at a flow rate of 11-12 ml͞min. Single-channel currents were recorded using the patchclamp technique in the cell-attached mode, as reported (18)(19)(20), from the animal pole of the oocytes. 5HT-HCl was dissolved just before an experiment.…”

Section: Methodsmentioning

confidence: 99%

Neuronal nicotinic threonine-for-leucine 247 α ₇ mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Palma

Maggi

Eusebi

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Mutation of the highly conserved leucine residue (Leu-247) converts 5-hydroxytryptamine (5HT) from an antagonist into an agonist of neuronal homomeric ␣ 7 nicotinic acetylcholine receptor expressed in Xenopus oocytes. We show here that acetylcholine (AcCho) activates two classes of single channels with conductances of 44 pS and 58 pS, similar to those activated by 5HT. However, the mean open time of AcCho-gated ion channels (11 ms) is briefer than that of 5HT-gated ion channels (18 ms). Furthermore, whereas the open time of AcCho channels lengthens with hyperpolarization, that of 5HT channels is decreased. In voltage-clamped oocytes, the apparent affinity of the ␣ 7 mutant receptor for 5HT is not modified by the presence of dihydro-␤-erythroidine, which acts on the AcCho binding site in a competitive manner. This indicates a noncompetitive action of 5HT on nicotinic acetylcholine receptors. Considered together, our findings show that AcCho gates ␣ 7 mutant channels with similar conductance but with different kinetic profile than the channels gated by 5HT, suggesting that the two agonists act on different docking sites. These results will help to understand the crosstalk between cholinergic and serotonergic systems in the central nervous system.

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“…12,[24][25][26] Borosilicate-glass patch pipettes (2-4 M⍀ tip resistance) were filled with oocyte Ringer's medium containing ACh (0.2 M) or ACh (0.2 M) plus fluoxetine (10 M). Unitary channel activity was recorded using an Axopatch 200B amplifier (Axon Instruments).…”

Section: Cell-attached Recordingsmentioning

confidence: 99%

Effects of fluoxetine on wild and mutant neuronal α7 nicotinic receptors

et al. 1998

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Fluoxetine is used in the treatment of a variety of clinical disorders including depression and obesity, and of cocaine detoxification or alcoholism. It is generally believed that fluoxetine exerts its clinical effects because it selectively blocks 5-hydroxytryptamine (5HT) reuptake into nerve terminals. In here we describe that fluoxetine antagonized the neuronal homomeric ␣ 7 nicotinic acetylcholine receptors (nAChR) expressed in Xenopus oocytes, with an IC 50 of 43 M, when fluoxetine was coapplied with ACh, and of 1.6 M when the oocytes were pretreated briefly with fluoxetine. A similar block occurred in oocytes expressing L247T ␣ 7 mutant nAChR. Furthermore, blockage of mutant ␣ 7 receptors appeared non-competitive and was stronger with cell membrane hyperpolarization. Cell-attached single channel recordings in oocytes expressing L247T ␣ 7 mutant nAChR showed that the voltage-dependence of the blockage by fluoxetine could be due to a drastic decrease in channel opening frequency accompanied by marked channel flickering and reduced channel conductance. We conclude that fluoxetine behaves as a reversible blocker of both wild and mutant ␣ 7 receptors; and that the Leu-247T mutation in the channel domain renders the blockage of ␣ 7 nAChR by fluoxetine voltagedependent. These effects of fluoxetine on ␣ 7 receptors may be clinically important.A great deal of interest has recently focused on the neuronal nicotinic ␣ 7 receptor due to its special structural and functional characteristics as compared to other members of the nicotinic receptor family. In particular, the ␣ 7 subunit forms functional homomeric receptors; 1,2 and the substitution of the conserved leucine 247 for threonine, in the M2 channel domain, changes drastically their function. [3][4][5] Fluoxetine is a potent inhibitor of 5HT uptake that is used extensively in clinical practice; and it was thought that its therapeutic effects were essentially due to blocking of the 5HT-transporter. 6 However, we have recently shown that fluoxetine is a potent blocker of nicotinic receptors, inhibiting not only heteromeric muscular but also neuronal nicotinic receptors expressed in Xenopus oocytes. 7 The experiments reported here were done in oocytes injected with ␣ 7 subunit cDNA to investigate whether fluoxetine would also block homomeric ␣ 7 nAChR. Fluoxetine was also used as a tool to investigate further the role of the Leu-247 residue as a determinant of the functional and pharmacological profiles of the ␣ 7 receptor.Oocytes injected with the wild-type ␣ 7 subunit cDNA had a membrane potential of −38 ± 4 mV and responded to ACh with an inward current (I ACh ) whose peak amplitude depended on transmitter concentration. In oocytes held at −60 mV, 150 M ACh, a concentration close to the EC 50 for ␣ 7 nAChR, 1,8-10 elicited a mean acetylcholine-current (I ACh ) of −77 ± 13 nA (mean ± sem n = 15; two donors, range −20 nA to −177 nA), that decayed to 90% (T 0.1 ) in 0.81 ± 0.01 s. Fluoxetine alone (100 M) did not elicit obvious current responses in either non-inject...

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Protein kinase C modulates exogenous acetylcholine current in Xenopus oocytes

Cited by 8 publications

References 34 publications

Selective effects of a 4‐oxystilbene derivative on wild and mutant neuronal chick α7 nicotinic receptor

Selective effects of a 4‐oxystilbene derivative on wild and mutant neuronal chick α7 nicotinic receptor

Neuronal nicotinic threonine-for-leucine 247 α ₇ mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Effects of fluoxetine on wild and mutant neuronal α7 nicotinic receptors

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Protein kinase C modulates exogenous acetylcholine current in Xenopus oocytes

Cited by 8 publications

References 34 publications

Selective effects of a 4‐oxystilbene derivative on wild and mutant neuronal chick α7 nicotinic receptor

Selective effects of a 4‐oxystilbene derivative on wild and mutant neuronal chick α7 nicotinic receptor

Neuronal nicotinic threonine-for-leucine 247 α 7 mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine

Effects of fluoxetine on wild and mutant neuronal α7 nicotinic receptors

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Neuronal nicotinic threonine-for-leucine 247 α ₇ mutant receptors show different gating kinetics when activated by acetylcholine or by the noncompetitive agonist 5-hydroxytryptamine