Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Charnet, Pierre; Labarca, Cesar; Cohen, Bruce; Davidson, Norman; Lester, Henry A.; Pilar, G.

doi:10.1113/jphysiol.1992.sp019132

Cited by 49 publications

(51 citation statements)

References 38 publications

(66 reference statements)

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“…9, A-J; Table 3). Consistent with previous reports (Charnet et al, 1992;Kuryatov et al, 1997;Figl et al, 1998), the rat ␣4␤2 wild-type channels displayed multiple conductance states in 0 mM added Ca 2ϩ (Fig. 9, A-D; Table 3).…”

Section: Receptorsupporting

confidence: 80%

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Rodrigues-Pinguet¹,

Pinguet²,

Figl³

et al. 2005

Mol Pharmacol

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Extracellular Ca2ϩ robustly potentiates the acetylcholine response of ␣4␤2 nicotinic receptors. Rat orthologs of five mutations linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE)-␣4(S252F), ␣4(S256L), ␣4(ϩL264), ␤2(V262L), and ␤2(V262M)-reduced 2 mM Ca 2ϩ potentiation of the ␣4␤2 1 mM acetylcholine response by 55 to 74%. To determine whether altered allosteric Ca 2ϩ activation or enhanced Ca 2ϩ block caused this reduction, we coexpressed the rat ADNFLE mutations with an ␣4 N-terminal mutation, ␣4(E180Q), that abolished ␣4␤2 allosteric Ca 2ϩ activation. In each case, Ca 2ϩ inhibition of the double mutants was less than that expected from a Ca 2ϩ blocking mechanism. In fact, the effects of Ca 2ϩ on the ADNFLE mutations near the intracellular end of the M2 region-␣4(S252F) and ␣4(S256L)-were consistent with a straightforward allosteric mechanism. In contrast, the effects of Ca 2ϩ on the ADNFLE mutations near the extracellular end of the M2 region-␣4(ϩL264)␤2, ␤2(V262L), and ␤2(V262M)-were consistent with a mixed mechanism involving both altered allosteric activation and enhanced block. However, the effects of 2 mM Ca 2ϩ on the ␣4␤2, ␣4(ϩL264)␤2, and ␣4␤2(V262L) single-channel conductances, the effects of membrane potential on the ␤2(V262L)-mediated reduction in Ca 2ϩ potentiation, and the effects of eliminating the negative charges in the extracellular ring on this reduction failed to provide any direct evidence of mutant-enhanced Ca 2ϩ block. Moreover, analyses of the ␣4␤2, ␣4(S256L), and ␣4(ϩL264) Ca 2ϩ concentration-potentiation relations suggested that the ADNFLE mutations reduce Ca 2ϩ potentiation of the ␣4␤2 acetylcholine response by altering allosteric activation rather than by enhancing block.

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Section: Receptorsupporting

confidence: 80%

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Rodrigues-Pinguet¹,

Pinguet²,

Figl³

et al. 2005

Mol Pharmacol

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“…The largest WT conductance state was similar to the 46 pS value for recombinantly expressed human á4â2 nicotinic receptors (Buisson et al 1996). The smaller WT conductance state (34 pS) was similar to a previous value (34 pS) for rat á4â2 nicotinic receptors expressed in oocytes (Charnet et al 1992 In the single patch without 49 pS openings, we assigned a percentage area of zero to the 49 pS component and 100% to the 34 pS component. The WT channels spent 60 ± 11 % (mean ± s.e.m., n = 6 patches) of the total open time in the 34 pS state and 40 ± 11 % (n = 6) of the total open time in the 49 pS state.…”

Section: Mutations Affect Single-channel Conductancementioning

confidence: 87%

Two mutations linked to nocturnal frontal lobe epilepsy cause use‐dependent potentiation of the nicotinic ACh response

Figl

Viseshakul

Shafaee

et al. 1998

The Journal of Physiology

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Two mutations in the M2 region of the human á4 neuronal nicotinic subunit -á4(S248F) and á4(776ins3) -have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) (Steinlein et al. 1995(Steinlein et al. , 1997. The á4(S248F) mutation is a serine to phenylalanine substitution at position 248 in the human á4 nicotinic subunit. The á4(776ins3) mutation is a 3-base pair insertion that adds a leucine at position 259 in the amino acid sequence of the human á4 subunit. Photo-affinity labelling and structurefunction experiments show that the M2 region of the nicotinic subunits forms the conducting pore of the receptor (reviewed in Karlin & Akabas, 1995). Thus, both ADNFLE mutations lie in the conducting pore of the nicotinic receptor. ADNFLE patients suffer from brief and occasionally violent nocturnal seizures . However, the physiological mechanism responsible for these seizures has not been established. Previous studies show that the predominant brain nicotinic receptor subtype is á4â2 (Whiting & Lindstrom, 1987;Flores et al. 1991;Whiting et al. 1991). Receptors formed by co-expressing á4(S248F) or á4(776ins3) subunits with wild-type (WT) â2 subunits in Xenopus oocytes (Weiland et al. 1996; Steinlein et al. 1997;Kuryatov et al. 1997) differ from the WT receptor in several ways but no common effects of the two mutations on the acetylcholine (ACh) response have been reported previously. To determine whether the ADNFLE mutations have any common effects on the ACh response, we constructed two rat homologues (S252F and +L264) of the human ADNFLE mutations á4(S248F) and á4(777ins3), co-expressed them with rat â2 subunits in Xenopus oocytes, and studied the properties of the expressed receptors. We also constructed the rat double mutation V247I:S252F, which combined the S252F mutation with a second V247I mutation that converted the only rat/human residue substitution in the á4 M2 region to the corresponding human residue. All three Journal of Physiology (1998) 1. We constructed rat homologues (S252F and +L264) of two human á4 nicotinic mutations -á4(S248F) and á4(777ins3) -that have been linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) and co-expressed them with wild-type rat â2 subunits in Xenopus oocytes. 2. The S252F and +L264 mutations had three common effects on the ACh response. First, they caused use-dependent potentiation of the response during a train of brief 100 nÒ ACh pulses. Second, they delayed the rise times of the 5-15 nÒ (+L264) and 30 nÒ (S252F) ACh responses. Third, they reduced extracellular Ca¥-induced increases in the 30 ìÒ ACh response. 3. Beside these shared effects, the S252F mutation also reduced the channel burst duration measured from voltage-jump relaxations, enhanced steady-state desensitization and reduced the single-channel conductance. In contrast, the +L264 mutation prolonged the channel burst duration, did not affect desensitization and slightly increased single-channel conductance. Neither mutation affected the number of surface receptors measured b...

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“…Therefore, it is likely that at some types of receptors 5_HT acts on the agonist binding sites and at others within the ionic channel. This would resemble the effects of hexamethonium, which appears to inhibit muscle AChRs competitively (Rang & Rylett, 1984) but neuronal receptors non-competitively (Charnet et al 1992). However, we have reported that in oocytes expressing the á2â4 neuronal AChR subtype the ACh-current is inhibited by 5_HT in a noncompetitive and voltage-dependent fashion (Garc úa-Colunga & Miledi, 1994, which is similar to the effects of 5_HT on chromaffin cells (Vijayaraghavan et al 1993) and on á7 homomeric AChR (Palma et al 1997).…”

Section: Effects Of Serotonergic Agents On Muscle Achrsmentioning

confidence: 99%

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Garcı́a-Colunga¹,

Miledi²

1999

Exp. Physiol.

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summaryXenopus laevis oocytes were used to analyse the effects of serotonin (5-hydroxytryptamine, 5_HT) and serotonergic agents on ionic currents elicited by the activation of mammalian muscle nicotinic acetylcholine receptors (AChRs). 5_HT as well as other serotonergic agents, such as ketanserin, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), methysergide, spiperone, or fluoxetine alone (up to 1 mÒ), did not elicit membrane currents in Xenopus oocytes expressing AChRs, but they reversibly reduced the current elicited by acetylcholine (ACh-current). Serotonin was applied before, together with or after ACh application, and its effects were examined on desensitizing and non-desensitizing ACh-currents. 5_HT reduced the amplitude and accelerated the desensitization of the desensitizing currents. In contrast, non-desensitizing currents were reduced in amplitude but their time course was not significantly affected. With the same concentration of 5_HT the inhibition was stronger on desensitizing than on non-desensitizing ACh-currents. For example, 100 ìÒ 5_HT reduced the peak of a desensitizing ACh-current to 0·48 ± 0·06 (peak current ratio) and after 40 s the current was reduced to a ratio of 0·25 ± 0·04, whereas a non-desensitizing ACh-current was reduced to a ratio of 0·66 ± 0·01. All the serotonergic agents tested inhibited the ACh-currents rapidly and reversibly, suggesting that they are acting directly on the AChRs. The half-inhibitory concentration, IC50, of 5_HT acting on nondesensitizing currents elicited by 250 nÒ ACh was 247 ± 26 ìÒ and the Hill coefficient was •0·88, suggesting a single site for the interaction of 5_HT with the receptor. It appears that 5_HT inhibits AChRs non-competitively because neither the half-effective concentration of ACh, EC50, for ACh-current nor the Hill coefficient were affected by 5_HT. Furthermore, the extent of inhibition of 5_HT on AChRs did not depend on the nicotinic agonist (suberyldicholine, ACh or nicotine). The inhibition of AChRs by serotonergic agents was voltage-dependent. The electrical distance of the binding site for 5_HT was •0·75, whereas for the other serotonergic agents tested it was •0·22, suggesting that ketanserin, 8-OH-DPAT, methysergide, spiperone and fluoxetine act within the ion channel, but at a site more external than that for 5_HT. These substances inhibited the AChcurrent more potently than 5_HT. We conclude that 5_HT and serotonergic agents inhibit, in a noncompetitive manner, the ACh-current in muscle AChRs by blocking the open receptor-channel complex. Moreover, 5_HT appears to promote the desensitized state of the receptor when the current is elicited by high ACh concentrations. introduction The muscle nicotinic acetylcholine receptor (AChR), a neurotransmitter-gated ion channel, is a member of a gene superfamily that includes GABAA, glycine and 5_HT× receptors (Cockcroft et al. 1995). Structurally, the AChR is a pentamer composed of four different subunits (á, â, ã or å, and ä) with a stoichiometry of áµâãä (Karlin & Akabas, 1995; Unwin, ...

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Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Cited by 49 publications

References 38 publications

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Two mutations linked to nocturnal frontal lobe epilepsy cause use‐dependent potentiation of the nicotinic ACh response

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

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Pharmacological and kinetic properties of alpha 4 beta 2 neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes.

Cited by 49 publications

References 38 publications

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca2+ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca2+ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Two mutations linked to nocturnal frontal lobe epilepsy cause use‐dependent potentiation of the nicotinic ACh response

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

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Product

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Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor

Mutations Linked to Autosomal Dominant Nocturnal Frontal Lobe Epilepsy Affect Allosteric Ca²⁺ Activation of the α4β2 Nicotinic Acetylcholine Receptor