Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface

Research output: Contribution to journal › Journal article › Research › peer-review

Standard

Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. / Ahring, Philip K; Olsen, Jeppe A; Nielsen, Elsebet Ø; Peters, Dan; Pedersen, Martin H F; Rohde, Line A; Kastrup, Jette Sandholm Jensen; Shahsavar, Azadeh; Indurthi, Dinesh C; Chebib, Mary; Gajhede, Michael; Balle, Thomas.

In: Neuropharmacology, Vol. 92, 2015, p. 135-45.

Research output: Contribution to journal › Journal article › Research › peer-review

Harvard

Ahring, PK, Olsen, JA, Nielsen, EØ, Peters, D, Pedersen, MHF, Rohde, LA, Kastrup, JSJ, Shahsavar, A, Indurthi, DC, Chebib, M, Gajhede, M & Balle, T 2015, 'Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface', Neuropharmacology, vol. 92, pp. 135-45. https://doi.org/10.1016/j.neuropharm.2014.12.035

APA

Ahring, P. K., Olsen, J. A., Nielsen, E. Ø., Peters, D., Pedersen, M. H. F., Rohde, L. A., Kastrup, J. S. J., Shahsavar, A., Indurthi, D. C., Chebib, M., Gajhede, M., & Balle, T. (2015). Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. Neuropharmacology, 92, 135-45. https://doi.org/10.1016/j.neuropharm.2014.12.035

Vancouver

Ahring PK, Olsen JA, Nielsen EØ, Peters D, Pedersen MHF, Rohde LA et al. Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. Neuropharmacology. 2015;92:135-45. https://doi.org/10.1016/j.neuropharm.2014.12.035

Author

Ahring, Philip K ; Olsen, Jeppe A ; Nielsen, Elsebet Ø ; Peters, Dan ; Pedersen, Martin H F ; Rohde, Line A ; Kastrup, Jette Sandholm Jensen ; Shahsavar, Azadeh ; Indurthi, Dinesh C ; Chebib, Mary ; Gajhede, Michael ; Balle, Thomas. / Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface. In: Neuropharmacology. 2015 ; Vol. 92. pp. 135-45.

Bibtex

@article{15bf1546e2024f17b277afb71615d62b,

title = "Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface",

abstract = "The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-β2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2(HQT)), which converts the β(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(β2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-β2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors.",

author = "Ahring, {Philip K} and Olsen, {Jeppe A} and Nielsen, {Elsebet {\O}} and Dan Peters and Pedersen, {Martin H F} and Rohde, {Line A} and Kastrup, {Jette Sandholm Jensen} and Azadeh Shahsavar and Indurthi, {Dinesh C} and Mary Chebib and Michael Gajhede and Thomas Balle",

year = "2015",

doi = "10.1016/j.neuropharm.2014.12.035",

language = "English",

volume = "92",

pages = "135--45",

journal = "Neuropharmacology",

issn = "0028-3908",

publisher = "Pergamon Press",

}

RIS

TY - JOUR

T1 - Engineered α4β2 nicotinic acetylcholine receptors as models for measuring agonist binding and effect at the orthosteric low-affinity α4-α4 interface

AU - Ahring, Philip K

AU - Olsen, Jeppe A

AU - Nielsen, Elsebet Ø

AU - Peters, Dan

AU - Pedersen, Martin H F

AU - Rohde, Line A

AU - Kastrup, Jette Sandholm Jensen

AU - Shahsavar, Azadeh

AU - Indurthi, Dinesh C

AU - Chebib, Mary

AU - Gajhede, Michael

AU - Balle, Thomas

PY - 2015

Y1 - 2015

N2 - The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-β2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2(HQT)), which converts the β(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(β2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-β2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors.

AB - The nicotinic acetylcholine receptor α4β2 is important for normal mammalian brain function and is known to express in two different stoichiometries, (α4)2(β2)3 and (α4)3(β2)2. While these are similar in many aspects, the (α4)3(β2)2 stoichiometry differs by harboring a third orthosteric acetylcholine binding site located at the α4-α4 interface. Interestingly, the third binding site has, so far, only been documented using electrophysiological assays, actual binding affinities of nicotinic receptor ligands to this site are not known. The present study was therefore aimed at determining binding affinities of nicotinic ligands to the α4-α4 interface. Given that epibatidine shows large functional potency differences at α4-β2 vs. α4-α4 interfaces, biphasic binding properties would be expected at (α4)3(β2)2 receptors. However, standard saturation binding experiments with [(3)H]epibatidine did not reveal biphasic binding under the conditions utilized. Therefore, an engineered β2 construct (β2(HQT)), which converts the β(-) face to resemble that of an α4(-) face, was utilized to create (α4)3(β2(HQT))2 receptors harboring three α4-α4 interfaces. With this receptor, low affinity binding of epibatidine with a Kd of ∼5 nM was observed in sharp contrast to a Kd value of ∼10 pM observed for wild-type receptors. A strong correlation between binding affinities at the (α4)3(β2(HQT))2 receptor and functional potencies at the wild-type receptor of a range of nicotinic ligands highlighted the validity of using the mutational approach. Finally, large differences in activities at α4-β2 vs. α4-α4 interfaces were observed for structurally related agonists underscoring the need for establishing all binding parameters of compounds at α4β2 receptors.

U2 - 10.1016/j.neuropharm.2014.12.035

DO - 10.1016/j.neuropharm.2014.12.035

M3 - Journal article

C2 - 25595102

VL - 92

SP - 135

EP - 145

JO - Neuropharmacology

JF - Neuropharmacology

SN - 0028-3908

ER -

ID: 138521372