Human 5-HT3A -- Glu 224

Species Original Mutated to Mutation
Human Glu 224 Asp

E224D

Rat Equivalent Glu 229    
Mouse Equivalent Asp 229

Glu

Ala

D229E

D229A

 

hGlu224Asp

In a triplet mutation of hM223I, hE224D and hS225I, the potentcy of (+)-Tubocurarine increased by 5-fold. When the triplet mutation was also combined with hY217Q and hR219K, there was a 26-fold increase in the IC50 of (+)-Tubocurarine relative to the human wild type. When this quintuple mutation was combined with hY228S and hV237I, there was a further decrement of 2-fold of the IC50 value of (+)-Tubocurarine relative to the quintuple mutant.

 

Hope AG, Belelli D, Mair ID, Lambert JJ, Peters JA (1999) Molecular Determinants of (+)- Tubocurarine Binding at Recombinant 5-Hydroxytryptamine3A Receptor Subunits. Molecular Pharmacology 55:1037-1043

go back to top
go back to 5-HT3A

 

mAsp229Glu

In a single point mutation of mD229E, a substantial increase in IC50 (9-fold) was obtained.

In a triplet mutation of mI228M, mD229E and mI230S, the antagonist potentcy of (+)-Tubocurarine shifted toward that observed for the human. This produced a 20-fold increase in IC50 relative to that observed for the WT m5-HT3A. When this triplet mutation was also combined with mQ222Y and mK224R there was a 54-fold decrease in the IC50 of (+)-Tuocurarine relative to the mouse wild type. When this quintuple mutation was combined with mS233Y and mI242V, there was a further increment of 3-fold of the IC50 value of the (+)-Tubocurarine relative to the quintuple mutant.

Hope AG, Belelli D, Mair ID, Lambert JJ, Peters JA (1999) Molecular Determinants of (+)- Tubocurarine Binding at Recombinant 5-Hydroxytryptamine3A Receptor Subunits. Molecular Pharmacology 55:1037-1043

 

Twenty six residues (I71, Y73,W90, R92, N128, E129, Y143, Y153, T179, T181, S182,W183, L184, W195, V201, R202, S203, S206, I207, F226, I228, D229, I230, Y234, E236, K238) thought to be within 5A of the 5-HT binding site were substituted with either alanine or a residue with somewhat similar physicochemical properties to the wildtype residue. The effects of these substitutions were investigated by measuring the binding of [3H]granisetron. The results were used to map the granisetron binding site within an homology model of the mouse 5-HT3A receptor based on the crystal structure of the AChBP. Mouse 5-HT3A receptors were expressed in HEK293 cells for radioligand binding.

The D229A (Kd=3.80 +/- 0.26 nM) mutant 5-HT3A receptor bound [3H]granisetron with a reduced affinity. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The D229E (Kd = 0.11 +/- 0.03 nM) mutation increased the affinity of [3H]granisetron binding.Glutamate, like aspartate is an acid residue which is presumably required for the correct structure of the antagonist binding site.

Receptor [3H]granisetron binding (Kd)
WT 0.31+/-0.04
W90A*

NB

W90Y* 0.90 +/- 0.06
R92A* 1.80 +/- 0.40
R92K 1.00 +/- 0.30
E129A* NB
E129D* NB
Y153A* 2.36 +/- 0.53
Y153F 0.90 +/- 0.20
T179A* 3.20 +/- 0.10
T179S 0.38 +/- 0.20
S181A* 0.12 +/- 0.04
S181S 0.58 +/- 0.10
S182A* 1.00 +/- 0.20
S182T* 1.80 +/- 0.09
W183A*/Y* NB
L184A* 4.11 +/- 0.94
L184I* 0.71 +/- 0.05
W195A* 5.08 +/- 0.88
W195Y* 8.70 +/- 2.40
S203A* 0.08 +/-0.02
S203T 0.26 +/- 0.11
S206A* 1.67 +/- 0.27
S206T* 4.40 +/- 0.49
I228A* 1.40 +/- 0.30
I228N 0.30 +/- 0.05
D229A* 3.80 +/- 0.26
D229E* 0.11 +/- 0.03
I230A 0.30 +/- 0.10
I230N* 1.70 +/- 0.40
Y234A* NB
Y234F 1.30 +/- 0.36

NB, No binding

* significantly different from the WT 5-HT3A receptor

 

Thompson AJ, Price KL, Reeves DC, Chan SL, Chau PL, Lummis SC(2005). Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding.The Journal of Biological Chemistry 280(21):20476-82

go back to top
go back to 5-HT3A

 

mAsp229Ala

In this study amino acids from E225 to Y234 of a murine 5-HT3ASR were individually mutated to alanine. Conservative mutations were made if the initial alanine mutation showed no binding and/or function. The mutated receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies.

The D229A mutation caused and 8-fold reduction in the Kd value for [3H]granisetron, with no significant changes to Bmax. There was a 140-fold reduction in the Ki value for 5-HT, with a 12.5-fold reduction in the EC50 value and no significant changes in Imax. There was no significant change in the Ki value for mCPBG, and no significant changes in the EC50 and Imax values.

Suryanarayanan A, Joshi PR, Bikádi Z, Mani M, Kulkarni TR, Gaines C, Schulte MK. (2005) The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide. Biochemistry 44(25):9140-9.

go back to top
go back to 5-HT3A

 

Twenty six residues (I71, Y73,W90, R92, N128, E129, Y143, Y153, T179, T181, S182,W183, L184, W195, V201, R202, S203, S206, I207, F226, I228, D229, I230, Y234, E236, K238) thought to be within 5A of the 5-HT binding site were substituted with either alanine or a residue with somewhat similar physicochemical properties to the wildtype residue. The effects of these substitutions were investigated by measuring the binding of [3H]granisetron. The results were used to map the granisetron binding site within an homology model of the mouse 5-HT3A receptor based on the crystal structure of the AChBP. Mouse 5-HT3A receptors were expressed in HEK293 cells for radioligand binding.

The D229A (Kd=3.80 +/- 0.26 nM) mutant 5-HT3A receptor bound [3H]granisetron with a reduced affinity. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The D229E (Kd = 0.11 +/- 0.03 nM) mutation increased the affinity of [3H]granisetron binding.Glutamate, like aspartate is an acid residue which is presumably required for the correct structure of the antagonist binding site.

Receptor [3H]granisetron binding (Kd)
WT 0.31+/-0.04
W90A*

NB

W90Y* 0.90 +/- 0.06
R92A* 1.80 +/- 0.40
R92K 1.00 +/- 0.30
E129A* NB
E129D* NB
Y153A* 2.36 +/- 0.53
Y153F 0.90 +/- 0.20
T179A* 3.20 +/- 0.10
T179S 0.38 +/- 0.20
S181A* 0.12 +/- 0.04
S181S 0.58 +/- 0.10
S182A* 1.00 +/- 0.20
S182T* 1.80 +/- 0.09
W183A*/Y* NB
L184A* 4.11 +/- 0.94
L184I* 0.71 +/- 0.05
W195A* 5.08 +/- 0.88
W195Y* 8.70 +/- 2.40
S203A* 0.08 +/-0.02
S203T 0.26 +/- 0.11
S206A* 1.67 +/- 0.27
S206T* 4.40 +/- 0.49
I228A* 1.40 +/- 0.30
I228N 0.30 +/- 0.05
D229A* 3.80 +/- 0.26
D229E* 0.11 +/- 0.03
I230A 0.30 +/- 0.10
I230N* 1.70 +/- 0.40
Y234A* NB
Y234F 1.30 +/- 0.36

NB, No binding

* significantly different from the WT 5-HT3A receptor

 

Thompson AJ, Price KL, Reeves DC, Chan SL, Chau PL, Lummis SC(2005). Locating an antagonist in the 5-HT3 receptor binding site using modeling and radioligand binding.The Journal of Biological Chemistry 280(21):20476-82

 

back to top
go back to 5-HT3A

Previous Next