Human 5-HT3A -- Trp 178

Species Original Mutated to Mutation
Human Trp 178  

 

Rat Equivalent Trp 183    
Mouse Equivalent Trp 183

Tyr

Ser

Ala

W183Y

W183S

W183A

 

mTrp183Tyr

Binding of both [3H]granisetron and [3H]mCPBG was ablated by this mutation.

  wild-type W183Y
5-HT3 (EC50) 2.10 +/- 0.40 194 +/- 19.2
5-HT3 (nH) 2.22 +/- 0.20 1.92 +/- 0.26
mCPBG (EC50) 0.81 +/- 0.09

19.6 +/- 2.8

mCPBG (nH) 1.97 +/- 0.20 2.05 +/- 0.18

 

 

Spier AD, Lumis SCR (2000) The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain. The Journal of Biological Chemistry 275(8): 5620-5625

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.

In addition to [3H]granisetron binding, immunofluorescence was used to examine whether non-binding mutant receptors reached the cell surface. This approach demonstrated that W90A, E129A, E129D, W183A, W183Y and Y234A mutant receptors were expressed at the cell membrane even though there was no [3H]granisetron binding. The table below lists those mutations that significantly affected [3H]granisetron binding.

The W183A mutant 5-HT3A receptor did not bind to [3H]granisetron. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The W183Y mutant did not bind to [3H]granisetron. Tyrosine, like tryptophan contains an aromatic ring 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

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mTrp183Ser

There was no binding of [3H]granisetron, [3H]mCPBG, 5-HT or mCPBG.

 

Spier AD, Lumis SCR (2000) The Role of Tryptophan Residues in the 5-Hydroxytryptamine3 Receptor Ligand Binding Domain. The Journal of Biological Chemistry 275(8): 5620-5625

 

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mTrp183Ala

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.

In addition to [3H]granisetron binding, immunofluorescence was used to examine whether non-binding mutant receptors reached the cell surface. This approach demonstrated that W90A, E129A, E129D, W183A, W183Y and Y234A mutant receptors were expressed at the cell membrane even though there was no [3H]granisetron binding. The table below lists those mutations that significantly affected [3H]granisetron binding.

The W183A mutant 5-HT3A receptor did not bind to [3H]granisetron. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The W183Y mutant did not bind to [3H]granisetron. Tyrosine, like tryptophan contains an aromatic ring 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

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