Human 5-HT3A -- Trp 190

Species Original Mutated to Mutation
Human Trp 190  

 

Rat Equivalent Trp 195    
Mouse Equivalent Trp 195

Tyr

Ser

Ala

W195Y

W195S

W195A

 

mTrp195Tyr

Wild- Type W195Y
[3H] Granisetron (Kd) 0.17 +/- 0.003

8.7 +/- 2.4

[3H] Granisetron (nH) 1.03 +/- 0.011 1.24 +/- 0.18
[3H] mCPBG (Kd) 1.48 +/- 0.09

2.66 +/- 0.62

[3H] mCPBG (nH) 1.07 +/- 0.02 0.84 +/- 0.22
5-HT3 (EC50)

2.10 +/- 0.40

8.09 +/- 0.93
5-HT3 (nH) 2.22 +/- 0.20 2.74 +/- 0.25
mCPBG (EC50) 0.81 +/- 0.09 1.68 +/- 0.38
mCPBG (nH) 1.97 +/- 0.20 1.58 +/- 0.20

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.

The W195A (Kd=5.08 +/- 0.88 nM) mutant 5-HT3A receptor bound [3H]granisteron with a reduced affinity. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The W195Y (Kd = 8.70 +/- 2.40 nM) mutation reduced the affinity of [3H]granisetron binding.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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mTrp195Ser

  Wild-Type W195S
[3H] Granisetron (Kd) 0.17 +/- 0.003 1.35 +/- 0.07
[3H] Granisetron (nH) 1.03 +/- .011 1.37 +/- 0.14
[3H] mCPBG (Kd) 1.48 +/- 0.09 2.47 +/- 0.28
[3H] mCPBG (nH) 1.07 +/- 0.02

0.72 +/- 0.17

5-HT3 (EC50) 2.10 +/- 0.40 18.8 +/- 2.45
5-HT3 (nH) 2.22 +/- 0.20 2.58 +/- 0.15
mCPBG (EC50) 0.81 +/- 0.09 2.15 +/- 0.16
mCPBG (nH) 1.97 +/- 0.20 2.19 +/- 0.27

 

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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mTrp195Ala

 

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 W195A (Kd=5.08 +/- 0.88 nM) mutant 5-HT3A receptor bound [3H]granisteron with a reduced affinity. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The W195Y (Kd = 8.70 +/- 2.40 nM) mutation reduced the affinity of [3H]granisetron binding.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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