Human 5-HT3A -- Trp 85

Species Original Mutated to Mutation
Human Trp 85  

 

Rat Equivalent Trp 90    
Mouse Equivalent Trp 90

Ala

Phe

Tyr

Ser

W90A

W90F

W90Y

W90S

 

mTrp90Ala

Cells with W90A receptors did not produce whole cell currents with high concentrations of 5-HT concentrations nor was there detectable specific binding of the antagonist [3H]granisetron.

 

Yan D, Schulte MK, Bloom KE, White MM (1999) Structural Features of the Ligand-binding Domain of the Serotonin 5HT3 Receptor. The Journal of Biological Chemistry 274 (9): 5537-5541

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 W90A mutant 5-HT3A receptor does not bind to [3H]granisteron. The wild type receptor bound [3H]granisteron with a Kd = 0.31 +/- 0.04 nM. The W90Y (Kd = 0.90 +/- 0.06 nM) mutant bound [3H]granisetron with a reduced affinity. 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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mTrp90Phe

 

inhibition of
curare (10nM)
binding of [3H]
granisetron (Kd)
Curare Inhibition
(Ki)
MDL 72222 Serotonin phenylbiguanide m-chlorophynylbiguanide
wild-type 85% 1.3 +/- 0.1nM 138 +/- 22nM        
W90F 15% 10.9 +/- 2.9nM 1063 +/- 179 nM similar reduction
as curare
no significant
difference in affinity
no significant
difference in affinity
no significant
difference in affinity

Nine residues (Thr 86, Thr 87, Tyr 88, Ile 89, Tyr 91, Arg 92, Gln 93, Tyr 94, Trp 95) were mutated individually to alanine. The ligands [3H]granisetron, curare, and serotonin were used to examine the properites of 5-HT3 receptor binding sites.

W90F was analyzed instead of W90A, and it proved to be the only one that significantly affected the interaction of curare with the receptor. R92A was the only substitution that altered the affinity of the agonist serotonin. W90F, R92A, and Y94A all reduced the affinity of [3H]granisetron. The periodicity of the effect suggests the involvement of a beta-strand.

 

Yan D, Schulte MK, Bloom KE, White MM (1999) Structural Features of the Ligand-binding Domain of the Serotonin 5HT3 Receptor. The Journal of Biological Chemistry 274 (9): 5537-5541

 

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mTrp90Tyr

  Wild-Type W90Y
[3H] Granisetron (Kd) 0.17 +/- 0.003 0.92 +/- 0.06
[3H] Granisetron (nH) 1.03 +/- 0.11 0.92 +/- 0.03
[3H] mCPBG (Kd) 1.48 +/- 0.09 8.24 +/- 0.73
[3H] mCPBG (nH) 1.07 +/- 0.02 1.00 +/- 0.003
mCPBG (EC50) 0.81 +/- 0.09 2.00 +/- 0.10
mCPBG (nH) 1.97 +/- 0.20 2.14 +/- 0.13
5-HT3 (EC 50)

2.10 +/- 0.40

8.02 +/- 0.30
5-HT3 (nH) 2.22 +/- 0.20 2.02 +/- 0.11
2-Me-5-HT (EC50) 12.7 +/- 0.45 uM 12.9 +/- 1.98 uM
2-Me-5-HT (nH)   no significant change

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 W90A mutant 5-HT3A receptor does not bind to [3H]granisteron. The wild type receptor bound [3H]granisteron with a Kd = 0.31 +/- 0.04 nM. The W90Y (Kd = 0.90 +/- 0.06 nM) mutant bound [3H]granisetron with a reduced affinity. 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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mTrp90Ser

The mutation of W90S did not show any response to [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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