Human 5-HT3A -- Thr 174

Species Original Mutated to Mutation
Human Thr 174  

 

Rat Equivalent Thr 179    
Mouse Equivalent Thr 179

Ala

Ser

T179A

T179S

mTyr179Ala mTyr179Ser

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 T179A (Kd= 3.20 +/- 0.10 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 T179S (Kd = 0.38 +/- 0.20 nM) mutation did not significantly alter the affinity of [3H]granisetron binding. Serine, like threonine contains a polar 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