Human 5-HT3A -- Glu 231

Species Original Mutated to Mutation
Human

Glu 231

Gln

Asp

E231Q

E231D

Rat Equivalent Glu 236    
Mouse Equivalent Glu 236

Ala

Asp

E236A

E236D

 

hGlu231Gln

EC50 (uM) Granisetron Inhibition (IC50) (nM) [3H]mCPBG (Kd) [3H]MCPBG (Granisetron Inhibition Ki) (nM) [3H] GR65630 (Kd) [3H]GR65630 (Granisetron Inhibition Ki) (nM) [3H]GR65630 (mCPBG Inhibition Ki)
Wild Type 2.1 +/- 0.6 0.14 +/- 0.1 12.7 +/- 1.9 2.0 +/- 0.6 1.4 +/- 0.3 1.4 +/- 0.4 11 +/- 4
E231Q 44 +/- 12 12 +/- 5 >60 >22 >20 12 +/- 5 150 +/- 20

 

Schreiter C, Hovius R, Costioli M, Pick H, Kellenberger S, Schild L, Vogel H (2003) Characterization of the Ligand-Binding Site of the Serotonin 5-HT3 Receptor. The Journal of Biological Chemistry 278(25):22709-22716


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hGlu231Asp

EC50 (uM) Granisetron Inhibition (IC50) (nM) [3H]mCPBG (Kd) [3H]MCPBG (Granisetron Inhibition Ki) (nM) [3H] GR65630 (Kd) [3H]GR65630 (Granisetron Inhibition Ki) (nM) [3H]GR65630 (mCPBG Inhibition Ki)
Wild Type 2.1 +/- 0.6 0.14 +/- 0.1 12.7 +/- 1.9 2.0 +/- 0.6 1.4 +/- 0.3 1.4 +/- 0.4 11 +/- 4
E231D >2*104 not relevant no binding detected not relevant no binding detected not relevant not relevant

 

Schreiter C, Hovius R, Costioli M, Pick H, Kellenberger S, Schild L, Vogel H (2003) Characterization of the Ligand-Binding Site of the Serotonin 5-HT3 Receptor. The Journal of Biological Chemistry 278(25):22709-22716


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mGlu236Ala mGlu236Asp

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) were replaced by either alanine or a residue with similar physicochemical properties to the native residue, to examine their roles in docking of [3H]granisetron into the 5-HT3A receptor binding site. Mouse 5-HT3A receptors were expressed in HEK293 cells and homology modeling, ligand-docking and radioligand binding were used.

The E236A and E236D mutations did not significantly alter the affinity of the antagonist [3H]granisetron. The table below lists conservative and non-conservative substitutions that either both, or individually affected [3H]granisetron binding.

 

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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