Human 5-HT3A -- Met 223

Species Original Mutated to Mutation
Human Met 223 Ile

M223I

Rat Equivalent Ile 228    
Mouse Equivalent Ile 228

Met

Ala

Asn

I228M

I228A

I228N

 

hMet223Ile

In a triplet mutation of hM223I, hE224D and hS225I, the potentcy of (+)-Tubocurarine increased by 5-fold. When the triplet mutation was also combined with hY217Q and hR219K, there was a 26-fold increase in the IC50 of (+)-Tubocurarine relative to the human wild type. When this quintuple mutation was combined with hY228S and hV237I, there was a further decrement of 2-fold of the IC50 value of (+)-Tubocurarine relative to the quintuple mutant.

Hope AG, Belelli D, Mair ID, Lambert JJ, Peters JA (1999) Molecular Determinants of (+)- Tubocurarine Binding at Recombinant 5-Hydroxytryptamine3A Receptor Subunits. Molecular Pharmacology 55:1037-1043

 

The mutation was made in the human 5-HT3A subunit at Met223. This residue was numbered Met200 in the paper in which numbering began at the end of the signal sequence.

In order to identify the potency of meta-chlorophenylbiguanide (mCPBG) in human 5-HT3A receptors, the recombinant human and rat 5-HT3A receptors were expressed in Xenopus Oocytes by cRNA injection and 5-HT-evoked currents were recorded using two-electorde voltage-clamp. The effect of replacing human Leu215, Pro216, Tyr217, Arg219, Met223, Ser225 and Tyr228 (numbering from the beginning of the signal sequence) by the equivalent residues (Phe, Thr, Lys,Gln, Ile, Thr and Ser) in the rat was to increase the potency of mCPBG as an agonist at the mutant human 5-HT3A receptor by 13-fold.

Mochizuki S.,Miyake A.,Furuichi K. (1999) Identification of a domain affecting agonist potency of meta-chlorophenylbiguanide in 5-HT3 receptors. European Journal of Pharmacology 369:125-132

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mIle228Met

In a single point mutation of I228M, the IC50 value for (+)- Tubocurarine was indistinguishable from the wildtype.

In a triplet mutation of mI228M, mD229E and mI230S, the antagonist potentcy of (+)-Tubocurarine shifted toward that observed for the human. This produced a 20-fold increase in IC50 relative to that observed for the WT m5-HT3A. When this triplet mutation was also combined with mQ222Y and mK224R there was a 54-fold decrease in the IC50 of (+)-Tuocurarine relative to the mouse wild type. When this quintuple mutation was combined with mS233Y and mI242V, there was a further increment of 3-fold of the IC50 value of the (+)-Tubocurarine relative to the quintuple mutant.

Hope AG, Belelli D, Mair ID, Lambert JJ, Peters JA (1999) Molecular Determinants of (+)- Tubocurarine Binding at Recombinant 5-Hydroxytryptamine3A Receptor Subunits. Molecular Pharmacology 55:1037-1043

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mIle228Ala

In this study amino acids from E225 to Y234 of a murine 5-HT3ASR were individually mutated to alanine. Conservative mutations were made if the initial alanine mutation showed no binding and/or function. The mutated receptors were evaluated using radioligand binding, two-electrode voltage clamp, and immunofluorescence studies.

The I228A mutation caused a 4.5-fold reduction in the Kd value for [3H]granisetron, with no significant changes to Bmax. There was a 235-fold reduction in the Ki value for 5-HT, with a 10-fold reduction in the EC50 value and a significant decrease in Imax. There was a 5-fold reduction in the Ki value for mCPBG, with a 16-fold reduction in the EC50 value and no significant changes in Imax.

Suryanarayanan A, Joshi PR, Bikádi Z, Mani M, Kulkarni TR, Gaines C, Schulte MK. (2005) The loop C region of the murine 5-HT3A receptor contributes to the differential actions of 5-hydroxytryptamine and m-chlorophenylbiguanide. Biochemistry 44(25):9140-9.

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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 I228A (Kd=1.40 +/- 0.30 nM) mutant 5-HT3A receptor bound [3H]granisetron with a reduced affinity. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The I228N (Kd = 0.30 +/- 0.05 nM) mutation did not significantly alter the affinity of the [3H]granisetron binding. Asparagine, like isoleucine is an uncharge residue appears to be 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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mIle228Asn

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 I228A (Kd=1.40 +/- 0.30 nM) mutant 5-HT3A receptor bound [3H]granisetron with a reduced affinity. The wild type receptor bound [3H]granisetron with a Kd = 0.31 +/- 0.04 nM. The I228N (Kd = 0.30 +/- 0.05 nM) mutation did not significantly alter the affinity of the [3H]granisetron binding. Asparagine, like isoleucine is an uncharge residue appears to be 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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