Human 5-HT3A -- Tyr 138

Species Original Mutated to Mutation
Human Tyr 138  

 

Rat Equivalent Tyr 143    
Mouse Equivalent Tyr 143

Phe

Ala

Ser

Y143F

Y143A

Y143S

mTyr143Ala

The mutation in the mouse 5-HT3A at residue Y143A was numbered Y142A in this paper .

This study examined the roles of 13 residues in the binding of several agonists and antagonists to the mouse 5-HT3A receptor. Three mutations caused a large change in binding affinity of 5-HT, mCPBG, d-tubocurarine and lerisetron: Y141A (termed 140), Y143A (termed 142) and Y153A (termed 152). The Ki values for these compounds (determined by their abilities to displace [3H]granisetron) are listed in the table below. The authors also examined the potency of 5-HT as an agonist at the 5-HT3A Y152A mutant receptor.

The other mutations V142A termed (termed 141), V144A (termed 143), H146A (termed 145), R147A (termed 146), E149A (termed 148), Q151A (termed 150), N152A (termed 151), K154A (termed 153) have less binding of [3H]granisetron. Mutations G148A (termed 147) and V150A (termed 149) abolished [3H]granisetron binding.

Ki WT 5-HT3A Y141A Y143A Y153A
5-HT 74 ± 17 nM 97 ± 20 nM 8 ± 2.0 mM 1.8 ± 0.4 mM
mCPBG 3.0 ± 0.55 nM 22 ± 6.1 nM 470 ± 90 nM 73 ± 26 nM
d-Tubocurarine 6 ± 2.1 nM

300 ± 120 nM

39 ± 19 nM 60 ± 19 nM

Lerisetron

0.8 ± 0.19 nM 2.1 ± 0.33 nM 130 ± 28 nM 150 ± 36 nM

5-HT EC50

2.7 ± 0.25 μM NT NT 370 ± 27 μM

Venkataraman P,Venkatachalan SP,Joshi PR, Muthalagi M and Schulte MK (2002) Identification of critical residues in loop E in the 5-HT3ASR binding site.BMC Biochemistry 3:15

Eleven residues (Tyr 50, Tyr 73, Tyr 88, Tyr 91, Tyr 94, Tyr 141, Tyr 143, Tyr 153, Tyr 167, Tyr 234, Tyr 240) were mutated individually for alanine, serine and some residues for phenylalanine. The ligands [3H]granisetron and 5-HT were used to examine the properities of ligand binding sites and function of the 5-HT3A receptor, respectively (see Table below).

Mouse 5-HT3A receptors were expressed in Xenopus Oocytes (cRNA injection) and studied using two-electrode voltage-clamp technique. HEK293 cells were used in radioligand binding and calcium imaging assays to study the affinity of [3H]granisetron and the potency of 5-HT, respectively.

Y143A, Y143F and Y143S mutant mouse 5-HT3A receptors bound the antagonist [3H]granisetron. The Y143S mutant required 5-HT concentrations above 500 mM for a significant response.

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT 0.32 +/- 0.035 ++ 1.47 +/-0.42 2.39 +/- 0.23
Y50F NB - NR NR
Y50A NB -/+ NR NR
Y50S NB -/+ NR 1.59 +/- 0.18
Y73A/S YES NT NT NT
Y88A/S YES NT NT NT
Y91F YES NT 2.13 +/- 0.51 4.22 +/-0.25
Y91A NO - NR 13.7 +/-1.25*
Y91S NO + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NO ++ NR NT
Y143F YES NT NT NT
Y143A YES NT NR NT
Y143S YES NT >500* NT
Y153A YES NT 74.3 +/- 8.9* NT
Y153S NO ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NO ++ NR NT
Y234S NO + NR NT
Y240A/S YES NT NT NT

NT, Not tested

++, Presence of cell surface receptors revealed by immunocytochemistry

-/+, Reduced cell surface receptor expression compared to WT 5-HT3A

-, Absence of cell surface receptors

* significantly different from the WT 5-HT3A receptor

Price KL, Lummis SCR (2004). The role of Tyrosine residues in the extracellular domain of the 5-Hydroxytryptamine3 receptor.The Journal of Biological Chemistry 279(22):23294-23301

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 Y143A and Y143F 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

 

mTyr143Phe

Eleven residues (Tyr 50, Tyr 73, Tyr 88, Tyr 91, Tyr 94, Tyr 141, Tyr 143, Tyr 153, Tyr 167, Tyr 234, Tyr 240) were mutated individually for alanine, serine and some residues for phenylalanine. The ligands [3H]granisetron and 5-HT were used to examine the properities of ligand binding sites and function of the 5-HT3A receptor, respectively (see Table below).

Mouse 5-HT3A receptors were expressed in Xenopus Oocytes (cRNA injection) and studied using two-electrode voltage-clamp technique. HEK293 cells were used in radioligand binding and calcium imaging assays to study the affinity of [3H]granisetron and the potency of 5-HT, respectively.

Y143A, Y143F and Y143S mutant mouse 5-HT3A receptors bound the antagonist [3H]granisetron. The Y143S mutant required 5-HT concentrations above 500 mM for a significant response.

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT 0.32 +/- 0.035 ++ 1.47 +/-0.42 2.39 +/- 0.23
Y50F NB - NR NR
Y50A NB -/+ NR NR
Y50S NB -/+ NR 1.59 +/- 0.18
Y73A/S YES NT NT NT
Y88A/S YES NT NT NT
Y91F YES NT 2.13 +/- 0.51 4.22 +/-0.25
Y91A NO - NR 13.7 +/-1.25*
Y91S NO + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NO ++ NR NT
Y143F YES NT NT NT
Y143A YES NT NR NT
Y143S YES NT >500* NT
Y153A YES NT 74.3 +/- 8.9* NT
Y153S NO ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NO ++ NR NT
Y234S NO + NR NT
Y240A/S YES NT NT NT

NT, Not tested

++, Presence of cell surface receptors revealed by immunocytochemistry

-/+, Reduced cell surface receptor expression compared to WT 5-HT3A

-, Absence of cell surface receptors

* significantly different from the WT 5-HT3A receptor

Price KL, Lummis SCR (2004). The role of Tyrosine residues in the extracellular domain of the 5-Hydroxytryptamine3 receptor.The Journal of Biological Chemistry 279(22):23294-23301

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 Y143A and Y143F mutations did not significantly altered 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

 

mTyr143Ser

Eleven residues (Tyr 50, Tyr 73, Tyr 88, Tyr 91, Tyr 94, Tyr 141, Tyr 143, Tyr 153, Tyr 167, Tyr 234, Tyr 240) were mutated individually for alanine, serine and some residues for phenylalanine. The ligands [3H]granisetron and 5-HT were used to examine the properities of ligand binding sites and function of the 5-HT3A receptor, respectively (see Table below).

Mouse 5-HT3A receptors were expressed in Xenopus Oocytes (cRNA injection) and studied using two-electrode voltage-clamp technique. HEK293 cells were used in radioligand binding and calcium imaging assays to study the affinity of [3H]granisetron and the potency of 5-HT, respectively.

Y143A, Y143F and Y143S mutant mouse 5-HT3A receptors bound the antagonist [3H]granisetron. The Y143S mutant required 5-HT concentrations above 500 mM for a significant response.

Receptor [3H]granisetron binding (Kd) Cell membrane Calcium imaging (EC50) mM Electrophysiology (EC50) mM
WT 0.32 +/- 0.035 ++ 1.47 +/-0.42 2.39 +/- 0.23
Y50F NB - NR NR
Y50A NB -/+ NR NR
Y50S NB -/+ NR 1.59 +/- 0.18
Y73A/S YES NT NT NT
Y88A/S YES NT NT NT
Y91F YES NT 2.13 +/- 0.51 4.22 +/-0.25
Y91A NO - NR 13.7 +/-1.25*
Y91S NO + NR 57.7 +/-7.16 *
Y94A/S YES NT NT NT
Y141F YES NT NT NT
Y141A YES NT NR NT
Y141S NO ++ NR NT
Y143F YES NT NT NT
Y143A YES NT NR NT
Y143S YES NT >500* NT
Y153A YES NT 74.3 +/- 8.9* NT
Y153S NO ++ 59.2 +/- 7.3* NT
Y167A/S YES NT NT NT
Y234F YES NT NT NT
Y234A NO ++ NR NT
Y234S NO + NR NT
Y240A/S YES NT NT NT

NT, Not tested

++, Presence of cell surface receptors revealed by immunocytochemistry

-/+, Reduced cell surface receptor expression compared to WT 5-HT3A

-, Absence of cell surface receptors

* significantly different from the WT 5-HT3A receptor

 

Price KL, Lummis SCR (2004). The role of Tyrosine residues in the extracellular domain of the 5-Hydroxytryptamine3 receptor.The Journal of Biological Chemistry 279(22):23294-23301

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