Human 5-HT3A -- Tyr 136

Species Original Mutated to Mutation
Human Tyr 136  

 

Rat Equivalent Tyr 135    
Mouse Equivalent Tyr 141

Phe

Ala

Ser

Y141F

Y141A

Y141S

mTyr141Ala

The mutation in the mouse 5-HT3A at residue Y141A was numbered Y140A 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.

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.

Mutants Y141F and Y141A are critical for antagonist [3H] granisetron binding of the mouse 5-HT3A receptor. The affinity of the antagonist [3H] granisetron decrease in Y141A by 28 folds due to presence of the ring structure of this residue. No specific binding of [3H]granisetron was observed with Y141S mutant.

 

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

 

 

mTyr141Phe

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.

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.

Mutants Y141F and Y141A are critical for antagonist [3H] granisetron binding of the mouse 5-HT3A receptor. The affinity of the antagonist [3H] granisetron decrease in Y141A by 28 folds due to presence of the ring structure of this residue. No specific binding of [3H]granisetron was observed with Y141S mutant.

 

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

 

mTyr141Ser

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.

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.

Mutants Y141F and Y141A are critical for antagonist [3H] granisetron binding of the mouse 5-HT3A receptor. The affinity of the antagonist [3H] granisetron decrease in Y141A by 28 folds due to presence of the ring structure of this residue. No specific binding of [3H]granisetron was observed with Y141S mutant.

 

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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