Human 5-HT3A -- Tyr 86

Species Original Mutated to Mutation
Human Tyr 86  

 

Rat Equivalent Tyr 91    
Mouse Equivalent Tyr 91

Phe

Ala

Ser

Y91F

Y91A

Y91S

 

mTyr91Ala

Nine residues (Thr 86, Thr 87, Tyr 88, Ile 89, Tyr 91, Arg 92, Gln 93, Tyr 94, Trp 95) were mutated individually to alanine (alanine scanning mutagenesis). The ligands [3H]granisetron, curare, and serotonin were used to examine the properites of the 5-HT3 binding site.

W90F was analyzed instead of W90A, and it proved to be the only one that significantly affected the interaction of curare with the receptor. R92A was the only substitution that altered the affinity of the agonist serotonin. W90F, R92A, and Y94A all reduced the affinity of [3H] granisetron. The periodicity of the effect suggest the involvement of a beta-strand.

Yan D, Schulte MK, Bloom KE, White MM (1999) Structural Features of the Ligand-binding Domain of the Serotonin 5HT3 Receptor. The Journal of Biological Chemistry 274 (9): 5537-5541

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.

The Y91 residue plays an important role in assembly and function of the mouse 5-HT3A receptor. No specific binding of [3H]granisetron was observed with Y91A and Y91S mutants. The Y91F mutant bound [3H]granisetron and the mutation did not significantly affect the potency of 5-HT. The Y91A and Y91S mutations significantly reduced the potency of 5-HT (see table below).

 

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

 

mTyr91Phe

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.

The Y91 residue plays an important role in assembly and function of the mouse 5-HT3A receptor. No specific binding of [3H]granisetron was observed with Y91A and Y91S mutants. The Y91F mutant bound [3H]granisetron and the mutation did not significantly affect the potency of 5-HT. The Y91A and Y91S mutations significantly reduced the potency of 5-HT (see table below).

 

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

 

mTyr91Ser

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.

The Y91 residue plays an important role in assembly and function of the mouse 5-HT3A receptor. No specific binding of [3H]granisetron was observed with Y91A and Y91S mutants. The Y91F mutant bound [3H]granisetron and the mutation did not significantly affect the potency of 5-HT. The Y91A and Y91S mutations significantly reduced the potency of 5-HT (see table below).

 

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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