Human 5-HT3A -- Tyr 228

Species Original Mutated to Mutation
Human Tyr 228 Ser

Y228S

Rat Equivalent Ser 227    
Mouse Equivalent Ser 233 Tyr

Ala

Thr

S233Y

S233A

S233T

 

hTyr228Ser

When the quintuple mutation of hM223I, hE224D, hS225I, hY217Q and hR219K 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 Tyr228. This residue was numbered Tyr205 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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mSer233Tyr

In a single point mutation of mS233Y, a modest increase in IC50 (2.5-3.0 fold) was obtained.

When the quintuple mutation of mI228M, mD229E, mI230S, mQ222Y and mK224R 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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mSer233Ala

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 S233A mutation abolished binding to [3H]granisetron. The EC50 value could not be determined as the receptor was not functional.

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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mSer233Thr

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 S233T mutation did not significantly change either the Kd value for [3H]granisetron, or the Bmax value. There was a 4-fold reduction in the Ki value for 5-HT, with a -1.5-fold change in the EC50 value and no significant change in Imax. No significant changes were seen in the Ki value for mCPBG. However, there was a -1.6-fold change in the EC50 value and no significant change 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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