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Identification of critical residues in loop E in the 5-HT3ASR binding site.

Venkataraman P, Venkatachalan SP, Joshi PR, Muthalagi M, Schulte MK - BMC Biochem. (2002)

Bottom Line: All members of this family share a large degree of sequence homology and presumably significant structural similarity.Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands.Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Northwestern University, Evanston, IL 60201. USA. padma964@hotmail.com

ABSTRACT

Background: The serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.

Results: Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic alpha7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.

Conclusion: Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

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Inhibition of [3H]granisetron binding by 5-HT3R ligands. The data represent the combined results of at least four experiments. Assays were conducted as described in Materials and Methods. Fractional response is the fraction of [3H]granisetron binding obtained in the presence and absence of inhibitor. An IC50 value was determined from the data using non-linear curve fitting as described in the methods and the Ki value calculated using the Cheng-Prusoff equation, the Kd as determined in Figure 3 and the concentration of ligand used to obtain the inhibition data. Kd values are reported in Table 3. A. 5-HT B. mCPBG C. dtC D. Lerisetron
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Figure 4: Inhibition of [3H]granisetron binding by 5-HT3R ligands. The data represent the combined results of at least four experiments. Assays were conducted as described in Materials and Methods. Fractional response is the fraction of [3H]granisetron binding obtained in the presence and absence of inhibitor. An IC50 value was determined from the data using non-linear curve fitting as described in the methods and the Ki value calculated using the Cheng-Prusoff equation, the Kd as determined in Figure 3 and the concentration of ligand used to obtain the inhibition data. Kd values are reported in Table 3. A. 5-HT B. mCPBG C. dtC D. Lerisetron

Mentions: A more detailed analysis of the competition binding data obtained for the Y140A, Y142A and Y152A mutations is shown in Figure 4. For 5-HT, the Y142A mutation produced a 110 fold increase in Ki and Y152A produced a 24 fold increase. No change in Ki was observed for the Y140A mutation (Figure 4A). The 5-HT3R agonistmCPBG showed a similar profile for the changes in Ki values resulting from mutations of the three tyrosines (Figure 4B). As was observed for 5-HT, the Y142A mutation produced a large increase in Ki (160 fold) while the Y14 0A and Y152A mutations produced only 7 and 24 fold changes respectively.


Identification of critical residues in loop E in the 5-HT3ASR binding site.

Venkataraman P, Venkatachalan SP, Joshi PR, Muthalagi M, Schulte MK - BMC Biochem. (2002)

Inhibition of [3H]granisetron binding by 5-HT3R ligands. The data represent the combined results of at least four experiments. Assays were conducted as described in Materials and Methods. Fractional response is the fraction of [3H]granisetron binding obtained in the presence and absence of inhibitor. An IC50 value was determined from the data using non-linear curve fitting as described in the methods and the Ki value calculated using the Cheng-Prusoff equation, the Kd as determined in Figure 3 and the concentration of ligand used to obtain the inhibition data. Kd values are reported in Table 3. A. 5-HT B. mCPBG C. dtC D. Lerisetron
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC117120&req=5

Figure 4: Inhibition of [3H]granisetron binding by 5-HT3R ligands. The data represent the combined results of at least four experiments. Assays were conducted as described in Materials and Methods. Fractional response is the fraction of [3H]granisetron binding obtained in the presence and absence of inhibitor. An IC50 value was determined from the data using non-linear curve fitting as described in the methods and the Ki value calculated using the Cheng-Prusoff equation, the Kd as determined in Figure 3 and the concentration of ligand used to obtain the inhibition data. Kd values are reported in Table 3. A. 5-HT B. mCPBG C. dtC D. Lerisetron
Mentions: A more detailed analysis of the competition binding data obtained for the Y140A, Y142A and Y152A mutations is shown in Figure 4. For 5-HT, the Y142A mutation produced a 110 fold increase in Ki and Y152A produced a 24 fold increase. No change in Ki was observed for the Y140A mutation (Figure 4A). The 5-HT3R agonistmCPBG showed a similar profile for the changes in Ki values resulting from mutations of the three tyrosines (Figure 4B). As was observed for 5-HT, the Y142A mutation produced a large increase in Ki (160 fold) while the Y14 0A and Y152A mutations produced only 7 and 24 fold changes respectively.

Bottom Line: All members of this family share a large degree of sequence homology and presumably significant structural similarity.Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands.Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neurobiology, Northwestern University, Evanston, IL 60201. USA. padma964@hotmail.com

ABSTRACT

Background: The serotonin type 3 receptor (5-HT3R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family.

Results: Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic alpha7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR.

Conclusion: Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

Show MeSH
Related in: MedlinePlus