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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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Changes in Ki or Kd (for binding of [3H]granisetron) as a result of alanine mutations. The values shown are the ratio of the Kd or Ki for wildtype and mutant receptors. The length of each bar illustrates the magnitude of change in either the Kd or Ki relative to wildtype while the direction indicates an increase (up) or decrease (down) in the Kd or Ki. A. Most alanine mutations produced little change in Kd. No binding was detected for the G147A and V149A receptors therefore no Kd could be determined. B. Changes in Ki were small for most receptors although large changes were observed for select ligands at Y140A, Y142A and Y152A receptors. The relative increases in Ki for these amino acids are as follows: 5-HT: Y140A 1.3 ± 0.30 fold, Y142A 110 ± 25 fold, Y152A 24 ± 5.6 fold mCPBG: Y140A 7.3 ± 1.7 fold, Y142A 160 ± 36 fold, Y152A 24 ± 5.6 fold d-tc: Y140A 50 ± 12 fold, Y142A 6.5 ± 1.5, Y152A 10.0 ± 2.3 fold lerisetron: Y140A 4.6 ± 1.1 fold, Y142A 160 ± 37 fold, Y152A 190 ± 43 fold
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Figure 2: Changes in Ki or Kd (for binding of [3H]granisetron) as a result of alanine mutations. The values shown are the ratio of the Kd or Ki for wildtype and mutant receptors. The length of each bar illustrates the magnitude of change in either the Kd or Ki relative to wildtype while the direction indicates an increase (up) or decrease (down) in the Kd or Ki. A. Most alanine mutations produced little change in Kd. No binding was detected for the G147A and V149A receptors therefore no Kd could be determined. B. Changes in Ki were small for most receptors although large changes were observed for select ligands at Y140A, Y142A and Y152A receptors. The relative increases in Ki for these amino acids are as follows: 5-HT: Y140A 1.3 ± 0.30 fold, Y142A 110 ± 25 fold, Y152A 24 ± 5.6 fold mCPBG: Y140A 7.3 ± 1.7 fold, Y142A 160 ± 36 fold, Y152A 24 ± 5.6 fold d-tc: Y140A 50 ± 12 fold, Y142A 6.5 ± 1.5, Y152A 10.0 ± 2.3 fold lerisetron: Y140A 4.6 ± 1.1 fold, Y142A 160 ± 37 fold, Y152A 190 ± 43 fold

Mentions: All mutant receptors were tested for their ability to bind the 5-HT3R antagonist [3H]granisetron. Table 2 shows the Kd values for wildtype mouse 5-HT3ASRs and the 13 alanine mutations we evaluated. [3H]granisetron is a potent antagonist of the wt 5-HT3R (Kd = 0.98 ± 0.12 nM). This value agrees with published data for this compound [19,20]. Bmax values range from the 5.5 pmoles/mg protein observed for E148A to 0.30 pmoles/mg protein for the K153A mutation, indicating some variability in expression of the different receptors. In general, however, receptor expression was similar to that reported by other laboratories [1,20-22]. No detectable binding was observed for G147A and V149A mutant receptors. For all other mutants, decreases in binding affinity (increased Kd) were observed although the magnitude of the change was less than 10 fold in all cases. A bar graph showing the change in Kd value resulting from each alanine mutation is shown in Figure 2A. The largest decreases in binding affinity were observed for Y142A (4.6 fold, Figure 3), E148A (5.3 fold) and Q150A – K153A (6 – 8 fold).


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)

Changes in Ki or Kd (for binding of [3H]granisetron) as a result of alanine mutations. The values shown are the ratio of the Kd or Ki for wildtype and mutant receptors. The length of each bar illustrates the magnitude of change in either the Kd or Ki relative to wildtype while the direction indicates an increase (up) or decrease (down) in the Kd or Ki. A. Most alanine mutations produced little change in Kd. No binding was detected for the G147A and V149A receptors therefore no Kd could be determined. B. Changes in Ki were small for most receptors although large changes were observed for select ligands at Y140A, Y142A and Y152A receptors. The relative increases in Ki for these amino acids are as follows: 5-HT: Y140A 1.3 ± 0.30 fold, Y142A 110 ± 25 fold, Y152A 24 ± 5.6 fold mCPBG: Y140A 7.3 ± 1.7 fold, Y142A 160 ± 36 fold, Y152A 24 ± 5.6 fold d-tc: Y140A 50 ± 12 fold, Y142A 6.5 ± 1.5, Y152A 10.0 ± 2.3 fold lerisetron: Y140A 4.6 ± 1.1 fold, Y142A 160 ± 37 fold, Y152A 190 ± 43 fold
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Figure 2: Changes in Ki or Kd (for binding of [3H]granisetron) as a result of alanine mutations. The values shown are the ratio of the Kd or Ki for wildtype and mutant receptors. The length of each bar illustrates the magnitude of change in either the Kd or Ki relative to wildtype while the direction indicates an increase (up) or decrease (down) in the Kd or Ki. A. Most alanine mutations produced little change in Kd. No binding was detected for the G147A and V149A receptors therefore no Kd could be determined. B. Changes in Ki were small for most receptors although large changes were observed for select ligands at Y140A, Y142A and Y152A receptors. The relative increases in Ki for these amino acids are as follows: 5-HT: Y140A 1.3 ± 0.30 fold, Y142A 110 ± 25 fold, Y152A 24 ± 5.6 fold mCPBG: Y140A 7.3 ± 1.7 fold, Y142A 160 ± 36 fold, Y152A 24 ± 5.6 fold d-tc: Y140A 50 ± 12 fold, Y142A 6.5 ± 1.5, Y152A 10.0 ± 2.3 fold lerisetron: Y140A 4.6 ± 1.1 fold, Y142A 160 ± 37 fold, Y152A 190 ± 43 fold
Mentions: All mutant receptors were tested for their ability to bind the 5-HT3R antagonist [3H]granisetron. Table 2 shows the Kd values for wildtype mouse 5-HT3ASRs and the 13 alanine mutations we evaluated. [3H]granisetron is a potent antagonist of the wt 5-HT3R (Kd = 0.98 ± 0.12 nM). This value agrees with published data for this compound [19,20]. Bmax values range from the 5.5 pmoles/mg protein observed for E148A to 0.30 pmoles/mg protein for the K153A mutation, indicating some variability in expression of the different receptors. In general, however, receptor expression was similar to that reported by other laboratories [1,20-22]. No detectable binding was observed for G147A and V149A mutant receptors. For all other mutants, decreases in binding affinity (increased Kd) were observed although the magnitude of the change was less than 10 fold in all cases. A bar graph showing the change in Kd value resulting from each alanine mutation is shown in Figure 2A. The largest decreases in binding affinity were observed for Y142A (4.6 fold, Figure 3), E148A (5.3 fold) and Q150A – K153A (6 – 8 fold).

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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