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Loop B is a major structural component of the 5-HT3 receptor.

Thompson AJ, Lochner M, Lummis SC - Biophys. J. (2008)

Bottom Line: Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor.The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein.Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, United Kingdom.

ABSTRACT
The 5-HT(3) receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT(3) receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [3H]granisetron binding affinity (K(d)) and 5-HT EC(50) were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC(50) values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

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Example data for receptor function and radioligand binding at wild-type and mutant receptors. (A) Channel function was measured using a voltage-sensitive fluorometric dye on a FlexStation. Values from a series of experiments were normalized, averaged and fitted with a four-parameter logistic equation. The calculated EC50 values are shown in Table 2. (B) Kd values were estimated using the 5-HT3 antagonist [3H]granisetron. The examples show binding for single experiments, fitted with a one site binding equation. Kd values for a series of experiments were averaged for each mutant, and are presented in Table 1.
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fig2: Example data for receptor function and radioligand binding at wild-type and mutant receptors. (A) Channel function was measured using a voltage-sensitive fluorometric dye on a FlexStation. Values from a series of experiments were normalized, averaged and fitted with a four-parameter logistic equation. The calculated EC50 values are shown in Table 2. (B) Kd values were estimated using the 5-HT3 antagonist [3H]granisetron. The examples show binding for single experiments, fitted with a one site binding equation. Kd values for a series of experiments were averaged for each mutant, and are presented in Table 1.

Mentions: All 15 amino acids within a contiguous sequence of the 5-HT3A receptor subunit (Fig. 1) were mutated to either Ala or an amino acid with properties similar to the wild-type amino acid (subsequently referred to as a conserved amino acid change). Mutant receptors were characterized using [3H]granisetron binding to explore changes in the ligand binding site (Table 1, Fig. 2), and 5-HT induced changes in membrane potential were measured using a voltage-sensitive fluorescent dye (Table 2, Fig. 2). Wild-type receptors had a [3H]granisetron binding affinity (Kd) of 0.5 nM (n = 11) and 5-HT EC50 of 0.24 μM (n = 12), values that are similar to those previously published using the same techniques (15,24,25).


Loop B is a major structural component of the 5-HT3 receptor.

Thompson AJ, Lochner M, Lummis SC - Biophys. J. (2008)

Example data for receptor function and radioligand binding at wild-type and mutant receptors. (A) Channel function was measured using a voltage-sensitive fluorometric dye on a FlexStation. Values from a series of experiments were normalized, averaged and fitted with a four-parameter logistic equation. The calculated EC50 values are shown in Table 2. (B) Kd values were estimated using the 5-HT3 antagonist [3H]granisetron. The examples show binding for single experiments, fitted with a one site binding equation. Kd values for a series of experiments were averaged for each mutant, and are presented in Table 1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2599823&req=5

fig2: Example data for receptor function and radioligand binding at wild-type and mutant receptors. (A) Channel function was measured using a voltage-sensitive fluorometric dye on a FlexStation. Values from a series of experiments were normalized, averaged and fitted with a four-parameter logistic equation. The calculated EC50 values are shown in Table 2. (B) Kd values were estimated using the 5-HT3 antagonist [3H]granisetron. The examples show binding for single experiments, fitted with a one site binding equation. Kd values for a series of experiments were averaged for each mutant, and are presented in Table 1.
Mentions: All 15 amino acids within a contiguous sequence of the 5-HT3A receptor subunit (Fig. 1) were mutated to either Ala or an amino acid with properties similar to the wild-type amino acid (subsequently referred to as a conserved amino acid change). Mutant receptors were characterized using [3H]granisetron binding to explore changes in the ligand binding site (Table 1, Fig. 2), and 5-HT induced changes in membrane potential were measured using a voltage-sensitive fluorescent dye (Table 2, Fig. 2). Wild-type receptors had a [3H]granisetron binding affinity (Kd) of 0.5 nM (n = 11) and 5-HT EC50 of 0.24 μM (n = 12), values that are similar to those previously published using the same techniques (15,24,25).

Bottom Line: Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor.The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein.Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Cambridge, Cambridge, CB2 1QW, United Kingdom.

ABSTRACT
The 5-HT(3) receptor belongs to a family of therapeutically important neurotransmitter-gated receptors whose ligand binding sites are formed by the convergence of six peptide loops (A-F). Here we have mutated 15 amino acid residues in and around loop B of the 5-HT(3) receptor (Ser-177 to Asn-191) to Ala or a residue with similar chemical properties. Changes in [3H]granisetron binding affinity (K(d)) and 5-HT EC(50) were determined using receptors expressed in human embryonic kidney 293 cells. Substitutions at all but one residue (Thr-181) altered or eliminated binding for one or both mutants. Receptors were nonfunctional or EC(50) values were altered for all but two mutants (S182T, I190L). Homology modeling indicates that loop B contributes two residues to a hydrophobic core that faces into the beta-sandwich of the subunit, and the experimental data indicate that they are important for both the structure and the function of the receptor. The models also show that close to the apex of the loop (Ser-182 to Ile-190), loop B residues form an extensive network of hydrogen bonds, both with other loop B residues and with adjacent regions of the protein. Overall, the data suggest that loop B has a major role in maintaining the structure of the region by a series of noncovalent interactions that are easily disrupted by amino acid substitutions.

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