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Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding.

Lee WY, Sine SM - J. Gen. Physiol. (2004)

Bottom Line: However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh.Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89.The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Mayo Clinic College of Medicine, 200 First St., SW, MSB 1-35, Rochester, MN 55905, USA.

ABSTRACT
We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.

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[125I]α-Bungarotoxin binding to wild-type and mutant AChRs expressed in BOSC 23 cells. (A) Cell surface expression of receptors containing mutations at αD89. (B) Cell surface expression of receptors containing mutations at αT148 and αT150. (C) Cell surface expression of receptors containing double mutations. Combination of the optimal single mutations yielded the hydrophobic pair with optimal cell surface expression, αT148L plus αT150A.
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fig8: [125I]α-Bungarotoxin binding to wild-type and mutant AChRs expressed in BOSC 23 cells. (A) Cell surface expression of receptors containing mutations at αD89. (B) Cell surface expression of receptors containing mutations at αT148 and αT150. (C) Cell surface expression of receptors containing double mutations. Combination of the optimal single mutations yielded the hydrophobic pair with optimal cell surface expression, αT148L plus αT150A.

Mentions: We substituted a series of residues with polar and nonpolar side chains at αD89 and measured cell surface expression with radiolabeled α-bungarotoxin (Fig. 8 A). The Ala substitution abolishes receptor expression on the cell surface, while Glu, Asn, Ser, Thr, His, and Lys all give low but measurable expression. Of these, the αD89T and αD89E mutations were examined for their effects on single channel kinetics. The Thr substitution, which neutralizes the negative charge but maintains a small side chain, impairs the kinetics of receptor activation even more than αD89N; it causes a more pronounced slowing of the rate of ACh association but does not affect the kinetics of channel gating (Fig. 9; Table II). For the αD89T mutation, adequate fitting could only be achieved by explicitly including two desensitized states, using the method described for the wild-type receptor, presumably because of the more pronounced slowing of ACh association and consequent overlap of closed time components due to activation with those due to desensitization.


Invariant aspartic Acid in muscle nicotinic receptor contributes selectively to the kinetics of agonist binding.

Lee WY, Sine SM - J. Gen. Physiol. (2004)

[125I]α-Bungarotoxin binding to wild-type and mutant AChRs expressed in BOSC 23 cells. (A) Cell surface expression of receptors containing mutations at αD89. (B) Cell surface expression of receptors containing mutations at αT148 and αT150. (C) Cell surface expression of receptors containing double mutations. Combination of the optimal single mutations yielded the hydrophobic pair with optimal cell surface expression, αT148L plus αT150A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2234004&req=5

fig8: [125I]α-Bungarotoxin binding to wild-type and mutant AChRs expressed in BOSC 23 cells. (A) Cell surface expression of receptors containing mutations at αD89. (B) Cell surface expression of receptors containing mutations at αT148 and αT150. (C) Cell surface expression of receptors containing double mutations. Combination of the optimal single mutations yielded the hydrophobic pair with optimal cell surface expression, αT148L plus αT150A.
Mentions: We substituted a series of residues with polar and nonpolar side chains at αD89 and measured cell surface expression with radiolabeled α-bungarotoxin (Fig. 8 A). The Ala substitution abolishes receptor expression on the cell surface, while Glu, Asn, Ser, Thr, His, and Lys all give low but measurable expression. Of these, the αD89T and αD89E mutations were examined for their effects on single channel kinetics. The Thr substitution, which neutralizes the negative charge but maintains a small side chain, impairs the kinetics of receptor activation even more than αD89N; it causes a more pronounced slowing of the rate of ACh association but does not affect the kinetics of channel gating (Fig. 9; Table II). For the αD89T mutation, adequate fitting could only be achieved by explicitly including two desensitized states, using the method described for the wild-type receptor, presumably because of the more pronounced slowing of ACh association and consequent overlap of closed time components due to activation with those due to desensitization.

Bottom Line: However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh.Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89.The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Biophysics, Mayo Clinic College of Medicine, 200 First St., SW, MSB 1-35, Rochester, MN 55905, USA.

ABSTRACT
We examined functional contributions of interdomain contacts within the nicotinic receptor ligand binding site using single channel kinetic analyses, site-directed mutagenesis, and a homology model of the major extracellular region. At the principal face of the binding site, the invariant alphaD89 forms a highly conserved interdomain contact near alphaT148, alphaW149, and alphaT150. Patch-clamp recordings show that the mutation alphaD89N markedly slows acetylcholine (ACh) binding to receptors in the resting closed state, but does not affect rates of channel opening and closing. Neither alphaT148L, alphaT150A, nor mutations at both positions substantially affects the kinetics of receptor activation, showing that hydroxyl side chains at these positions are not hydrogen bond donors for the strong acceptor alphaD89. However substituting a negative charge at alphaT148, but not at alphaT150, counteracts the effect of alphaD89N, demonstrating that a negative charge in the region of interdomain contact confers rapid association of ACh. Interpreted within the structural framework of ACh binding protein and a homology model of the receptor ligand binding site, these results implicate main chain amide groups in the domain harboring alphaW149 as principal hydrogen bond donors for alphaD89. The specific effect of alphaD89N on ACh association suggests that interdomain hydrogen bonding positions alphaW149 for optimal interaction with ACh.

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