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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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Plot of log likelihood (LL) against the channel opening rate constant β2 obtained from a global fit of Scheme II to the data for receptors containing αD89N. LL plateaus at a β2 of around 40,000 s−1 (open symbol and arrow).
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fig6: Plot of log likelihood (LL) against the channel opening rate constant β2 obtained from a global fit of Scheme II to the data for receptors containing αD89N. LL plateaus at a β2 of around 40,000 s−1 (open symbol and arrow).

Mentions: Opening rate constant is a lower bound (see text and Fig. 6).


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

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

Plot of log likelihood (LL) against the channel opening rate constant β2 obtained from a global fit of Scheme II to the data for receptors containing αD89N. LL plateaus at a β2 of around 40,000 s−1 (open symbol and arrow).
© Copyright Policy
Related In: Results  -  Collection

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

fig6: Plot of log likelihood (LL) against the channel opening rate constant β2 obtained from a global fit of Scheme II to the data for receptors containing αD89N. LL plateaus at a β2 of around 40,000 s−1 (open symbol and arrow).
Mentions: Opening rate constant is a lower bound (see text and Fig. 6).

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.

Show MeSH