Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine<sub>3</sub>Receptor Revealed by Unnatural Amino Acid Mutagenesis

Beene, Darren L.; Price, Kerry L.; Lester, Henry A.; Dougherty, Dennis A.; Lummis, Sarah C. R.

doi:10.1523/jneurosci.2429-04.2004

Cited by 89 publications

(91 citation statements)

References 25 publications

(50 reference statements)

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“…Thus, Lys145 could play a role in both binding and gating of homomeric nAChRs, as reported for some other residues located at the extracellular domain of a variety of Cys-loop receptors (Galzi et al, 1991;Chen et al, 1995;Sine et al, 2002;Grutter et al, 2003;Beene et al, 2004;Newell et al, 2004). An interaction between Lys145 and the binding segments may be an initial trigger for ion channel activation, transducing a change into the neighboring Cys-loop that is an important part of the channel opening mechanism (Chakrapani et al, 2004;Sala et al, 2005).…”

Section: Nicotinic Receptor Binding and Gating Properties 1675mentioning

confidence: 55%

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

Criado

Mulet²,

Bernal³

et al. 2005

Mol Pharmacol

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Activation of nicotinic acetylcholine receptors is initiated by binding of agonists, and as a consequence, specific domains transmit the chemical signal to the channel gate through a sequence of conformational changes. Recent high-resolution structural data from a snail acetylcholine binding protein have shown that the side chain of a lysine residue, located in the ␤-strand ␤7 and strictly conserved in ␣ subunits of nicotinic receptors, systematically moves upon agonist binding, suggesting that it might be involved in both binding and gating. To test this hypothesis in neuronal nicotinic receptors, Lys145 was substituted by other amino acids in the ␣7 nicotinic receptor, and expression levels and electrophysiological responses for several nicotinic agonists and antagonists were determined. Substitutions of Lys145 showed a variety of functional effects: 1) strong reductions in the functional responses to acetylcholine, nicotine, and dimethylphenylpiperazinium, the latter becoming an antagonist; 2) increases in the agonist EC 50 values (up to 80-fold with acetylcholine); 3) heterogeneous behavior of the different agonists, with epibatidine and cytisine being less affected by the substitutions; 4) decreases of agonist affinities for the desensitized receptors; and 5) small changes in the affinity of nicotinic antagonists. It is concluded that the presence of a polar or positively charged side chain at this position improves the gating function with acetylcholine and nicotine, although the lysine side chain seems to be necessary for retaining the binding properties of acetylcholine. The results are compatible with the involvement of Lys145 in the early steps of channel activation by acetylcholine.Nicotinic acetylcholine receptors (nAChRs) mediate fast synaptic transmission in nerve and muscle cells, and they are members of the Cys-loop family of ligand-gated ion channels, which includes 5-hydroxytryptamine 3 , ␥-GABA A receptors, and glycine receptors . Such receptors are allosteric proteins because the signal generated at the binding site region must be transmitted to the channel gate located some distance away, and this process involves a more than local conformational change. Site-directed mutagenesis has been useful in identifying several residues and domains involved in the mechanisms of coupling agonist binding to channel gating, but the precise molecular mechanisms underlying channel activation remain mainly unclear. Unwin and coworkers have proposed a model of activation in which the interaction of the agonist with the binding site generates a 15°clockwise rotation of the ␣ subunits that is transmitted to the gate through rearrangements of several extracellular structures, mainly loops 2 and 7 (Cys-loop) and the M2-M3 linker (Miyazawa et al., 2003;Unwin, 2005). This hypothesis is supported by functional data obtained in several Cys-loop receptors, such as GABA A receptors (Sigel et al., 1999;Bera et al., 2002;Kash et al., 2003), glycine receptors (Rajendra et al., 1995;Lynch et al., 1997;Absalom et a...

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Section: Nicotinic Receptor Binding and Gating Properties 1675mentioning

confidence: 55%

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

Criado

Mulet²,

Bernal³

et al. 2005

Mol Pharmacol

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“…The procedure used here has been described previously (Beene et al, 2004). In short, unnatural amino acids (as shown in Fig.…”

Section: Methodsmentioning

confidence: 99%

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Pless

Hanek²,

Price³

et al. 2011

Mol Pharmacol

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Cation-interactions have been demonstrated to play a major role in agonist-binding in Cys-loop receptors. However, neither the aromatic amino acid contributing to this interaction nor its location is conserved among Cys-loop receptors. Likewise, it is not clear how many different agonists of a given receptor form a cation-interaction or, if they do, whether it is with the same aromatic amino acid as the major physiological agonist. We demonstrated previously that Phe159 in the glycine receptor (GlyR) ␣1 subunit forms a strong cation-interaction with the principal agonist, glycine. In the current study, we investigated whether the lower efficacy agonists of the human GlyR ␤-alanine and taurine also form cation-interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-interactions between Phe159 and the amino groups of ␤-alanine and taurine. The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that ␤-alanine and taurine are orientated subtly differently in the binding pocket, with their amino groups further from Phe159 than that of glycine. These data therefore show that similar agonists can have similar but not identical orientations and interactions in the binding pocket and provide a possible explanation for the lower potencies of ␤-alanine and taurine.

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“…The OpusXpress system is an integrated work station that allows electrophysiological recordings to be made from up to eight oocytes simultaneously. This system has been used previously to examine the function of ion channels, including nicotinic acetylcholine and serotonin 5HT3 receptors as well as CFTR (23)(24)(25). Oocyte impalement is automated, and compound delivery is performed by a computer-controlled fluid handler; compounds are removed from 96-well plates using disposable pipette tips and applied to individual oocytes.…”

Section: Methodsmentioning

confidence: 99%

Small Molecule Activator of the Human Epithelial Sodium Channel

Lü¹,

Echeverri²,

Kalabat³

et al. 2008

Journal of Biological Chemistry

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The epithelial sodium channel (ENaC), a heterotrimeric complex composed of ␣, ␤, and ␥ subunits, belongs to the ENaC/ degenerin family of ion channels and forms the principal route for apical Na ؉ entry in many reabsorbing epithelia. Although high affinity ENaC blockers, including amiloride and derivatives, have been described, potent and specific small molecule ENaC activators have not been reported. Here we describe compound S3969 that fully and reversibly activates human ENaC (hENaC) in an amiloride-sensitive and dose-dependent manner in heterologous cells. Mechanistically, S3969 increases hENaC open probability through interactions requiring the extracellular domain of the ␤ subunit. hENaC activation by S3969 did not require cleavage by the furin protease, indicating that nonproteolyzed channels can be opened. Function of ␣␤G37S␥ hENaC, a channel defective in gating that leads to the salt-wasting disease pseudohypoaldosteronism type I, was rescued by S3969. Small molecule activation of hENaC may find application in alleviating human disease, including pseudohypoaldosteronism type I, hypotension, and neonatal respiratory distress syndrome, when improved Na ؉ flux across epithelial membranes is clinically desirable.

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Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine₃Receptor Revealed by Unnatural Amino Acid Mutagenesis

Cited by 89 publications

References 25 publications

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Small Molecule Activator of the Human Epithelial Sodium Channel

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Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine3Receptor Revealed by Unnatural Amino Acid Mutagenesis

Cited by 89 publications

References 25 publications

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

Mutations of a Conserved Lysine Residue in the N-Terminal Domain of α7 Nicotinic Receptors Affect Gating and Binding of Nicotinic Agonists

A Cation-π Interaction at a Phenylalanine Residue in the Glycine Receptor Binding Site Is Conserved for Different Agonists

Small Molecule Activator of the Human Epithelial Sodium Channel

Contact Info

Product

Resources

About

Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamine₃Receptor Revealed by Unnatural Amino Acid Mutagenesis