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Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity.

ABSTRACT: Nicotinic acetylcholine receptor (nAChR) ?4 and ?2 subunits assemble in two alternate stoichiometries to produce (?4?2)(2)?4 and (?4?2)(2)?2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at ?4(+)/?2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either ?4 or ?2 as auxiliary subunits produces isoform-specific ?4(+)/?4(-) or ?2(+)/?2(-) interfaces. Using fully concatenated (?4?2)(2)?4 nAChRs in conjunction with structural modeling, chimeric receptors, and functional mutagenesis, we have identified an additional site at the ?4(+)/?4(-) interface that accounts for isoform-specific agonist sensitivity of the (?4?2)(2)?4 nAChR. The additional site resides in a region that also contains a potentiating Zn(2+) site but is engaged by agonists to contribute to receptor activation. By engineering ?4 subunits to provide a free cysteine in loop C at the ?4(+)?4(-) interface, we demonstrated that the acetylcholine responses of the mutated receptors are attenuated or enhanced, respectively, following treatment with the sulfhydryl reagent [2-(trimethylammonium)ethyl]methanethiosulfonate or aminoethyl methanethiosulfonate. The findings suggest that agonist occupation of the site at the ?4(+)/(?4(-) interface leads to channel gating through a coupling mechanism involving loop C. Overall, we propose that the additional agonist site at the ?4(+)/?4(-) interface, when occupied by agonist, contributes to receptor activation and that this additional contribution underlies the agonist sensitivity signature of (?4?2)(2)?4 nAChRs.

SUBMITTER: Mazzaferro S

PROVIDER: S-EPMC3162463 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity.

Mazzaferro Simone S Benallegue Naïl N Carbone Anna A Gasparri Federica F Vijayan Ranjit R Biggin Philip C PC Moroni Mirko M Bermudez Isabel I

The Journal of biological chemistry 20110714 35

Nicotinic acetylcholine receptor (nAChR) α4 and β2 subunits assemble in two alternate stoichiometries to produce (α4β2)(2)α4 and (α4β2)(2)β2, which display different agonist sensitivities. Functionally relevant agonist binding sites are thought to be located at α4(+)/β2(-) subunit interfaces, but because these interfaces are present in both receptor isoforms, it is unlikely that they account for differences in agonist sensitivities. In contrast, incorporation of either α4 or β2 as auxiliary subu ...[more]

PMID: 21757735

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Project description:The neuronal alpha4beta2 nicotinic acetylcholine receptor (nAChR) is one of the most widely expressed nAChR subtypes in the brain. Its subunits have high sequence identity (54 and 46% for alpha4 and beta2, respectively) with alpha and beta subunits in Torpedo nAChR. Using the known structure of the Torpedo nAChR as a template, the closed-channel structure of the alpha4beta2 nAChR was constructed through homology modeling. Normal-mode analysis was performed on this closed structure and the resulting lowest frequency mode was applied to it for a "twist-to-open" motion, which increased the minimum pore radius from 2.7 to 3.4 A and generated an open-channel model. Nicotine could bind to the predicted agonist binding sites in the open-channel model but not in the closed one. Both models were subsequently equilibrated in a ternary lipid mixture via extensive molecular dynamics (MD) simulations. Over the course of 11 ns MD simulations, the open channel remained open with filled water, but the closed channel showed a much lower water density at its hydrophobic gate comprised of residues alpha4-V259 and alpha4-L263 and their homologous residues in the beta2 subunits. Brownian dynamics simulations of Na+ permeation through the open channel demonstrated a current-voltage relationship that was consistent with experimental data on the conducting state of alpha4beta2 nAChR. Besides establishment of the well-equilibrated closed- and open-channel alpha4beta2 structural models, the MD simulations on these models provided valuable insights into critical factors that potentially modulate channel gating. Rotation and tilting of TM2 helices led to changes in orientations of pore-lining residue side chains. Without concerted movement, the reorientation of one or two hydrophobic side chains could be enough for channel opening. The closed- and open-channel structures exhibited distinct patterns of electrostatic interactions at the interface of extracellular and transmembrane domains that might regulate the signal propagation of agonist binding to channel opening. A potential prominent role of the beta2 subunit in channel gating was also elucidated in the study.

Dataset Information

Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity.

Publications

Additional acetylcholine (ACh) binding site at alpha4/alpha4 interface of (alpha4beta2)2alpha4 nicotinic receptor influences agonist sensitivity.

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