Abstract
We have uncovered a significant allosteric response of the D2 dopamine receptor to physiologically relevant concentrations of sodium (140 mM), characterized by a sodium-enhanced binding affinity for a D4-selective class of agonists and antagonists. This enhancement is significantly more pronounced in a D2-V2.61(91)F mutant and cannot be mimicked by an equivalent concentration of the sodium replacement cation N-methyl-d-glucamine. This phenomenon was explored computationally at the molecular level by analyzing the effect of sodium binding on the dynamic properties of D2 receptor model constructs. Normal mode analysis identified one mode (M19), which is involved in the open/closed motions of the binding cleft as being particularly sensitive to the sodium effect. To examine the consequences for D2 receptor ligand recognition, one of the ligands, L-745,870 [3-{[4-(4-chlorophenyl) piperazin-1-yl]-methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenylpiperazinyl methylazaindole], was docked into conformers along the M19 trajectory. Structurally and pharmacologically well established ligand-receptor interactions, including the ionic interaction with D3.32(114) and interactions between the ligand aryl moieties and V2.61(91)F, were achieved only in “open” phase conformers. The docking of (-)-raclopride [3,5-dichloro-N-(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-6-methoxybenzamide] suggests that the same binding cleft changes in response to sodium-binding perturbation account as well for the enhancements in binding affinity for substituted benzamides in the wild-type D2 receptor. Our findings demonstrate how key interactions can be modulated by occupancy at an allosteric site and are consistent with a mechanism in which sodium binding enhances the affinity of selected ligands through dynamic changes that increase accessibility of substituted benzamides and 1,4-DAP ligands to the orthosteric site and accessibility of 1,4-DAPs to V2.61(91)F.
Footnotes
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This study was supported by the National Institutes of Health [Grant R01-MH063162], the Cofrin Center for Biomedical Information in the HRH Prince Alwaleed Bin Talal Bin Abdulaziz Alsaud Institute for Computational Biomedicine at Weill Medical College of Cornell University, and National Institutes of Health, National Institute on Drug Abuse [Postdoctoral Training Grant T32 DA007274].
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.108.141531.
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ABBREVIATIONS: 1,4-DAP, 1,4-disubstituted aromatic piperidine/piperazine; NMA, normal mode analysis; NET-856, [3H]methylspiperone; (-)-quinpirole, (4aR-trans)-4,4a,5,6,7,8,8a,9-octahydro-5-propyl-1H-pyrazolo[3,4-g]quinoline; TMS, transmembrane segment; HEK, human embryonic kidney; DMEM, Dulbecco's modified Eagle's media; MSP, methylspiperone, 8-[4-(4-fluorophenyl)-4-oxobutyl]-(3-methyl-1-phenyl)-1,3,8-triazaspiro[4,5]decan-4-one hydrochloride; Ro 20-1724, 4-[(3-butoxy-4-methoxyphenyl)-methyl]-2-imidazolidinone; ANOVA, analysis of variance; GROMACS, Groningen machine for chemical simulations; TM, transmembrane; NOMAD-Ref, normal mode analysis deformation and refinement; RMSD, root mean square deviation; L-745,870, 3-{[4-(4-chlorophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine or CPPMA, chlorophenylpiperazinyl methylazaindole; L-750,667, 3-{[4-(4-iodophenyl) piperazin-1-yl]methyl}-1H-pyrrolo[2,3-b]pyridine; NGD 94-1, 2-phenyl-4(5)-[4–92-pyrimidinyl)-piperazin-1-yl)-methyl]-imidazole; (-)-raclopride, 3,5-dichloro-N-(1-ethylpyrrolidin-2-ylmethyl)-2-hydroxy-6-methoxybenzamide; RBI-257, 1-[4-iodobenzyl]-4-[N-(3-isopropoxy-2-pyridinyl)-N-methyl]-aminopiperidine; PD168,077, N-[[4-(2-cyanophenyl)-1-piperazinyl]methyl]-3-methylbenzamide; FAUC213, 2-[4-(4-chlorophenyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine; Gpp(NH)p, 5′-guanylylimidodiphosphate; Ro61-6270, 2-amino-benzoic acid 1-benzyl-piperidin-4-yl ester; UK14304, 5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine; p-iodoclonidine, 2-[(2,6-dichloro-4-iodophenyl)imino]imidazoline.
- Received May 27, 2008.
- Accepted October 8, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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Ligand Selectivity of D2 Dopamine Receptors Is Modulated by Changes in Local Dynamics Produced by Sodium Binding
