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Abstract
The serotonin1A (5-HT1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We have examined the modulatory role of cholesterol on the ligand binding of the bovine hippocampal 5-HT1A receptor by cholesterol complexation in native membranes using digitonin. Complexation of cholesterol from bovine hippocampal membranes using digitonin results in a concentration-dependent reduction in specific binding of the agonist 8-OH-DPAT and antagonist p-MPPF to 5-HT1A receptors. The corresponding changes in membrane order were monitored by analysis of fluorescence polarization data of the membrane depth-specific probes, DPH and TMA-DPH. Taken together, our results point out the important role of membrane cholesterol in maintaining the function of the 5-HT1A receptor. An important aspect of these results is that non-availability of free cholesterol in the membrane due to complexation with digitonin rather than physical depletion is sufficient to significantly reduce the 5-HT1A receptor function. These results provide a comprehensive understanding of the effects of the sterol-complexing agent digitonin in particular, and the role of membrane cholesterol in general, on the 5-HT1A receptor function.
| Abbreviations | ||
| BCA | = | bicinchoninic acid |
| DMPC | = | dimyristoyl-sn-glycero-3-phosphocholine |
| DPH | = | 1,6-diphenyl-1,3,5-hexatriene |
| 5-HT | = | 5-hydroxytryptamine |
| 8-OH-DPAT | = | 8-hydroxy-2(di-N-propylamino)tetralin |
| p-MPPF | = | 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-fluorobenzamido]ethyl-piperazine |
| p-MPPI | = | 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-iodobenzamido]ethyl-piperazine |
| PMSF | = | phenylmethylsulfonyl fluoride |
| TMA-DPH | = | 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene |
| TLC | = | thin layer chromatography |
| Tris | = | tris-(hydroxymethyl)aminomethane |
| Abbreviations | ||
| BCA | = | bicinchoninic acid |
| DMPC | = | dimyristoyl-sn-glycero-3-phosphocholine |
| DPH | = | 1,6-diphenyl-1,3,5-hexatriene |
| 5-HT | = | 5-hydroxytryptamine |
| 8-OH-DPAT | = | 8-hydroxy-2(di-N-propylamino)tetralin |
| p-MPPF | = | 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-fluorobenzamido]ethyl-piperazine |
| p-MPPI | = | 4-(2′-methoxy)-phenyl-1-[2′-(N-2″-pyridinyl)-p-iodobenzamido]ethyl-piperazine |
| PMSF | = | phenylmethylsulfonyl fluoride |
| TMA-DPH | = | 1-[4-(trimethylammonio)phenyl]-6-phenyl-1,3,5-hexatriene |
| TLC | = | thin layer chromatography |
| Tris | = | tris-(hydroxymethyl)aminomethane |