title: Interaction of anesthetics with the Rho GTPase regulator Rho GDP dissociation inhibitor.
abstract: The physiological effects of anesthetics have been ascribed to their interaction with hydrophobic sites within functionally relevant CNS proteins. Studies have shown that volatile anesthetics compete for luciferin binding to the hydrophobic substrate binding site within firefly luciferase and inhibit its activity (Franks, N. P., and Lieb, W. R. (1984) Nature 310, 599-601). To assess whether anesthetics also compete for ligand binding to a mammalian signal transduction protein, we investigated the interaction of the volatile anesthetic, halothane, with the Rho GDP dissociation inhibitor (RhoGDIalpha), which binds the geranylgeranyl moiety of GDP-bound Rho GTPases. Consistent with the existence of a discrete halothane binding site, the intrinsic tryptophan fluorescence of RhoGDIalpha was quenched by halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) in a saturable, concentration-dependent manner. Bromine quenching of tryptophan fluorescence is short-range and W192 and W194 of the RhoGDIalpha are located within the geranylgeranyl binding pocket, suggesting that halothane binds within this region. Supporting this, N-acetyl-geranylgeranyl cysteine reversed tryptophan quenching by halothane. Short chain n-alcohols ( n < 6) also reversed tryptophan quenching, suggesting that RhoGDIalpha may also bind n-alkanols. Consistent with this, E193 was photolabeled by 3-azibutanol. This residue is located in the vicinity of, but outside, the geranylgeranyl chain binding pocket, suggesting that the alcohol binding site is distinct from that occupied by halothane. Supporting this, N-acetyl-geranylgeranyl cysteine enhanced E193 photolabeling by 3-azibutanol. Overall, the results suggest that halothane binds to a site within the geranylgeranyl chain binding pocket of RhoGDIalpha, whereas alcohols bind to a distal site that interacts allosterically with this pocket.
authors.affiliation: Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, USA.
authors.forename: Cojen
authors.initials: C
authors.lastname: Ho
authors.forename: Sivananthaperumal
authors.initials: S
authors.lastname: Shanmugasundararaj
authors.forename: Keith W
authors.initials: KW
authors.lastname: Miller
authors.forename: Steve A
authors.initials: SA
authors.lastname: Malinowski
authors.forename: Anthony C
authors.initials: AC
authors.lastname: Cook
authors.forename: Simon J
authors.initials: SJ
authors.lastname: Slater
mesh_terms: D000494:Allosteric Regulation / Q000187:drug effects; D018685:Anesthetics, Inhalation / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002460:Cell Line; D003545:Cysteine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism; D020730:Guanine Nucleotide Dissociation Inhibitors / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000235:genetics / Q000378:metabolism; D006221:Halothane / Q000494:pharmacology*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D018411:Spodoptera; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; D020742:rhoA GTP-Binding Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D018685:Anesthetics, Inhalation; D004224:Diterpenes; D020730:Guanine Nucleotide Dissociation Inhibitors; C095716:N-acetyl-S-geranylgeranyl-cysteine; D062947:rho-Specific Guanine Nucleotide Dissociation Inhibitors; C066737:RHOA protein, human; D006153:Guanosine Diphosphate; D020742:rhoA GTP-Binding Protein; D003545:Cysteine; D006221:Halothane