Mechanism of local anesthetic effect on mitochondrial ATP synthase as deduced from photolabelling and inhibition studies with phenothiazine derivatives.
The mode of interaction between mitochondrial ATP synthase and two phenothiazine derivatives, chlorpromazine (CPZ) and trifluoperazine (TFP), was studied as a model for the interaction of local anesthetic drugs with membrane proteins. Photolabelling experiments demonstrated that CPZ and TFP interact with various subunits of either the peripheral F1 moiety of the membrane-embedded F0 sector. Both drugs, however, labelled the membrane sector much more heavily. Qualitative differences in labelling were observed between CPZ and TFP, indicating non-identical sites of interaction. These diversities appeared related to the different hydrophobicities of the two drugs since: (a) TFP, which has a higher lipid/water partition coefficient, labelled the more hydrophobic subunits more markedly than CPZ; (b) reduced glutathione, a hydrophilic free radical scavenger that does not penetrate the membrane continuum, had a negligible effect on the labelling by TFP, whereas it reduced the labelling of various subunits by CPZ; (c) the labelling by [3H]TFP was poorly antagonized by cold CPZ, whereas it was almost totally prevented by fluphenazine, a phenothiazine similar to TFP in hydrophobic character. Consistently, double-inhibition experiments showed that TFP and fluphenazine are mutually exclusive inhibitors of mitochondrial ATP synthase, whereas TFP and CPZ are mutually nonexclusive. The nature of the phospholipid bilayer influenced neither the labelling nor the inhibition patterns. The complex of these data indicate that tertiary amine local anesthetics affect the activity of membrane proteins by interacting with a multiplicity of relatively aspecific hydrophobic sites located preferentially, but not exclusively, on the membrane-embedded domains. It is suggested that at least two phenothiazine derivatives of different hydrophobicities be used in photolabelling experiments, before any generalization is made, since the molecular targets of these drugs vary according to their hydrophobic character.
Dabbeni-Sala
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Dipartimento di Farmacologia, Istituto di Patologia Generale, Padova, Italy.
Schiavo
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Palatini
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D000345:Affinity Labels; D000779:Anesthetics, Local / Q000494:pharmacology*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002746:Chlorpromazine / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000201:enzymology*; D010777:Photochemistry; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors*; D014268:Trifluoperazine / Q000494:pharmacology*
D000345:Affinity Labels; D000779:Anesthetics, Local; D014268:Trifluoperazine; D006180:Proton-Translocating ATPases; D005978:Glutathione; D002746:Chlorpromazine