The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1 408), was isolated and sequenced. The mechanism of action of the phenothiazine tranquilizers has been the subject of numerous studies (42). Studies reported in the pharmacological literature suggest that the phenothiazines inhibit one of the two types of dopamine receptors (24, 48), a hypothesis consistent with the antipsychotic and Parkinson's disease-like effects. Reports in the biochemical literature suggest that the phenothiazines may function as inhibitors of calcium-binding proteins, especially calmodulin (50) and protein kinase C (25), and may affect calcium-or calmodulin-regulated processes in cells (47). This alternative model is consistent with the effect of these drugs in vivo, since neurons and muscle cells are two types of cells in the body that are dependent on calcium gradients or currents for function.The medical effectiveness of the phenothiazines is directly related to the hydrophobicity of the molecule and varies with the side-chain modifications to the planar ring (50). Trifluoperazine (TFP) is the most hydrophobic of the medically effective phenothiazines. The hydrophobic character of the phenothiazine tranquilizers confers two properties to the drugs: lipid solubility and membrane association. Therefore these drugs can cross the blood-brain barrier readily, can get into any cell type, and can associate with or intercalate into any membranous structure.Since TFP is a medically important drug that affects cells that are dependent upon calcium gradients for function in vivo and inhibits calcium-binding proteins with hydrophobic domains in vitro, we isolated spontaneous TFP-resistant mutants of the simple eucaryote Saccharomyces cerevisiae in an attempt to find mutations in genes coding for proteins involved in calcium transport or regulated pathways. Calcium-sensitive or -dependent mutants of S. cerevisiae have been isolated before (30)(31)(32), and calcium-related biochemical activities have been documented in S. cerevisiae (8, 10-* Corresponding author.12, 29, 38). TFP-resistant pseudorevertants of a calciumdependent mutation have been isolated and are recessive to the wild type for TFP resistance and show TFP-dependent growth at 37°C (30). In this report we describe the isolation and characterization of a spontaneous mutation to dominant TFP resistance and the sequence of the gene encoding it.MATERIALS AND METHODS Strains and plasmids. S. cerevisiae strains were of the S288c background. The wild-type haploids used in this work were NF134 (Mata his4-539 lys2-801 ura3-52) and NF147 (MATTa ade2-101 ura3-52) (43). NF408 was a spontaneous mutation of NF147 to dom...

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The antipsychotic drug trifluoperazine has been long considered a calmodulin inhibitor from in vitro studies but may function in vivo as a more general inhibitor by disturbing ion fluxes and altering the membrane potential. Resistance to trifluoperazine can arise in Saccharomyces cerevisiae cells by alterations in at least three distinct genetic loci. One locus, defined by a spontaneous dominant trifluoperazine resistance mutation (TFP1-408), was isolated and sequenced. The sequence of the TFP1-408 gene revealed a large open reading frame coding for a large protein of 1,031 amino acids with predicted hydrophobic transmembrane domains. A search of existing amino acid sequences revealed a significant homology with F0F1 ATP synthase. Mutant TFP1-408 cells did not grow efficiently in the presence of 50 mM CaCl2, whereas wild-type cells did. Wild-type cells became resistant to trifluoperazine in the presence of 50 mM CaCl2 or 50 mM MgCl2. Mutant cells showed a higher rate of calcium transport relative to wild-type cells. These data suggest that the TFP1 gene product codes for a transmembrane ATPase-like enzyme possibly involved in Ca2+ transport or in generating a transmembrane ion gradient between two cellular compartments.

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