Antifungal Activity of Amiodarone Is Mediated by Disruption of Calcium Homeostasis
2003; Elsevier BV; Volume: 278; Issue: 31 Linguagem: Inglês
10.1074/jbc.m303300200
ISSN1083-351X
AutoresSoma Sen Gupta, Van‐Khue Ton, Veronica G. Beaudry, Samuel Rulli, Kyle W. Cunningham, Rajini Rao,
Tópico(s)Cholinesterase and Neurodegenerative Diseases
ResumoThe antiarrhythmic drug amiodarone was recently demonstrated to have novel broad range fungicidal activity. We provide evidence that amiodarone toxicity is mediated by disruption of Ca2+ homeostasis in Saccharomyces cerevisiae. In mutants lacking calcineurin and various Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1/Mid1 and Yvc1), and exchangers (Vcx1), amiodarone sensitivity correlates with cytoplasmic calcium overload. Measurements of cytosolic Ca2+ by aequorin luminescence demonstrate a biphasic response to amiodarone. An immediate and extensive calcium influx was observed that was dose-dependent and correlated with drug sensitivity. The second phase consisted of a sustained release of calcium from the vacuole via the calcium channel Yvc1 and was independent of extracellular Ca2+ entry. To uncover additional cellular pathways involved in amiodarone sensitivity, we conducted a genome-wide screen of nearly 5000 single-gene yeast deletion mutants. 36 yeast strains with amiodarone hypersensitivity were identified, including mutants in transporters (pmr1, pdr5, and vacuolar H+-ATPase), ergosterol biosynthesis (erg3, erg6, and erg24), intracellular trafficking (vps45 and rcy1), and signaling (ypk1 and ptc1). Of three mutants examined (vps45, vma3, and rcy1), all were found to have defective calcium homeostasis, supporting a correlation with amiodarone hypersensitivity. We show that low doses of amiodarone and an azole (miconazole, fluconazole) are strongly synergistic and exhibit potent fungicidal effects in combination. Our findings point to the potentially effective application of amiodarone as a novel antimycotic, particularly in combination with conventional antifungals. The antiarrhythmic drug amiodarone was recently demonstrated to have novel broad range fungicidal activity. We provide evidence that amiodarone toxicity is mediated by disruption of Ca2+ homeostasis in Saccharomyces cerevisiae. In mutants lacking calcineurin and various Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1/Mid1 and Yvc1), and exchangers (Vcx1), amiodarone sensitivity correlates with cytoplasmic calcium overload. Measurements of cytosolic Ca2+ by aequorin luminescence demonstrate a biphasic response to amiodarone. An immediate and extensive calcium influx was observed that was dose-dependent and correlated with drug sensitivity. The second phase consisted of a sustained release of calcium from the vacuole via the calcium channel Yvc1 and was independent of extracellular Ca2+ entry. To uncover additional cellular pathways involved in amiodarone sensitivity, we conducted a genome-wide screen of nearly 5000 single-gene yeast deletion mutants. 36 yeast strains with amiodarone hypersensitivity were identified, including mutants in transporters (pmr1, pdr5, and vacuolar H+-ATPase), ergosterol biosynthesis (erg3, erg6, and erg24), intracellular trafficking (vps45 and rcy1), and signaling (ypk1 and ptc1). Of three mutants examined (vps45, vma3, and rcy1), all were found to have defective calcium homeostasis, supporting a correlation with amiodarone hypersensitivity. We show that low doses of amiodarone and an azole (miconazole, fluconazole) are strongly synergistic and exhibit potent fungicidal effects in combination. Our findings point to the potentially effective application of amiodarone as a novel antimycotic, particularly in combination with conventional antifungals. The emergence of drug-resistant fungi poses an increasing threat to the treatment of opportunistic fungal infections in patients with compromised immune systems, as occurs commonly in cancer and AIDS. This can only be countered by the discovery of new antifungal agents, particularly those that target different molecular pathways, and a better understanding of their mode of action. Recently, the antiarrhythmic drug amiodarone (AMD) 1The abbreviations used are: AMD, amiodarone; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetracetic acid; FLUC, fluconazole; MIC, miconazole; RLU, relative luminescence unit(s); WT, wild type; SC, synthetic complete.1The abbreviations used are: AMD, amiodarone; BAPTA, 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetracetic acid; FLUC, fluconazole; MIC, miconazole; RLU, relative luminescence unit(s); WT, wild type; SC, synthetic complete. was shown to have potent fungicidal activity against not only Saccharomyces but also pathogenic yeasts such as Candida, Cryptococcus, Fusarium, and Aspergillus (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar). Viability of Cryptococcus neoformans after exposure to 10 μm AMD fell dramatically, with a 10-fold loss of viable cells after1hof treatment and only 0.001% viable cells remaining after 1 day. The cytotoxic effect of AMD appeared to be mediated by calcium, as evidenced by the ability of high (millimolar) concentrations of extracellular Ca2+ to ameliorate drug toxicity (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar) and by an increase in cytosolic Ca2+ upon drug application (2Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (75) Google Scholar). There is emerging evidence that cytosolic Ca2+ entry in yeast is critical for survival under a variety of cell stresses, including hyper- and hypo-osmotic shock, protein unfolding agents, and antifungal drugs (3Cruz M.C. Goldstein A.L. Blankenship J.R. Del Poeta M. Davis D. Cardenas M.E. Perfect J.R. McCusker J.H. Heitman J. EMBO J. 2002; 21: 546-559Crossref PubMed Scopus (272) Google Scholar, 4Matsumoto T.K. Ellsmore A.J. Cessna S.G. Low P.S. Pardo J.M. Bressan R.A. Hasegawa P.M. J. Biol. Chem. 2002; 277: 33075-33080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, 5Bonilla M Nastase K.K. Cunningham K.W. EMBO J. 2002; 21: 2343-2353Crossref PubMed Scopus (207) Google Scholar, 6Edlind T. Smith L. Henry K. Katiyar S. Nickels J. Mol. Microbiol. 2002; 46: 257-268Crossref PubMed Scopus (88) Google Scholar). The release of calcium from intracellular stores must be compensated by stimulation of extracellular calcium influx, a phenomenon commonly known as capacitative calcium entry (7Locke E.G. Bonilla M. Liang L. Takita Y. Cunningham K.W. Mol. Cell. Biol. 2000; 20: 6686-6694Crossref PubMed Scopus (178) Google Scholar). Thus, one possibility is that the cytosolic Ca2+ increase in response to AMD promotes cell survival and is due to capacitative calcium entry, as has been suggested by Courchesne and Ozturk (2Courchesne W.E. Ozturk S. Mol. Microbiol. 2003; 47: 223-234Crossref PubMed Scopus (75) Google Scholar). Paradoxically, excessive or unregulated levels of calcium in the cytoplasm also lead to cell death and are implicated in the cytotoxicity of several drugs (8Andjelic S. Khanna A. Suthanthiran M. Nikolic-Zugic J. J. Immunol. 1997; 158: 2527-2534PubMed Google Scholar) as well as fungal toxins (9Kurzweilova H. Sigler K. Folia Microbiol. 1993; 38: 524-526Crossref PubMed Scopus (8) Google Scholar, 10Lecourieux D. Mazars C. Pauly N. Ranjeva R. Pugin A. Plant Cell. 2002; 14: 2627-2641Crossref PubMed Scopus (305) Google Scholar). To better understand the role of calcium in AMD toxicity, it is important to distinguish between a capacitative calcium entry mechanism triggered by store depletion or a loss in calcium homeostasis, possibly due to an effect of the drug on ion channels and transporters. The experiments described in this work address these two possibilities and begin to elucidate the cellular basis of AMD toxicity. We present a detailed investigation of how AMD disrupts Ca2+ homeostasis in Saccharomyces cerevisiae. The roles of different Ca2+ transporters, including pumps (Pmr1 and Pmc1), channels (Cch1, Mid1, and Yvc1), and exchangers (Vcx1), were evaluated in cells lacking these proteins and exposed to relatively low, therapeutically relevant levels of AMD. Pmr1 is an ATP-driven pump that sequesters Ca2+ and Mn2+ into the Golgi/secretory pathway and maintains cellular ion homeostasis under normal growing conditions (11Sorin A. Rosas G. Rao R. J. Biol. Cell. 1997; 272: 9895-9901Scopus (217) Google Scholar, 12Marchi V. Sorin A Wei Y. Rao R. FEBS Lett. 1999; 454: 181-186Crossref PubMed Scopus (54) Google Scholar). Homologues of Pmr1 are widely distributed in organisms including Caenorhabditis elegans, Drosophila, and humans and constitute the newly recognized SPCA subtype of Ca2+-ATPases (13Ton V.-K. Mandal D. Vahadji C. Rao R. J. Biol. Chem. 2002; 277: 6422-6427Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Pmc1 is the yeast Ca2+ pump related to mammalian plasma membrane Ca2+-ATPases that is induced under calcium stress (14Cunningham K.W. Fink G.R. Mol. Cell. Biol. 1996; 16: 2226-2237Crossref PubMed Scopus (375) Google Scholar) or in the absence of Pmr1 (12Marchi V. Sorin A Wei Y. Rao R. FEBS Lett. 1999; 454: 181-186Crossref PubMed Scopus (54) Google Scholar) and serves to detoxify excess Ca2+ by sequestration into the vacuole. Vacuolar calcium sequestration is also accomplished by the H+/Ca2+ exchanger, Vcx1 (15Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (354) Google Scholar, 16Pozos T.C. Sekler I. Cyert M.S. Mol. Cell. Biol. 1996; 16: 3730-3741Crossref PubMed Scopus (126) Google Scholar, 17Miseta A. Kellermayer R. Aiello D.P. Fu L. Bedwell D.M. FEBS Lett. 1999; 451: 132-136Crossref PubMed Scopus (116) Google Scholar), which requires the proton electrochemical gradient generated by the vacuolar H+-ATPase. Calcium release from the vacuole is mediated by Yvc1, a transient receptor potential-like Ca2+ channel (18Palmer C.P. Zhou X.L. Lin J. Loukin S.H. Kung C. Saimi Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7801-7805Crossref PubMed Scopus (168) Google Scholar). Cch1 is the yeast homologue of mammalian voltage-gated calcium channels and, together with the stretch-activated Ca2+ channel Mid1, constitutes a Ca2+ entry channel at the plasma membrane (19Paidhungat M. Garrett S. Mol. Cell. Biol. 1997; 17: 6339-6347Crossref PubMed Scopus (160) Google Scholar). There is experimental evidence for at least one other calcium influx channel of unknown molecular identity (5Bonilla M Nastase K.K. Cunningham K.W. EMBO J. 2002; 21: 2343-2353Crossref PubMed Scopus (207) Google Scholar, 20Muller E.M. Locke E.G. Cunningham K.W. Genetics. 2001; 159: 1527-1538Crossref PubMed Google Scholar). Together, these proteins regulate cellular calcium levels and are critical for proper functioning of the calcium signaling cascade. Calcium activation of calmodulin and the consequent activation of the calcium- and calmodulin-activated protein phosphatase, calcineurin, are believed to lead to a number of transcriptional and post-translational signals that mediate a variety of different cellular responses to extracellular cues, including cell cycle progression and the protein kinase C-mediated cell wall integrity pathway (reviewed in Refs. 21Cyert M.S. Annu. Rev. Genet. 2001; 35: 647-672Crossref PubMed Scopus (149) Google Scholar and 22Fox D.S. Heitman J. Bioessays. 2002; 24: 894-903Crossref PubMed Scopus (110) Google Scholar). The conservation of several components of the calcium homeostatic machinery with higher eukaryotes makes yeast an excellent model to study the role of calcium signaling in AMD toxicity. The results of our analysis of deletion mutants point to a requirement for Pmr1-mediated Ca2+ homeostasis in the growth response to AMD. Similar to recent observations on other drug resistance mechanisms (3Cruz M.C. Goldstein A.L. Blankenship J.R. Del Poeta M. Davis D. Cardenas M.E. Perfect J.R. McCusker J.H. Heitman J. EMBO J. 2002; 21: 546-559Crossref PubMed Scopus (272) Google Scholar, 6Edlind T. Smith L. Henry K. Katiyar S. Nickels J. Mol. Microbiol. 2002; 46: 257-268Crossref PubMed Scopus (88) Google Scholar), we also demonstrate a critical role for calcineurin in AMD tolerance. Analyses of cytoplasmic Ca2+ levels by aequorin luminescence assays indicate a correlation between AMD-induced cytosolic Ca2+ increase and growth toxicity. Specifically, AMD seems to cause both Ca2+ influx at the cell membrane and release from internal stores, including the vacuole. To identify additional factors that mediate drug sensitivity, we screened nearly 5000 single-gene deletion mutants for hypersensitivity to AMD. We identify 36 mutants, implicated in known and novel pathways that may be important for drug resistance and detoxification. The results of this study indicate that AMD kills yeast by a mechanism different from that used by conventional antifungals such as the azoles and the polyenes. Finally, we show that low concentrations of AMD and an azole (miconazole and fluconazole) are strikingly synergistic in combination, suggesting that supplementation of conventional antifungal treatment with AMD may be a practical means of converting the fungistatic effect of the azoles into more effective fungicides. In summary, our findings provide important insights into the cellular effects of AMD and its potential efficacy in the treatment of fungal infections. Yeast Strains and Media—Isogenic sets of yeast deletions were all derived from W303 and have been described before (15Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (354) Google Scholar). The MATα S. cerevisiae deletion library was purchased from Research Genetics (Invitrogen). The Q783A and D53A mutations of PMR1 have been described (23Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar, 24Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Candida albicans SC5314 (25Fonzi W.A. Irwin M.Y. Genetics. 1993; 134: 717-728Crossref PubMed Google Scholar) was obtained from the laboratory of Brendan Cormack at The Johns Hopkins University School of Medicine, and C. neoformans (JEC21) (26Cruz M.C. Goldstein A.L. Blankenship J. Poeta M.D. Perfect J.R. McCusker J.M. Bennani Y.L. Cardenas M.E. Heitman J. Antimicrob. Agents. Chemother. 2001; 45: 3162-3170Crossref PubMed Scopus (111) Google Scholar) was a gift from the laboratory of Joseph Heitman at Duke University Medical Center. All yeast strains were grown in synthetic complete (SC) medium (Bio 101, Inc., Vista, CA) or standard YPD medium (2% Difco yeast extract, 1% bacto-peptone, 2% dextrose) at 30 or 37 °C as specified in the legends to Figs. 1, 5, and 6.Fig. 5Synergism between amiodarone and miconazole in S. cerevisiae. A, wild type S. cerevisiae (BY4742) was grown in SC medium in the absence or presence of AMD (4 μm), or MIC (1 μm), individually or in combination. After 24 h, the cells were stained with methylene blue and counted as described under "Experimental Procedures." Viability was calculated as the number of unstained cells divided by the total number of cells counted (about 250–300), expressed as a percentage. The average of three separate experiments is shown. B and C, wild type cells (BY4742) were grown overnight in SC medium without (B) and with both drugs (C) and then stained with 4 μm FUN-1 for 1 h at 30 °C. Final concentrations of AMD and MIC were 4 μm and 1 μm, respectively. Excitation wavelength was 488 nm, and emission was at 520 and 600 nm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 6Synergism between amiodarone and fluconazole in pathogenic fungi. A, C. albicans SC5314 was grown in 1 ml of YPD supplemented with 2 μm and 4 μm AMD in the presence or absence of 8 μg/ml FLUC at 37 °C. After 20 h, 5 μl of cells were diluted into 1.5 ml of drug-free YPD and grown at 37 °C for another 20 h. Growth (absorbance at 600 nm) was plotted as a percentage of control (no drug). Data points are averages of duplicates, with S.D. less than 5%. B, C. neoformans JEC21 was grown in 5 ml of YPD at 30 °C, shaken vigorously for 20 h. The medium had 4 μm AMD with or without 16 μg/ml FLUC. 5 μl of cells exposed to AMD alone or 20 μl of cells exposed to both drugs were diluted into 10 ml of YPD and grown for 24–48 h at 30 °C until saturation. Growth was measured as optical density at 600 nm and then plotted as a percentage of control (no drug). Data points are averages of duplicates, with S.D. equal to or less than 10%.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Growth Assays for Drug Sensitivity—AMD was purchased from Sigma, dissolved in dimethyl sulfoxide as a 20 mm stock, and stored at –20 °C. 10 μl of saturated seed cultures were inoculated in 3 ml of growth medium containing different concentrations of AMD (0–15 μm). Cultures were grown in a 30 °C shaker for 24 h, and growth was determined by optical density at 600 nm. To test the effect of divalent cations, AMD sensitivity was monitored in the presence of 10–25 mm CaCl2 or 10 mm MgCl2. Alternatively, the cation chelator 1,2-bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetracetic acid (BAPTA; Sigma) was added to final concentrations of 0.5 and 2 mm. To examine the role of calcineurin inhibition, FK506 was diluted from a stock of 0.2 mg/ml to a final concentration of 1 μg/ml and added to cell cultures grown in stationary 96-well plates at 30 °C. Miconazole and fluconazole were diluted from a stock of 5 mm (in Me2SO) or 2 mg/ml (in water), respectively. In the fluconazole screen with C. albicans, the seed culture was diluted by 1:100 and added to 1 ml of YPD containing different drug concentrations in a 24-well plate. After a 24–48-h incubation at 37 °C, 5 μl of cells were diluted into 1.5 ml of YPD containing no drugs and grown overnight. Growth was measured by absorbance at 600 nm. With C. neoformans, 50 μl of saturated seed culture was added to 5 ml of YPD supplemented with various drug concentrations. Cells were shaken vigorously for 20 h at 30 °C. 5 μl of cells exposed to AMD alone or 20 μl of cells exposed to both drugs were then diluted in 10 ml YPD and grown to saturation for 24–48 h. Growth was measured by absorbance at 600 nm. Genome-wide AMD Sensitivity Screen—200 μl of SC medium supplemented with 7 μm AMD was added to a 96-well plate and inoculated with 5 μl of freshly grown stationary phase yeast culture derived from single-gene deletion strains from the ResGen MATα deletion library (Invitrogen). Control cultures contained an equal volume of dimethyl sulfoxide. The plates were incubated at room temperature for a period of 12–24 h. Cultures were resuspended with a multichannel pipettor, and growth was monitored by measuring the absorbance at 600 nm in a SPECTRAmax 340 microplate reader (Molecular Devices). The relative growth of each strain was expressed as a percentage of A 600 of the control culture (i.e. no AMD). Strains with enhanced sensitivity to AMD were selected based on growth inhibition of 80% or greater relative to control. All candidate strains were retested for enhanced sensitivity in 4 and 8 μm AMD. Aequorin Luminescence Assay—Yeast strains were transformed with plasmid pEVP11-Aeq-89 (4Matsumoto T.K. Ellsmore A.J. Cessna S.G. Low P.S. Pardo J.M. Bressan R.A. Hasegawa P.M. J. Biol. Chem. 2002; 277: 33075-33080Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) carrying the aequorin gene and then grown to midlog phase in SC medium lacking leucine. Cells were harvested, incubated with 0.25 mg/ml coelenterazine (Molecular Probes, Inc., Eugene, OR) in the dark for 20 min, washed and resuspended in medium, and then incubated at 30 °C for 90 min to allow recovery. AMD (at specified concentrations) was added to 0.3 ml of cells in a cuvette through an injector, and luminescence was immediately recorded every second for 10 min in a luminometer (Lumat LB 9507). The maximal luminescence (L max) was determined as described (26Cruz M.C. Goldstein A.L. Blankenship J. Poeta M.D. Perfect J.R. McCusker J.M. Bennani Y.L. Cardenas M.E. Heitman J. Antimicrob. Agents. Chemother. 2001; 45: 3162-3170Crossref PubMed Scopus (111) Google Scholar). Specifically, cells were lysed with 1% digitonin in the presence of 1 m CaCl2, and a peak of RLU was recorded. Calcium concentration was calculated with Equation 1, [Ca2+]=((L/Lmax)1/3+[118(L/Lmax)1/3]-1)/(7×106-[7×106(L/Lmax)1/3])(Eq. 1) where L represents the RLU at a given time point. The base-line calcium concentration of pmc1 in SC and Me2SO was ∼0.38 μm, and that of pmr1 was about 0.52 μm. Methylene Blue Viability Assay—S. cerevisiae cells grown in media with and without the specified concentrations of AMD and miconazole were stained with methylene blue (Sigma) taken from a stock of 0.1 mg/ml. 250–300 cells were counted under a light microscope. Dead cells stain blue. FUN-1 Confocal Microscopy—The Live/Dead yeast viability kit was purchased from Molecular Probes. 50 μl of cells grown overnight in the presence or absence of drugs (as specified in Fig. 5) were incubated in SC medium at 30 °C for 1 h with 4 μm FUN-1 dye, which was diluted in SC from a stock of 200 μm in Me2SO. Immediately after incubation, the cells were examined under a confocal laser-scanning microscope (PerkinElmer UltraView LCI System) equipped with an inverted × 100 oil immersion objective lens. The fluorescent dye was excited at 488 nm by the krypton/argon laser. Conversion of FUN-1 into cylindrical intravacuolar structures was monitored by recording fluorescent micrographs at emission wavelengths of 600 nm (metabolically active and inactive cells) or 520 nm (metabolically inactive cells only). Pseudocolorization was done with Adobe Photoshop software (Adobe Systems Inc.). Hypersensitivity of the pmr1 Mutant to Amiodarone—To investigate the role of various calcium transport pathways in mediating drug sensitivity, we compared the growth of individual, isogenic gene deletion mutants in media supplemented with micromolar concentrations of AMD. As shown in Fig. 1A, the pmr1 mutant was strikingly more sensitive to AMD; in contrast, the pmc1 null strain showed a small but reproducible tolerance to the drug, relative to wild type. A survey of deletion mutants of the vacuolar H+/Ca2+ exchanger (vcx1) and all known Ca2+ channels in yeast (yvc1, cch1, and mid1) revealed a modest exacerbation of AMD sensitivity in both cch1 and mid1 mutants but not in the others (not shown). Taken together, these results suggest that some aspect of Ca2+ signaling, maintained in large part by the ATP-dependent calcium efflux pump, Pmr1, is important in mediating the growth toxicity of AMD. Extracellular Calcium Modulates Amiodarone Toxicity—Extracellular calcium has been reported to confer a dose-dependent abrogation of susceptibility to KP4 fungal toxin in the yeast Ustilago maydis (28Gage M.J. Bruenn J. Fischer M. Sanders D. Smith T.J. Mol. Microbiol. 2001; 41: 775-785Crossref PubMed Scopus (45) Google Scholar). Here, 10 mm CaCl2 or MgCl2 appeared to protect cell growth against AMD toxicity, whereas BAPTA, a membrane-impermeant cation chelator, enhanced AMD toxicity, consistent with the removal of Ca2+ (Fig. 1B). Interestingly, the protective effect of increasing Ca2+ concentrations (10–25 mm) on AMD sensitivity was most significant in strains lacking Pmr1 (not shown), which may reflect, at least in part, the known dependence of this strain on extracellular Ca2+ for optimum growth (29Durr G. Strayle J. Plemper R. Elbs S. Klee S.K. Catty P. Wolf D.H. Rudolph H.K. Mol. Biol. Cell. 1998; 9: 1149-1162Crossref PubMed Scopus (345) Google Scholar). Mn2+ can be a surrogate for Ca2+ in a number of physiological processes but was ineffective in conferring protection (data not shown). Monovalent ions (K+ and Na+) were previously reported to have little or no effect on AMD toxicity to yeast (1Courchesne W.E. J. Pharmacol. Exp. Ther. 2002; 300: 195-199Crossref PubMed Scopus (81) Google Scholar). Ion Selectivity Mutants of Pmr1 Confirm the Specific Role of Calcium in Amiodarone Toxicity—Since Pmr1 mediates the high affinity transport of both Ca2+ and Mn2+ into the Golgi (11Sorin A. Rosas G. Rao R. J. Biol. Cell. 1997; 272: 9895-9901Scopus (217) Google Scholar, 24Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), it was of interest to determine whether the enhanced sensitivity of pmr1 mutant to AMD was a consequence of a loss of Ca2+ or Mn2+ transport. We have previously described two ion selectivity mutants of Pmr1, Q783A and D53A, which show nearly exclusive transport of either Ca2+ or Mn2+, respectively (23Mandal D. Woolf T. Rao R. J. Biol. Cell. 2000; 273: 23933-23938Google Scholar, 24Wei Y. Chen J. Rosas G. Tompkins D.A. Holt A. Rao R. J. Biol. Chem. 2000; 275: 23927-23932Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). These mutants were expressed in a yeast strain devoid of endogenous Ca2+ pumps, with the additional deletion of calcineurin to maintain viability (pmr1pmc1cnb1) (11Sorin A. Rosas G. Rao R. J. Biol. Cell. 1997; 272: 9895-9901Scopus (217) Google Scholar, 15Cunningham K.W. Fink G.R. J. Cell Biol. 1994; 124: 351-363Crossref PubMed Scopus (354) Google Scholar). Selective loss of Ca2+ transport in Pmr1 mutant D53A resulted in loss of tolerance to AMD, similar to the host strain lacking Pmr1 and Pmc1 (Fig. 1C). In contrast, selective loss of Mn2+ transport of mutant Q783A did not alter AMD sensitivity. These results are consistent with the inability of extracellular Mn2+ to protect against AMD toxicity, and they establish the specific role of Ca2+ ions in mediating the cellular effects of the drug. Loss of Calcineurin Function Enhances Toxicity of Amiodarone—Calcineurin has been shown to be nonessential for normal growth but critical for survival during membrane stress in C. albicans (3). The enhanced AMD sensitivity of the triple mutant, pmr1pmc1cnb1 (Fig. 1C) relative to the individual gene deletions (Fig. 1A) suggested that loss of calcineurin may be synergistic with loss of the Golgi Ca2+ pump Pmr1. To confirm this, we investigated the effect of FK506 (1 μg/ml), a potent inhibitor of calcineurin, on the AMD sensitivity of wild type and pmr1 strains (Fig. 1D). As predicted, the addition of FK506 exacerbated the AMD sensitivity of the pmr1 mutant. Additionally, the cnb1 mutant, lacking functional calcineurin, displayed increased sensitivity to AMD at higher concentrations of the drug (Fig. 1D). Taken together, these data confirm the importance of calcineurin in the Ca2+ signaling pathway involved in drug sensitivity. Amiodarone Triggers Calcium Influx in Yeast Cells—Next, we investigated whether AMD induced calcium entry into yeast and whether calcium entry correlated with drug sensitivity. Calcium-dependent luminescence of the aequorin-coelenterazine photoprotein complex was monitored in the first few minutes immediately following drug addition. In all strains examined, application of AMD resulted in a biphasic elevation of cytoplasmic calcium that was dependent on AMD concentration (Fig. 2, A–C). The first peak of calcium occurred extremely fast (within 1–1.5 min) and was followed by a sustained rise that lasted for the duration of the assay. The magnitude of both phases, as well as the kinetics of the initial elevation was steeply dependent on AMD concentration, with maximal increase observed between 10 and 20 μm AMD. Elevation of cytosolic calcium was found to correlate with AMD sensitivity; thus, the addition of 10 μm AMD elicited the largest luminescence changes in the pmr1 mutant, whereas the pmc1 mutant showed similar changes relative to wild type (Fig. 2D). Estimated cytosolic calcium concentrations of the initial peak, based on the calibration described under "Experimental Procedures," reached 0.7 μm in wild type and pmc1 and close to 1 μm in pmr1 cells; the second sustained rise remained around 0.5 μm for WT and pmc1 but stayed at 0.75 μm for pmr1. Ca2 + Entry in Response to AMD Comes from Extracellular and Intracellular Stores—It was of interest to determine the relative contribution of extracellular and intracellular Ca2+ stores to the biphasic elevations of cytosolic Ca2+ observed in Fig. 2. When we used the membrane-impermeant cation chelator BAPTA (5 mm) to deplete extracellular Ca2+, the first peak of AMD-induced aequorin luminescence in wild type cells was eliminated, indicating that external calcium contributed to the rapid, initial rise in cytoplasmic calcium (Fig. 3A). Following a short lag, a broad, sustained rise was observed in BAPTA-treated cells that probably corresponded to the second phase of luminescence seen in the absence of BAPTA. These results implied that Ca2+ release from intracellular stores must contribute to cytosolic elevation of Ca2+ and, further, that Ca2+ entry from extracellular sources is not required to trigger release from stores. The transient receptor potential-like channel Yvc1 has been shown to mediate Ca2+ release from the vacuole in response to hypertonic shock (30Denis V. Cyert M.S. J. Cell Biol. 2002; 156: 29-34Crossref PubMed Scopus (228) Google Scholar). We investigated the contribution of vacuolar Ca2+ release by examining aequorin luminescence in the yvc1 mutant (Fig. 3A). The changes in intracellular Ca2+ in the yvc1 mutant were essentially similar to wild type in the absence of BAPTA. The first peak of wild type cells reached ∼0.8–0.9 μm, and that of yvc1 was about 0.7 μm. The second sustained rise in both strains ranged from 0.59 to 0.66 μm of free calcium. Strikingly, BAPTA treatment completely abolished Ca2+ elevation in the yvc1 mutant. These results indicate that whereas the firs
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