Nonselective TRPC channel inhibition and suppression of aminoglycoside-induced premature termination codon readthrough by the small molecule AC1903
2022; Elsevier BV; Volume: 298; Issue: 2 Linguagem: Inglês
10.1016/j.jbc.2021.101546
ISSN1083-351X
AutoresAlireza Baradaran‐Heravi, Claudia Bauer, Isabelle B. Pickles, Sara Hosseini-Farahabadi, Aruna D. Balgi, Kunho Choi, Deborah M. Linley, David J. Beech, Michel Roberge, Robin S. Bon,
Tópico(s)Insect behavior and control techniques
ResumoNonsense mutations, which occur in ∼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4–C1, and TRPC5–C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8–18 μM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake. Nonsense mutations, which occur in ∼11% of patients with genetic disorders, introduce premature termination codons (PTCs) that lead to truncated proteins and promote nonsense-mediated mRNA decay. Aminoglycosides such as G418 permit PTC readthrough and so may be used to address this problem. However, their effects are variable between patients, making clinical use of aminoglycosides challenging. In this study, we tested whether TRPC nonselective cation channels contribute to the variable PTC readthrough effect of aminoglycosides by controlling their cellular uptake. Indeed, a recently reported selective TRPC5 inhibitor, AC1903, consistently suppressed G418 uptake and G418-induced PTC readthrough in the DMS-114 cancer cell line and junctional epidermolysis bullosa (JEB) patient-derived keratinocytes. Interestingly, the effect of AC1903 in DMS-114 cells was mimicked by nonselective TRPC inhibitors, but not by well-characterized inhibitors of TRPC1/4/5 (Pico145, GFB-8438) or TRPC3/6/7 (SAR7334), suggesting that AC1903 may work through additional or undefined targets. Indeed, in our experiments, AC1903 inhibited multiple TRPC channels including TRPC3, TRPC4, TRPC5, TRPC6, TRPC4–C1, and TRPC5–C1, as well as endogenous TRPC1:C4 channels in A498 renal cancer cells, all with low micromolar IC50 values (1.8–18 μM). We also show that AC1903 inhibited TRPV4 channels, but had weak or no effects on TRPV1 and no effect on the nonselective cation channel PIEZO1. Our study reveals that AC1903 has previously unrecognized targets, which need to be considered when interpreting results from experiments with this compound. In addition, our data strengthen the hypothesis that nonselective calcium channels are involved in aminoglycoside uptake. Nonsense mutations account for ∼11% of all genetic lesions in patients with inherited diseases (1Mort M. Ivanov D. Cooper D.N. Chuzhanova N.A. A meta-analysis of nonsense mutations causing human genetic disease.Hum. Mutat. 2008; 29: 1037-1047Google Scholar). They introduce a TAG, TGA, or TAA premature termination codon (PTC) and typically result in production of mRNAs with decreased stability as well as defective truncated proteins. PTC readthrough is a mechanism by which ribosomes recognize nonsense mutations as sense codons, enabling synthesis of the full-length and functional protein rather than truncated product. Aminoglycoside antibiotics were the first and are still among the most active chemicals discovered to induce PTC readthrough in yeast (2Singh A. Ursic D. Davies J. Phenotypic suppression and misreading Saccharomyces cerevisiae.Nature. 1979; 277: 146-148Google Scholar), mammalian cells (3Burke J.F. Mogg A.E. Suppression of a nonsense mutation in mammalian cells in vivo by the aminoglycoside anthiotics G-418 and paromomycin.Nucleic Acids Res. 1985; 13: 6265-6272Google Scholar), animal models (4Du M. Jones J.R. Lanier J. Keeling K.M. Lindsey R.J. Tousson A. Bebök Z. Whitsett J.A. Dey C.R. Colledge W.H. Evans M.J. Sorscher E.J. Bedwell D.M. Aminoglycoside suppression of a premature stop mutation in a Cftr-/- mouse carrying a human CFTR-G542X transgene.J. Mol. Med. 2002; 80: 595-604Google Scholar, 5Barton-Davis E.R. Cordier L. Shoturma D.I. Leland S.E. Sweeney H.L. Aminoglycoside antibiotics restore dystrophin function to skeletal muscles of mdx mice.J. Clin. Invest. 1999; 104: 375-381Google Scholar), and patients (6Clancy J.P. Bebök Z. Ruiz F. King C. Jones J. Walker L. Greer H. Hong J. Wing L. Macaluso M. Lyrene R. Sorscher E.J. Bedwell D.M. Evidence that systemic gentamicin suppresses premature stop mutations in patients with cystic fibrosis.Am. J. Respir. Crit. Care Med. 2001; 163: 1683-1692Google Scholar, 7Malik V. Rodino-Klapac L.R. Viollet L. Wall C. King W. Al-Dahhak R. Lewis S. Shilling C.J. Kota J. Serrano-Munuera C. Hayes J. Mahan J.D. Campbell K.J. Banwell B. Dasouki M. et al.Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy.Ann. Neurol. 2010; 67: 771-780Google Scholar). These compounds bind at the decoding center of eukaryotic ribosomes and facilitate pairing of near-cognate aminoacyl-tRNAs to the PTCs resulting in formation of full-length protein (8François B. Russell R.J.M. Murray J.B. Aboul-ela F. Masquida B. Vicens Q. Westhof E. Crystal structures of complexes between aminoglycosides and decoding A site oligonucleotides: Role of the number of rings and positive charges in the specific binding leading to miscoding.Nucleic Acids Res. 2005; 33: 5677-5690Google Scholar, 9Garreau De Loubresse N. Prokhorova I. Holtkamp W. Rodnina M.V. Yusupova G. Yusupov M. Structural basis for the inhibition of the eukaryotic ribosome.Nature. 2014; 513: 517-522Google Scholar). Several nonaminoglycoside readthrough compounds have also been identified, including negamycin, tylosin, RTC13, RTC14, GJ71, GJ72, and ataluren (10Gatti R.A. SMRT compounds correct nonsense mutations in primary immunodeficiency and other genetic models.Ann. N. Y. Acad. Sci. 2012; 1250: 33-40Google Scholar, 11Du M. Liu X. Welch E.M. Hirawat S. Peltz S.W. Bedwell D.M. PTC124 is an orally bioavailable compound that promotes suppression of the human CFTR-G542X nonsense allele in a CF mouse model.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 2064-2069Google Scholar, 12Du L. Jung M.E. Damoiseaux R. Completo G. Fike F. Ku J.M. Nahas S. Piao C. Hu H. Gatti R.A. A new series of small molecular weight compounds induce read through of all three types of nonsense mutations in the ATM gene.Mol. Ther. 2013; 21: 1653-1660Google Scholar, 13Zilberberg A. Lahav L. Rosin-Arbesfeld R. Restoration of APC gene function in colorectal cancer cells by aminoglycoside- and macrolide-induced read-through of premature termination codons.Gut. 2010; 59: 496-507Google Scholar, 14Arakawa M. Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice.J. Biochem. 2003; 134: 751-758Google Scholar). However, these compounds induce PTC readthrough at low rates and often at or below the detection limit of Western blotting for endogenous protein expression. In addition to their severe in vivo toxicity, aminoglycosides induce variable levels of PTC readthrough in cell lines as well as patients, making long-term clinical administration of these drugs challenging (15Martorell L. Cortina V. Parra R. Barquinero J. Vidal F. Variable readthrough responsiveness of nonsense mutations in hemophilia A.Haematologica. 2020; 105: 508-518Google Scholar, 16Pranke I. Bidou L. Martin N. Blanchet S. Hatton A. Karri S. Cornu D. Costes B. Chevalier B. Tondelier D. Girodon E. Coupet M. Edelman A. Fanen P. Namy O. et al.Factors influencing readthrough therapy for frequent cystic fibrosis premature termination codons.ERJ Open Res. 2018; 4: 00080-02017Google Scholar, 17Linde L. Boelz S. Nissim-Rafinia M. Oren Y.S. Wilschanski M. Yaacov Y. Virgilis D. Neu-Yilik G. Kulozik A.E. Kerem E. Kerem B. Nonsense-mediated mRNA decay affects nonsense transcript levels and governs response of cystic fibrosis patients to gentamicin.J. Clin. Invest. 2007; 117: 683-692Google Scholar, 18Baradaran-Heravi A. Balgi A.D. Zimmerman C. Choi K. Shidmoossavee F.S. Tan J.S. Bergeaud C. Krause A. Flibotte S. Shimizu Y. Anderson H.J. Mouly V. Jan E. Pfeifer T. Jaquith J.B. et al.Novel small molecules potentiate premature termination codon readthrough by aminoglycosides.Nucleic Acids Res. 2016; 44: 6583-6598Google Scholar, 19Wargo K.A. Edwards J.D. Aminoglycoside-induced nephrotoxicity.J. Pharm. Pract. 2014; 27: 573-577Google Scholar, 20Guthrie O.W. Aminoglycoside induced ototoxicity.Toxicology. 2008; 249: 91-96Google Scholar). In general, variation in drug response is caused by differential local drug concentrations (pharmacokinetics) or drug actions (pharmacodynamics) and to a great extent is attributed to genetic variations (21Roden D.M. McLeod H.L. Relling M.V. Williams M.S. Mensah G.A. Peterson J.F. Van Driest S.L. Pharmacogenomics.Lancet. 2019; 394: 521-532Google Scholar). Variations in aminoglycoside-induced PTC readthrough could be in part related to the PTC and its surrounding sequence as well as its distance to the poly-A tail sequence. Moreover, differential cellular uptake of aminoglycosides and consequent variable intracellular concentrations of these compounds may underlie mechanisms of PTC readthrough variation. However, the correlation of genetic variations in genes involved in cellular uptake of aminoglycosides and their contribution to PTC readthrough in human cells is not well understood. Endocytosis (22Hashino E. Shero M. Endocytosis of aminoglycoside antibiotics in sensory hair cells.Brain Res. 1995; 704: 135-140Google Scholar, 23Hashino E. Shero M. Salvi R.J. Lysosomal targeting and accumulation of aminoglycoside antibiotics in sensory hair cells.Brain Res. 1997; 777: 75-85Google Scholar) and permeation through nonselective cation channels (including TRP channels) (24Karasawa T. Wang Q. Fu Y. Cohen D.M. Steyger P.S. TRPV4 enhances the cellular uptake of aminoglycoside antibiotics.J. Cell Sci. 2008; 121: 2871-2879Google Scholar, 25Myrdal S.E. Steyger P.S. TRPV1 regulators mediate gentamicin penetration of cultured kidney cells.Hear. Res. 2005; 204: 170-182Google Scholar, 26Makabe A. Kawashima Y. Sakamaki Y. Maruyama A. Fujikawa T. Ito T. Kurima K. Griffith A.J. Tsutsumi T. Systemic fluorescent gentamicin enters neonatal mouse hair cells predominantly through sensory mechanoelectrical transduction channels.J. Assoc. Res. Otolaryngol. 2020; 21: 137-149Google Scholar, 27Marcotti W. van Netten S.M. Kros C.J. The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels.J. Physiol. 2005; 567: 505-521Google Scholar, 28Lee J.H. Park C. Kim S.J. Kim H.J. Oh G.S. Shen A. So H.S. Park R. Different uptake of gentamicin through TRPV1 and TRPV4 channels determines cochlear hair cell vulnerability.Exp. Mol. Med. 2013; 45e12Google Scholar) are the main proposed mechanisms for cellular uptake of aminoglycosides. In this study, we addressed the hypothesis that TRPC channels may contribute to variable effects of aminoglycoside-mediated PTC readthrough. Following the observation that low PTC readthrough in response to treatment with the aminoglycoside G418 (which is cationic at physiological pH) was correlated with a mutation in TRPC5, we tested the effects of selected TRPC channel inhibitors on G418-induced PTC readthrough. We found that the 2-aminobenzimidazole derivative AC1903, recently reported as a selective TRPC5 channel inhibitor (29Zhou Y. Castonguay P. Sidhom E.H. Clark A.R. Dvela-Levitt M. Kim S. Sieber J. Wieder N. Jung J.Y. Andreeva S. Reichardt J. Dubois F. Hoffmann S.C. Basgen J.M. Montesinos M.S. et al.A small-molecule inhibitor of TRPC5 ion channels suppresses progressive kidney disease in animal models.Science. 2017; 358: 1332-1336Google Scholar, 30Sharma S.H. Pablo J.L. Montesinos M.S. Greka A. Hopkins C.R. Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound, AC1903.Bioorg. Med. Chem. Lett. 2019; 29: 155-159Google Scholar), suppresses cellular uptake of the aminoglycoside G418 and G418-induced PTC readthrough in the DMS-114 cell line and in JEB patient-derived keratinocytes. PTC readthrough suppression in DMS-114 cells could be reproduced with the nonselective calcium channel modulators SKF96365 and 2-APB, but not with the well-characterized, selective TRPC1/4/5 inhibitors Pico145 and GFB-8438, suggesting that AC1903 may have additional targets. We subsequently found that, in contrast to previous reports, AC1903 is not a selective inhibitor of TRPC5 (or TRPC1/4/5) channels, but that it inhibits multiple TRP channels, including those formed by TRPC3, TRPC4, TRPC6, and TRPV4. Our study underlines that investigations of TRPC5 channel biology with nonselective inhibitors such as AC1903 need to be supported carefully by orthogonal chemical or genetic approaches. In addition, our results are consistent with the hypothesis that multiple nonselective (TRP) cation channels can mediate cellular aminoglycoside uptake, which may trigger new investigations into the use of aminoglycosides for the treatment of genetic diseases. Variation in PTC readthrough response has been reported among individuals or cell lines with different nonsense mutations. Although this in part highlights the importance of the stop codon and its surrounding sequence as well as the physical location of the nonsense mutation in the mRNA sequence, understanding the contribution of other underlying genetic variations becomes more complicated. To rule out the effect of the sequence and position of nonsense mutations on response variation to potential PTC readthrough modulators, we studied two cancer cell lines, DMS-114 and TC-71, with identical homozygous nonsense mutation R213X in their TP53 gene. Exposure of DMS-114 cells to increasing concentrations of the aminoglycoside G418 for 24 h resulted in strong, concentration-dependent increase in production of full-length p53 (the readthrough product) up to 144-fold compared with untreated cells (Fig. 1A). In contrast, increasing concentrations of G418 only elicited a small increase in PTC readthrough in TC-71 cells, up to eightfold compared to untreated cells (Fig. 1A). To find out whether the reduced response to G418 in TC-71 cells was associated with lower intracellular levels of G418, we measured concentrations of G418 in both cell lines at 0, 8 and 24 h post exposure to G418 (100 μg/ml). Compared with DMS-114, intracellular G418 levels were slightly but significantly lower in TC-71 cells at 8 h and 24 h post G418 exposure, by ∼11% and ∼9%, respectively (Fig. 1B). These data suggest that reduced intracellular G418 level in TC-71 cells versus DMS-114 may be partially responsible for the difference in G418-induced PTC readthrough. To find out the possible contribution of genetic variants to this variation, we searched exome sequencing data in COSMIC (https://cancer.sanger.ac.uk/cosmic) and identified a hemizygous p.R175H missense mutation in TRPC5 in the TC-71 cell line not present in the DMS-114 cell line. This gene encodes the protein TRPC5, which forms homo- and heterotetrameric, nonselective cation channels permeable by Ca2+ and Na+ (31Clapham D.E. TRP channels as cellular sensors.Nature. 2003; 426: 517-524Google Scholar, 32Abramowitz J. Birnbaumer L. Physiology and pathophysiology of canonical transient receptor potential channels.FASEB J. 2009; 23: 297-328Google Scholar, 33Montell C. The TRP superfamily of cation channels.Sci. Signal. 2005; 2005re3Google Scholar, 34Wang H. Cheng X. Tian J. Xiao Y. Tian T. Xu F. Hong X. Zhu M.X. TRPC channels: Structure, function, regulation and recent advances in small molecular probes.Pharmacol. Ther. 2020; 209: 107497Google Scholar, 35Bon R.S. Beech D.J. In pursuit of small molecule chemistry for calcium-permeable non-selective TRPC channels - mirage or pot of gold?.Br. J. Pharmacol. 2013; 170: 459-474Google Scholar, 36Minard A. Bauer C.C. Wright D.J. Rubaiy H.N. Muraki K. Beech D.J. Bon R.S. Remarkable progress with small-molecule modulation of TRPC1/4/5 channels: Implications for understanding the channels in health and disease.Cells. 2018; 7: 52Google Scholar, 37Bon R.S. Wright D.J. Beech D.J. Sukumar P. Pharmacology of TRPC channels and its potential in cardiovascular and metabolic medicine.Annu. Rev. Pharmacol. Toxicol. 2022; 62: 427-446Google Scholar). Based on several pathogenicity prediction algorithms including SIFT (38Sim N.L. Kumar P. Hu J. Henikoff S. Schneider G. Ng P.C. SIFT web server: Predicting effects of amino acid substitutions on proteins.Nucleic Acids Res. 2012; 40: W452-W457Google Scholar) and PolyPhen-2 (39Adzhubei I.A. Schmidt S. Peshkin L. Ramensky V.E. Gerasimova A. Bork P. Kondrashov A.S. Sunyaev S.R. A method and server for predicting damaging missense mutations.Nat. Methods. 2010; 7: 248-249Google Scholar), this mutation is considered tolerable and benign. R175 of TRPC5 is located in a putative zinc-binding domain—consisting of H172, C176, C178, and C181—that is conserved between TRPC proteins (40Wright D.J. Simmons K.J. Johnson R.M. Beech D.J. Muench S.P. Bon R.S. Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.Commun. Biol. 2020; 3: 704Google Scholar, 41Song K. Wei M. Guo W. Quan L. Kang Y. Wu J.-X. Chen L. Structural basis for human TRPC5 channel inhibition by two distinct inhibitors.Elife. 2021; 10e63429Google Scholar). This domain has also been implicated in TRPC5 regulation by S-glutathionylation (42Hong C. Seo H. Kwak M. Jeon J. Jang J. Jeong E.M. Myeong J. Hwang Y.J. Ha K. Kang M.J. Lee K.P. Yi E.C. Kim I.G. Jeon J.H. Ryu H. et al.Increased TRPC5 glutathionylation contributes to striatal neuron loss in Huntington's disease.Brain. 2015; 138: 3030-3047Google Scholar), as well as S-palmitoylation required for TRPC5 trafficking to the plasma membrane (43Hong C. Choi S.H. Kwak M. Jeong B. Ko J. Park H.J. Choi S. Jun J.Y. So I. TRPC5 channel instability induced by depalmitoylation protects striatal neurons against oxidative stress in Huntington's disease.Biochim. Biophys. Acta Mol. Cell Res. 2020; 1867: 118620Google Scholar). Because other nonselective cation channels of the TRP family have been reported to contribute to aminoglycoside import (24Karasawa T. Wang Q. Fu Y. Cohen D.M. Steyger P.S. TRPV4 enhances the cellular uptake of aminoglycoside antibiotics.J. Cell Sci. 2008; 121: 2871-2879Google Scholar, 25Myrdal S.E. Steyger P.S. TRPV1 regulators mediate gentamicin penetration of cultured kidney cells.Hear. Res. 2005; 204: 170-182Google Scholar, 26Makabe A. Kawashima Y. Sakamaki Y. Maruyama A. Fujikawa T. Ito T. Kurima K. Griffith A.J. Tsutsumi T. Systemic fluorescent gentamicin enters neonatal mouse hair cells predominantly through sensory mechanoelectrical transduction channels.J. Assoc. Res. Otolaryngol. 2020; 21: 137-149Google Scholar, 27Marcotti W. van Netten S.M. Kros C.J. The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels.J. Physiol. 2005; 567: 505-521Google Scholar, 28Lee J.H. Park C. Kim S.J. Kim H.J. Oh G.S. Shen A. So H.S. Park R. Different uptake of gentamicin through TRPV1 and TRPV4 channels determines cochlear hair cell vulnerability.Exp. Mol. Med. 2013; 45e12Google Scholar), we decided to test the potential role of TRPC5 and other TRPC channels in this process. To test whether TRPC5 or other TRPC channels may contribute to G418-mediated PTC readthrough, we screened a panel of TRPC inhibitors in DMS-114 cells. To this end, we selected compounds reported as selective TRPC channel inhibitors: AC1903 (TRPC5) (29Zhou Y. Castonguay P. Sidhom E.H. Clark A.R. Dvela-Levitt M. Kim S. Sieber J. Wieder N. Jung J.Y. Andreeva S. Reichardt J. Dubois F. Hoffmann S.C. Basgen J.M. Montesinos M.S. et al.A small-molecule inhibitor of TRPC5 ion channels suppresses progressive kidney disease in animal models.Science. 2017; 358: 1332-1336Google Scholar, 30Sharma S.H. Pablo J.L. Montesinos M.S. Greka A. Hopkins C.R. Design, synthesis and characterization of novel N-heterocyclic-1-benzyl-1H-benzo[d]imidazole-2-amines as selective TRPC5 inhibitors leading to the identification of the selective compound, AC1903.Bioorg. Med. Chem. Lett. 2019; 29: 155-159Google Scholar); GFB-8438 (TRPC4/5) (44Yu M. Ledeboer M.W. Daniels M. Malojcic G. Tibbitts T.T. Coeffet-Le Gal M. Pan-Zhou X.-R. Westerling-Bui A. Beconi M. Reilly J.F. Mundel P. Harmange J.-C. Discovery of a potent and selective TRPC5 inhibitor, efficacious in a focal segmental glomerulosclerosis model.ACS Med. Chem. Lett. 2019; 10: 1579-1585Google Scholar); or SAR7334 (TRPC3/6/7) (45Maier T. Follmann M. Hessler G. Kleemann H.-W. Hachtel S. Fuchs B. Weissmann N. Linz W. Schmidt T. Löhn M. Schroeter K. Wang L. Rütten H. Strübing C. Discovery and pharmacological characterization of a novel potent inhibitor of diacylglycerol-sensitive TRPC cation channels.Br. J. Pharmacol. 2015; 172: 3650-3660Google Scholar). For the simultaneous inhibition of all TRPC channels, we either used a combination of GFB-8438 and SAR7334, or the nonselective calcium channel modulators SKF96365 or 2-APB, each of which affects a broad spectrum of TRP channels and inhibits TRPC channels. Compounds were screened at two concentrations, reflecting their relative potencies against various TRPC channels. In our experiments, PTC readthrough induced by 400 μg/ml G418 was suppressed by SKF96365 (∼33%) and especially by 2-APB (∼66%), but GFB-8438, SAR7334, or their combination had no effect (Fig. 1C). Interestingly, AC1903 had an effect similar to SKF96365 when used at 50 μM (i.e., about three times its reported IC50 for TRPC5 currents (29Zhou Y. Castonguay P. Sidhom E.H. Clark A.R. Dvela-Levitt M. Kim S. Sieber J. Wieder N. Jung J.Y. Andreeva S. Reichardt J. Dubois F. Hoffmann S.C. Basgen J.M. Montesinos M.S. et al.A small-molecule inhibitor of TRPC5 ion channels suppresses progressive kidney disease in animal models.Science. 2017; 358: 1332-1336Google Scholar)), suppressing G418-induced PTC readthrough by ∼27% (Fig. 1C). Therefore, we decided to investigate the effects of AC1903 in more detail. To further investigate AC1903 as a suppressor of G418-induced PTC readthrough, we exposed DMS-114 and TC-71 cells to various concentrations of AC1903 for 3 h prior to exposure to 400 μg/ml G418 for another 3 h, followed by media replacement and incubation for another 18 h. Exposure to AC1903 alone did not affect PTC readthrough in either cell line (Fig. 2, A and B). As expected, G418 elicited strong PTC readthrough in DMS-114 but not in TC-71 cells (Fig. 2, A and B). In DMS-114 but not in TC-71 cells, combination of AC1903 (50 or 150 μM) with G418 reduced PTC readthrough by 20% and 74%, respectively, compared with G418 alone (Fig. 2, A and B). In order to better understand the effects of AC1903 on G418 uptake and PTC readthrough, we exposed DMS-114 cells and JEB01 keratinocytes, derived from a junctional epidermolysis bullosa (JEB) patient with a homozygous nonsense mutation (p.R688X) in the COL17A1 gene, to increasing concentrations of G418 without or with pretreatment with 150 μM AC1903. Exposure of DMS-114 cells to G418 alone resulted in a concentration-dependent increase in full-length p53, which decreased ∼70% when cells were additionally treated with AC1903 (Fig. 2C). This reduction in PTC readthrough was correlated with significantly decreased intracellular G418 levels in the presence of AC1903 (Fig. 2D). Similarly, JEB01 keratinocytes produced full-length Collagen XVII in the presence of G418 alone, which declined ∼45–80% in combination with AC1903 (Fig. 2E). Again, reduction of PTC readthrough in JEB01 cells exposed to AC1903 was correlated with significantly declined intracellular G418 levels (Fig. 2F). These data suggest that AC1903 suppresses cellular uptake of G418 and G418-induced PTC readthrough in DMS-114 and JEB01 cells, and the findings could indicate a possible role of TRPC5 channels in cellular regulation of G418 uptake. However, because the effect of AC1903 on G418-induced PTC readthrough could not be reproduced by the TRPC4/5 channel inhibitor GFB-8438 (Fig. 1C), further investigation was required. In an attempt to further probe the potential role of TRPC5 channels in the regulation of intracellular G418 levels by AC1903, we exposed DMS-114 cells to the xanthine derivative Pico145 in the presence of 400 μg/ml G418. Pico145 (also named HC-608) is a well-characterized, highly selective chemical probe of TRPC5, TRPC4, and heteromeric TRPC1/4/5 channels with picomolar to nanomolar potency (40Wright D.J. Simmons K.J. Johnson R.M. Beech D.J. Muench S.P. Bon R.S. Human TRPC5 structures reveal interaction of a xanthine-based TRPC1/4/5 inhibitor with a conserved lipid binding site.Commun. Biol. 2020; 3: 704Google Scholar, 46Rubaiy H.N. Ludlow M.J. Henrot M. Gaunt H.J. Miteva K. Cheung S.Y. Tanahashi Y. Hamzah N. Musialowski K.E. Blythe N.M. Appleby H.L. Bailey M.A. McKeown L. Taylor R. Foster R. et al.Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels.J. Biol. Chem. 2017; 292: 8159-8173Google Scholar, 47Just S. Chenard B.L. Ceci A. Strassmaier T. Chong A. Blair N.T. Gallaschun R.J. Camino D. Cantin S. Amours M.D. Eickmeier C. Fanger C.M. Hecker C. Hessler D.P. Hengerer B. et al.Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice.PLoS One. 2018; 13e0191225Google Scholar, 48Bauer C.C. Minard A. Pickles I.B. Simmons K.J. Chuntharpursat-Bon E. Burnham M.P. Kapur N. Beech D.J. Muench S.P. Wright M.H. Warriner S.L. Bon R.S. Xanthine-based photoaffinity probes allow assessment of ligand engagement by TRPC5 channels.RSC Chem. Biol. 2020; 1: 436-448Google Scholar) that has been used successfully to inhibit endogenous TRPC1/4/5 channels in cells (46Rubaiy H.N. Ludlow M.J. Henrot M. Gaunt H.J. Miteva K. Cheung S.Y. Tanahashi Y. Hamzah N. Musialowski K.E. Blythe N.M. Appleby H.L. Bailey M.A. McKeown L. Taylor R. Foster R. et al.Picomolar, selective, and subtype-specific small-molecule inhibition of TRPC1/4/5 channels.J. Biol. Chem. 2017; 292: 8159-8173Google Scholar, 49Muraki K. Ohnishi K. Takezawa A. Suzuki H. Hatano N. Muraki Y. Hamzah N. Foster R. Waldmann H. Nussbaumer P. Christmann M. Bon R.S. Beech D.J. Na+ entry through heteromeric TRPC4/C1 channels mediates (-)Englerin A-induced cytotoxicity in synovial sarcoma cells.Sci. Rep. 2017; 7: 16988Google Scholar, 50Lepannetier S. Gualdani R. Tempesta S. Schakman O. Seghers F. Kreis A. Yerna X. Slimi A. de Clippele M. Tajeddine N. Voets T. Bon R.S. Beech D.J. Tissir F. Gailly P. Activation of TRPC1 channel by metabotropic glutamate receptor mGluR5 modulates synaptic plasticity and spatial working memory.Front. Cell. Neurosci. 2018; 12: 318Google Scholar, 51Arboit A. Reboreda A. Yoshida M. Involvement of TRPC4 and 5 channels in persistent firing in hippocampal CA1 pyramidal cells.Cells. 2020; 9: 365Google Scholar, 52Martín-Aragón Baudel M.A.S. Shi J. Large W.A. Albert A.P. Obligatory role for PKCδ in PIP2-mediated activation of store-operated TRPC1 channels in vascular smooth muscle cells.J. Physiol. 2020; 598: 3911-3925Google Scholar), tissues (50Lepannetier S. Gualdani R. Tempesta S. Schakman O. Seghers F. Kreis A. Yerna X. Slimi A. de Clippele M. Tajeddine N. Voets T. Bon R.S. Beech D.J. Tissir F. Gailly P. Activation of TRPC1 channel by metabotropic glutamate receptor mGluR5 modulates synaptic plasticity and spatial working memory.Front. Cell. Neurosci. 2018; 12: 318Google Scholar, 53Blum T. Moreno-Pérez A. Pyrski M. Bufe B. Arifovic A. Weissgerber P. Freichel M. Zufall F. Leinders-Zufall T. Trpc5 deficiency causes hypoprolactinemia and altered function of oscillatory dopamine neurons in the arcuate nucleus.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 15236-15243Google Scholar, 54Yerna X. Schakman O. Ratbi I. Kreis A. Lepannetier S. de Clippele M. Achouri Y. Tajeddine N. Tissir F. Gualdani R. Gailly P. Role of the TRPC1 channel in hippocampal long-term depression and in spatial memory extinction.Int. J. Mol. Sci. 2020; 21: 1712Google Scholar), and animals (47Just S. Chenard B.L. Ceci A. Strassmaier T. Chong A. Blair N.T. Gallaschun R.J. Camino D. Cantin S. Amours M.D. Eickmeier C. Fanger C.M. Hecker C. Hessler D.P. Hengerer B. et al.Treatment with HC-070, a potent inhibitor of TRPC4 and TRPC5, leads to anxiolytic and antidepressant effects in mice.PLoS One. 2018; 13e0191225Google Scholar, 54Yerna X. Schakman O. Ratbi I. Kreis A. Lepannetier S. de Clippele M. Achouri Y. Tajeddine N. Tissir F. Gualdani R. Gailly P. Role of the TRPC1 channel in hippocampal long-term depression and in spatial memory extinction.Int. J. Mol. Sci. 2020; 21: 1712Google Scholar, 55Cheung S.Y. Henrot M. Al-Saad M. Baumann M. Muller H. Unger A. Rubaiy H.N. Mathar I. Dinkel K. Nussbaumer P. Klebl B. Freichel M. Rode B. Trainor S. Clapcote S.J. et al.TRPC4/TRPC5 channels m
Referência(s)