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• W2065600512 abstract "The K+ channels encoded by the human Ether-a-gogo Related Gene-1 (hERG1) are crucially involved in controlling heart and brain excitability and are selectively influenced by reactive oxygen species (ROS). To localize the molecular regions involved in ROS-induced modulation of hERG1, segmental exchanges between the ROS-sensitive hERG1 and the ROS-insensitive bovine ether-a-gogo gene (bEAG) K+ channels were generated, and the sensitivity of these chimeric channels to ROS was studied with the two-microelectrode voltage-clamp technique upon their expression in Xenopus oocytes. Substitution of the S5-S6 linker of hERG1 with the corresponding bEAG region removed channel sensitivity to ROS, whereas the reverse chimeric exchange introduced ROS sensitivity into bEAG. Mutation of each of the two hERG1 histidines at positions 578 and 587 within the S5-S6 linker generated K+ channels insensitive to modulation by ROS. In addition, the two iron chelators desferrioxamine (1 mm) and o-phenanthroline (0.2 mm) significantly inhibited hERG1 outward K+ currents and prevented hERG1 inhibition induced by the ROS-scavenging enzyme catalase (1000 units/ml). Finally, the hERG1-inhibitory effect exerted by the iron chelators was prevented by the hERG1 H578D/H587Y double mutation. Collectively, the results obtained suggest that histidines at positions 578 and 587 in the S5-S6 linker region of hERG1 K+channels are crucial players in ROS-induced modulation of hERG1 K+ channels. The K+ channels encoded by the human Ether-a-gogo Related Gene-1 (hERG1) are crucially involved in controlling heart and brain excitability and are selectively influenced by reactive oxygen species (ROS). To localize the molecular regions involved in ROS-induced modulation of hERG1, segmental exchanges between the ROS-sensitive hERG1 and the ROS-insensitive bovine ether-a-gogo gene (bEAG) K+ channels were generated, and the sensitivity of these chimeric channels to ROS was studied with the two-microelectrode voltage-clamp technique upon their expression in Xenopus oocytes. Substitution of the S5-S6 linker of hERG1 with the corresponding bEAG region removed channel sensitivity to ROS, whereas the reverse chimeric exchange introduced ROS sensitivity into bEAG. Mutation of each of the two hERG1 histidines at positions 578 and 587 within the S5-S6 linker generated K+ channels insensitive to modulation by ROS. In addition, the two iron chelators desferrioxamine (1 mm) and o-phenanthroline (0.2 mm) significantly inhibited hERG1 outward K+ currents and prevented hERG1 inhibition induced by the ROS-scavenging enzyme catalase (1000 units/ml). Finally, the hERG1-inhibitory effect exerted by the iron chelators was prevented by the hERG1 H578D/H587Y double mutation. Collectively, the results obtained suggest that histidines at positions 578 and 587 in the S5-S6 linker region of hERG1 K+channels are crucial players in ROS-induced modulation of hERG1 K+ channels. reactive oxygen species, NO⋅, nitric oxide reactive nitrogen species ⋅OH, hydroxyl radical hydrogen peroxide bovine ether-a-gogo gene, hERG1, humanEther-a-gogo Related Gene-1 rat ether-a-gogo related genes 2 and3 malondialdehyde diethylenetetraamine NONOate iron- and ascorbate-containing solution desferrioxamine ortho-phenanthroline extracellular K+concentrations catalase Oxidation and reduction reactions occurring during aerobic respiration can trigger the formation of reactive oxygen species (ROS),1 a family of molecules that includes superoxide ( O2⨪), hydroxyl radical (⋅OH), and hydrogen peroxide (H2O2), each having specific half-life, diffusibility, and biological reactivity (1Yu B.P. Physiol. Rev. 1994; 74: 139-162Crossref PubMed Scopus (2223) Google Scholar). ROS have been proposed as crucial regulators of cellular responses in several pathophysiological states, such as cardiovascular (2Kaneko M. Matsumoto Y. Hayashi H. Kobayashi A. Yamazaki N. Mol. Cell. Biochem. 1994; 139: 91-100Crossref PubMed Scopus (85) Google Scholar) and neurodegenerative disorders (3Coyle J.T. Puttfarcker P. Science. 1993; 262: 689-695Crossref PubMed Scopus (3533) Google Scholar), senescence (4Sohal R.S. Weindruch R. Science. 1996; 273: 59-63Crossref PubMed Scopus (2631) Google Scholar), and programmed cell death (5Korsmeyer S.J. Yin X.M. Oltvai Z.N. Veis-Novak D.J. Linette G.P. Biochim. Biophys. Acta. 1995; 1271: 63-66Crossref PubMed Scopus (254) Google Scholar). Oxidative stress refers to the imbalance between ROS production and cellular antioxidant defense systems (6Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine.2nd Ed. Clarendon Press, Oxford1989: 1-81Google Scholar). Iron ions have a primary role in the induction of oxidative stress, acting as catalysts in the Fenton reaction, which leads to the conversion of the highly diffusible, slow reacting H2O2 into the highly reactive and potent oxidant ⋅OH (7Halliwell B. J. Neurochem. 1992; 59: 1609-1623Crossref PubMed Scopus (2659) Google Scholar). In addition, oxidative stress is also influenced by nitric oxide (NO⋅) and other reactive nitrogen species (RNS) (8Gross S.S. Wolin M.S. Annu. Rev. Physiol. 1995; 57: 737-769Crossref PubMed Scopus (821) Google Scholar), which have been shown to exert both pro- and antioxidant effects during ischemia-reperfusion injury, depending on their cellular sources and on the stage of evolution of the ischemic process (9Darley-Usmar V. Wiseman H. Hallywell B. FEBS Lett. 1995; 369: 131-135Crossref PubMed Scopus (524) Google Scholar, 10Iadecola C. Trends Neurosci. 1997; 20: 132-139Abstract Full Text Full Text PDF PubMed Scopus (955) Google Scholar). Changes in protein function induced by ROS has been recognized as being crucial for oxidative stress-mediated pathophysiological changes. Maximal sensitivity to ROS is conferred by amino acids containing sulfur atoms (cysteine and methionine), hydroxyl groups (tyrosine), or aromatic rings (histidine, phenylalanine, and tryptophan) (11Stadtman E.R. Annu. Rev. Biochem. 1993; 62: 797-821Crossref PubMed Scopus (1266) Google Scholar). Interestingly, histidines in proteins are often associated with transition metals, particularly with redox-active iron ions, and histidines themselves are vulnerable to metal-catalyzed free radical reactions (12Chevion M. Free Radic. Biol. Med. 1988; 5: 27-37Crossref PubMed Scopus (453) Google Scholar). Oxidative modification of histidine residues may lead to their conversion to asparagine, aspartate, or 2-oxo-histidine (13Uchida K. Kawakishi S. FEBS Lett. 1993; 332: 208-210Crossref PubMed Scopus (120) Google Scholar). K+ channels play a crucial role in shaping the electrical activity of neuronal and cardiac cells, and modification of K+ channel activity by ROS and RNS may lead to drastic changes in the excitability of these tissues, such as those occurring during ischemia-reperfusion events (14Martin R.L. Lloyd H.G. Cowan A.I. Trends Neurosci. 1994; 17: 251-257Abstract Full Text PDF PubMed Scopus (320) Google Scholar); furthermore, the heterogeneity of the K+ channel subsets expressed in specific cells has also been suggested to underlie their different response patterns to hypoxic/anoxic episodes (15Kourie J.I. Am. J. Physiol. 1998; 275: C1-C24Crossref PubMed Google Scholar). The K+ channels encoded by the human Ether-a-gogo Related Gene-1 (hERG1) play a crucial role in excitable tissues (16Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar). In fact, in cardiac tissue, hERG1 encodes for a K+ current having the biophysical and pharmacological properties of native cardiacI Kr, one of the action potential repolarizing currents (17Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2152) Google Scholar). Alteration in hERG1 K+ channels function prompted by drugs and/or gene defects are responsible for the cardiac arrhythmias occurring during the Long QT syndrome (18Curran M.E. Splawski I. Timothy K.W. Vincent G.M. Green E.D. Keating M.T. Cell. 1995; 80: 795-803Abstract Full Text PDF PubMed Scopus (1996) Google Scholar). In neuronal cells, hERG1 K+ channels have been implicated in the changes of the resting membrane potential associated with the cell cycle (19Arcangeli A. Bianchi L. Becchetti A. Faravelli L. Coronnello M. Mini E. Olivotto M. Wanke E. J. Physiol. (Lond.). 1995; 489: 455-471Crossref Scopus (217) Google Scholar), in the control of neuritogenesis and differentiation (20Faravelli L. Arcangeli A. Olivotto M. Wanke E. J. Physiol. (Lond.). 1996; 469: 13-23Crossref Scopus (92) Google Scholar), and in spike-frequency adaptation (21Chiesa N. Rosati B. Arcangeli A. Olivotto M. Wanke E. J. Physiol. (Lond.). 1997; 501: 313-318Crossref Scopus (149) Google Scholar). Recent studies from our laboratory suggest that hERG1 K+channels are influenced by ROS and NO⋅ (22Taglialatela M. Castaldo P. Iossa S. Pannaccione A. Fresi A. Ficker E. Annunziato L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11698-11703Crossref PubMed Scopus (77) Google Scholar, 23Taglialatela M. Pannaccione A. Castaldo P. Iossa S. Annunziato L. Mol. Pharmacol. 1999; 56: 1298-1308Crossref PubMed Scopus (38) Google Scholar). In particular, the outward currents carried by hERG1 K+ channels heterologously expressed in Xenopus oocytes were enhanced by perfusion with a solution containing iron sulfate and ascorbic acid (Fe/Asc), a widely used experimental condition to promote oxidative stress (1Yu B.P. Physiol. Rev. 1994; 74: 139-162Crossref PubMed Scopus (2223) Google Scholar), and were suppressed by the ROS-detoxifying enzyme catalase. In addition, both endogenously produced or pharmacologically delivered NO⋅ was able to inhibit resting hERG1 outward currents and prevented their stimulation by Fe/Asc. These effects appeared to be indirect actions of the gaseous mediator on hERG1 currents, attributable to the potent antioxidant properties of NO⋅ (1Yu B.P. Physiol. Rev. 1994; 74: 139-162Crossref PubMed Scopus (2223) Google Scholar,24Kanner J. Harel S. Granit R. Arch. Biochem. Biophys. 1991; 289: 130-136Crossref PubMed Scopus (394) Google Scholar). The biophysical mechanism by which ROS and RNS modulated hERG1 outward currents without affecting the inward current component was a depolarizing and hyperpolarizing shift, respectively, of the voltage dependence of the steady-state inactivation curve (22Taglialatela M. Castaldo P. Iossa S. Pannaccione A. Fresi A. Ficker E. Annunziato L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11698-11703Crossref PubMed Scopus (77) Google Scholar, 23Taglialatela M. Pannaccione A. Castaldo P. Iossa S. Annunziato L. Mol. Pharmacol. 1999; 56: 1298-1308Crossref PubMed Scopus (38) Google Scholar, 25Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (668) Google Scholar). Among the K+ channels investigated, the described modulation by ROS appears to be highly selective for hERG1. In fact, ROS did not affect any channels that were only distantly related to hERG1 (rKv2.1, rKv3.1 and mKIR 2.1) or more closely related to hERG1 (bEAG, rERG2, and rERG3) (16Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar, 26Shi W. Wymore R.S. Wang H.S. Pan Z. Cohen I.S. McKinnon D. Dixon J.E. J. Neurosci. 1997; 17: 9423-9432Crossref PubMed Google Scholar). In the present experiments, to localize the molecular regions involved in ROS-induced modulation of hERG1, we have taken advantage of the similarities between the primary sequence of hERG1 and the ROS-insensitive channel bEAG to generate several chimeras encompassing different regions of the genes encoding for these two K+ channel subunits. The results obtained suggested that a critical region for ROS-induced modulation was localized in a 30-amino acid stretch located within the S5-S6 linker region of hERG1. Within this region, the substitution of each of the two histidines at positions 578 and 587 in hERG1 with the corresponding bEAG amino acid removed channel sensitivity to ROS-induced modulation, thus highlighting their participation in the important regulatory mechanism of hERG1 K+ channels. Xenopus oocytes dissociation, maintenance, and microinjection followed standard procedures (23Taglialatela M. Pannaccione A. Castaldo P. Iossa S. Annunziato L. Mol. Pharmacol. 1999; 56: 1298-1308Crossref PubMed Scopus (38) Google Scholar). Briefly, ovarian lobes were surgically removed from adult female Xenopus laevis frogs (Rettili di Schneider, Varese, Italy) and placed in 100-mm Petri dishes containing a Ca2+-free solution of the following composition (in millimolar): 82.5 NaCl, 2 KCl, 1MgCl2, 5 HEPES, 2.5 pyruvic acid, 100 units/ml penicillin, and 100 μg/ml streptomycin, pH 7.5, with NaOH. After four extensive washes, the oocytes (stages V–VI) were dissociated by collagenase treatment (type IA, 45–80 min at a concentration of 2 mg/ml). Dissociated oocytes were then placed in a Ca2+-containg solution of the following composition (in millimolar): 100 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, 2.5 pyruvic acid, 100 units/ml penicillin, and 100 μg/ml streptomycin, pH 7.5, with NaOH, in a 19 °C incubator and used for the experiments on the following day. Lipid peroxidation in Xenopus oocytes was determined by assaying the intracellular malondialdehyde (MDA) production by means of the 2-thiobarbituric acid test (22Taglialatela M. Castaldo P. Iossa S. Pannaccione A. Fresi A. Ficker E. Annunziato L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11698-11703Crossref PubMed Scopus (77) Google Scholar), using previously described procedures (27Esterbauer H. Cheeseman K.H. Methods Enzymol. 1990; 186: 407-421Crossref PubMed Scopus (2869) Google Scholar). MDA, in the cell homogenate, was measured using a PerkinElmer Life Sciences LS5B spectrophotofluorometer (excitation 495 nm, emission 530 nm). The cloning ofhERG1 from human hippocampus (16Warmke J.W. Ganetzky B. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3438-3442Crossref PubMed Scopus (866) Google Scholar) (GenBankTMaccession number 04270) and of the bovine isoform of EAGfrom brain tissue (28Frings S. Brull N. Dzeja C. Angele A. Hagen V. Kaupp U.B. Baumann A. J. Gen. Physiol. 1998; 111: 583-599Crossref PubMed Scopus (74) Google Scholar) (bEAG, GenBankTMaccession number Y13430) has been already described. BothbEAG and hERG1 cDNAs were subcloned into a modified pSP64 vector. The engineering of some of the constructs used in the present study has been already described (29Ficker E. Jarolimek W. Kiehn J. Baumann A. Brown A.M. Circ. Res. 1998; 82: 386-395Crossref PubMed Scopus (264) Google Scholar). Briefly, for engineering of the chimeric constructs hERG1 (bEAG S 1 /S 6), hERG1 (bEAG S 4 -S 5 linker/S 6), and bEAG (hERG1 S 4 -S 5 linker/S 6), the S1-S6 core regions of hERG1 and bEAG were subcloned into pBluescript as BstEII-XhoI andBstBI-KpnI fragments, respectively. By use of codon redundancy, the following silent restriction sites were introduced into hERG1 BstEII-XhoI:NarI (A to G at hERG1 1359),MluI (G to A at hERG1 1782), and KpnI (C to G at hERG1 2199). In addition, an NarI site was destroyed at hERG1 1329 (C to G). The MluI and KpnI sites in hERG1 were introduced in positions equivalent to the naturally occurring MluI andKpnI restriction sites in bEAG. InbEAG BstBI-KpnI, a silentNarI site (A to G at bEAG 803) was engineered in a position equivalent to the one introduced into hERG1 sequence. For the construction of hERG1 (bEAG S 1 -S 6), theNarI-KpnI fragment was excised from thebEAG BstBI-KpnI construct in pBluescript and swapped with the corresponding hERG1fragment in hERG1-pBluescript. In a final step, theBstEII-XhoI fragment was excised and subcloned into full-length hERG1-pSP64 from which the wild-typeBstEII-XhoI fragment had been removed. For construction of bEAG (hERG1 S 4 -S 5 linker/S 6) andhERG1 (bEAG S 4 -S 5 linker/S 6), MluI-KpnI fragments were excised and subcloned into the opposite pBluescript plasmids. In a second step, BstEII-XhoI and BstBI-KpnI fragments were excised and subcloned into full-length hERG1-pSP64 and bEAG-pSP64, respectively. Chimeric constructs hERG1 (bEAG S 5 -S 6 linker),hERG1 (bEAG 573/602), and point mutationshERG1 H578D, hERG1 H587Y, and hERG1H578D/H587Y were generated by overlap extension polymerase chain reaction using the BstEII-XhoI hERG1cassettes generated in pBluescript in which the MluI andKpnI had been introduced as previously described. For all these constructs, the entire MluI-KpnI cassettes were manually sequenced before to subcloning into full-lengthhERG1-pSP64. cDNAs from all these constructs were linearized with the restriction enzymes EcoRI or EcoRV, and cRNAs were in vitro transcribed from linearized cDNAs by means of commercially available kits (mCAP, Stratagene), using SP6 RNA polymerase. RNAs were stored in a stock solution (250 ng/μl) at −20 °C in 0.1 m KCl. One day after isolation,Xenopus oocytes were microinjected with 50 nl of the respective cRNA stock solution or appropriate dilutions. 2–10 days after the cRNA microinjection, K+ currents expressed were measured by the two microelectrode voltage-clamp technique using a commercially available amplifier (Warner OC-725A, Warner Instrument Corp.). Current and voltage electrodes were filled with 3 m KCl, 10 mm HEPES (pH 7.4; ∼1 MΩ of resistance). The bath solution contained (in millimolar): 88 NaCl, 10 KCl, 2.6 MgCl2, 0.18 CaCl2, 5 HEPES, pH 7.5 (ND88). This solution was perfused in the recording chamber at a rate of about 0.2 ml/min. Data were stored on the hard disc of a 486 IBM compatible computer for off-line analysis. The pCLAMP (version 6.0.2, Axon Instruments, Burlingame, CA) software was used for data acquisition and analysis. Currents were recorded at room temperature. Oocytes that showed signs of membrane deterioration during the experiment (an increase in the holding current at −90 mV of more than 200 nA) were excluded from the electrophysiological analysis. All the materials used were purchased from Sigma Chemical Co. (Milan, Italy); the NO⋅ donor diethylenetetraamine NONOate (NOC) was obtained from Cayman Chemical (Ann Arbor, MI). Iron sulfate and ascorbate stock solutions (10 and 25 mm, respectively) were prepared daily and stored in light-protected tubes to avoid spontaneous oxidation. All solutions were prepared fresh daily, before the execution of the experiments. Statistical significance between the data was obtained by means of the Student t test. When appropriate, data are expressed as the mean ± S.E. In the figures asterisks denote values statistically different from the controls (p < 0.05). hERG1 K+channels expressed in Xenopus oocytes were activated by depolarizing pulses above −60 mV, displayed pronounced inward rectification at positive potentials (>0 mV) due to a fast C-type inactivation process (17Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2152) Google Scholar, 25Smith P.L. Baukrowitz T. Yellen G. Nature. 1996; 379: 833-836Crossref PubMed Scopus (668) Google Scholar), and were specifically modulated by ROS (22Taglialatela M. Castaldo P. Iossa S. Pannaccione A. Fresi A. Ficker E. Annunziato L. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11698-11703Crossref PubMed Scopus (77) Google Scholar). In fact, perfusion of hERG1-expressing oocytes with the ROS-producing Fe/Asc solution (25 and 50 μm, respectively) increased by ∼30% hERG1 outward K+currents evoked by depolarizing steps from −80 mV to +40 mV from a holding voltage of −90 mV (Fig. 1). The increase of hERG1 outward current induced by Fe/Asc was independent on extracellular K+ concentrations ([K+]e), because it was observed with [K+]e ranging from 2 (data not shown) to 42 mm; with 42 mm [K+]e, the outward currents at 0 mV were increased by 26 ± 9% in the presence of Fe/Asc (p > 0.05 versus that observed with 10 mm [K+]e;n = 5). By contrast, the K+ channels encoded by bEAG gave rise to delayed rectifier-like outward currents with activation kinetics strongly dependent on the holding potential and an extremely fast current deactivation at more hyperpolarized membrane potentials. Interestingly, the currents carried by bEAG channels were completely insensitive to Fe/Asc perfusion (Fig. 1), suggesting therefore that bEAG channels are resistant to ROS-induced modulation. To localize the molecular regions involved in ROS-induced modulation of hERG1 K+ channels, we took advantage of the fact that the bEAG K+ channels were insensitive to ROS; thus, segmental exchanges between hERG1 and bEAG K+ channel subunits were performed. Replacement of the “core” region (from the beginning of S1 to the end of S6) of hERG1 with the corresponding region of bEAG (chimeric construct hERG1 (bEAG S1/S6)), led to the expression of K+-selective channels that were insensitive to Fe/Asc perfusion. Similarly, exchange of the region spanning from the beginning of the S4-S5 linker to the end of S6 of hERG1 with the corresponding region of bEAG (hERG1 (bEAG S4-S5 linker/S6)), also led to the disappearance of the channel sensitivity to ROS. Interestingly, the reverse chimeric exchange, namely the replacement of the region between the beginning of the S4-S5 linker region to the end of S6 of bEAG with the corresponding hERG1 sequence (bEAG (hERG1 S4-S5linker/S6)), generated channels having K+currents that were significantly potentiated by Fe/Asc perfusion (Fig.1). These results suggested that the ROS-induced modulation of hERG1 K+ channels required the presence of a specific amino acid sequence in the region located between the S4-S5 linker and the S6transmembrane domain. Within this region, a smaller chimera substituting only the S5-S6 linker of hERG1 with that of bEAG (hERG1 (bEAG S5-S6 linker)) generated K+ channels that were still insensitive to Fe/Asc-induced modulation, suggesting that the molecular determinants for ROS sensitivity of hERG1 are located within the S5-S6 linker. To more specifically localize the amino acids involved in ROS sensitivity within the S5-S6 linker region of hERG1, a smaller chimera (hERG1 (bEAG 573/602)) was generated. In this construct, a 30-amino acid sequence of hERG1 (from the end of the putative S5transmembrane segment to the GGPS amino acid sequence present in both hERG1 and bEAG) was substituted with the corresponding 40-amino acid stretch encoded by bEAG (Fig. 2). The K+ channels encoded by this chimeric construct were completely insensitive to the effects of Fe/Asc (25/50 μm) perfusion (Fig. 1), supporting the idea that residues located within this 30-amino acid sequence of hERG1 are crucially involved in determining the channel sensitivity to ROS. The currents carried by the hERG1 (bEAG 573/602) chimera displayed a selectivity for K+ ions identical to that of wild-type hERG1 channels. With 10 mm K+ ions in the extracellular solution, the reversal potential for the currents carried by wild-type hERG1 channels was −60 ± 0.6 mV (n = 6), whereas that for hERG1 (bEAG 573/602) channels was −57.6 ± 1.6 mV (n = 5) (p > 0.05). Furthermore, the midpoint voltage of channel activation and the slope of the activation curves, calculated as described in the legend for Fig. 3, were, respectively: −32.4 ± 1.07 mV and 8.77 ± 0.8 (n = 4) for wild-type hERG1 and −31.75 ± 1.5 mV and 8.1 ± 0.43 (n = 4) for hERG1 (bEAG 573/602) (p > 0.05). Interestingly, the midpoint voltage of channel inactivation was significantly affected by the mutation, because it was −61.8 ± 1.0 mV (n = 13) for wild-type hERG1 and −68.5 ± 0.6 mV (n = 4) for hERG1 (bEAG 573/602) (p < 0.05). The slopes of the inactivation curves were, respectively, 17.3 ± 0.3 mV and 18.0 ± 0.7 (p > 0.05) for the two channels (Fig.3 A). In addition, the currents carried by the hERG1 (bEAG 573/602) chimeric channels were insensitive not only to perfusion with Fe/Asc but also with the ROS-detoxifying enzyme catalase (1000 units/ml) (Fig.3 B); furthermore, the same chimeric substitution also removed the channel sensitivity to the NO⋅-donor NOC (0.3 mm) (Fig. 3 B). Fig. 3 (C andD) shows the effects of a 5-min perfusion with catalase and NOC, respectively, on the outward K+ currents carried by the channels encoded by wild-type hERG1 and hERG1 (bEAG 573/602). Given the results obtained with the hERG1 (bEAG 573/602) chimera, we engineered a reverse chimeric exchange by transplanting the hERG1 573–602 region into bEAG, to investigate whether this chimeric replacement was sufficient to introduce ROS modulation into ROS-insensitive bEAG channels. Unfortunately, injection ofXenopus oocytes with the cRNA encoded by this chimeric cDNA construct did not lead to the expression of functional channels (data not shown). The results presented showed that the 30-amino acid region in the S5-S6 linker of hERG1 channels substituted in the hERG1 (bEAG 573/602) chimera contains the molecular determinants responsible for the channel sensitivity to ROS-induced modulation. As shown in the alignment of Fig. 2, within this 30-amino acid region, the hERG1 sequence contains two histidines at position 578 and 587, which are not present in the ROS-insensitive bEAG, rERG2, and rERG3 K+ channels. Therefore, the possible involvement of these two histidine residues in the modulation of hERG1 channels by ROS was investigated. As shown in Fig. 4, single point mutations at positions 578 or 587 introducing in hERG1 the corresponding bEAG residues (hERG1 H578D and hERG1 H587Y), as well as the double-substitution hERG1 H578D/H587Y, completely removed the channel sensitivity to the stimulatory effect exerted by Fe/Asc (25/50 μm, respectively). In addition, the inhibition of the outward hERG1 K+ currents caused by catalase (1000 units/ml) and by the NO⋅ donor NOC (0.3 mm) was completely abolished in these histidine-lacking mutant channels. The removal of each of the two histidines caused a leftward shift in the channel voltage dependence of inactivation, without affecting the activation process. In fact, the midpoint voltage of channel activation and the slope of the activation curves were, respectively, −35 ± 0.3 mV and 9.18 ± 0.2 (n = 8) for wild-type hERG1, −37 ± 0.19 mV and 9.1 ± 0.04 (n = 6) for hERG1 H578D, −35.2 ± 0.77 mV and 9.13 ± 0.09 (n = 6) for hERG1 H587Y, and −37 ± 0.6 mV and 8.6 ± 0.08 (n= 7) for hERG1 H578D/H587Y (p > 0.05). On the other hand, the midpoint voltage of channel inactivation and the slope of the inactivation curves were, respectively, −61.8 ± 1.0 mV and 17.3 ± 0.3 (n = 13) for wild-type hERG1, −87.1 ± 1.8 mV (p < 0.05 versushERG1) and 20 ± 0.8 (n = 7) for hERG1 H578D, −69.0 ± 1.2 mV (p < 0.05 versushERG1) and 17.4 ± 0.5 (n = 7) for hERG1 H587Y, and −72.2 ± 0.7 mV (p < 0.05 versushERG1) and 15.3 ± 0.2 (n = 6) for hERG1 H578D/H587Y. To gain more insight into the molecular mechanism by which histidines at position 578 and 587 participate in hERG1 channel modulation by ROS, the possible involvement of iron ions has also been investigated. To this aim, the effect exerted on hERG1 channels by the two iron chelators desferrioxamine (DFX) (6Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine.2nd Ed. Clarendon Press, Oxford1989: 1-81Google Scholar, 30Lloyd J.B. Cable H. Rice-Evans C. Biochem. Pharmacol. 1991; 41: 1361-1363Crossref PubMed Scopus (148) Google Scholar) ando-phenanthroline (PHE) (31Boumans H. van Gaalen M.C. Grivell L.A. Berden J.A. J. Biol. Chem. 1997; 272: 16753-16760Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), were studied. Perfusion of hERG1-expressing oocytes for 5 min with DFX (1 mm) or PHE (0.2 mm) significantly inhibited the outward K+currents at all the potentials tested between −40 and +40 mV (Fig.5 A), without affecting either the amplitude or the kinetics of the inward currents (data not shown). Interestingly, the inhibitory effect of DFX on hERG1 currents was not reversible upon washout of the iron chelator from the perfusion medium for up to 20 min; however, the presence of 25 μmFeSO4 (with or without 50 μm ascorbic acid) readily increased hERG1 outward currents back to their resting value (Fig. 5 B). These results suggest that the inhibition of hERG1 outward K+ currents by DFX was due to the drug ability to specifically chelate iron ions, rather than being the consequence of an unspecific effect of the molecule or its ability to chelate other metal ions, which are known to influence hERG1 channel function (32Ho W.K. Kim I. Lee C.O. Youm J.B. Lee S.H. Earm Y.E. Biophys. J. 1999; 76: 1959-1971Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Furthermore, DFX (1 mm) completely counteracted the stimulatory effect of Fe/Asc (25/50 μm) on the outward K+ currents carried by hERG1 (Fig. 5 C). Interestingly, DFX (1 mm) was also able to prevent the inhibitory action of the ROS-detoxifying enzyme catalase (1000 units/ml) on hERG1 outward K+currents under resting conditions (Fig. 5 D). To test more directly the hypothesis that the iron chelators DFX and PHE might interfere with the oxidating process promoted by iron ions, the effects of DFX and PHE on resting and Fe/Asc-enhanced intracellular malondialdehyde (MDA) production, a direct index of lipid peroxidation, were measured. Both DFX (0.1–1 mm) and PHE (0.2 mm) effectively decreased the basal concentration of MDA (Fig. 6). Furthermore, DFX (1 mm) was able to prevent the Fe/Asc-induced increase in MDA production, confirming that, in the presence of the iron chelator, iron ions are unable to participate in the Fenton reaction and to trigger oxidative stress. To investigate the possible participation of hERG1 histidines at position 578 and 587 in iron-dependent channel modulation by ROS, the effects of the iron chelators DFX and PHE on the histidine-lacking channels hERG1 H578D/H587Y and bEAG were compared with those occurring in wild-type hERG1 channels. Both DFX (1 mm) and PHE (0.2 mm) were without any effect i" @default.
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• W2065600512 title "Histidines 578 and 587 in the S5-S6Linker of the Human Ether-a-gogo Related Gene-1K+ Channels Confer Sensitivity to Reactive Oxygen Species" @default.
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