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• W2078646599 abstract "Mutations in the humanether-a-gogo-related gene (HERG) K+ channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduceI Kr currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K+ channels without block. Mutations in the humanether-a-gogo-related gene (HERG) K+ channel gene cause chromosome 7-linked long QT syndrome type 2 (LQT2), which is characterized by a prolonged QT interval in the electrocardiogram and an increased susceptibility to life-threatening cardiac arrhythmias. LQT2 mutations produce loss-of-function phenotypes and reduceI Kr currents either by the heteromeric assembly of non- or malfunctioning channel subunits with wild type subunits at the cell surface or by retention of misprocessed mutant HERG channels in the endoplasmic reticulum. Misprocessed mutations often encode for channel proteins that are functional upon incorporation into the plasma membrane. As a result the pharmacological correction of folding defects and restoration of protein function are of considerable interest. Here we report that the trafficking-deficient pore mutation HERG G601S was rescued by a series of HERG channel blockers that increased cell surface expression. Rescue by these pharmacological chaperones varied directly with their blocking potency. We used structure-activity relationships and site-directed mutagenesis to define the binding site of the pharmacological chaperones. We found that binding occurred in the inner cavity and correlated with hydrophobicity and cationic charge. Rescue was domain-restricted because the trafficking of two misprocessed mutations in the C terminus, HERG F805C and HERG R823W, was not restored by channel blockers. Our findings represent a first step toward the design of pharmacological chaperones that will rescue HERG K+ channels without block. The cardiac potassium channel gene HERG 1The abbreviations used are:HERGhumanether-a-gogo-related geneLQT2human long QT syndrome type 2I Krrapidly activating delayed rectifier potassium currentERendoplasmic reticulumTEAtetraethylammoniumalkyl-TEAalkyltriethylammoniumHEKhuman embryonic kidneyWTwild typeTMAtrimethylammoniumalkyl-TMAalkyltrimethylammoniumC6-C8-, C10-, N-hexyl, N-octyl, N-decyl, respectivelyRC50half-maximal rescue concentrationTBAtetrabutylammoniumpFpico Farad 1The abbreviations used are:HERGhumanether-a-gogo-related geneLQT2human long QT syndrome type 2I Krrapidly activating delayed rectifier potassium currentERendoplasmic reticulumTEAtetraethylammoniumalkyl-TEAalkyltriethylammoniumHEKhuman embryonic kidneyWTwild typeTMAtrimethylammoniumalkyl-TMAalkyltrimethylammoniumC6-C8-, C10-, N-hexyl, N-octyl, N-decyl, respectivelyRC50half-maximal rescue concentrationTBAtetrabutylammoniumpFpico Farad (KCNH2) is mutated in the long QT syndrome type 2 (LQT2), a familial, autosomal dominant cardiac disease associated with prolongation of the QT interval and torsade de pointes (1Curran 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 (1978) Google Scholar, 2Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2135) Google Scholar). LQT2 mutations are, with only one exception, loss-of-function mutations that reduce the repolarizing cardiac potassium current I Kr thereby prolonging the cardiac action potential (3Sanguinetti M.C. Curran M.E. Spector P.S. Keating M.T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2208-2212Crossref PubMed Scopus (390) Google Scholar, 4Lees-Miller J.P. Duan Y. Teng G.Q. Thorstad K. Duff H.J. Circ. Res. 2000; 86: 507-513Crossref PubMed Scopus (72) Google Scholar). When expressed in heterologous cells loss of function is caused either by mal- or nonfunctioning tetrameric channels inserted into the plasma membrane or by trafficking-deficient mutant channels retained in the endoplasmic reticulum (ER) (5Zhou Z. Gong Q. Epstein M.L. January C.T. J. Biol. Chem. 1998; 273: 21061-21066Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 6Ficker E. Thomas D. Viswanathan P.C. Dennis A.T. Priori S.G. Napolitano C. Memmi M. Wible B.A. Kaufman E.S. Iyengar S. Schwartz P.J. Rudy Y. Brown A.M. Am. J. Physiol. 2000; 279: H1748-H1756Crossref PubMed Google Scholar, 7Ficker E. Dennis A.T. Obejero-Paz C.A. Castaldo P. Taglialatela M. Brown A.M. J. Mol. Cardiol. 2000; 32: 2327-2337Abstract Full Text PDF PubMed Scopus (92) Google Scholar, 8Kagan A., Yu, Z. Fishman G.I. McDonald T.V. J. Biol. Chem. 2000; 275: 11241-11248Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). humanether-a-gogo-related gene human long QT syndrome type 2 rapidly activating delayed rectifier potassium current endoplasmic reticulum tetraethylammonium alkyltriethylammonium human embryonic kidney wild type trimethylammonium alkyltrimethylammonium C8-, C10-, N-hexyl, N-octyl, N-decyl, respectively half-maximal rescue concentration tetrabutylammonium pico Farad humanether-a-gogo-related gene human long QT syndrome type 2 rapidly activating delayed rectifier potassium current endoplasmic reticulum tetraethylammonium alkyltriethylammonium human embryonic kidney wild type trimethylammonium alkyltrimethylammonium C8-, C10-, N-hexyl, N-octyl, N-decyl, respectively half-maximal rescue concentration tetrabutylammonium pico Farad Mutations in hereditary channelopathies including cystic fibrosis, nephrogenic diabetes insipidus, and episodic ataxia (9Kopito R.R. Physiol. Rev. 1999; 79: S167-S173Crossref PubMed Scopus (374) Google Scholar, 10Tamarappoo B.K. Verkman A.S. J. Clin. Invest. 1998; 101: 2257-2267Crossref PubMed Scopus (286) Google Scholar, 11Zerr P. Adelman J.P. Maylie J. J. Neurosci. 1998; 18: 2842-2848Crossref PubMed Google Scholar) may produce misfolded proteins that are recognized and retained by quality control mechanisms in the ER. In cystic fibrosis the most common mutation, ΔF508, is trafficking-deficient and can be rescued by lowering the incubation temperature or by using chemical chaperones such as glycerol, dimethyl sulfoxide or trimethylamine N-oxide (12Denning G.M. Anderson M.P. Amara J.F. Marshall J. Smith A.E. Welsh M.J. Nature. 1992; 258: 761-764Crossref Scopus (1057) Google Scholar, 13Sato S. Ward C.L. Krouse M.E. Wine J.J. Kopito R.R. J. Biol. Chem. 1996; 271: 635-639Abstract Full Text Full Text PDF PubMed Scopus (464) Google Scholar, 14Brown C.R. Hong-Brown Q. Biwersi J. Verkman A.S. Welch W.J. Cell Stress Chaperones. 1996; 1: 117-125Crossref PubMed Scopus (358) Google Scholar). These results have stimulated interest in the development of methods to correct folding defects and restore protein function. However, low incubation temperatures are of practical value only “in vitro,” and chemical chaperones work at high concentrations (10–1000 mm) that may restrict their use in patients. A more promising pharmacological strategy has been introduced for misfolded P-glycoprotein mutants using specific substrates and blockers to rescue the target protein (15Loo T.W. Clarke D.M. J. Biol. Chem. 1997; 272: 709-712Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). More recently, pharmacological strategies have been validated further with the rescue of misfolded vasopressin receptors mutants using small organic V2 receptor antagonists (16Morello J.-P. Salahpour A. Laperriere A. Bernier V. Arthus M.-F. Lonergan M. Petaejae-Repo U. Angers S. Morin D. Bichet D.G. Bouvier M. J. Clin. Invest. 1999; 105: 887-895Crossref Scopus (469) Google Scholar). In LQT2, expression of two trafficking-deficient mutations, HERG N470D and HERG R752W, at low temperature resulted in incorporation of functional channels in the plasma membrane (6Ficker E. Thomas D. Viswanathan P.C. Dennis A.T. Priori S.G. Napolitano C. Memmi M. Wible B.A. Kaufman E.S. Iyengar S. Schwartz P.J. Rudy Y. Brown A.M. Am. J. Physiol. 2000; 279: H1748-H1756Crossref PubMed Google Scholar, 17Zhou Z. Gong Q. January C.T. J. Biol. Chem. 1999; 274: 31123-31126Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Incubation with chemical chaperones restored trafficking and rescued functional channels for HERG N470D, a mutation in the transmembrane domain, but was completely ineffective for HERG R752W, a mutation in the cyclic nucleotide binding domain. Similarly, pharmacological rescue of functional channel protein has been reported for HERG N470D when synthesized in the presence of the methanesulfonanilide E4031, the antihistamine astemizole, or the prokinetic drug cisapride, whereas E4031 was completely ineffective when incubated with HERG R752W (6Ficker E. Thomas D. Viswanathan P.C. Dennis A.T. Priori S.G. Napolitano C. Memmi M. Wible B.A. Kaufman E.S. Iyengar S. Schwartz P.J. Rudy Y. Brown A.M. Am. J. Physiol. 2000; 279: H1748-H1756Crossref PubMed Google Scholar,17Zhou Z. Gong Q. January C.T. J. Biol. Chem. 1999; 274: 31123-31126Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). How blocking molecules interact with HERG channels to stabilize certain mutant conformations for export to the plasma membrane but fail to stabilize others is unknown. Whether block and rescue rely on the same structural determinants of the channel protein and whether pharmacological rescue can be separated from block are also unknown. In the present work we used a series of channel blockers to probe the binding site for drug-induced rescue of protein trafficking in HERG G601S. HERG G601S is a temperature-sensitive missense mutation in the pore region of the channel protein with most channel protein retained in the ER when expressed at physiological temperatures (18Furutani M. Trudeau M.C. Hagiwara N. Seki A. Gong Q. Zhou Z. Imamura S. Nagashima H. Kasanuki H. Takao A. Momma K. January C.T. Robertson G.A. Matsuoka R. Circulation. 1999; 99: 2290-2294Crossref PubMed Scopus (164) Google Scholar). In our experiments we were guided by more recent insights into the structure of the methanesulfonanilide binding site of HERG K+channels (19Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (855) Google Scholar, 20Lees-Miller J.P. Duan Y. Teng G.Q. Duff H.J. Mol. Pharmacol. 2000; 57: 367-374PubMed Google Scholar). We used the known structure-activity relationship of the noncardiac drug astemizole to compare the relative efficacies of channel block and channel rescue. We extended our observations by comparing block produced by alkyl-TEA derivatives with their ability to rescue the mutant channels. We show that the inner vestibule of the HERG K+ channel forms the receptor sites for compounds that produce both rescue and block and demonstrate that both sites share the properties of the internal quaternary ammonium receptor site present in most K+ channels. HERG mutations were generated by overlap extension PCR, verified by sequencing, and subcloned into full-length HERG-pcDNA3 as described previously (6Ficker E. Thomas D. Viswanathan P.C. Dennis A.T. Priori S.G. Napolitano C. Memmi M. Wible B.A. Kaufman E.S. Iyengar S. Schwartz P.J. Rudy Y. Brown A.M. Am. J. Physiol. 2000; 279: H1748-H1756Crossref PubMed Google Scholar). The polyclonal HERG antibody used in Western blots and immunostaining was generated in rabbits against a glutathioneS-transferase fusion protein containing the last 112 amino acids of HERG (residues 1048–1159). HERG antiserum was purified on an affinity column consisting either of a short C-terminal peptide corresponding to HERG residues 1102–1121 (TLTLDSLSQVSQFMACEELP) or the entire fusion protein. HEK293 cells maintained in Dulbecco's modified Eagle's medium, 10% fetal bovine serum plus penicillin/streptomycin at 37 °C, 5% CO2, were transiently transfected with 2 μg of HERG wild type (WT) or HERG mutant cDNA in 60-mm culture dishes using LipofectAMINE/Plus as recommended by manufacturer (Invitrogen). Some experiments were performed using COS-7 cells. After transfection, cells were incubated for 48 h at either 26 or 37 °C. For Western blot experiments, HERG channel blockers were diluted in prewarmed culture medium and added for 12–16 h to cells 36 h post-transfection. Trafficking of HERG WT channels was studied using a stable cell line established in HEK293 cells. Cells were solubilized for 1 h at 4 °C in lysis buffer (50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 1 mm EDTA, 1% Triton X-100), containing protease inhibitor mix (Complete, Roche Biochemicals). Protein concentrations were determined by the BCA method (Pierce). N-Glycosidase F treatments (NEB P0704S) were performed as recommended by the manufacturer. For Western blotting, proteins were separated on 7.5% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. Membranes were blocked overnight with 5% nonfat dry milk in phosphate-buffered saline plus 0.1% Tween and immunoblotted with rabbit polyclonal HERG antibody (1:100 dilution; 1 h at room temperature) followed by horseradish peroxidase-conjugated secondary antibody (1:3,000; 1 h at room temperature; Amersham Biosciences, Inc.). ECL Plus (Amersham Biosciences, Inc.) was used for blot development. Immunoblot images were captured directly on a Storm PhosphorImager to quantify image densities of HERG protein bands. For densitometric analysis pixel densities of fully glycosylated HERG protein bands were integrated from equal areas after background subtraction using ImageQuant (Molecular Dynamics). Image densities were normalized for maximal densities measured during treatment with increasing concentrations of astemizole, cisapride, E4031, and quinidine. Image densities measured in vehicle-treated (ethanol or dimethyl sulfoxide) HERG G601S controls were defined as zero protein expression levels. Densitometric analysis of experiments done with quaternary ammonium ions were normalized to fully glycosylated protein bands detected after incubation with maximal rescue concentrations (5 μm) of either astemizole or E4031. In these experiments, protein isolated in parallel from either astemizole- or E4031-treated cells was run on the same gel and immunoblotted together with experimental samples treated with quaternary ammonium ions to get independent measures of differences in rescue efficacy. Rescue concentration response curves were constructed from three or four independent experiments covering the entire concentration range by fitting Hill equations (see below) to mean values ± S.E. For electrophysiological experiments HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and penicillin/streptomycin at 37 °C, 5% CO2. Transient transfections were carried out overnight in 35-mm culture dishes using a modified Ca2+phosphate method. We transfected 2 μg of channel cDNA together with 0.5 μg of enhanced green fluorescent protein cDNA to allow for identification of successfully transfected cells. One day after transfection, HEK cells were replated onto coverslips and incubated with drug-containing culture medium. On the day of the experiment, cells were transferred to control medium 2 h before current recordings were started. Dose-response relationships for C6-TEA, C8-TEA, C10-TEA and C8-TMA (quaternary ammonium) were measured using HEK cells stably transfected with HERG WT. In these experiments IC50 values were determined in cells held at 0 mV to simulate the ER membrane potential. Channel blockade was measured by analyzing reinactivating HERG current amplitudes on return to 0 mV after a 10-ms voltage clamp pulse to −140 mV. Concentration-response relationships for quaternary ammonium block were fit to Hill equations of the form shown in Equation 1, Idrug/Icontrol=1/(1+(D/IC50)m)Equation 1 where I indicates current, D is drug concentrations, n is the Hill coefficient, and IC50 is the concentration necessary for half-maximal block. Patch pipettes were filled with (in mm): 100 potassium aspartate, 20 KCl, 2 MgCl2, 1 CaCl2, 10 EGTA, 10 HEPES (pH 7.2). The extracellular solution had the following composition (in mm): 140 NaCl, 5 KCl, 1 MgCl2, 1.8 CaCl2, 10 HEPES, 10 glucose (pH 7.4). No leak subtraction was applied. All current recordings were performed at room temperature (20–22 °C). To analyze changes in current densities membrane capacitances were measured using the analog compensation circuit of an Axon 200B patch clamp amplifier. Pclamp software (Axon Instruments) was used for generation of voltage clamp protocols and data acquisition. Whenever possible data are presented as mean ± S.E. of n experiments. Astemizole and norastemizole were kindly provided by Janssen (Beerse, Belgium). E4031 was a gift from Eisai (Tokyo, Japan). Cisapride was obtained from Research Diagnostics (Flanders, NJ); quinidine was purchased from Sigma Chemical. C8-TMA was bought as bromide salt from Fluka and TCI America. Asymmetrical tetraethyl ammonium compounds (C-TEA) were synthesized as described previously (21Jarolimek W. Soman K.V. Alam M. Brown A.M. Pflügers Arch. 1995; 430: 672-681Crossref PubMed Scopus (16) Google Scholar). Briefly, one part of triethylamine was added to one part alkylbromide in the presence of absolute ethanol and heated under gentle refluxing for 8–10 h. Recrystallized alkyl-TEA compounds were tested for identity by NMR and elementary analysis. HERG G601S is located in the S5-pore helix linker and was described as a trafficking-deficient, hypomorphic LQT2 mutant expressing greatly reduced, kinetically unaltered currents when expressed in mammalian cells at physiological temperature (18Furutani M. Trudeau M.C. Hagiwara N. Seki A. Gong Q. Zhou Z. Imamura S. Nagashima H. Kasanuki H. Takao A. Momma K. January C.T. Robertson G.A. Matsuoka R. Circulation. 1999; 99: 2290-2294Crossref PubMed Scopus (164) Google Scholar). Much larger currents were recorded on expression in Xenopusoocytes, indicating restoration of protein trafficking at lower incubation temperatures. The trafficking defect was reflected in Western blots in which the channel protein was detected only as a core-glycosylated immature protein of 135 kDa when isolated from transiently transfected cells incubated at 37 °C (Fig.1 B1). On incubation at 26 °C an additional band at 155 kDa appeared representing the fully mature channel protein (see Fig. 5 B). These results showed that HERG G601S is a temperature-sensitive trafficking mutant with few channels evading quality control at 37 °C.Figure 5Structure-function studies of pharmacological rescue using astemizole and norastemizole. Panel A, Western blot analysis of equal amounts (20 μg) of total protein from cells expressing HERG G601S. Protein processing is analyzed in the presence of increasing amounts of astemizole (A) and norastemizole (NA). Panel B, HERG G601S trafficking is restored by incubation at lower temperature (26 °C);A5.0 indicates rescue by 5 μm astemizole. The double mutated channel HERG G601S/F656C is rescued at a lower incubation temperature (26 °C) but not by incubation with 5 μm astemizole. Panel C, HERG 601S and HERG G601S/F656C are shown in the presence of increasing amounts of astemizole (A). Markers indicate core-glycosylated protein at 135 kDa and mature, fully glycosylated protein at about 155 kDa. All drugs were applied in the micromolar concentration range with individual concentrations indicated above the immunoblots. Panel D, rescue-response curves for HERG G601S incubated with increasing concentrations of astemizole and norastemizole and for HERG G601S/F656C with astemizole. Half-maximal rescue concentrations RC50 were 0.06 ± 0.03 μm for HERG G601S and astemizole, 1.4 ± 0.5 μm for HERG G601S and norastemizole. The efficacy of rescue is reduced dramatically for HERG G601S treated with norastemizole and for HERG G601S/F656C treated with astemizole (n = 3 or 4).View Large Image Figure ViewerDownload (PPT) We tested three HERG channel blockers for restoration of G601S trafficking at 37 °C. These blockers appear to interact with different portions of the extended binding site for methanesulfonanilides in the inner vestibule of HERG (19Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (855) Google Scholar). We used E4031, a methanesulfonanilide drug in which the methanesulfonyl group is thought to bind to a pocket formed between the pore helix and the S6 helix and its aromatic piperidine ring to aromatic amino acid residues at Tyr-652 and Phe-656. We also tested the prokinetic drug cisapride, which is thought to interact with Tyr-652 and Phe-656 but not with the S6/pore helix pocket. Finally we tested the antiarrhythmic drug quinidine, which is thought to interact mainly with Phe-656 but not with the inactivated state of the HERG channel (20Lees-Miller J.P. Duan Y. Teng G.Q. Duff H.J. Mol. Pharmacol. 2000; 57: 367-374PubMed Google Scholar). In electrophysiological experiments HERG currents were blocked in mammalian cells by E4031 with an IC50 of 7–10 nm (22Abbott G.W. Sesti F. Splawski I. Buck M.E. Lehmann M.H. Timothy K.W. Keating M.T. Goldstein S.A. Cell. 1999; 97: 175-187Abstract Full Text Full Text PDF PubMed Scopus (1170) Google Scholar, 23Zhou Z. Gong Q., Ye, B. Fan Z. Makielski J.C. Robertson G.A. January C.T. Biophys. J. 1998; 74: 230-241Abstract Full Text Full Text PDF PubMed Scopus (623) Google Scholar). Cisapride blocked HERG currents with an IC50 of 6–7 nm (24Rampe D. Roy M. Dennis A. Brown A.M. FEBS Lett. 1997; 417: 28-32Crossref PubMed Scopus (278) Google Scholar, 25Mohammad S. Zhou Z. Gong Q. January C.T Am. J. Physiol. 1997; 273: H2534-H2538PubMed Google Scholar), and quinidine blocked with an IC50 of about 1 μm (26Poo S.S. Wang D.W. Yang I.C. Johnson J.P. Nie L. Bennett P.B. J. Cardiovasc. Pharmacol. 1999; 33: 181-185Crossref PubMed Scopus (74) Google Scholar, 27Yang T. Roden D.M. Circulation. 1996; 93: 407-411Crossref PubMed Scopus (379) Google Scholar). We tested whether all of these drugs restored HERG G601S trafficking in a concentration-dependent manner and whether the difference in interaction with the methanesulfonanilide binding site was reflected in their ability to rescue HERG G601S. HEK293 cells were transiently transfected with HERG G601S cDNA, incubated for 36 h at 37 °C, and then exposed overnight to different concentrations of cisapride (Fig. 1 B1), quinidine (Fig. 1 C), and E4031 (Fig. 1 D) before immunoblotting. In vehicle-treated control cells only a core-glycosylated protein band of 135 kDa could be detected (Fig. 1,B–D). With increasing drug concentrations an additional 155 kDa band appeared which could be reduced to a smaller form of about 130 kDa by treatment with N-glycosidase F, an enzyme used to remove all carbohydrate residues from glycoproteins (Fig.1 B2). The fully glycosylated 155-kDa form of the channel protein which could be detected after exposure of G601S cells to the different channel blockers was quantified using a PhosphorImager, normalized, and plotted as a function of drug concentration (Fig.1 E). All three drugs restored trafficking of HERG G601S, with half-maximal rescue concentrations (RC50) of 0.6 ± 0.1 μm for cisapride, 1.1 ± 0.2 μmfor E4031, and 1.7 ± 0.4 μm for quinidine (n = 3 or 4). For WT protein both the 135- and the 155-kDa forms are synthesized at 37 °C (Fig.2 A, con), and expression of the mature fully glycosylated channel protein at 155 kDa is not modified significantly when cells expressing HERG WT are exposed to channel blockers at concentrations that maximally restore trafficking in HERG G601S (Fig. 2 B). To test whether the increased production of fully glycosylated mature G601S protein by HERG blockers was accompanied by increased expression of functional channels we measured G601S currents in vehicle-treated control cells and in cells exposed 48 h to saturating concentrations of cisapride (5 μm), E4031 (5 μm) and quinidine (100 μm). In these experiments the different drugs were washed out 2 h before the start of patch clamp recordings. Fig.3 A illustrates current recordings from a control cell expressing G601S at 37 °C and from a cell treated for 12 h with 5 μm cisapride. Rescued G601S channels showed no alteration in kinetic properties compared with untreated mutant channels (data not shown). However, current densities increased in the presence of saturating concentrations of cisapride, E4031, and quinidine from 54 ± 7 pA/pF (control) to 116 ± 17, 147 ± 39 and 96 ± 8 pA/pF, respectively (Fig. 3 B). Despite a significant increase, current densities remained smaller in G601S than in cells transiently transfected with equal amounts of WT cDNA (382 ± 50 pA/pF). This difference was not the result of incomplete washout of HERG blockers because in control experiments about 80% of WT current density was recovered after treatment with 5 μm astemizole (309 ± 32 pA/pF, Fig. 3 B). Astemizole is a second generation antihistamine that blocked HERG with an IC50 of 0.9 nm (28Zhou Z. Vorperian V.R. Gong Q. Zhang S. January C.T. J. Cardiovasc. Electrophysiol. 1999; 10: 836-843Crossref PubMed Scopus (196) Google Scholar). In humans it undergoes oxidative N-alkylation generating norastemizole (Fig.4) reported to have a reduced IC50 of 27.7 nm with about 50% of HERG current resistant to block by norastemizole (28Zhou Z. Vorperian V.R. Gong Q. Zhang S. January C.T. J. Cardiovasc. Electrophysiol. 1999; 10: 836-843Crossref PubMed Scopus (196) Google Scholar). This observation suggests that hydrophobic side chains attached to tertiary amines in noncardiac HERG-blocking antihistamines are important for block (29Zhang M.Q. Curr. Med. Chem. 1997; 4: 171-184Google Scholar). We asked whether these side chains were also important for rescue of HERG G601S. In immunoblotting experiments the RC50 for G601S with the parent drug astemizole was determined to be 0.06 ± 0.03 μm (Fig. 5 D). For norastemizole we measured a RC50 of 1.4 ± 0.5 μm with maximally 30% of the fully glycosylated protein recovered compared with rescue experiments using astemizole (Fig. 5,A and D). This result provided evidence that efficacies for both block and rescue correlate with each other. Given this assumption we hypothesized that mutations in the drug binding site of HERG should similarly abolish restoration of trafficking. Recently it has been reported that high affinity drug binding in HERG is dominated by a phenylalanine in position 656 (19Mitcheson J.S. Chen J. Lin M. Culberson C. Sanguinetti M.C. Proc. Natl. Acad. U. S. A. 2000; 97: 12329-12333Crossref PubMed Scopus (855) Google Scholar, 20Lees-Miller J.P. Duan Y. Teng G.Q. Duff H.J. Mol. Pharmacol. 2000; 57: 367-374PubMed Google Scholar). We mutated the Phe-656 to Cys-656. This mutation did not modify HERG currents (data not shown) but dramatically reduced the affinity for astemizole (Fig.3 C). HERG F656C was not trafficking-deficient but was retained in the ER when combined with the trafficking mutation 601S (HERG G601S/F656C) with trafficking being restored by incubation at 26 °C (Fig. 5 B). In contrast to G601S, however, the affinity of the double mutant for astemizole block was dramatically reduced (Figs. 3 C and 5 B). In line with our hypothesis we found that the double mutated channel was not rescued by astemizole (Fig. 5, C and D). Even at high concentrations only about 15% of fully glycosylated protein was recovered compared with HERG G601S. The suppression of pharmacological rescue by the F656C mutation provides further evidence that just as for block, rescue involves interactions with the inner vestibule of the HERG channel. For antihistamines and class III antiarrhythmic blockers of HERG a general structure has been proposed consisting of aromatic benzene rings, which are connected via a short chain to a basic nitrogen atom with the basic nitrogen carrying additional hydrophobic substituents (29Zhang M.Q. Curr. Med. Chem. 1997; 4: 171-184Google Scholar). In this model the tertiary amine at physiological pH is mainly in the protonated quaternary form and resembles quaternary ammonium ions such as TEA, which are known to be potent potassium channel blockers. We used alkyl-TEA derivatives to probe the molecular characteristics of the rescue site in HERG G601S. In squid axon it was shown that block by TEA compounds increased with increasing alkyl chain length and that at a given chain length block decreased with changes in the size of the remaining N-substituents (30Armstrong C.M. J. Gen. Physiol. 1971; 58: 413-437Crossref PubMed Scopus (693) Google Schol" @default.
• W2078646599 created "2016-06-24" @default.
• W2078646599 creator A5030990489 @default.
• W2078646599 creator A5031529358 @default.
• W2078646599 creator A5066088212 @default.
• W2078646599 creator A5086423794 @default.
• W2078646599 date "2002-02-01" @default.
• W2078646599 modified "2023-10-16" @default.
• W2078646599 title "The Binding Site for Channel Blockers That Rescue Misprocessed Human Long QT Syndrome Type 2 ether-a-gogo-related Gene (HERG) Mutations" @default.
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