Native ventricular IKr channels are heteromers containing both ERG1a and ERG1b alpha subunits, which associate in vivo and localize to the T tubules of ventricular myocytes.
Native ventricular IKr channels are heteromers containing ERG1a and ERG1b subunits, suggesting the hERG1b-specific exon is a novel target for screening mutations causing type 2 long QT syndrome.
Previous studies suggest native cardiac IKr channels are composed of alpha subunits encoded solely by the 1a transcript of the ERG1 gene. Using isoform-specific ERG1 antibodies, we have new evidence that subunits encoded by an alternate transcript, ERG1b, are also expressed in rat, canine, and human heart. The ERG1a and -1b subunits associate in vivo where they localize to the T tubules of ventricular myocytes. These data indicate native ventricular IKr channels are heteromers containing two α subunit types, ERG1a and -1b. The hERG1b-specific exon thus represents a novel target to screen for mutations causing type 2 long QT syndrome. These findings also suggest phenotypic analyses of existing type 2 long QT syndrome mutations, especially those exclusive to the hERG1a amino terminus, should be carried out in systems expressing both subunits. Previous studies suggest native cardiac IKr channels are composed of alpha subunits encoded solely by the 1a transcript of the ERG1 gene. Using isoform-specific ERG1 antibodies, we have new evidence that subunits encoded by an alternate transcript, ERG1b, are also expressed in rat, canine, and human heart. The ERG1a and -1b subunits associate in vivo where they localize to the T tubules of ventricular myocytes. These data indicate native ventricular IKr channels are heteromers containing two α subunit types, ERG1a and -1b. The hERG1b-specific exon thus represents a novel target to screen for mutations causing type 2 long QT syndrome. These findings also suggest phenotypic analyses of existing type 2 long QT syndrome mutations, especially those exclusive to the hERG1a amino terminus, should be carried out in systems expressing both subunits. Long QT syndrome (LQTS) 1The abbreviations used are: LQTS, long QT syndrome; HEK, human embryonic kidney; PBS, phosphate-buffered saline.1The abbreviations used are: LQTS, long QT syndrome; HEK, human embryonic kidney; PBS, phosphate-buffered saline. is an inherited or acquired disease associated with episodic ventricular arrhythmias and sudden death. One form of inherited LQTS (LQTS-2) results from mutations in the human Ether-a-go-go-Related Gene 1 (hERG1or KCNH2) (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). hERG1 encodes a potassium channel with biophysical and pharmacological properties similar to those of cardiac IKr, thus explaining the underlying cause of LQTS-2 as a defect in this repolarizing current (2Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2136) Google Scholar, 3Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1088) Google Scholar). In mammalian heart, two ERG 2The human isoform is termed hERG; ERG is the more general term applied to lower mammals.2The human isoform is termed hERG; ERG is the more general term applied to lower mammals. transcripts, 1a and 1b, encode proteins differing in their amino-terminal sequence (see Fig. 1A) and gating properties (4London B. Trudeau M.C. Newton K.P. Beyer A.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Satler C.A. Robertson G.A. Circ. Res. 1997; 81: 870-878Crossref PubMed Scopus (246) Google Scholar, 5Lees-Miller J.P. Kondo C. Wang L. Duff H.J. Circ. Res. 1997; 81: 719-726Crossref PubMed Scopus (174) Google Scholar). Expressed in Xenopus oocytes, these subunits preferentially form heteromultimers (4London B. Trudeau M.C. Newton K.P. Beyer A.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Satler C.A. Robertson G.A. Circ. Res. 1997; 81: 870-878Crossref PubMed Scopus (246) Google Scholar). However, despite high levels of ERG1b transcript (4London B. Trudeau M.C. Newton K.P. Beyer A.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Satler C.A. Robertson G.A. Circ. Res. 1997; 81: 870-878Crossref PubMed Scopus (246) Google Scholar), first generation ERG1 antibodies against a common epitope identified only ERG1a protein in native tissue (6Pond A.L. Scheve B.K. Benedict A.T. Petrecca K. Van Wagoner D.R. Shrier A. Nerbonne J.M. J. Biol. Chem. 2000; 275: 5997-6006Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 7Finley M.R. Li Y. Hua F. Lillich J. Mitchell K.E. Ganta S. Gilmour R.F. Freeman L.C. Am. J. Physiol. 2002; 283: H126-H138Crossref PubMed Scopus (76) Google Scholar), suggesting that ERG1b subunits do not contribute to cardiac IKr channels. Here we provide the first direct evidence for ERG1b protein expression, localization, and co-assembly with ERG1a in cardiac ventricular myocytes. These findings indicate cardiac IKr channels are minimally composed of ERG1a and -1b α subunits. Cell Lines and Antibodies—Human embryonic kidney 293 (HEK-293) cell lines stably expressing wild-type hERG1a have been described previously (8Zhou 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, 9Furutani M. Trudeau M.C. Hagiwara N. Seki A. Gong Q.M. Zhou A.F. 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). Cell lines stably expressing hERG1a and -1b were prepared by transfection of HEK-293/hERG1a stable cells with hERG1b containing a Kozak consensus sequence (4London B. Trudeau M.C. Newton K.P. Beyer A.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Satler C.A. Robertson G.A. Circ. Res. 1997; 81: 870-878Crossref PubMed Scopus (246) Google Scholar) cloned into the BamHI/EcoRI sites of pcDNA3.1z (Invitrogen). Separate cell colonies were selected after plating cells at low density and grown in media containing 100 μg/ml Zeocin, 500 μg/ml neomycin for selection. All HEK-293 cells were cultured in Dulbecco's modified Eagle's medium at 37 °C. The pan-ERG1 antibody, ERG1-KA, has been described previously (10Roti Roti E.C. Myers C.D. Ayers R.A. Boatman D.E. Delfosse S.A. Chan E.K.L. Ackerman M.J. January C.T. Robertson G.A. J. Biol. Chem. 2002; 277: 47779-47785Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). ERG1 isoform-specific antibodies were produced by Bethyl Laboratories (Montgomery, TX) in rabbits. Antisera were affinity-purified using the same peptides employed in immunization. The sequence for the ERG1b peptide is amino acids 12–25 (GALRPRAQKGRVRR), and the sequence for ERG1a is amino acids 140–153 (SPAHDTNHRG-PPTS) (Neoclone, Madison, WI). In addition, a hERG1a-specific antibody raised in goat (HERG N-20) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-coupled secondary antibodies were purchased from Pierce and Santa Cruz Biotechnology. Fluorophore-coupled secondary antibodies were purchased from Molecular Probes (Lake Oswego, OR). Myosin binding protein C (MyBP-C) was a gift from Drs. Richard Moss and Samantha Harris (University of Wisconsin, Madison). Cardiac Tissue Preparation—Human male ventricular lysate was purchased from ProSci, Inc. (Poway, CA). Human male ventricular tissue was a gift from Dr. Timothy Kamp, (Dept. of Medicine, University of Wisconsin, Madison). Canine ventricular myocytes, a gift from the laboratory of Dr. Rob Haworth (Dept. of Surgery, University of Wisconsin, Madison), were isolated from mongrel males and enzymatically treated as previously described (11He J.Q. Conklin M.W. Foell J.D. Wolff M.R. Haworth R.A. Coronado R. Kamp T.J. Cardiovasc. Res. 2001; 49: 298-307Crossref PubMed Scopus (245) Google Scholar). Sprague-Dawley rat ventricles were excised from anesthetized adult males after injection of sodium pentobarbital (100 mg/kg body weight intraperitoneal) as described previously (11He J.Q. Conklin M.W. Foell J.D. Wolff M.R. Haworth R.A. Coronado R. Kamp T.J. Cardiovasc. Res. 2001; 49: 298-307Crossref PubMed Scopus (245) Google Scholar). Rat ventricular myocytes were prepared using the same procedure as described for the canine tissue. All procedures have been approved by the Research Animal Resources Center at University of Wisconsin, Madison. Cell Membrane Protein Preparations—Membranes were prepared from myocytes or ventricular tissue after suspension in homogenization buffer (25 mm Tris-HCl, pH 7.4, 10 mm NaEGTA, 20 mm NaEDTA). All buffers used in this procedure contained the following protease inhibitor mixture: 5 μg/ml aprotinin, 50 μg/ml 1,10-phenanthroline, 0.7 μg/ml pepstatin A, 1.56 μg/ml benzamidine, and 1× Complete minitab (Roche Applied Science). Suspensions were homogenized using a Polytron homogenizer at setting 6 for two bursts of 15 s each followed by sonication on ice twice at an amplitude of 20 for 20 s each. Suspensions were spun at 2,000 × g at 4 °C for 10 min to remove cellular debris. The supernatants were subject to further centrifugation at 40,000 × g for 30 min at 4 °C. The resultant pellet was solubilized on a rotary shaker at 4 °C for 2 h in either Triton buffer (150 mm NaCl, 25 mm Tris-HCl, pH 7.4, 20 mm NaEDTA, 10 mm NaEGTA, 5 mm glucose, and 1% (v/v) Triton X-100), or RIPA buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.4, 1 mm NaEDTA, and 1% (v/v) Triton X-100, 1% (v/v) sodium deoxycholate, 0.1% (v/v) sodium dodecylsulfate). Samples were then spun at 10,000 × g to remove insoluble material. Cell line membrane pellets were prepared by washing plates gently with PBS, aspirating, and adding either Triton buffer or RIPA buffer. Cells were then scraped, collected in a microcentrifuge tube, and sonicated on ice twice at an amplitude of 20 for 20 s each. The suspension was rotated at 4 °C for 2 h and then centrifuged at 10,000 × g for 10 min to remove insoluble material. Protein concentrations of all samples were determined using a modified Bradford assay (DC Protein Assay, Bio-Rad). Biochemical Analysis—Membrane proteins were deglycosylated using PNGase F and endoglycosidase H (Roche Applied Science) as described previously (8Zhou 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, 12Zhou Z. Gong Q. Epstein M.L. January C.T. J. Biol. Chem. 1998; 273: 21061-21066Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Proteins were denatured at 60 °C to avoid thermal aggregation at higher temperatures. To determine which proteins were expressed on the surface membrane, proteins were surface-biotinylated using sulfo-NHS-LC-Biotin reagent as described previously (10Roti Roti E.C. Myers C.D. Ayers R.A. Boatman D.E. Delfosse S.A. Chan E.K.L. Ackerman M.J. January C.T. Robertson G.A. J. Biol. Chem. 2002; 277: 47779-47785Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Briefly, 100 mm tissue culture dishes with growth at 70–80% confluency were rinsed three times with cold PBS and incubated with freshly prepared biotin reagent (5 mg/ml) in PBS for 45 min at 4 °C. Cells were then rinsed once with 25 mm Tris-HCl, pH 7.5, to quench the reaction, followed by three washes with cold PBS. Membrane proteins were prepared as indicated above. Western Blot Analysis—Membrane proteins (cell lines 2–10 μg/lane, heart lysates 30–50 μg/lane) were electrophoresed on 7.5% SDS-polyacrylamide gels along with prestained molecular weight markers (Bio-Rad) and then transferred to Immobilon-P polyvinylidene difluoride membranes (Millipore, Bedford, MA) for 1 h at 100 mV. Western blots were blocked, probed, and analyzed as described earlier (10Roti Roti E.C. Myers C.D. Ayers R.A. Boatman D.E. Delfosse S.A. Chan E.K.L. Ackerman M.J. January C.T. Robertson G.A. J. Biol. Chem. 2002; 277: 47779-47785Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). For peptide block experiments 5 μl of antibody was incubated with 10 μg of peptide in 100 μl of TBS (150 mm NaCl, 25 mm Tris-HCl, pH 7.4) for 6 h at 4 °C and then centrifuged at 10,000 × g for 20 min. The supernatant was carefully removed and used to probe Western blots. Western blot controls include probing blots with secondary antibody alone and peptide block of primary antibody. In the case of heart lysates, a lane containing hERG1a/1b cell membrane preparation was included as a positive control. Co-immunoprecipitation—Membrane lysates (cell lines 100–200 μg/reaction, heart lysates 500–1000 μg/reaction) in 1 ml of TBS were cleared with 50 μl of protein A- or G-Sepharose beads (Amersham Biosciences) depending on the origin of the immunoprecipitating antibody; protein A was used for rabbit and protein G for goat immunoprecipitating antibodies. Cleared lysates were incubated with antibody (ERG1b at 1:100 or N-20 at 1:20) on a rotating platform for 3–16 h at 4 °C. 50 μl of protein A- or G-coupled beads were added and samples were incubated at 4 °C for an additional 1–3 h. Beads were collected by centrifugation at 10,000 × g and washed three times with 150 mm NaCl, 25 mm Tris-HCl, pH 7.4, 5 mm NaEDTA, 1% (v/v) Trition X-100, followed by one wash with 150 mm NaCl, 25 mm Tris-HCl, pH 7.4. Proteins were eluted with 200 ng/ml antibody-specific peptide for 1 h at 4 °C. Samples were centrifuged at 10,000 × g, and the supernatant was collected. 100 μl of LSB (25 mm Tris-HCl, pH 6.8, 2% (v/v) sodium dodecylsulfate, 10% glycerol) was added to the beads to elute any proteins that remained bound. Additional controls included lysates processed without antibody. Eluted proteins were Western blotted as described above. Immunohistochemistry—Isolated canine myocytes were fixed in 2% paraformaldehyde-PBS, pH 7.4, for 10 min at room temperature and washed three times in PBS, pH 7.4. Myocytes were then either stored at 4 °C (for up to 8 weeks) or processed immediately. Myocytes were washed once in PBS, pH 7.4, + 1% Triton X-100 and permeabilized in PBS, pH 7.4, + 0.5% Triton X-100 for 10 min at room temperature followed by incubation in 0.75% glycine-PBS (pH 7.4) for 10 min at room temperature to quench any free aldehydes and incubation in blocking buffer (PBS, pH 7.4, + 0.1% Tween-20 + 10% donkey serum + 2% bovine serum albumin) for 2 h at 4 °C with rotation. Cells were washed three times with PBS, pH 7.4, + 0.1% Tween-20 and divided into 0.5-ml aliquots. Each myocyte aliquot was incubated overnight at 4 °C in diluted primary antibody. ERG1b antibodies were diluted 1:1000, ERG1a antibodies (N-20) were diluted 1:10, and myosin binding protein C antibodies were diluted 1:500. Myocytes were washed three times for 1 h in PBS, pH 7.4, + 0.1% Tween-20. Secondary antibodies were diluted in PBS, pH 7.4, + 0.1% Tween-20, + 5% bovine serum albumin, and spun to remove any aggregates. Myocytes were suspended in 0.5 ml of diluted secondary antibody and incubated in the dark 2 h at room temperature with rotation. Donkey anti-rabbit Alexa 488 and donkey anti-goat Alexa 568 antibodies were diluted 1:1000. Myocytes were washed briefly 3x with PBS, pH 7.4, + 0.1% Tween-20 followed by two 1-h washes with PBS, pH 7.4, and were stored at 4 °C until viewed on a Zeiss Axiovert 200 microscope with a ×63 objective. Optical sectioning was accomplished using the Apotome, and was for Alexa 488 and Alexa 568 do not of secondary antibodies was by cells with one primary with secondary antibody raised against the was each secondary is Secondary alone controls were also used to the was an on a Western blot of rat heart tissue we with a ERG1 antibody termed as a for a (10Roti Roti E.C. Myers C.D. Ayers R.A. Boatman D.E. Delfosse S.A. Chan E.K.L. Ackerman M.J. January C.T. Robertson G.A. J. Biol. Chem. 2002; 277: 47779-47785Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). in Fig. the antibody identified three at and The two higher molecular are in with and rat (6Pond A.L. Scheve B.K. Benedict A.T. Petrecca K. Van Wagoner D.R. Shrier A. Nerbonne J.M. J. Biol. Chem. 2000; 275: 5997-6006Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). The is in with ERG1b protein produced in systems (see not previously in native tissue. The hERG1 transcript that a protein of the same S. D.J. A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) not the sequence against which the antibody was To the that the represents ERG1b, we antibodies to the ERG1a and ERG1b amino Fig. 2 the of the two and the pan-ERG1 antibody on Western blots of membrane proteins prepared from HEK-293 cells stably expressing hERG1a and -1b. The antibody at and with previously results these as and hERG1 (8Zhou 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). In addition, three lower molecular at and The and are with to hERG1b in cell lines L. M. A. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (174) Google Scholar) and expressed in cells (6Pond A.L. Scheve B.K. Benedict A.T. Petrecca K. Van Wagoner D.R. Shrier A. Nerbonne J.M. J. Biol. Chem. 2000; 275: 5997-6006Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar). blots with the antibody the and not the three The and were incubation of the ERG1a with the 1a peptide to probing the blots The antibody the and not the two higher hERG1a These were by of the with the peptide These data that both 1a and and that ERG1a and -1b are for their Membrane proteins from stable HEK-293 hERG1a/1b cell lines were incubated with to determine the hERG1b on Western blots to as previously for the hERG1a and (8Zhou 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). all from the hERG1b proteins by membrane with PNGase F the higher molecular hERG1b to a with endoglycosidase which only that are in the not processed in the the to the the represents the hERG1b the represents the and the represents the To determine the hERG1b is expressed on the cell where contribute to hERG1 surface proteins were to cell proteins were with and with only the hERG1b protein was hERG1b is expressed on the cell surface in HEK-293 we the that the lower molecular in native cardiac tissue to the ERG1b protein using the and In Western blots from two human ventricular membrane the antibody at and The and are with from human tissue (6Pond A.L. Scheve B.K. Benedict A.T. Petrecca K. Van Wagoner D.R. Shrier A. Nerbonne J.M. J. Biol. Chem. 2000; 275: 5997-6006Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar) and the and The antibody the and that ERG1b protein is expressed in human was also in canine ventricular tissue. the antibody proteins at and and at and Fig. that high molecular weight at and were by both and ERG1a antibodies and thus ERG1a 1 and The ERG1b antibody the at and which were also by ERG1-KA, that these ERG1b 1 and These data that both ERG1a and -1b proteins are expressed the a of mammalian To determine ERG1a and -1b are associated in native were carried out in canine cardiac membrane Fig. that ERG1a protein was with the antibody and that ERG1b protein was with the antibody. results were from membrane of human myocytes with antibody not These data ERG1a and -1b proteins associate in mammalian ventricular myocytes in with their ERG1a and ERG1b to the same using the 1a and antibody in permeabilized canine myocytes a of a T and (11He J.Q. Conklin M.W. Foell J.D. Wolff M.R. Haworth R.A. Coronado R. Kamp T.J. Cardiovasc. Res. 2001; 49: 298-307Crossref PubMed Scopus (245) Google Scholar). To ERG1 more we myocytes with ERG1a and myosin binding protein C (MyBP-C) M.L. Moss Circ. Res. 2002; PubMed Scopus Google Scholar). were from a of in as a of by of that the of the cell to the R. R. B. Biol. PubMed Scopus Google the ERG1a in is in to and lines and T tubules are in the A.F. R. PubMed Scopus Google Scholar). The ERG1a in from the cell surface to the as of a T where the These data indicate ERG1 in canine myocytes is with a T In this we have ERG1b protein is produced in mammalian heart. The of ERG1b protein in studies is to a lower of the antibodies employed to the common epitope in ERG1a and -1b. by M.R. Li Y. Hua F. Lillich J. Mitchell K.E. Ganta S. Gilmour R.F. Freeman L.C. Am. J. Physiol. 2002; 283: H126-H138Crossref PubMed Scopus (76) Google Scholar) in and M. Am. J. Physiol. PubMed Scopus Google Scholar) in rat the of an in of cardiac In is to suggest these of the ERG1b also ERG1a and -1b proteins are associated in ventricular myocytes. In ERG1a and -1b with biophysical properties that be by the of two of channels (4London B. Trudeau M.C. Newton K.P. Beyer A.K. Copeland N.G. Gilbert D.J. Jenkins N.A. Satler C.A. Robertson G.A. Circ. Res. 1997; 81: 870-878Crossref PubMed Scopus (246) Google Scholar). The properties of these channels more those of native IKr channels A. D.J. Circ. Res. 870-878Crossref PubMed Scopus Google Scholar). of ERG1a and -1b to T in canine ventricular myocytes is with studies in rat myocytes ERG1 protein to the T tubules where at the of F. C. Circ. Res. PubMed Scopus Google Scholar). These data suggest native IKr which are to T both ERG1a and -1b subunits. cardiac IKr and the disease of and acquired LQTS the native in systems as as minimally of hERG1a and -1b. In addition, findings have for amino-terminal mutations causing of LQTS-2 mutations in the amino of hERG1a where they the gating cause J. A. Splawski I. Keating M.T. M.C. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. L. J. M. Chem. 2001; PubMed Scopus Google Scholar, A. A. D.J. N. J. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, Kamp T.J. January C.T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, S. January C.T. Circ. Res. PubMed Scopus Google Scholar). ERG1a and -1b are alternate produced by the ERG1 mutations in the ERG1a amino are not to the of wild-type ERG1b from this gene. These findings for LQTS for mutations in the hERG1b-specific exon and the disease of all mutations in systems in which hERG1a and -1b are of the Robertson for and Dr. for with antibodies, Dr. Haworth for canine heart Dr. human heart Drs. Moss and Samantha Harris for antibodies, Drs. January and for tissue culture and Drs. and and of their laboratory for
Jones et al. (Wed,) conducted a other in Long QT syndrome (context). Isoform-specific ERG1 antibodies was evaluated on Expression and localization of ERG1a and ERG1b subunits. Native ventricular IKr channels are heteromers containing both ERG1a and ERG1b alpha subunits, which associate in vivo and localize to the T tubules of ventricular myocytes.