Endoplasmic Reticulum Retention and Rescue by Heteromeric Assembly Regulate Human ERG 1a/1b Surface Channel Composition

Defects in the trafficking of subunits encoded by the human ether-à-go-go-related gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in IKr-mediated repolarization. Native IKr channels are composed of two α subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used Western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and to determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an “RXR” ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting that hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels. Defects in the trafficking of subunits encoded by the human ether-à-go-go-related gene (hERG1) can lead to catastrophic arrhythmias and sudden cardiac death due to a reduction in IKr-mediated repolarization. Native IKr channels are composed of two α subunits, hERG 1a and 1b. In heterologous expression systems, hERG 1b subunits efficiently produce current only in heteromeric combination with hERG 1a. We used Western blot analysis and electrophysiological recordings in HEK-293 cells and Xenopus oocytes to monitor hERG 1b maturation in the secretory pathway and to determine the factors regulating surface expression of hERG 1b subunits. We found that 1b subunits expressed alone were largely retained in the endoplasmic reticulum (ER), thus accounting for the poor functional expression of homomeric 1b currents. Association with hERG 1a facilitated 1b ER export and surface expression. We show that hERG 1b subunits fail to mature because of an “RXR” ER retention signal specific to the 1b N terminus of the human sequence and not conserved in other species. Mutating the RXR facilitated maturation and functional expression of homomeric hERG 1b channels in a charge-dependent manner. Co-expression of the 1b RXR mutants with hERG 1a did not further enhance 1b maturation, suggesting that hERG 1a promotes 1b trafficking by overcoming the RXR-mediated retention. Thus, selective trafficking mechanisms regulate subunit composition of surface hERG channels. Voltage-gated potassium (K+) channels encoded by the human ether-à-go-go-related gene (hERG1 or KCNH2) mediate the repolarizing cardiac current IKr (1Sanguinetti M.C. Jiang C. Curran M.E. Keating M.T. Cell. 1995; 81: 299-307Abstract Full Text PDF PubMed Scopus (2135) Google Scholar, 2Trudeau M.C. Warmke J.W. Ganetzky B. Robertson G.A. Science. 1995; 269: 92-95Crossref PubMed Scopus (1087) Google Scholar). Perturbation of IKr due to mutations in the hERG1 gene or drug block of hERG 2The abbreviations used are: hERGhuman ether-à-go-go-related geneLQTSlong QT syndromeERendoplasmic reticulumHEKhuman embryonic kidney.2The abbreviations used are: hERGhuman ether-à-go-go-related geneLQTSlong QT syndromeERendoplasmic reticulumHEKhuman embryonic kidney. channels can cause sudden cardiac death associated with long QT syndrome (LQTS) (3Curran 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). human ether-à-go-go-related gene long QT syndrome endoplasmic reticulum human embryonic kidney. human ether-à-go-go-related gene long QT syndrome endoplasmic reticulum human embryonic kidney. Native IKr channels are composed of hERG 1a and 1b α subunits encoded by alternate transcripts of the hERG1 gene (4Jones E.M. Roti Roti E.C. Wang J. Delfosse S.A. Robertson G.A. J. Biol. Chem. 2004; 279: 44690-44694Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). hERG 1a and 1b subunits have identical transmembrane and C-terminal sequences but divergent N termini (5London 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, 6Lees-Miller J.P. Kondo C. Wang L. Duff H.J. Circ. Res. 1997; 81: 719-726Crossref PubMed Scopus (174) Google Scholar), which interact to promote heteromeric assembly early in channel biogenesis (7Phartiyal P. Jones E.M. Robertson G.A. J. Biol. Chem. 2007; 282: 9874-9882Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). When expressed heterologously, hERG 1a homomeric and 1a/1b heteromeric channels yield robust currents but with distinct properties because of the divergent N termini of the constituent subunits (5London 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, 8Wang J. Trudeau M.C. Zappia A.M. Robertson G.A. J. Gen. Physiol. 1998; 112: 637-647Crossref PubMed Scopus (145) Google Scholar, 9Viloria C.G. Barros F. Giraldez T. Gomez-Varela D. de la Pena P. Biophys. J. 2000; 79: 231-246Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). However, hERG 1b homomers produce undetectable or very small currents (5London 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). Why hERG 1b functional expression is inefficient, or how hERG 1a promotes 1b surface expression, is unknown. In this study we found that hERG 1b protein efficiently exited the ER only in the presence of hERG 1a. We tested the hypothesis that hERG 1b subunits possess ER retention/retrieval signals that are inactivated upon association with the 1a subunit, thus favoring surface expression of the heteromeric channel. Of two RXR motifs (two arginines separated by any single residue) in the 1b N terminus, surprisingly only the one motif specific to the human sequence and not conserved in other organisms subserved ER retention. Moreover, mutations within this signal promoted surface expression in a charge-dependent manner. Co-expression of the 1b RXR mutant with 1a revealed that heteromerization with hERG 1a facilitates hERG 1b maturation by preventing RXR-mediated ER retention/retrieval. Reagents and Antibodies–HEK-293 cell culture, transfection, lysis, co-immunoprecipitation procedures, and analyses of Western blots have been described previously (7Phartiyal P. Jones E.M. Robertson G.A. J. Biol. Chem. 2007; 282: 9874-9882Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Whole-cell Patch Clamp–Wild-type hERG 1b and the mutant cDNAs (1.5 μg) were transiently expressed along with the enhanced green fluorescent protein, and cells with fluorescence were chosen for electrical recordings. Whole-cell patch clamp recordings were performed using an Axopatch 200B amplifier (Axon Instruments, Inc.). The bath solution contained (in mm) 137 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 10 glucose, 5 tetraethylammonium, and 10 HEPES (pH 7.4 with NaOH). Tetraethylammonium (5 mm) was included in the bath solution to block endogenous voltage-gated potassium channels. Whole-cell currents were recorded with a fire-polished pipette tip of ∼1–2 μm with a resistance of 2–4 megohms. Internal pipette solution contained (in mm) 130 KCl, 1 MgCl2, 5 EGTA, 5 MgATP, 10 HEPES (pH 7.2 with KOH). Currents were digitized at 2 kHz unfiltered. Series resistance compensation was typically 60–70%, such that voltage errors were less than 5 mV. Leak subtraction was not used. Current recordings were carried out at room temperature (22 °C). Two-electrode Voltage Clamp–For electrophysiological recordings in oocytes, cRNA preparation and injection were carried out as described previously (8Wang J. Trudeau M.C. Zappia A.M. Robertson G.A. J. Gen. Physiol. 1998; 112: 637-647Crossref PubMed Scopus (145) Google Scholar). Briefly, hERG 1b and mutant DNA were cloned into pGH19 expression vector. cRNA transcripts were purified through a G-50 column (Amersham Biosciences), and 30 ng of cRNA was injected into oocytes. Currents were recorded 2–3 days after injection using a two-electrode voltage clamp. Oocytes were held at a potential of –80 mV. Currents were evoked by 480-ms pulses ranging from –80 to +40 mV in increments of +20 mV followed by a repolarizing step to –105 mV for 480 ms. Statistical Analysis–X-ray films of Western blots were quantified by measuring optical density using LabWorks image and acquisition analysis software (Upland, CA). We ensured signals were within a linear range of detection for the ECL reagent and the x-ray film by probing increasing amounts of lysate with the relevant antibody. Data were analyzed using PRISM 2.0 software (GraphPad, San Diego, CA). All Western blotting and electrophysiology data from the RXR mutants were compared with wild-type 1b using one-way analysis of variance and Bonferroni's multiple comparison post-test. A p value of 0.01 was used to assess significance. In Figs. 1 and 3, 4 5, 6, a single asterisk denotes p < 0.01, and double asterisks denote p < 0.001. A post-test for linear trend was used to assess whether charge-dependent improvement in 1b maturation and function was significant.FIGURE 3The nonconserved R15XR motif in the hERG 1b N terminus controls its trafficking. A, maturation of 1bN15XN and 1bN15XN/N22XN mutants assessed by Western blot analysis. α-CT, C-terminal pan-hERG antibody; α-PDI, protein-disulfide isomerase antibody. B, optical density measurements of 1b signal from Western blots of R15XR mutants (n = 5; **, p < 0.001). For this set of experiments comparing single and double mutant effects on maturation, the 1bN15XN mutation caused an ∼5-fold increase in maturation; across all experiments in which 1b and 1bN15XN were compared, the difference was 4-fold (n = 11; **, p < 0.001). C, representative whole-cell patch clamp recordings (normalized to cell capacitance) from wild-type 1b, 1bN15XN, and 1bN15XN/N22XN. Arrows indicate hERG peak tail currents. D, histogram comparing current densities of R15XR mutants with wild-type 1b (*, p < 0.01). The number of cells used for current recordings for each condition is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 4ER retention of 1b by the active R15XR signal is dependent on its charge. A, maturation of 1b was assessed by Western blotting of charge-conserved (K15XK), charged-neutralized (N15XN), and charge-reversed (D15XD) mutants. α-CT, C-terminal pan-hERG antibody; α-PDI, protein-disulfide isomerase antibody. B, optical density was measured for wild-type and R15XR mutants from blots such as shown in A to determine 1b maturation (n ≥ 6; *, p < 0.01; **, p < 0.001). C, representative two-electrode voltage clamp current recordings from oocytes expressing R15XR mutants. D, 1.25 μg of ethidium bromide-stained cRNA for each of the 1b constructs showing cRNA integrity and comparability. E, peak hERG 1b tail currents from three batches of oocytes. The number of oocytes used for current recordings is indicated (**, p < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Rescue of 1b trafficking by hERG 1a and mutation of RXR are not additive. A, Western blot of cell lysates expressing 1b, 1bN15XN, or 1bN15XN with 1a, probed with hERG C-terminal antibody. α-CT, C-terminal pan-hERG antibody; α-PDI, protein-disulfide isomerase antibody. B, protein signals from 1b and 1bNXN quantified by densitometry and plotted as mature/total protein fraction (n = 9; **, p < 0.001). n.s., not significant. C, lysate co-expressing 1a and 1bN15XN (lane 1), immunoprecipitated with 1a-specific antibody (lane 2), and Western-blotted with C-terminal antibody. M, mature glycoforms; I, immature glycoforms.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 6Analysis of 1a N-terminal deletions on hERG 1b trafficking. A, left panel, Western blot showing maturation profiles of sequential N-terminal deletions of 1a probed with C-terminal pan-hERG antibody (α-CT). Lysate from cells expressing hERG 1a or 1b is shown for comparison. Only 1a and 1aΔ200 proteins exhibited a mature protein band; the other four deletions failed to exit the ER. Right panel, Western blot analysis of 5 μg of lysate from cells co-expressing hERG 1b with the 1a N-terminal deletions. 5 μg of lysate from cells co-expressing 1b with 1a is shown as a positive control. An increased quantity (25 μg) of lysate from cells expressing only 1b (last lane) is also shown for comparison of mature (M) and immature (I) 1b protein bands. The mature/total 1b ratio failed to improve when 1b was co-expressed with the trafficking-impaired 1a N-terminal deletion constructs. Co-expression of 1b with the only trafficking-competent N-terminal deletion, 1aΔ200, improved 1b maturation significantly (n = 5; p < 0.01). The protein for 1aΔ250 migrated at the same size as mature 1b protein; expression studies were used to assess maturation in this case (see B and C). B, two-electrode voltage clamp recordings from Xenopus oocytes show 1aΔ200 expressed efficiently, whereas 1aΔ250 did not. C, histogram comparing the peak tail current from 1aΔ200 and 1aΔ250 shows significant differences in expression levels (n = 7; **, p<0.001). D, based on the above data from 1aΔ200 and 1aΔ250, an internal deletion between residues 200 and 250 of the 1a N terminus was made to determine whether this region contained the masking domain for the 1b N terminus. Unfortunately, the internally deleted 1aΔ203–228 protein was itself trafficking-incompetent and therefore also did not improve maturation of 1b. α-PDI, protein-disulfide isomerase antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT) hERG 1a Promotes Maturation of 1b–To investigate the biochemical basis for functional rescue of 1b subunits by 1a, we monitored maturation of the 1b protein with or without 1a. Like other glycoproteins (10Helenius A. Aebi M. Science. 2001; 291: 2364-2369Crossref PubMed Scopus (1963) Google Scholar), both hERG 1a and 1b subunits are detected as two protein bands on a Western blot: a core-glycosylated, immature form and a complexly glycosylated, mature form (4Jones E.M. Roti Roti E.C. Wang J. Delfosse S.A. Robertson G.A. J. Biol. Chem. 2004; 279: 44690-44694Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 11Zhou 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). The mature glycoform represents the fraction of core-glycosylated protein that has exited the ER and has been further processed in the Golgi. When expressed alone, 1b exhibited low levels of maturation, with functional expression previously (5London 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). In the presence of 1a, 1b maturation was enhanced Thus, hERG 1a facilitates ER exit and maturation of 1b subunits. to of 1b tested the hypothesis that ER retention signals in the divergent 1b N terminus maturation and trafficking to the cell We two RXR motifs in the hERG 1b N the at is only in the human 1b the at is conserved across We the arginines of the conserved motif to to assess its in preventing maturation of hERG 1b. Western blot analysis of HEK-293 cells revealed significant improvement in trafficking of wild-type 1b (n = 5; p B and C). whole-cell patch clamp recordings in HEK-293 cells also failed to significant functional rescue of mutants the typically low wild-type expression levels and R15XR hERG 1b the arginines of the R15XR motif to residues this RXR motif is not conserved other we were to the mutation maturation of hERG 1b 3, A, 1 and and A double mutation of both RXR motifs in the 1b N terminus did not further improve maturation of 1b that of the single mutation 3, A, 3, and expression and function of and the double as assessed by whole-cell patch clamp recordings in HEK-293 the biochemical with an increase in current density to 3, and Thus, mutations in the nonconserved R15XR signal enhance 1b maturation and functional surface expression. R15XR as a of 1b trafficking. also the that 1b subunits and can produce potassium currents in the of hERG 1a or expressed subunits as long as can to the cell of 1b by R15XR by into the by which R15XR 1b we the residues to conserved and with did not improve maturation whereas 1bN15XN and mutations enhanced 1b trafficking to ∼5-fold A and The increase in maturation of R15XR mutants was as by an analysis of variance post-test for linear trend (see We assessed whether improved maturation in 1b mutants into functional expression at the cell For this we recorded currents using a two-electrode voltage clamp in Xenopus oocytes, the of injected cRNA for each Oocytes expressing mutant 1b currents to than wild-type 1b in a charge-dependent and with biochemical maturation profiles in HEK-293 cells differences were in the or voltage of currents by the four 1b the that currents are due to increased channel number at the cell surface and not to hERG 1a 1b by RXR-mediated whether the enhanced maturation of 1b by mutation of R15XR was to the by which 1a promotes 1b An on maturation of R15XR mutant 1b by with 1a by than one trafficking Western blot analysis the increase in 1bN15XN maturation was not further enhanced in the presence of 1a 5, A and suggesting that the by which 1a and RXR mutations promote 1b trafficking from the ER is the The of an on 1bN15XN maturation is not due to a of 1a and 1bN15XN to as can using hERG 1a-specific antibody from lysate that association with 1a the 1b N-terminal RXR retention signal to promote trafficking. We to a region in the 1a N terminus for masking the 1b RXR using deletion analysis We a of constructs deleted by increments of Of only the 1aΔ200 protein exhibited a mature protein with wild-type 1a as Western blot analysis left panel, 1 and The 1aΔ200 was the only to currents (n = 7; p < 6, B and C). When co-expressed with hERG 1b, the 1aΔ200 promoted 1b maturation (n = 5; p < panel, 1 and 1aΔ200 exhibited maturation and promoted 1b maturation, whereas 1aΔ250 did not 6, we to further the masking region between residues in the 1a N terminus. Unfortunately, an internal deletion in this region was trafficking-incompetent and therefore also did not improve maturation of 1b We that the masking region in the 1a N terminus of experiments using to its In this study we a by which heteromeric association of subunits an N-terminal ER retention motif to regulate subunit composition of hERG surface channels. We ER retention of hERG 1b subunits to a single N-terminal RXR The of retention is by its as by in 1b maturation and surface expression as the is and a mutation of the RXR motif in hERG 1b promotes trafficking and functional surface expression, that of the 1b subunits to its ER retention. homomeric 1b channels are from trafficking. with 1a the RXR-mediated ER retention and promotes 1b Thus, a for surface expression of hERG 1a/1b is encoded in the of the N terminus that hERG 1b. We that the 1a N terminus the 1b ER retention signal or a that from the ER a masking from a with that the 1a and 1b N termini interact in a as by in of purified N-terminal (7Phartiyal P. Jones E.M. Robertson G.A. J. Biol. Chem. 2007; 282: 9874-9882Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). ER retention signals have also been in channels and B and subunit ER retention/retrieval signals in homomeric proteins B. Full Text Full Text PDF PubMed Scopus Google Scholar, M. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, K.W. J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). We that hERG 1a subunits the 200 residues are to and promote 1b to further a masking region in the hERG 1a N terminus were by the of 1a deletion mutants to the 1a N-terminal masking domain also such that in its both 1a and 1b RXR signals are and the subunits are to the ER. the 1b N terminus two RXR only one is conserved across but the nonconserved R15XR ER retention. In other R15XR is by that 1b subunits are at current (5London 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), is to its the human RXR to the 1b subunit in the ER. a motif in subunits, as an ER retention signal M. C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the nonconserved R15XR in hERG 1b is by a based on studies in to the retention of an RXR signal B. Full Text Full Text PDF PubMed Scopus Google Scholar). The that the effects on 1b maturation by R15XR mutations and with 1a are not that heteromerization with 1a ER retention of 1b. that association of 1bN15XN with 1a channels in that the a potential by the 1a subunits. However, on the of maturation that the 1bN15XN mutant has not its maturation potential not further mature when associated with 1a. mutation an ER export Science. 1997; PubMed Scopus Google we that both the mutation and the presence of 1a the retention in 1b, the mutation to an function as a trafficking than mutations have been to the hERG 1a of all the mutations to in for the of the hERG1 mutations thus fail to mature D.J. Gong Q. Z. January C.T. PubMed Scopus Google Scholar, D. C. M. A.M. J. Physiol. 2000; 279: PubMed Google Scholar, M. Trudeau M.C. A. Gong Q. Z. A. January C.T. Robertson G.A. PubMed Scopus Google Scholar). the mechanisms are in mutations lead to protein and the ER Q. Jones Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Q. M. Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In one of the terminus by a mutation an C-terminal RXR a of ER retention for T. Wang Z. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, in to hERG 1b, wild-type 1a subunits form functional homomeric and therefore is that 1a RXR motifs are or as of 1a subunit of ER retention motifs for other to The of hERG 1b in function and has not been A specific of 1b, which 1a, cardiac J.P. J. Duff H.J. Cell. Biol. PubMed Scopus Google Scholar), a for the 1b subunit in cardiac 1a/1b currents less and currents in to a voltage clamp that the suggesting the of 1b in is to in IKr J. and A. therefore to for mutations in the hERG and to study of hERG 1a-specific mutations in the presence of hERG 1b. We not the of the hERG 1a and 1b subunits in channels expressed in heterologous or an alternate (5London 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, 6Lees-Miller J.P. Kondo C. Wang L. Duff H.J. Circ. Res. 1997; 81: 719-726Crossref PubMed Scopus (174) Google Scholar), 1b to that or studies of hERG 1b on IKr function and the mechanisms by which its cardiac We and for and of the Robertson for and Ganetzky for an of the