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Circulation Research. 1997;81:870-878

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(Circulation Research. 1997;81:870-878.)
© 1997 American Heart Association, Inc.

Articles

Two Isoforms of the Mouse Ether-a-go-go–Related Gene Coassemble to Form Channels With Properties Similar to the Rapidly Activating Component of the Cardiac Delayed Rectifier K+ Current

Barry London, Matthew C. Trudeau, Kimberly P. Newton, Anita K. Beyer, Neal G. Copeland, Debra J. Gilbert, Nancy A. J enkins, Carol A. Satler, , Gail A. Robertson

From the Division of Cardiology (B.L., K.P.N., A.K.B.), University of Pittsburgh (Pa) Medical Center; the Department of Physiology (M.C.T., G.A.R.), University of Wisconsin Medical School, Madison; the Mammalian Genetics Laboratory (N.G.C., D.J.G., N.A.J.), ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Md; and the Central Research Division (C.A.S.), Pfizer Inc, Groton, Conn.

Correspondence to Barry London, MD, PhD, Division of Cardiology, University of Pittsburgh Medical Center, BST 1744, 200 Lothrop St, Pittsburgh, PA 15213-2582. E-mail london{at}card2.cath.upmc.edu


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract HERG, the human ether-a-go-go–related gene, encodes a K+-selective channel with properties similar to the rapidly activating component of the delayed rectifier K+ current (IKr). Mutations of HERG cause the autosomal-dominant long-QT syndrome (LQTS), presumably by disrupting the normal function of IKr. The current produced by HERG is not identical to IKr, however, and the mechanism by which HERG mutations cause LQTS remains uncertain. To better define the role of Erg in the heart, we cloned Merg1 from mouse genomic and cardiac cDNA libraries. Merg1 has 16 exons and maps to mouse chromosome 5 in an area syntenic to human chromosome 7q, the map locus of HERG. We isolated three cardiac isoforms of Merg1: Merg1a is homologous to HERG and is expressed in heart, brain, and testes, Merg1a' lacks the first 59 amino acids of Merg1a and is not expressed abundantly, and Merg1b has a markedly shorter divergent N-terminal cytoplasmic domain and is expressed specifically in the heart. The Merg1 isoforms, like HERG, produce inwardly rectifying E-4031–sensitive currents when heterologously expressed in Xenopus oocytes. Merg1a and HERG produce currents with slow deactivation kinetics, whereas Merg1a' and Merg1b currents deactivate more rapidly. Merg1b coassembles with Merg1a to form channels with deactivation kinetics that are more rapid than those of Merg1a or HERG and nearly identical to IKr. In addition, a homologue of Merg1b is present in human cardiac and smooth muscle. Thus, we have identified a novel N-terminal Erg isoform that is expressed specifically in the heart, has rapid deactivation kinetics, and coassembles with the longer isoform in Xenopus oocytes. This N-terminal Erg isoform may determine the properties of IKr and contribute to the pathogenesis of LQTS.


Key Words: K+ channel • coassembly • long-QT syndrome • rapidly activating component of delayed rectifier K+ current • HERG


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The human ether-a-go-go–related gene, HERG, was originally cloned by homology to the Drosophila K+ channel gene eag.1,2 It has six putative membrane-spanning domains (S1 to S6), a conserved putative pore domain, and a putative CNB domain. HERG is known to contain introns,3 but the entire gene structure has not been published. It is expressed abundantly in cardiac atrial and ventricular tissues of many mammalian species.4,5 When expressed in Xenopus oocytes, HERG channels produce an inwardly rectifying K+-selective current that is blocked by methanesulfonanilides, such has E-4031 or dofetilide, and is enhanced by extracellular K+.6,7 The inward rectification of HERG is due to a fast inactivation mechanism with similarities to C-type inactivation in Shaker.8,9

IKr is one component of the delayed rectifier K+ current that contributes to the repolarization phase of the mammalian cardiac action potential. IKr has been identified and characterized in atrial and ventricular myocytes from several mammalian species, including mice and humans.4,10-17 Electrophysiological and pharmacological similarities between IKr and the currents produced by HERG channels in vitro have led to widespread acceptance of the hypothesis that Erg channels produce IKr.6,7,18-20 The deactivation kinetics of IKr in mouse AT-1 cells13 and guinea pig ventricular myocytes,10 however, are significantly faster than those of HERG channels expressed in Xenopus oocytes or mammalian cell lines. Along with a recent report suggesting that HERG subunits may associate with other channel polypeptides,21 these findings suggest that the relationship of HERG to IKr is not fully understood, and a role for other channel subunits in IKr remains possible.

Mutations of HERG cause the autosomal-dominant LQTS (Romano-Ward syndrome) in families in which the affected individuals are linked to the LQT2 locus on human chromosome 7q35-q36.3 LQTS was first described in the early 1960s and is characterized clinically by recurrent syncope, a prolonged QT interval with an abnormal T-wave morphology on the surface ECG, torsade de pointes (polymorphic ventricular tachycardia with a rotating axis), and sudden death.22 The exact mechanism by which mutations of HERG subunits cause LQTS remains uncertain.

A more complete understanding of HERG is essential to the understanding of its role in the electrophysiology of the normal heart and the pathophysiology of disease. We report in the present study the cloning of Merg1, the mouse homologue of HERG, the characterization of its gene structure, and the identification of a novel cardiac-specific N-terminal truncated isoform that may contribute to the cardiac current IKr.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
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 down arrowReferences
 
Cloning of Merg1
Standard methods for library screening and molecular cloning were used.23 Using a radiolabeled PCR-generated fragment from the hydrophobic core of HERG (T7 QuickPrime, Pharmacia Biotech), we screened mouse and human heart cDNA libraries (Stratagene) and isolated a partial-length mouse cDNA clone homologous to HERG (CM3), which was {approx}3550 bp in length and extended to the poly-A tail. We rescreened the mouse library with a more 5' fragment cloned from the human cDNA library and identified an {approx}750-bp cDNA clone (CMH2), which started {approx}40 nucleotides downstream from the putative start codon of HERG, and an {approx}990-bp cDNA clone (CMH1) that had a novel {approx}95 bp at its 5' end and was then homologous to HERG, starting {approx}75 nucleotides downstream from the putative start codon of HERG. Using the inserts of CMH2 and CM3 as templates for randomly primed probes, we then screened the mouse heart library and identified a full-length clone homologous to HERG (H1, {approx}4215 bp, designated Merg1a) and a second shorter clone with a novel 5' end (H7, {approx}3135 bp, designated Merg1b). Both H1 and H7 contain in-frame stop codons in the 5' untranslated region. RACE (indicating rapid amplification of cDNA ends) was performed using nested antisense primers in exon 6, which is common to both isoforms (GIBCO-BRL). A clone that extended 30 nucleotides further upstream than the H7 cDNA clone was isolated; the extra nucleotides correspond to the genomic clone (see below). All cDNA clones were double-strand–sequenced.

Using PCR and cDNA fragments of HERG and Merg1a, we screened an SV129 mouse genomic library ({lambda}-FIX, Stratagene) and isolated several genomic clones, of which two were subcloned and sequenced. The first, Me3, contained exons 3 through 15 (including exon 1b); the second, 2GMBg6, contained exons 1a, 1a', and 2. There was no overlap detected between the clones, and the size of the gap between exons 2 and 3 is not known. With the exception of this gap, the portion of each clone between exons 1 and 15 was single-strand–sequenced; all exons and the regions flanking the exons were double-strand–sequenced. Eight nucleotide differences were detected between the cDNA and genomic clones of Merg1, of which five caused changes in the amino acid sequence (Fig 1Down). We also isolated and single-strand–sequenced the exons from two human genomic clones that contained HERG exons 1 through 6.



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  		Figure 1. Amino acid sequence of the Merg1 and HERG isoforms. The Merg1 sequences were determined from cDNA clones and compared with the sequence of HERG (Warmke and Ganetzky2 in 1994) and the sequence of the putative isoform HERGb determined from a HERG genomic clone (see text). Standard single-letter amino acid codes are used, and the code is capitalized for those amino acids in which the sequences of the Merg1 and HERG isoforms are identical. The amino acid positions of Merg1a, Merg1b, HERG, and HERGb are given to the right of the sequence. The putative initiator methionines, for translation of the Merg1a and Merg1a' isoforms, are indicated. The location of transmembrane domains S1 to S6, the putative pore domain, and the putative CNB are indicated above the sequences. Consensus protein kinase A phosphorylation sites are highlighted. The locations of exon boundaries in Merg1 are indicated by a caret below the aligned sequence. A total of eight single nucleotide differences were noted between Merg1 cDNA (BALB/c library, {lambda}-ZAP II, Stratagene) and genomic clones (SV129 library, {lambda}-FIX II, Stratagene). Five of these led to a change in the amino acid code and are underlined. They included R186H, T455A, Y752C, and N1006D in Merg1a and T5A in Merg1b; four of these alternate amino acids in the genomic clones are identical to the those found in HERG.

Mapping of Merg1 to Mouse Chromosome 5
Interspecific backcross progeny were generated by mating (C57BL/6JxMus spretus) F1 females and C57BL/6J males as described by Copeland and Jenkins.24 A total of 205 N2 mice were used to map the Merg1 locus. DNA was isolated, digested with several enzymes, and analyzed by Southern blot hybridization for informative RFLPs.25 Blots were prepared with Hybond-N+ nylon membrane (Amersham). The 3' untranslated region probe, an {approx}600-bp Sac I fragment of the mouse genomic clone, was labeled using a random-primed kit (Stratagene) and washed to a final stringency of 0.8x SSC and 0.1% SDS at 65°C. A fragment of {approx}23 kb was detected in Sca I–digested C57BL/6J DNA, and a fragment of 8.0 kb was detected in Sca I–digested M spretus DNA. The presence or absence of the 8.0-kb Sca I–digested M spretus–specific fragment was followed in backcross mice. A description of the probes and RFLPs for the loci linked to Merg1, including Gnail, En2, and Il6, has been reported previously.26 Recombination distances were calculated as described27 using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns. We also compared our interspecific map of chromosome 5 with a composite mouse linkage map that reports the map location of many uncloned mouse mutations (provided from Mouse Genome Database, a computerized database maintained at The Jackson Laboratory, Bar Harbor, Me).

Construction of Merg1 Isoform Expression Vectors
The expression construct for Merg1a was made by subcloning fragments of clones CM3 and CMH2 into the expression vector pGH19K and adding either a 5' fragment of clone H1 or a PCR-generated 5' fragment containing a consensus Kozak sequence (TCCGCCACCATG)28 at the putative initiator methionine (Merg1a). The Kpn I site in exon 3 was used for subcloning. pGH19K is a modification of the pGEMHE vector.29 The expression clone for Merg1a' was constructed using clones CM3 and CMH1. The expression clone for Merg1b was constructed by replacing the 5' end of the Merg1a expression vector with either the 5' end of clone H7 (Merg1bK-) or with a PCR-generated fragment containing a consensus Kozak sequence at the putative initiator ATG codon (Merg1b). The Spe I site in exon 9 was used for subcloning. Pfu polymerase (Stratagene) was used for PCR, and artifacts were excluded in all constructs by double-strand sequencing.

Oocyte Preparation and Electrophysiology
Xenopus oocytes were isolated and injected with RNA as described previously,7,30 except that some oocytes were defolliculated using osmotic shock, a technique that expedites the defolliculation procedure.31 No differences in electrophysiological responses were observed using the two methods. RNA was synthesized in vitro from the T7 promoter of the Merg1 cDNA expression constructs using the Message Machine kit (Ambion). The different clones exhibited markedly different expression levels. Because our goal was to compare different isoforms at similar expression levels, we "calibrated" individual transcription reactions according to expression level and diluted the RNA to a concentration that would produce currents in the range of 0.5 to 8.0 µA to ensure excellent voltage-clamp control. For coexpression, half the solution volume of each RNA species at 2x concentration used for independent expression was mixed and injected.

Currents were recorded with a two-electrode voltage clamp (OC-725C, Warner). Recording electrodes were made with a Sutter P-97 micropipette puller (Novato) from borosilicate glass (World Precision Instruments) and had resistances of 0.5 to 1.0 M{Omega} when filled with 2 mol/L KCl. Data acquisition and analysis were performed using pClamp6 (Axon Instruments) and Origin4 software (Microcal). Experiments were performed at room temperature (22°C to 24°C).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Three Isoforms of Merg1 Cloned From the Mouse Heart
We cloned Merg1, the mouse ether-a-go-go–related gene, from a mouse heart cDNA library (Stratagene) and identified three cDNA isoforms. Fig 1Up shows the amino acid sequences of the isoforms aligned with the previously published HERG sequence. Merg1a, the longest isoform, has an 1162–amino acid open reading frame and is 96% identical to HERG at the amino acid level. There is >99% identity between Merg1a and HERG in the region between the S1 and CNB domains.

Merg1a' has an alternate 5' untranslated region and is identical to Merg1a from amino acid 25 onward. The alternate 5' end has an in-frame stop codon and no downstream translation initiation codon. This requires that translation begin at or downstream from the second in-frame ATG codon of Merg1a, and the Merg1a' channel thus lacks the first 59 amino acids of Merg1a.

Merg1b, the shortest isoform, has an 820–amino acid open reading frame, is identical to Merg1a from S1 to the carboxy-terminal end, and has a novel, markedly shorter amino-terminal cytoplasmic domain. The 36 amino acids at the amino-terminal end of Merg1b show no significant homology to any previously cloned genes and lack a number of the consensus protein kinase phosphorylation sites found in Merg1a.

Structure and Location of MERG1
Fig 2ADown shows the genomic organization of Merg1, including the location of the exons and the alternate isoforms. Two closely related polyadenylation sites correspond to two 3' untranslated regions identified on cDNA clones. Merg1a is encoded by 15 exons and spans >25 kb of genomic DNA. MERG1a' appears to use an alternate exon-1 splice donor site that is 226 nucleotides downstream from the site used by the Merg1a isoform. Merg1b, on the other hand, has a different first exon (designated 1b), which is located between exons 5 and 6 of Merg1a. The regions immediately upstream from both exons 1a and 1b are extremely GC rich, with each containing three SP1 sites. The region upstream from exon 1b (between exons 5 and 6) may contain an alternate transcription initiation site that is used for the Merg1b isoform.



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  		Figure 2. Genomic structure and chromosome localization of Merg1. A, Intron/exon structure of Merg1 (top) and of the cDNA isoforms Merg1a, Merg1a', and Merg1b (bottom). The first in-frame ATG codons (*), the location of transmembrane domains, and the polyadenylation signals (#) are indicated. B, Merg1 was placed on mouse chromosome 5 by interspecific backcross analysis. The segregation patterns of Merg1 and flanking genes in 166 backcross animals are shown on the left. Each column represents the chromosome identified in the backcross progeny that was inherited from the C57BL/6JxM spretus F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and the white boxes represent the presence of an M spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome-5 linkage map showing the location of Merg1 in relation to linked genes is shown on the right. For individual pairs of loci, 166 or more animals were typed, and each locus was analyzed in pairwise combination using the additional data. The number of recombinant N2 animals is presented over the total number of N2 animals typed to the left of the chromosome maps between each pair of loci. The recombination frequencies, expressed as genetic distance in centimorgans (±1 SE) are also shown. Merg1 is mapped {approx}3.6 centimorgans distal to Gnail and 0.6 centimorgans proximal to En2 on the proximal portion of mouse chromosome 5. This region is homologous to human chromosome 7, and En2 has been placed on human 7q36. The positions of loci in human chromosomes are shown to the right. References for the human map positions of loci cited in the present study can be obtained from GDB (Genome Data Base), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The John Hopkins University (Baltimore, Md).

The chromosomal location of Merg1 was determined using interspecific backcrosses and is shown schematically in Fig 2BUp. Merg1 is linked to the loci Gnail, En2, and Il6 on the proximal part of mouse chromosome 5; this region shares homology with human chromosome 7q35-q36, the map locus of HERG.3 No mouse mutations linked to this area of chromosome 5 have a phenotype that might be expected for an alteration in the Merg1 locus.

Tissue Expression of the Merg1 Isoforms
Tissue-specific RNA expression of the Merg1 isoforms was determined using exon-specific probes on a mouse multitissue Northern blot and is shown in Fig 3Down. Probes from the 3' end of Merg1 in the region common to all three isoforms hybridized to a 4.4-kb transcript, which is abundant in heart, brain, and testes, along with a 3.4-kb transcript, which is abundant only in the heart. Probes upstream of exon 5 yielded only the larger 4.4-kb transcript in heart, brain, and testes. A probe specific for Merg1a' did not hybridize abundantly to RNA from any of the tissues tested (data not shown). A probe specific for Merg1b yielded the smaller 3.4-kb transcript, which is abundant only in the heart. Thus, Merg1b is expressed in a cardiac-specific manner and is likely coexpressed with Merg1a in mouse cardiac myocytes, although we cannot exclude the possibility of nonoverlapping expression of the two isoforms in different cells within the heart.



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  		Figure 3. Tissue-specific expression of Merg1 isoforms. Two mouse Northern blots (Clontech) were probed and washed at high stringency (ExpressHybe protocol, Clontech) using exon-specific probes. The panel at the left was probed with an Xho I– EcoRI restriction fragment from Merg1 cDNA clone CM3 and included the portion of the channel from exon 8 through the poly-A tail. Reprobing the blot with a PCR-generated fragment specific to exon 6 yielded identical results (not shown). The center panel was probed with a restriction fragment of a HERG cDNA clone that included exons 2 and 3 and extended to the Pst I site near the 5' end of exon 4. Reprobing the blot with a Merg1 genomic fragment from exon 1a or with a Merg1 cDNA fragment that included all of exon 4 yielded similar results (data not shown). The panel at the right was probed with a 300-bp Pst I restriction fragment from the 5' end of the Merg1 cDNA clone H7 and included only exon 1b.

N-Terminal Isoforms of HERG
Human muscle may also contain at least one other N-terminal isoform of HERG. A region between exons 5 and 6 of HERG is homologous to exon 1b of Merg1 (>90% identity at the nucleotide level). Other intronic regions of the HERG genomic clone, including the area corresponding to exon 1a' of Merg1, show much less conservation. Fig 1Up shows the sequence of the putative human homologue of Merg1b (HERGb). Northern blots probed with a DNA fragment from the 3' end of Merg1 show a 4.4-kb transcript in RNA from human brain, heart, and smooth muscle along with a 4.0-kb transcript specific to the heart (see Reference 33 ) and a 3.4-kb transcript in cardiac and smooth muscle. Reprobing the blot with a fragment from human exon 1b confirms low levels of expression of the shorter isoforms in human cardiac and smooth muscle tissues (data not shown).

Heterologous Expression of the Merg1 Isoforms in Xenopus Oocytes
Expression of the Merg1 isoforms in Xenopus produces currents with pronounced inward rectification (Fig 4ADown to 4C) similar to those produced by HERG.6,7 The outward current amplitude declines at more positive voltages as a consequence of rapid inactivation, giving rise to the hallmark negative slope conductance on the current-voltage plot (Fig 4DDown). On repolarization, the channels first recover from inactivation and then deactivate (close), producing a "hook" in the tail current. The most apparent difference among the isoforms is the rate of deactivation of this tail current (see below).



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  		Figure 4. Families of Merg1 K+ currents expressed in Xenopus oocytes. A, B, and C, Merg1a (A), Merg1a' (B), and Merg1b (C) currents elicited by 1-second pulses from -80 to 70 mV in 10-mV increments, followed by a 750-millisecond repolarization to -100 mV. Holding potential was -80 mV. Expression clones Merg1a and Merg1b contained an upstream Kozak consensus sequence, whereas Merg1a' lacked the Kozak consensus sequence. An expression clone for Merg1a with the native 5' untranslated region (without the Kozak sequence) yielded similar results. D, Current (I)–voltage (V) relation generated by measuring the current 1 second after depolarization and normalizing to the maximum current (I/Imax) obtained during the above protocol for each oocyte. Thus, I/Imax is proportional to the probability of channel opening, assuming no difference in single-channel conductance among the isoforms (Merg1a, n=4; Merg1a', n=3; Merg1b, n=5; and Merg1a/Merg1b, n=5). External Ringers' solution contained 4 mmol/L KCl, 94 mmol/L NaCl, 1.0 mmol/L MgCl2, 0.3 mmol/L CaCl2 (unless otherwise noted), and 5.0 mmol/L HEPES, pH 7.4. Scale bars are 1 µA and 250 milliseconds.

The marked variability in the levels of current expression between the isoforms was useful in demonstrating that Merg1a and Merg1b coassemble to form heteromultimers in oocytes (Fig 5ADown to 5D). A Merg1b construct that lacked an upstream Kozak consensus sequence (Merg1bK-) did not produce measurable currents (Fig 5BDown). However, on coexpression of Merg1bK- with Merg1a (Fig 5CDown), the resulting currents had deactivation kinetics that were faster than those of Merg1a expressed alone (Fig 5ADown). Thus, Merg1bK- expression can be rescued by coassembly with Merg1a and contributes significantly to the deactivation rate in the heteromer. The construct Merg1b, which contains a consensus Kozak sequence before the initiator methionine, expressed currents independently (Figs 4CUp and 5DDown). When Merg1b was coexpressed with Merg1a, the resulting deactivating tail currents exhibited intermediate kinetics and could not be predicted by the sum of any ratio of tail currents from homomeric Merg1b and Merg1a channels (Fig 5EDown). This supports the conclusion that Merg1a and Merg1b coassemble to form heteromultimeric channels with novel gating properties.



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  		Figure 5. Evidence for coassembly of Merg1a and Merg1b subunits. A, Expression of Merg1a is shown. B, Expression of Merg1bK- (lacking an upstream Kozak consensus sequence) did not give rise to measurable currents (n=12). C, Coexpression of Merg1a and Merg1bK- resulted in currents with faster deactivation kinetics than observed for Merg1a. D, Expression of Merg1b (same as Merg1bK- but containing a Kozak consensus sequence) produced currents with faster deactivation kinetics than observed for either Merg1a or Merg1a/Merg1b expressed together. Current decay on repolarization was best fit using the Chebyshev method in pClamp (Axon Instruments) with the sum of two exponentials: y=A0+(A1e-t/{tau}fast)+(A2e-t/{tau}slow). The time constant ({tau}) values (mean±SEM) are as follows: Merg1a, {tau}fast=202±24 milliseconds and {tau}slow=394±62 milliseconds (n=5); Merg1a/Merg1bK-, {tau}fast=59±4 milliseconds and {tau}slow=215±13 milliseconds (n=9). The voltage command was a depolarizing step to 20 mV for 1 second followed by a repolarizing step to -100 mV for 1.5 seconds, from a holding potential of -80 mV. E, The experimentally observed current from coexpression of Merg1a and Merg1b (observed 1a/1b, thick trace) cannot be described by a weighted algebraic summation of the Merg1a and Merg1b currents expressed independently. Current traces in panels A and D were normalized to the peak current and then weighted and summed as indicated in the figure. The same result was obtained in five other experiments.

The rapid deactivation kinetics of the short Merg1 isoforms and Merg1a/Merg1b heteromers are similar to those of native IKr (Fig 6Down). Two-exponential fits to the deactivating tail currents of the Erg isoforms show that Merg1a and HERG have similarly slow deactivation kinetics (Fig 6ADown and 6EDown to 6G). In contrast, the tail currents of Merg1a', Merg1b, and Merg1a/Merg1b heteromeric channels decay faster and are more like those of IKr measured from mouse AT-1 cells (Fig 6BDown to 6D, 6F, and 6G), a model system for native cardiac currents in the mouse.13



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  		Figure 6. Deactivation kinetics of Merg1 isoforms. A to E, Families of deactivation currents from Merg1 isoforms (A to C), coexpression of Merg1a and Merg1b isoforms (D), and HERG (E). After activation of channels by a 1-second pulse to 20 mV, deactivating currents were elicited by a series of 3-second pulses from 20 to -100 mV in 10-mV steps (voltage trace, bottom right). Current decay was fit with the sum of two exponentials as described above. F and G, Fast and slow time constants ({tau}fast and {tau}slow, respectively) plotted vs voltage (V). Isoforms were as follows: Merg1a (1a), n=6; Merg1a' (1a'), n=6; Merg1b (1b), n=11; Merg1a/Merg1b coexpression (1a/1b), n=8; and HERG, n=7. The time constants previously described by Yang et al13 for cardiac IKr are also plotted. H, Illustration of each Merg1 isoform subunit depicting that deactivation is modulated by the first 59 amino acids of the N-terminus. Scale bars are 1 µA and 1 second. Physiological Ca2+ Ringer's solution contained 4 mmol/L KCl, 94 mmol/L NaCl, 1.0 mmol/L MgCl2, 1.8 mmol/L CaCl2, and 5.0 mmol/L HEPES, pH 7.4.

The shorter N-terminal isoforms Merg1a' and Merg1b mimic the fast deactivation kinetics of HERG N-terminal deletions.8,32 Our findings provide a physiological counterpart to the deletion mutants and help to delineate a domain within the N-terminus important in modulating deactivation. Merg1a' lacks only the first 59 amino acids of Merg1a, thus restricting the region modulating deactivation to the extreme N-terminus (Fig 6HUp).

Like HERG channels, Merg1 isoforms and heteromers expressed in oocytes are blocked by the methanesulfonanilide E-4031. Figs 7ADown to 7C show the responses of Merg1a, Merg1b, and heteromeric Merg1a/1b currents evoked at 20 mV to 5 mmol/L E-4031. Dose-response curves indicate a similar range of sensitivity among the different Merg1 isoforms (Fig 7DDown) and HERG.7,18



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  		Figure 7. Block of Merg1 isoforms by E-4031. A, Merg1a. B, Merg1b. C, Merg1a and Merg1b coexpressed. The top trace in each panel represents current evoked in normal Ringer's solution. The lower trace in each panel was recorded in the presence of 5 µmol/L E-4031. To achieve steady-state block, oocytes were equilibrated in the presence of drug (100 nmol/L, 500 nmol/L, 1 µmol/L, and 5 µmol/L) and held for 10 minutes at -35 mV for Merg1a and Merg1a/Merg1b and at -30 mV for Merg1b (near the voltages of half-maximal activation; see Reference 18). A single step to +20 mV was then presented, followed by a step to -100 mV. D, Channel block measured as a reduction in the peak of the inward tail current, normalized to the unblocked tail currents, and plotted as a function of [E-4031]. The IC50 values (millimolar) and Hill coefficients, determined from a sigmoidal fit for each isoform, are as follows: Merg1a, 496±51 mmol/L and 1.4±0.2 (n=7); Merg1b, 315±48 mmol/L and 1.0±0.1 (n=6); and Merg1a/Merg1b, 532±78 mmol/L and 1.0±0.15 (n=6). Scale bars are 1 µA and 250 milliseconds.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
We have cloned three isoforms of Merg1 from the mouse heart and expressed them heterologously in Xenopus oocytes. Merg1a is structurally and functionally homologous to HERG. Merg1a' lacks the first 59 amino acids of Merg1a and has the rapid deactivation kinetics characteristic of N-terminal deletions of HERG.8,32 This suggests that one area of the channel that modulates the deactivation kinetics lies in these first 59 amino acids and likely requires translation of exon 1a. Merg1b begins at an alternate site downstream in the gene, has an N-terminal cytoplasmic domain that is distinct from that of Merg1a, and is expressed in a cardiac-specific manner. Western blots have raised the possibility of other ERG isoforms,33 but their size and tissue distribution are not consistent with Merg1b. Merg1b has rapid deactivation kinetics when expressed in Xenopus oocytes and coassembles with Merg1a to form heteromeric channels with rapid deactivation kinetics. Although a specific region of the N-terminal cytoplasmic domain of HERG has been shown to play a role in HERG subunit assembly,34 our results show that other sequences or domains must also control assembly of Merg1b into homomers and heteromers.

Merg1b expression levels in Xenopus oocytes were markedly lower than those of Merg1a. Although we cannot be certain that injection of similar amounts of RNA for each isoform produces similar quantities of channel protein, this finding may indicate that polypeptides containing shorter or truncated N-termini are less stably assembled as functional homomeric channels. In addition, Merg1b expression results in measurable currents only when the putative translation initiation site is preceded by a Kozak consensus sequence. This discrepancy may by explained by a lower expression level of the Merg1bK- construct, as would be expected in the absence of the Kozak consensus sequence. Alternatively, translation of the Merg1bK- construct may be initiated at AUG codons which lie downstream to amino acid 59 and result in even shorter isoforms. However, constructs both with and without the Kozak consensus sequence are apparently expressed sufficiently to coassemble with Merg1a and form heteromeric channels.

IKr generally has rapid deactivation kinetics.4,10-15,17 Rapid deactivation of IKr is essential for maintaining electrical excitability and homogeneity, especially at the high heart rates normally found in the mouse. Coexpression of Merg1b and Merg1a produces channels with deactivation kinetics that are much faster than those recorded for Merg1a or HERG alone and that match the physiological profile of IKr for mouse myocytes.13 This, along with the simultaneous expression of both isoform transcripts in the heart, supports the hypothesis that Merg1a and Merg1b coassemble in vivo to form the channel responsible for IKr in the mouse.

Differences in IKr deactivation kinetics have been reported between species.16 Our findings provide a plausible explanation for this variability in the properties of IKr. N-terminal truncated isoforms of Erg may be expressed at higher levels in cardiac myocytes that have a more rapid deactivation of IKr. Erg isoform expression may also contribute to gender and developmental differences in the QT interval of certain species.

A surprising degree of K+ channel heterogeneity is found in mammals. Factors contributing to this heterogeneity include (1) the existence of >50 genes encoding {alpha}-subunits,35-37 (2) alternate splicing of some K+ channel genes, including KvLQT1, which also causes LQTS,36,38 (3) coassembly of related {alpha}-subunits to form heteromultimers,39-42 and (4) ß-subunits that modify channel function.43-45 In the heart, it is likely that subunits encoded by different genes coassemble to form native channels, such as GIRK1 with CIR to produce the acetylcholine-sensitive K+ current (IKAch)46 and KVLQT1 with minK (IsK) to produce the slowly activating component of the delayed rectifier K+ current (IKa).47,48 In the present study, we describe coassembly of tissue-specific alternate isoforms of a single gene to form channels with properties similar to IKr. Coassembly of Merg1a with Merg1b produces channels with a unique kinetic profile restricted to the heart. Along with the examples cited above, our findings emphasize the caution required when equating a single gene product to a cardiac current.

The mechanism by which HERG mutations cause LQTS remains uncertain: possibilities include (1) a gene dosage effect, with heterozygotes having inadequate functional channels and outward repolarizing current, and (2) a dominant-negative mechanism, with mutated HERG subunits coassembling with wild-type subunits of HERG and possibly with subunits of related channels to form nonfunctional channels.3,34,49 A recent study showed that not all HERG mutations appeared to act in a dominant-negative manner when mutant and wild-type RNAs were coinjected into Xenopus oocytes.50 The present study suggests that at least two N-terminal HERG isoforms are present in the heart and may be important in determining the mechanism by which mutations cause LQTS. For example, one isoform may act in a dominant-negative manner even though another does not. In addition, although experimental screening bias may contribute, all HERG mutations associated with LQTS are in the region of the channel common to all of the isoforms.3,51-53 Thus, it may be necessary for a mutation to affect all of the isoforms to cause the autosomal-dominant disease phenotype. Alternatively, mutations that affect the expression level or functional properties of a cardiac-specific isoform similar to the one described in the present study may in some cases directly cause LQTS.


*    Selected Abbreviations and Acronyms
 
CNB = cyclic nucleotide binding
Erg = ether-a-go-go–related gene
HERG = human Erg
IKr = rapid component of cardiac delayed rectifier K+ current
LQTS = long-QT syndrome
Merg = mouse Erg
PCR = polymerase chain reaction
RFLP = restriction fragment–length polymorphism


*    Acknowledgments
 
This study was supported in part by National Heart, Lung, and Blood Institute grants K08 HL-02843 (Dr London) and R01 HL-55793 to 01 (Dr Robertson), a Child Health Research grant from the Charles H. Hood Foundation (Dr London), a predoctoral fellowship (96-F-Pre-23) from the American Heart Association, Wisconsin Affiliate, Inc (M. Trudeau), a gift from the Bayer Corp (Dr Robertson), and funding from the National Cancer Institute, Department of Health and Human Services, under contract with Advanced Bioscience Laboratories Inc (Dr Jenkins). Sequences for the Merg1 clones have been submitted to GenBank under the following accession numbers: AF012868 (Merg1a), AF012869 (Merg1b), and AF012870 and AF012871 (Merg1 genomic clones). We thank Mark R. Vesely and Richard Schell for their technical assistance; Arthur M. Feldman, Jinling Wang, Craig January, and Zhengfeng Zhou for their helpful discussions; Alexandre F. R. Stewart for his comments on the manuscript; and Eisai Research Laboratories (Japan) for their generous gift of E-4031.

Received May 6, 1997; accepted August 29, 1997.


*    References
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 *References
 

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