(Received for publication, June 24, 1996, and in revised form, October 1, 1996)
From the Department of Physiology and Department of
Neuroscience, The Johns Hopkins University School of Medicine,
Baltimore, Maryland 21205
HERG (uman
-
elated
ene)
encodes an inward-rectifier potassium channel formed by the assembly of
four subunits. Since the truncated HERG protein in patients
with long QT syndrome induces a dominant phenotype, that is, cardiac
sudden death, the assembly of nonfunctional complexes between wild-type
and mutated subunits was implicated in causing the disease. To
understand HERG-mediated cardiac sudden death at the
molecular level, it is important to determine which regions in the
HERG protein participate in subunit interaction. We
therefore report the identification of a subunit interaction domain,
NABHERG, that is localized at the hydrophilic cytoplasmic N
terminus and can form a tetramer in the absence of the rest of the
HERG protein. Truncated HERG proteins
containing NABHERG, including one that resulted from the
1261 human mutation, inhibit the functional expression of the
HERG channel in transfected cells. Together, these results support the notion that the expression of HERG in the human
heart may be decreased in the presence of the truncated subunit. Such a
decrease of potassium channel expression can contribute to the longer
QT intervals observed in the patients with the HERG
mutation.
Human eag-related gene (HERG) was first cloned on the basis of its homology to the Drosophila ether a go-go (eag) channel (1), a member of the eag potassium (K+) channel family. This group of proteins contains six putative transmembrane segments that are flanked by the cytoplasmic N-terminal and C-terminal domains (2, 3). Despite its overall topological similarity to the eag and Shaker-like outward K+ channels, electrophysiological studies have shown that HERG subunits form inward-rectifier K+ channels (4, 5), which are generally made up of a different class of K+ channel subunits with only two transmembrane segments (6). In contrast to the Shaker-like channels, little is known about the region or regions that mediate the subunit assembly for either eag or inward-rectifier K+ channels.
Recently, HERG mutations were found in patients with chromosome 7-linked long QT syndrome, and it was proposed that the HERG dysfunctions caused cardiac arrhythmia (7). Because the biophysical properties of the HERG channel expressed in Xenopus oocytes resemble a well-characterized, rapidly activating, delayed-rectifier K+ current (also called IKr) found in cardiac myocytes (8), HERG probably encodes subunits of cardiac IKr channels. Since a decrease in IKr current could induce the longer interval typical of that seen in patients with long QT syndrome, it was proposed that the dominant disease phenotype could result, at least in part, from the nonfunctional subunit assembly of the mutated HERG protein with its compatible functional subunits (9). Thus, identifying the region or regions in the HERG protein involved in subunit interaction may help us understand the cause of chromosome 7-linked long QT syndrome at the molecular level.
Expression of the glutathione S-transferase (GST)1 fusion protein was carried out using pGEX-4T2 vector (Pharmacia Biotech Inc.). Vectors in which exogenous gene expression was driven by human cytomegalovirus (CMV) immediate early promoter were used to carry out all transient transfection experiments, and standard recombinant DNA techniques (10, 11) were used for plasmid vector construction. The vectors that express partial cDNA fragments were constructed by the high fidelity polymerase chain reaction cloning strategy that we have previously described (12). The NABHERG coding sequence was obtained by using ML1095 (GGGTCGACAATGCCGGTGCGGAGG) and ML1129 (CAGGCGGCCGCCTACTTCTCCATCACCACC) primers. The coding sequence for N-Herg was obtained by using ML1103 (CAGGAATTCCTCAGGATGCCGGTGC) and ML1104 (CAGGAATTCGTGGATGCGCGGTGC) primers.
The CMV promoter in the pRc/CMV vector (Invitrogen) was used to express
cDNAs encoding the N-terminal domain of HERG (or N-Herg) and 1261. In the N-Herg expression vector, 12CA5 monoclonal epitope (PYDVPDYASL) was fused to the C terminus, while for
1261, 12CA5 monoclonal epitope was added to the N terminus. The expression vector
for intact HERG was constructed by first releasing the HERG full-length cDNA from pSP64.HERG (from Dr. Mark
Keating, University of Utah) (7, 8) with HindIII and
EcoRI restriction enzymes. The cDNA fragment was then
subcloned into the pRc/CMV vector (Invitrogen).
The 1261 mutation
was constructed by a polymerase chain reaction-based mutagenesis
strategy. The HERG coding sequence was amplified by
5
-ML1124 and 3
-ML1125 primers. The 3
-primer
(CAGGGATCCTCAGCAGGAAGGCAGCCGAGTAGGGTGTGAAGACAGCCGGTAGATGACCAGC) contains a single-base deletion at a position corresponding to position
1261 in HERG. Thus, the resultant fragment encodes a protein
with an amino acid sequence equivalent to that of
1261.
Fusion proteins and thrombin cleavage were obtained by standard protocol for GST fusions provided by manufacturer (Pharmacia Biotech Inc).
Gel Filtration ChromatographyProtein in 0.5 M NaCl was separated by FPLC on Superdex-200 in a running buffer containing 20 mM HEPES pH 7.5, 500 mM NaCl, 2 mM EDTA, 1 mM 2-mercaptoethanol, and 0.5 mM phenylmethylsulfonyl fluoride. In a typical separation, 100 µg of protein in 200 µl of running buffer was loaded. The column was developed at a flow rate of 0.5 ml/fraction/min.
Chemical Cross-linkingPurified fusion protein in a buffer containing 20 mM HEPES pH 7.5, 100 mM KCl, 2 mM EDTA, and 1 mM 2-mercaptoethanol was subjected to chemical cross-linking experiments using glutaraldehyde. The reaction was initiated by mixing an identical amount of protein (1 µg in 20 µl) with a stock solution of glutaraldehyde to a final concentration of 0.005% to 1%. After a 1-h incubation at room temperature, the reaction was quenched by adding 1 volume of 1 M glycine. The resultant protein preparations were separated by polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS-PAGE) according to standard procedures.
Tissue Culture, Transfection, and ImmunoblottingThe expression and immunoblot analysis were performed according to published protocols (13, 14).
Whole-cell Patch Clamp RecordingWhole-cell recordings were
carried out according to a published protocol (5, 14, 15). A typical
pulse protocol was designed according to the work of Smith et
al. (5). After the gigaohm seal was achieved, the cell was first
held at 77 mV, and the holding voltage was then jumped from this
potential up to a long depolarizing potential of +13 mV for 600 ms,
followed by decreasing test potentials from
57 mV to
137 mV at
20-mV increments. Current data were filtered at 1 kHz, digitized at 100-µs intervals, and stored in a computer (Dell 486/33) for later analysis. After basal leak current was subtracted, data were
transferred to SigmaPlot (Jandel Scientific Software) for final
analysis. The current density was then calculated from the
capacitance of the recorded cells and the peak amplitude at
117
mV.
One mutation found in patients with chromosome 7-linked long QT
syndrome is a single-base deletion at 1261 (1261), which introduces
a frameshift at amino acid 421 of HERG (7). The resultant
protein contains the first 420 amino acids of HERG (mostly its N-terminal hydrophilic domain), followed by a peptide of
RLSSHPTRLPSC. The protein is truncated at amino acid position 432 because of a stop codon. In a heterozygotic patient, this mutation
resulted in a dominant phenotype of cardiac sudden death (7). One
possible cellular mechanism for this phenomenon is that the truncated
1261 protein might associate with the intact subunit to alter the
expression or properties of the wild-type subunit. If this is the case,
it is likely that there is an embedded region or regions in the
1261 protein for subunit interaction. Amino acid comparison of
HERG and other eag-related genes has revealed
significant homology, including a stretch within the N-terminal region
(1). This conserved region, which we called NABHERG,
corresponds to amino acids 1 to 135 in HERG.
Since topologically comparable regions in the Shaker-like
K+ channels are involved in subunit assembly (12), it would
be interesting to test whether this region is involved in subunit interaction as well. We expressed this coding fragment as a GST fusion
protein in Escherichia coli. This fusion protein, with an
apparent molecular weight of 39,700 (consistent with the predicted size), was affinity-purified on glutathione-Sepharose beads (Fig. 1A, lane 2). Since the GST portion
and the conserved domain (NABHERG) are linked by a peptide
containing a cleavage site for a sequence-specific proteinase,
thrombin, the purified protein was digested with thrombin to yield two
polypeptides (29.7 and 12.8 kDa) corresponding to the GST protein and
NABHERG (Fig. 1A, lane 3). The thrombin digestion of this fusion protein was highly specific, since prolonged incubation with enzyme yielded no additional proteolytic species (Fig.
1A, lanes 3-6), and mock digestion revealed no
detectable protein degradation (Fig. 1A, lane
7).
If the conserved HERG region is involved in subunit interaction, it may be sufficient to form oligomers in the absence of the rest of the protein, similar to other subunit assembly domains of Kv1 to Kv4 subfamilies of Shaker-like channels (17). To test this hypothesis, we separated the GST-NABHERG on a Superdex-200 gel filtration column by FPLC and developed the column under high-salt conditions (0.5 M NaCl) to reduce nonspecific hydrophilic protein-protein interactions. The A280 chromatogram revealed a major peak corresponding to the Stoke's radius of a 160-kDa globular protein (Fig. 1B, open circles). When protein in each fraction was separated by SDS-PAGE followed by silver staining, the major peak was indeed the purified GST-NABHERG protein (39.7 kDa). This result indicates that GST-NABHERG is an oligomer.
Since the GST portion of the fusion protein may contribute to the protein-protein interaction, we performed similar experiments using pure NABHERG protein (12.8 kDa) after purification on a Mono Q column (Fig. 1A, lane 8; also see "Experimental Procedures"). The A280 chromatogram of the Superdex-200 separation showed only a single NABHERG protein peak corresponding to a globular protein with a molecular weight of 40,000 (Fig. 1B, closed circles). Thus, oligomer formation is mediated by NABHERG.
The uniform migration profile of GST-NABHERG and
NABHERG in the gel filtration column (Fig.
1B) supports the hypothesis that oligomers were formed via
these specific interactions. Since both Shaker-like K+
channels and inward-rectifier K+ channels are tetramers
(18, 19, 20), it is likely that the HERG homomultimeric channel
also contains four subunits. To test whether the oligomers observed in
gel filtration analysis are tetramers, we used either purified
GST-NABHERG or NABHERG protein to perform
chemical cross-linking experiments. Fig. 2A
shows SDS-PAGE analysis of the GST-NABHERG protein (39.7 kDa)
after incubation with different concentrations of glutaraldehyde (see
"Experimental Procedures"). As the concentration was increased, two
discrete additional protein species, with apparent molecular masses of
80.5 and 150 kDa (which agree with the sizes of dimers and tetramers),
were detected. Similarly, when NABHERG (12.8 kDa) was subjected
to the same analysis, we observed additional polypeptides with higher
molecular masses of 23.1 and 44.9 kDa, which correspond to the sizes of
dimers and tetramers (Fig. 2B). Taken together, these
results showed that oligomeric NABHERG is a tetramer.
A region involved in subunit interaction may participate in several
cellular processes including but not limited to subunit assembly.
Depending on the detailed assembly pathway of a specific oligomeric
protein, coexpression of a truncated form together with its intact
subunit or subunits can result in inhibition of the expression of
functional oligomers to different degrees, since the truncated protein
can compete for subunit interaction. This dominant suppression in
channel expression has been observed in transiently transfected cells
that express the Shaker-like K+ channels and the
acetylcholine receptor (21, 22). Since sequence data indicate that the
human 1261 mutation results in a truncated N-terminal domain of the
HERG subunit, it would be particularly interesting to test
whether this truncated protein containing NABHERG is capable of
inhibiting the functional expression of HERG.
We expressed HERG in COS cells using conventional transient
transfection procedures, and a whole-cell voltage clamp was used to
record the characteristic current induced by HERG (4, 5). The HERG channel was expressed alone or in the presence of
either the N-terminal domain of HERG (N-Herg, amino acids 1 to 396) or 1261 (see "Experimental Procedures" for detailed
amino acid positions). Within each group, we recorded from more than 29 transfected cells that expressed HERG currents. Consistent
with previous results obtained in Xenopus oocytes, the
electrophysiological properties of HERG channels showed no
detectable changes in the presence or absence of truncated proteins
(Fig. 3, A-D) (9).
When the current density within each group was determined and plotted
against the number of cells in the percentage of cells, we found that
either N-Herg or 1261 shifts the overall distribution of current
density to a lower level (Fig. 3, E-H). The current densities for these three groups are 42.7 ± 4.95 pA/pF (mean ± S.E., n = 33) for HERG + control plasmid,
29.1 ± 4.99 pA/pF (mean ± S.E., n = 29) for
HERG +
1261, and 22.5 ± 3.54 pA/pF (mean ± S.E., n = 29) for HERG + N-Herg. Although
transfection efficiency can vary by experiment, the distribution of
current density among cells positive for HERG current is
relatively consistent between different experiments. The decrease of
current density is not a result of alteration of cell surface area,
since there is no significant difference in capacitance among the three
groups. Therefore, both N-Herg and
1261 containing NABHERG
do inhibit the expression of HERG (p < 0.05 for
1261 and p < 0.001 for N-Herg), presumably by
competing for the functional subunit assembly of HERG
channels. The decrease of current density of HERG channels
could also result from nonselective inhibition by the
NH2-terminal domain of HERG. To test this, we
expressed HERG with
Kv
2-(39-316), a truncated Kv
2
subunit of Shaker-like potassium channel. Although the protein level of
Kv
2-(39-316) is comparable to that of N-Herg and higher than
that of
1261,
Kv
2-(39-316) did not inhibit the expression of
HERG (Fig. 3, J and I). To determine whether N-Herg and
1261 could nonspecifically inhibit surface expression of a membrane protein, we expressed membrane-bound CD4 (a T-cell surface antigen) with N-Herg. Using
fluorescence-activated cell sorting, we found comparable
CD4 surface expression in the presence or absence of N-Herg
(data not shown). Taken together, these results support the idea that
NABHERG plays a role in the subunit assembly of HERG
channels. The decrease of current density by
1261 seen in the
transfected cells may help explain the longer QT intervals seen in
patients with the HERG mutations.
The Shaker-like K+ channels and the HERG channels are clearly distinct classes of K+ channels in terms of both amino acid sequence and electrophysiological properties. Although the region or regions that mediate subunit assembly of either eag or eag-related channels are not known, it has been shown that the N-terminal domains of Shaker-like channels play critical roles in specifying the formation of heteromultimers (12). Systematic analysis of subunit interaction of the four major subfamilies (Kv1 to Kv4) of Shaker-like K+ channels has revealed the conserved motifs (NABKv1 to NABKv4) for the subfamily-specific interaction (17). Despite no detectable homology between NABHERG and that of Shaker-like K+ channels, the ability of NABHERG to form tetramers suggests remarkable similarity of assembly-domain arrangement between Shaker-like K+ and HERG channels. Given that NABHERG has considerable homology to comparable regions in eag and eag-like potassium (elk) channels (1), it would be interesting to see whether these regions play a role in the subunit interaction of the corresponding channels.
The decrease in K+ current density caused by either N-Herg
or 1261 supports the notion that NABHERG participates in
subunit assembly. One interesting question is why we did not observe a
more dramatic decrease in current density. Several mechanisms may
contribute to this apparent low-potency inhibition. From previous studies of Shaker-like K+ channels and acetylcholine
receptor, it is known that, in transfected cells, truncated subunits
without transmembrane segments have much lower potency in suppressing
functional channel expression than those with such segments (22). Since
truncated fragments without membrane-spanning segments are more likely
to be cytoplasmically soluble and non-membrane-bound, this difference
was generally attributed to the potential difference in their
targeting. In addition, both immunocytochemistry and immunoblot
analyses have shown that
1261 expressed considerably less than
N-Herg (Fig. 3J); yet
1261 and N-Herg in our assays have
similar inhibition potency.
How could a mutated HERG subunit alter the channel activity
in cardiac tissue to cause disease? A systematic approach to address this question would be to identify and examine all potential
perturbations, including both loss and gain of a function, caused by
the mutation. Perhaps a combination of changes alters certain aspects
of cardiac function, which in concert contribute to cardiac sudden
death. The effects of the 1261 mutant on HERG channel
expression have previously been examined in a Xenopus oocyte
system, and no suppression of channel expression was detected (9). It
would be interesting to try to determine what accounts for the
difference in expression between the mRNA-injected oocytes and the
DNA-transfected mammalian tissue culture cells. Further, previous
evidence has suggested that additional factors help define the
IKr, since the HERG channel expressed in oocytes
does not have the same pharmacological properties as IKr
does. One interesting example is the
N-methyl-D-aspartate receptor R1 (NR1), which by
itself is capable of forming a functional glutamate receptor channel,
with kinetic and pharmacological properties similar to those of native
channels (23). However, more detailed molecular and biochemical
analysis has revealed that the native N-methyl-D-aspartate receptors are in fact
mostly heteromultimers of NR1, NR1 splice variants, and various NR2
subunits (24, 25). Thus, future biochemical analysis of the native
HERG channel is essential to our understanding of the
HERG function in the human heart.
We thank Dr. Mark Keating for HERG
cDNA and Magdalena Bezanilla for N-Herg and Kv
2-(39-316)
constructs. We also thank Drs. Daniel Raben and Peter Gillespie and
members of the Li laboratory for helpful suggestions.