From the Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706 and the § Department of Biology, Brandeis University, Waltham, Massachusetts 02254
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ABSTRACT |
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Assembly of K channel subunits of the
Shaker (Sh) family occurs in a subfamily
specific manner. It has been suggested that subfamily specificity also
applies in the association of
subunits with Sh channels
(Rhodes, K. J., Keilbaugh, S. A., Barrezueta, N. X.,
Lopez, K. L., and Trimmer, J. S. (1995) J. Neurosci. 15, 5360-5371; Sewing, S., Roeper, J. and Pongs, O. (1996) Neuron 16, 455-463; Yu, W., Xu, J., and Li, M. (1996) Neuron 16, 441-453). Here we show that the
Drosophila
subunit homologue Hyperkinetic (Hk) associates with members of the ether à
go-go (eag), as well as Sh, families.
Anti-EAG antibody coprecipitates EAG and HK indicating a physical
association between proteins. Heterologously expressed Hk
dramatically increases the amplitudes of eag currents and
also affects gating and modulation by progesterone. Through their
ability to interact with a range of
subunits, the
subunits of
voltage-gated K channels are likely to have a much broader impact on
the signaling properties of neurons and muscle fibers than previously
suggested.
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INTRODUCTION |
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Voltage-gated ion channels can be profoundly affected by the
presence of auxiliary subunits (1). For voltage-gated K
(Kv) channels, affinity purification and
immunoprecipitation of mammalian -dendrotoxin-sensitive complexes
has identified two
subunits, Kv
1 and
Kv
2, that associate with
subunits of the
Sh family (2). Subsequent studies of
-
interactions
have suggested that coassembly with this class of
subunits is
restricted to the Kv1 subtype of
subunits (3-5).
Several Kv
subunits have been identified including
Drosophila, rat, ferret, bovine, and human subunits
(6-10).
The Drosophila Kv subunit Hk
shares ~42% amino acid identity with rat Kv
1 and
~48% identity with rat and bovine Kv
2 (6). Mutations
of the Hk locus result in ether-sensitive leg shaking and
hyperexcitability in nerve and muscle, characteristics that are shared
by Sh and eag mutants (11). SH:HK heteromultimers in vivo and expressed in oocytes exhibit currents that are
increased in amplitude with a voltage dependence that is shifted to
more negative values and more rapid kinetics when compared with the currents of SH alone (6, 12). It is not clear, however, whether these
changes are sufficient to account for the full range and severity of
the phenotypic defects observed in Hk mutants. For example,
the leg shaking of Hk mutants occurs in a cyclical pattern; this cyclical pattern is superimposed on the leg shaking of
Sh in Sh:Hk double mutants (13). We
therefore sought to determine whether HK can interact with other
subunits in addition to SH. In immunoprecipitation experiments, we
found that HK can associate with EAG
subunits. The functional
consequences of this association included an increase in eag
current amplitude, an acceleration of activation kinetics, and
"protection" from a down-modulation in eag current
amplitude that was otherwise observed in response to treatment with
progesterone. We also found that the ability to interact with
Hk was conserved in mouse eag (meag)
and the human eag-related gene, Herg. Some of the
data have been presented in abstract form (14).
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EXPERIMENTAL PROCEDURES |
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Generation of Antibodies--
For production of anti-EAG
antibody, fusion proteins containing the NH2-terminal
portion of EAG (residues 44-210) along with a six-histidine tag
(His-EAG-N) or glutathione S-transferase (GST-EAG-N) were
generated. Proteins were expressed in Escherichia coli and purified using standard protocols (15). 250 µg of GST-EAG-N was
injected into rabbits. Antibody was purified by passing the serum
through a nickle column to which His-EAG-N was bound and eluted
according to established procedures (Ref. 15, metal-chelate affinity
chromatography, 10.11B). For anti-Hk antibody, an Hk cDNA, HC208 (6), was modified by polymerase chain reaction (Taq polymerase, Promega) to add an EcoRI site
and a bacterial ribosomal binding site upstream of the start AUG
(GAAAGAATTCGAAGGAGAAGCACAATG, underlining indicates changes from the original cDNA; antisense primer ATGGGATCCGCCTTGTGGATGATG). The polymerase chain reaction product
was digested with EcoRI and EcoRV (New England
Biolabs) and subcloned into HC208 which was then inserted into the
bacterial expression vector pPROK-1 (CLONTECH
Laboratories, Inc.). After 4 h growth in 0.5 mM
isopropyl-1-thio--D-galactopyranoside, bacteria were
lysed and treated with Benzonase (400 units; EM Science). The soluble
fraction was precipitated with 75% NH4SO4
(w/v). Protein was resuspended in column buffer (100 mM
NaCl, 20 mM Tris, pH 7.0, 5% ethylene glycol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM
dithiothreitol) and run over a Macro-Prep 50 CM column (Bio-Rad). The
flow-through was precipitated (75% NH4SO4),
resuspended in 10 mM NaPO4 (pH 6.8), 1 mM
-mercaptoethanol, and then applied to an
hydroxylapatite column (Bio-Rad) and eluted with a 10-500 mM NaPO4 gradient. The fraction enriched for HK
(monitored by SDS-PAGE)1 was
reapplied to the hydroxylapatite column, washed with 10-100 mM NaPO4, and re-eluted using steps from
125-500 mM NaPO4. Flow-through from the 150 mM elution contained essentially homogenous HK. Rabbits were inoculated with homogenized gel slices containing purified HK. An
HK affinity column was prepared by incubating enriched HK fractions
with Affi-Gel 10 (Bio-Rad). Crude rabbit sera was applied to the
affinity column, washed with 1 M NaCl, eluted with 100 mM glycine (pH 2.5), 10% ethylene glycol, and collected in phosphate-buffered saline.
Immunoprecipitation-- For expression in tsA201 cells, the vector contained the simian cytomegalovirus IE94 promoter/enhancer sequence upstream from the coding regions. A tag of 6 copies of the MYC-epitope was fused to the NH2-terminal of eag. Cells were cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) with 10% cosmic calf serum (HyClone Laboratories, Inc.) and transfected by the calcium phosphate method. 15 µg of DNA per 10-cm plate was used for each construct. Transfected cells were lysed and solubilized in buffer containing (in mM): 20 Tris-HCl, pH 7.5, 150 NaCl, 5 EGTA, 5 EDTA, 1% CHAPS, 1 dithiothreitol, 1 phenylmethylsulfonyl fluoride, and leupeptin, aprotinin, and pepstatin A at 1 µg/ml. 700 µg of total protein from each supernatant was incubated overnight at 4 °C with anti-EAG antibody. The immunocomplex was precipitated with 50% rProtein A Avidgel F (BioProbe International, Inc.) and extensively washed.
Electrophysiology--
For expression in Xenopus
oocytes, constructs were placed in the pGH19 vector (provided by E. Goulding) (6, 16), a version of pGEMHE containing the 5'- and
3'-untranslated regions of the Xenopus -globin gene.
Hk- and
subunit-containing plasmids were linearized
using NheI and NotI, respectively, and capped RNA
transcribed in vitro using T7 RNA polymerase (Promega).
Stage V-VI oocytes, obtained from adult females (Nasco, Ft. Atkinson,
WI) as described previously (6), were maintained in L-15 media
(containing 50% L-15 (Life Technologies, Inc.), 15 mM
Hepes, 1 mM L-glutamine, 50 mg/ml gentamycin,
and 5 mg/ml bovine serum albumin, pH 7.4, NaOH) at 18 °C. Oocytes
were injected with ~30 nl of solution containing mRNA for the
subunit and Hk or RNA for the
subunit and an equal
amount of diethyl pyrocarbonate-treated water (~1-6 ng of
RNA/oocyte). A 2-fold excess of Hk RNA was used to maximize coassembly. Two- and 3-fold increases in the amount of
subunit RNA
produced linear increases in current amplitudes. Recordings were
performed using an OC-725B amplifier (Warner Instrument Corp.) and
pCLAMP 6 (Axon Instruments). Currents were filtered at 1-2 kHz (
3
dB, 8 pole Bessel) and sampled at 5-10 kHz. Linear leak and
capacitative currents were subtracted using P/n methods. The extracellular recording solution typically contained, in
mM: 140 NaCl, 2 KCl, 1 MgCl2, 10 Hepes (pH 7.1, NaOH). An extracellular solution high in K+ was used to
enhance tail currents during measurements of the voltage dependence of
activation and to record Herg currents; this solution
contained, in mM: 100 KCl, 1.8 CaCl2, 1 MgCl2, 5 Hepes (pH 7.4, KOH). Pipettes (2 M
KCl) had resistances of 0.5-2 M
. Experiments were performed at room
temperature (19-22 °C). Staurosporine, H7, phorbol 12-myristate
13-acetate, and caffeine (Alexis Corp.), D609 (Calbiochem), and
progesterone were prepared as stock solutions using dimethyl sulfoxide
or extracellular recording solution. The concentration of dimethyl
sulfoxide in the bath did not exceed 0.1%.
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RESULTS AND DISCUSSION |
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Eag shares only 12% amino acid identity with Sh and distinctive structural features define it as a member of a separate family (17). To examine whether there is a physical association between HK and EAG, six copies of the MYC-epitope were fused in-frame to the amino-terminal of EAG in a mammalian expression vector. tsA201 cells were transfected with either eag, Hk, or both constructs and the resulting complexes were immunoprecipitated from cell extracts using anti-EAG antibody. Equal amounts of total protein were used for each immunoprecipitation as judged by immunoblot analysis with anti-ACTIN antibody (Fig. 1A, bottom). Proteins were separated by SDS-PAGE and the immunoblot probed with an antibody (9E10) that recognizes the MYC-tag. EAG protein was evident as an ~139-kDa band that was observed in the lanes corresponding to cells transfected with the eag construct (Fig. 1A, top). After stripping, the immunoblot was reprobed with antibody to HK. HK was observed only in the lane corresponding to cells transfected with both constructs (Fig. 1A, middle). There was no obvious cross-reactivity between the anti-EAG antibody and HK (Fig. 1B). A separate immunoblot of the same whole cell extracts revealed no difference in the level of HK expression in Hk and Eag+Hk transfected cells (not shown).
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The above results suggest that there is a physical interaction between EAG and HK. To determine the physiological effect of the interaction, we examined currents in oocytes injected with eag RNA, either alone or together with RNA encoding Hk. Fig. 2 shows the results obtained on day 3 postinjection for one batch of oocytes. Expression with Hk produced a 3.9-fold increase in the mean current amplitudes recorded in response to test pulses to +40 mV (Fig. 2, A and B). Because the voltage dependence of activation did not change in the presence of Hk (Fig. 2E), the increase in amplitude is likely to be a result of an increase in the number of functional channels or an increase in the single channel conductance.
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Hk also accelerated the activation of eag
currents. For the oocytes in Fig. 2, the time to 80% of the maximum
current was decreased from the control value of 51.5 ± 3.5 ms
(n = 5) to 37.8 ± 3.4 ms (n = 4;
test pulse, 0 mV). The rising phase was best fit by a sum of two
exponentials, 1 and
2; both were
significantly decreased (Table I), an
effect that was most dramatic at lower potentials (Fig. 2, C
and D). The activation rate of eag also has been
shown to vary with changes in the voltage preceding the test pulse.
Activation occurs more rapidly with more positive prepulses, an effect
that is reminiscent of the Cole-Moore shift observed for K currents in
the crayfish axon (18, 19). As shown for
1 (Fig.
2f), in the presence of Hk activation was faster at all prepulse potentials. Finally, eag currents exhibited
an inactivating component at test pulse voltages above +20 mV.
Inactivation was well described by a single exponential that was slowed
from 45.5 ± 4.7 to 54.0 ± 2.9 ms in the presence of
Hk (n = 5 and 4, respectively; test pulse,
40 mV; Fig. 2G). A detailed summary of results, averaged
across the four batches of oocytes examined using the same recording
solution, is given in Table I. The above changes in kinetics were
observed in oocytes with currents ranging from 1 to 15 µA.
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Hk not only affected the properties, but also the modulation, of eag currents. To examine the role of Hk in modulation, we used progesterone which alters the activity of a number of intracellular messengers to trigger the transition from the G2 to M phase of the cell cycle in a process referred to as maturation (20). When recording from oocytes injected with eag alone, progesterone (30 µM) decreased current amplitudes by 20-50% (mean fractional current remaining in the presence of progesterone = 0.68 ± 0.03, n = 14; e.g. Fig. 3A). The decrease was initiated within the first minute following application and current levels stabilized at the new lower level within 5-10 min (e.g. Fig. 3A, bottom). Currents were restored following washout of progesterone (n = 7). In contrast, when Hk was coexpressed with eag, progesterone (30 µM) failed to decrease amplitudes in 10 of the 12 oocytes examined even when currents were monitored for 30 min (mean fractional current remaining = 0.96 ± 0.02; n = 12; e.g. Fig. 3B). Thus, the association with Hk either reduced the sensitivity of eag channels to progesterone or inhibited the response. To further identify the intracellular messengers involved in the response to progesterone, oocytes were incubated in staurosporine (1-2 µM) or H7 (50 µM), two nonspecific serine-threonine kinase inhibitors, for 30-90 min prior to the application of progesterone. Both staurosporine and H7 failed to block the effect (n = 6 and 9, respectively, not shown). In addition, caffeine (10 mM), which increases calcium levels by releasing calcium from intracellular stores, decreased eag:Hk, as well as eag, currents and therefore did not mimic the action of progesterone (n = 3 and 11, respectively, not shown). Phorbol 12-myristate 13-acetate (20-50 nM), a phorbol ester that activates protein kinase C, failed to produce a consistent change in eag currents (n = 12, not shown). We therefore attempted to block the response to progesterone at an earlier point in the signaling cascade. D609 is a specific inhibitor of phosphatidylcholine-specific phospholipase C (21) and, in oocytes, breakdown of phosphatidylcholine is responsible for the majority of diacylglyerol released following progesterone treatment (22). Preincubation of eag-expressing oocytes in bath solution containing D609 (50 µg/ml, 1 h) resulted in a complete block of the response to progesterone in 10 of 12 oocytes examined (mean fractional current remaining in the presence of progesterone = 0.89 ± 0.06, n = 12; e.g. Fig. 3c).
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To determine whether the interaction with Hk is specific to
eag or whether the interaction with Hk transcends
both species and eag subfamilies, we examined the effects of
Hk on two other members of the eag family which
have been isolated and physiologically characterized, meag
and Herg (16, 17, 19, 23). With the exception of
inactivation which is not observed for meag, the properties
of meag currents affected by Hk were the
properties affected in the case of eag, namely current
amplitudes and activation kinetics (Fig.
4, A and B, Table
I). Herg, in contrast to eag and meag,
carries a predominantly inward K current and represents a distinct
subtype within the eag family (16, 17). The inward rectification of Herg channels has been suggested to be the
result of a "C-type" inactivation that is much more rapid than
activation (24). In the comparison of Herg in the presence
and absence of Hk, little difference in current was detected
when recording at days 2 and 3 postinjection. By day 6, however,
oocytes coexpressing Hk could be clearly distinguished from
controls. Hk increased current amplitudes by 6.8-fold (test
pulse, 110 mV), the largest increase observed for any of the
subunits tested (Fig. 4, C and D). An estimate of
the activation rate was obtained for a subset of oocytes by varying the
duration of a prepulse to +10 mV. The amplitude of the inward current
during the subsequent hyperpolarization to
110 mV, corrected for
deactivation and normalized to the maximum observed (see
"Experimental Procedures"), was used to determine the fraction of
channels that had passed from the closed to open state during the
preceding depolarization. The resulting data were well fit by a single
exponential with a time constant of 81.5 ± 2.3 ms in controls
(n = 7, Fig. 4D). In the presence of
Hk the time constant was decreased to 65.9 ± 6.2 ms (n = 4), indicating that Hk accelerates the
activation of Herg, as well as eag and
meag currents. In addition, Hk slowed the
time-to-peak of the inward current from 46.3 ± 2.3 to 58.8 ± 3.0 ms (n = 8 and 9, respectively; test pulse
90
mV). Exponential fits revealed that the slowing was largely a result of
a slowing of deactivation (Fig. 4D). Hk also
produced a modest ~+4 mV shift in the midpoint of the
Boltzman curve describing the voltage dependence of
activation with little change in slope (Fig. 4D). A summary
of the results, averaged across oocyte batches, is given in Table
I.
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In conclusion, in a comparison with rat Kv1 whose
primary effect on Kv1.1 appears to be an acceleration of
inactivation (2), the primary role of Hk is to produce a
substantial increase in current amplitudes for each
subunit we have
examined, including Sh (6). In fact, an increase in
amplitude is the most common consequence of the interaction between
and
subunits for all voltage-gated channels (1). Our results also
demonstrate that coexpression with Hk can affect channel
modulation and suggest that the role of Hk and other
subunits of this class cannot be fully understood by an examination of
changes in the basal properties of
subunits. In this case, Hk
"protects" eag currents from the down-regulation in
amplitude that is otherwise observed in response to progesterone.
Intriguingly, the pathways activated by progesterone in oocytes may be
the same pathways whose activation at the Drosophila
neuromuscular junction results in a prolonged alteration in K currents
lasting many minutes (25).
Recent studies examining the interactions between the mammalian
Kv subunits and the
subunits of K channels have
indicated that the
subunits associate specifically with the
Kv1 subtype of
subunits (3-5). Our results suggest
that Kv
subunits may be more promiscuous and that they
associate with
subunits from at least two families. Of interest is
whether the interactions between Kv
subunits and
eag family members arise in vivo and whether the
potential to interact with Herg is conserved in any of the
mammalian Kv
subunits. The Herg gene is the
locus of one form of the inherited cardiac disorder known as long QT
syndrome (LQT-2) (26) and, in our experiments, was the most profoundly affected by Hk. Recent evidence suggests overlap in the
expression of the mammalian
and
counterparts (2, 3, 6-9, 17, 18, 26, 27) and some alleles of the seizure (sei)
locus, which encodes the Drosophila erg homologue (28, 29),
exhibit an enhanced temperature sensitivity in
Hk:sei, but not Sh:sei, double mutant combinations.2
Therefore, at least in Drosophila, there is an in
vivo interaction between Hk and sei that is
independent of the effects of Hk on Sh channels.
The average increase in Herg current amplitude produced by
Hk is substantially in excess of the recently reported
increase observed as a consequence of the association with minK (30). Thus, our results predict that this class of
subunits would make a
large contribution to cardiac repolarization.
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ACKNOWLEDGEMENTS |
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We thank K. McCormack and J. W. Hell for comments on the manuscript.
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FOOTNOTES |
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* This work was supported by grants from the Wisconsin Affiliate of the American Heart Association (to S. W. C. and G. F. W.), Muscular Dystrophy Association (to G. F. W.), and National Institutes of Health (to L. C. G. and B. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 445 Henry Mall,
Laboratory of Genetics, University of Wisconsin, Madison, WI 53706. Tel.: 608-265-9034; Fax: 608-262-2976.
1 The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesuflonic acid.
2 R. Kreber and B. Ganetzky, unpublished observations.
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REFERENCES |
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