1 Yale University School of Medicine and West Haven Veterans Administration Medical Center, New Haven, Connecticut 06510; and 2 University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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Voltage-gated K+ (Kv)
channels are heteromultimeric complexes consisting of pore-forming
-subunits and accessory
-subunits. Several
-subunits have been
identified and shown to interact with specific
-subunits to modify
their levels of expression or some of their kinetic properties. The aim
of the present study was to isolate accessory proteins for KCNA10, a
novel Kv channel
-subunit functionally related to Kv and cyclic
nucleotide-gated cation channels. Because one distinguishing feature of
KCNA10 is a putative cyclic nucleotide-binding domain located at the COOH terminus, the entire COOH-terminal region was used to probe a
human cardiac cDNA library using the yeast two-hybrid system. Interacting clones were then rescreened in a functional assay by
coinjection with KCNA10 in Xenopus oocytes. One of these
clones (KCNA4B), when injected alone in oocytes, produced no detectable current. However, when coinjected with KCNA10, it increased KCNA10 current expression by nearly threefold. In addition, the current became
more sensitive to activation by cAMP. KCNA4B can be
coimmunoprecipitated with the COOH terminus of KCNA10 and full-length
KCNA10. It encodes a soluble protein (141 aa) with no amino acid
homology to known
-subunits but with limited structural similarity
to the NAD(P)H-dependent oxidoreductase superfamily. KCNA4B
is located on chromosome 13 and spans ~16 kb, and its coding region
is made up of five exons. In conclusion, KCNA4B represents the first
member of a new class of accessory proteins that modify the properties
of Kv channels.
cyclic nucleotide; Xenopus oocyte; human; channel regulation
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INTRODUCTION |
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POTASSIUM CHANNELS are membrane proteins that regulate membrane potential and contribute to many important cellular processes. KCNA10 is a voltage-gated K+ (Kv) channel gene related to the Shaker superfamily (10). The encoded protein has 58% amino acid identity with Kv1.3, and its predicted secondary structure is identical to that of other Shaker-related K+ channels. Its most distinguishing feature is the presence of a putative cyclic nucleotide-binding (CNB) domain at the COOH terminus, suggesting that channel function might be regulated by cyclic nucleotides. The fact that a few other K+ channels also contain CNB domains hints at the existence of a new subclass of K+ channels with structural features common to Kv and cyclic nucleotide-gated (CNG) cation channels (6, 19, 20, 21). The best-studied of these is HERG, which binds cAMP, regulates cardiac repolarization, and is mutated in some cases of long QT syndrome (15, 16, 18). Other potential physiological roles for these proteins include arterial vasorelaxation (4), hormone secretion (8, 23), and neuronal membrane excitability (17).
We recently characterized the kinetics and pharmacological properties
of the -subunit of KCNA10 (10). When expressed in oocytes, it mediates Kv currents that exhibit minimal steady-state inactivation. Channels can be activated by depolarizations more positive than
30 mV, with half-activation at 3.5 ± 2.5 mV. The channel displays an unusual inhibitor profile, since in addition to
being blocked by classic K+ channel blockers, it is also
sensitive to inhibitors of the CNG cation channel, such as verapamil
and pimozide. Tail current analysis indicates that KCNA10 has a
K+-to-Na+ selectivity ratio of at least 15:1.
The single-channel conductance is ~11 pS, and channel activity is
inhibited by protein kinase C. These data reflect the properties of the
-subunit of KCNA10 expressed in Xenopus oocytes. It is
likely that they will be modified by additional subunits or even by a
different cellular environment. Numerous studies have shown that the
properties observed for cloned
-subunits very rarely match currents
known to exist in native tissues (7). Most often, it is
because the
-subunits interact with accessory proteins that modify
their kinetic and pharmacological properties. Furthermore, accessory
proteins often modulate the levels of expression of the
-subunits.
Several -subunits have been characterized. The Kv
genes encode
soluble proteins (367-404 aa), related to the NAD(P)H-dependent oxidoreductase superfamily, that interact with many Kv
-subunits (13). The KChIP genes encode Ca2+
sensors that modify the expression level and kinetic properties of
Kv4.2 and Kv4.3 (9). The KChAP proteins act as chaperones for Kv proteins such as Kv1.3, Kv2.1, Kv2.2, and Kv4.3 (9, 22). The MinK-related proteins also interact with Kv channels (1-3, 5, 15, 16).
Here we used the yeast two-hybrid method to identify an accessory
protein (KCNA4B), unrelated to other Kv -subunits, that binds to the
COOH terminus of KCNA10 and modifies the channel's kinetic properties.
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MATERIALS AND METHODS |
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Yeast two-hybrid library screen.
The yeast strain AH109, plasmids (pGBKT7:GAL4 DNA binding domain vector
and pACT2:GAL4 transcription activation domain vector), and human heart
cDNA library (cloned in pACT2) used in the two-hybrid experiments were
obtained from Clontech. The cDNA encoding the last 54 aa of KCNA10's
COOH terminus (C-TerKCNA10) was ligated in-frame into the
EcoRI/BamHI sites of pGBKT7. C-TerKCNA10/pGBKT7, together with the human heart cDNA library, was used to transform AH109. Transformants were plated on synthetic medium lacking
tryptophan, leucine, histidine, and adenine but containing
5-bromo-4-chloro-3-indoyl--D-galactopyranoside (20 mg/l) and 3-aminotriazole (7.5 mM). Blue colonies were selected after incubation for 3 days at 30°C. Plasmid DNA was isolated from
blue colonies and retested for interaction with the COOH terminus of
KCNA10 by retransformation into the AH109 strain. The cDNA inserts from
truly positive clones were sequenced by the dideoxy chain termination
method of Sanger et al. (14).
In vitro transcription, translation, and binding analysis of the
COOH terminus of KCNA10 and putative positive clones.
C-TerKCNA10/pGBKT7 also contains a T7 promoter and a 5' c-Myc epitope
tag. The plasmid was linearized with BamHI, and cRNA was
synthesized using T7 polymerase (Ambion). To prepare cRNA for the
library positives, each clone was amplified by PCR using a sense primer
(5-TCTTTAATACGACTCACTATAGGGCGAGAGATCTGTATGGCTTACCCATAC-3), which incorporated a T7 promoter sequence upstream of the
hemagglutinin (HA) epitope tag and an antisense primer provided by the
manufacturer. The PCR products were used as a template for cRNA
synthesis using T7 polymerase (Ambion). In vitro translation was
carried out for each individual cRNA using rabbit reticulocyte lysates
in the presence of 35S (Ambion). In some studies,
individual library positives and the COOH terminus of KCNA10 cRNAs were
mixed and translated together. A maximum of 500 ng of cRNA was used for
each 25-µl reaction. Protein-protein interactions were detected by
immunoprecipitation, as described below. Reactions were incubated
overnight at 4°C with 10 µl of 0.1 µg/µl anti-HA (Roche) or 10 µl of 0.1 µg/µl anti-c-Myc (Roche) antibody diluted in 0.5 ml of
IP buffer (150 mM NaCl, 50 mM Tris, pH 7.5, 1 mM dithiothreitol, 5 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml
leupeptin, 0.1% Tween 20). Immune complexes were then incubated with
20 µl of protein A-Sepharose (Roche) for 2 h at 4°C. After
four washes in 3 M NaCl, 50 mM Tris · HCl, pH 7.5, and 1%
Tween 20 buffer, bound proteins were eluted by boiling in SDS sample
buffer and analyzed on 12% SDS-polyacrylamide gels. The radiolabeled
proteins were detected by using an Instant Imager (Packard Instruments,
Meriden, CT) or by exposing film at 70°C. In control experiments,
we verified that the KCNA10 construct did not activate the reporter
gene (data not shown). We also confirmed that the construct could be
transcribed and translated in vitro and that a fusion protein of the
correct size could be immunoprecipitated using a commercially available monoclonal antibody raised against the c-Myc epitope tag (see Fig.
3A).
In vitro transcription and translation of full-length
KCNA10-positive clone KCNA4B.
The coding region of KCNA10 (accession no. U96110) was
amplified by PCR with a proofreading polymerase and a sense primer containing an HA epitope tag and XhoI site and an antisense
primer with a KpnI site:
5-GATCCTCGAGCCTAGAATGGATTACCCGTACGACGTT- CCGGACTACGCTGTGTGTGGCTGGAAAGAAAT-3 (sense) and 5-GATCCTCGAGCCTGGCTCCCCTAGAATGGATGTG-3
(antisense). The amplified product was ligated into PSD64TF, a
high-expression vector based on pGEM (Promega, Madison, WI)
containing 60 bp of the 5'-untranslated region (UTR) of
Xenopus -globin and 300 bp of the 3'-UTR
(10). In control studies, cRNA from HA-KCNA10 and
wild-type KCNA10 was synthesized and injected into Xenopus oocytes as described below. The resulting currents were compared to
ensure that addition of the HA epitope did not disrupt channel current
expression. The interaction of HA-KCNA10 and c-Myc-KCNA4B was then
examined as described in In vitro transcription, translation, and
binding analysis of the COOH terminus of KCNA10 and putative positive
clones.
Expression of KCNA10 in Xenopus oocytes.
The coding region of KCNA10 (accession no. U96110) was amplified by PCR
with a proofreading polymerase and a sense primer containing a
BamHI site and an antisense primer with an XbaI
site: 5'-CGGGGATCCCTCCCCTAGAATGGATGTG-3' (sense) and
5'-TCTAGAAAGAGACAGGATGGACCCAAGAAGCC-3' (antisense). The amplified
product was cut with BamHI and XbaI and ligated
into PSD64T7, a high-expression vector based on pGEM (Promega)
containing 60 bp of the 5'-UTR of Xenopus -globin and 300 bp of the 3'-UTR (10). The construct was sequenced to
ensure that mutations were not introduced during amplification.
Nucleotide and protein sequence analyses were carried out using
MacVector (Oxford Molecular Group) and Lasergene (DNASTAR, Madison,
WI). cRNA was synthesized with T7 polymerase after linearization with NotI.
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RESULTS |
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Identification of interacting subunits using the yeast two-hybrid
system.
The human heart cDNA library was screened at high stringency as
described in MATERIALS AND METHODS, and several
putative positive clones were isolated. Each library positive was
retested by cotransformation, along with C-TerKCNA10/pGBKT7, of AH109.
One of these clones (KCNA4B, accession no. AF262975) was
consistently positive and, therefore, was chosen for further study. It
contained a 1.4-kb insert, which was sequenced by the method of Sanger
et al. (14). A 600-bp sequence shown in Fig.
1 contains the longest open reading frame (ORF; nt 26-451). The ORF encodes a 141-aa protein (Fig.
1A). Structural analysis of the deduced amino acid sequence
was carried out using MacVector. There are consensus sites for protein
kinase A and protein kinase C and an endoplasmic reticulum
localization signal (Fig. 1A). KCNA4B is a soluble protein
with no significant amino acid homology to previously cloned
K+ channel -subunits, but with a conserved
aldoketoreductase motif (aa 76-92).
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Expression pattern of KCNA4B message.
The expression pattern of KCNA4B mRNA was determined by Northern
blot analysis. The 1.4-kb clone was labeled and used to probe a
multiple-tissue Northern blot containing poly(A)+ mRNA
isolated from a variety of human tissues. KCNA4B expression was detected in several tissues, including heart, skeletal muscle, kidney, small intestine, and placenta (Fig.
2). The predominant message has a
molecular weight of 0.9 kb, although a weaker band was detected at 1.1 kb. Message expression appears to be highest in heart and skeletal
muscle and notably absent in brain, liver, and lungs.
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In vitro association of KCNA4B and KCNA10.
These studies were carried out to confirm the interaction of the COOH
terminus of KCNA10 with KCNA4B and to determine whether KCNA4B also
interacted with full-length KCNA10. cRNAs for the various constructs
were translated in vitro using a rabbit reticulocyte lysate assay in
the presence of [35S]methionine. Monoclonal antisera
raised against the c-Myc or HA epitope were used for
immunoprecipitation as described in MATERIALS AND
METHODS. The COOH terminus of KCNA10 and KCNA4B did not
interact in a nonspecific manner with the Sepharose A column. Indeed,
as shown in Fig.
3A, lane
1, there are no visible protein bands in the absence of antibody.
On the other hand, the c-Myc antibody (specific for the COOH terminus
of KCNA10) immunoprecipitated KCNA4B and the COOH terminus of KCNA10
(Fig. 3A, lane 2), indicating that the two proteins interact
in vitro. KCNA4B can be immunoprecipitated alone using the HA
monoclonal antibody (Fig. 3A, lane 3), while the COOH
terminus of KCNA10 can be immunoprecipitated alone using the c-Myc
monoclonal antibody (Fig. 3A, lane 4). Taken together, these
data show that the COOH terminus of KCNA10 does interact with KCNA4B,
thereby confirming the yeast two-hybrid screen results.
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Functional consequence of KCNA10 and KCNA4B coexpression.
The Xenopus oocyte expression system was then used to
examine the functional significance of the interaction of KCNA10 with KCNA4B. We previously studied in detail the expression level, kinetic
properties, and pharmacological attributes of KCNA10 (10). Therefore, we compared currents expressed by KCNA10 alone with those
observed with KCNA10 and KCNA4B. Xenopus oocytes injected with only KCNA4B cRNA exhibited no significant current above background and were indistinguishable from water-injected oocytes (Fig.
4A). KCNA10 cRNA, on the other
hand, mediated Kv currents (peak current = 3.78 ± 0.48 µA
at +40 mV) that were identical to those previously observed (Fig.
4B, left). As shown in Fig. 4, B
(right) and C, coinjection of KCNA10 and KCNA4B
RNAs yielded Kv currents 2.8-fold larger than those observed with
KCNA10 alone: 10.53 ± 1.07 µA (n = 45) vs.
3.78 ± 0.48 µA (n = 36, P < 0.000003). The inhibitor profile of KCNA10 current was unaffected by
coexpression with KCNA4B (Table 1).
KCNA4B interacts with the COOH terminus (54 aa) of KCNA10. Although the
last 38 aa are specific for KCNA10A, the first 16 aa are conserved in
many Kv1.x channels. To test whether KCNA4B interacts with
other Kv1 channels, its effect on Kv1.3 (most closely related to
KCNA10) expression was examined. As shown in Fig. 4C,
coexpression of Kv1.3 and KCNA4B yielded current levels comparable to
those observed with Kv1.3 alone.
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DISCUSSION |
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We have identified a novel gene, KCNA4B, that encodes a
regulatory subunit of the Kv channel KCNA10. KCNA4B is a small
cytoplasmic protein (141 aa) with no significant homology to known
proteins and without any conserved functional domains, except a partial aldoketoreductase motif. The aldoketoreductase signature consists of
five elements and is present in a number of related NADPH-dependent oxidoreductases, such as aldehyde and aldose reductase, which have a
secondary structure consisting of a -
-
fold, characteristic of
nucleotide-binding proteins. Interestingly, the first class of Kv
-subunits cloned is homologous to members of the aldoketoreductase family (11). Although the significance of this observation
is unknown, these
-subunits are postulated to provide a link between the cell's redox potential and K+ channel
activity. Indeed, an oxidizing environment inhibits the effect of
Kv
on activation kinetics (12). We have no
evidence suggesting that KCNA4B confers such sensitivity to KCNA10
currents. Furthermore, since KCNA4B contains only one of the five
aldoketoreductase signature elements, it is unlikely to have
oxidoreductase activity.
KCNA4B modifies the properties of KCNA10 in two important ways. First, it increases current expression without changing the threshold for activation by voltage. The rise in channel current could be accounted for by a larger single-channel conductance (from 11 to 30 pS) or by an increase in the number of active channels at the plasma membrane. On the basis of the present understanding of Kv accessory proteins, it is more likely that KCNA4B upregulates the number of functional KCNA10 channels at the plasma membrane. We do not know whether KCNA4B remains bound to KCNA10 at the plasma membrane. If it does, it might increase KCNA10 current by stabilizing or activating KCNA10 channel proteins already inserted in the plasma membrane. Alternatively, KCNA4B might act as a chaperone to increase delivery of KCNA10 protein to the plasma membrane. This interaction might occur co- or posttranslationally, since the KCNA10 and KCNA4B proteins could be immunoprecipitated when the cRNAs were cotranslated or translated separately. KChAP is another Kv accessory subunit known to increase Kv channel activity through a chaperone-like effect. Unlike KCNA4B, it appears to mediate its action on Kv channels during translation, as evidenced by the fact that KChAP does not appear to interact with Kv channels when the cRNAs are translated separately (22).
In addition to increasing KCNA10 current, KCNA4B also alters its sensitivity to activation by cAMP. cGMP causes a small decrease in KCNA10 current, while cAMP has no significant effect (10). Coexpression with KCNA4B does not affect the action of cGMP but leads to significant upregulation by cAMP. The time course of activation is slow (5-20 min), suggesting that cAMP could mediate its action through protein phosphorylation. KCNA4B interacts with the COOH terminus of KCNA10, which is unique among Kv channels, in that it lacks significant amino acid homology to any of the cloned K+ channels and contains a putative CNB domain. Other K+ channels known to contain CNB domains (EAG, HERG, BCNG1, ATK1) do not appear to be regulated by cyclic nucleotides when expressed in heterologous systems, therefore raising the possibility that auxiliary subunits are necessary. Although the data suggest that KCNA4B represents such a protein, they do not address the molecular mechanisms mediating the increased sensitivity to cAMP. One possibility is that cAMP phosphorylates KCNA4B and potentiates its interaction with KCNA10. Alternatively, KCNA4B interacts with KCNA10 to cause a conformational change that favors the binding of cAMP to the CNB domain or protein phosphorylation.
KCNA4B appears to interact in a specific manner with KCNA10, since
Kv1.3 (the Kv channel most closely related to KCNA10) expression was
unaffected. This would be in agreement with the understanding that each
Kv -subunit interacts with a few specific
-subunits (13). KCNA10 currents are blocked not only by classic Kv
channel inhibitors, such as 4-aminopyridine and charybdotoxin, but also by antagonists of CNG channels (pimozide and verapamil). KCNA4B did not
affect the sensitivity to any of the inhibitors tested, including pore
blockers such as charybdotoxin, suggesting that the pore region is
unaffected by any conformational changes that may result from its
interaction with KCNA10. KCNA4B's message expression
parallels, to a large extent, that of KCNA10, with predominant
expression in heart, kidney, and skeletal muscle, but not in brain.
More precise localization of both proteins in kidney is awaiting the
development of suitable antibodies.
In summary, KCNA4B is a novel protein isolated in a yeast two-hybrid screen using the COOH terminus of the Kv channel KCNA10 as bait. KCNA4B binds to the COOH terminus of KCNA10 and can be immunoprecipitated in vitro and in vivo. The biological significance of this interaction is reflected in the fact that KCNA4B increases KCNA10 current expression by at least 2.8-fold and also alters its sensitivity to cAMP. KCNA4B represents the fourth class of accessory subunits thought to have biologically significant interactions with Kv channels.
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ACKNOWLEDGEMENTS |
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This work was supported by a Veterans Administration Merit Review Award and National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-48105B (both to G. V. Desir). G. V. Desir is an Established Investigator of the American Heart Association.
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FOOTNOTES |
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Address for reprint requests and other correspondence: G. V. Desir, Dept. of Medicine, Sect. of Nephrology, Yale University School of Medicine, West Haven VA Medical Center, 2073 LMP, 333 Cedar St., New Haven, CT 06510 (E-mail: gary.desir{at}yale.edu).
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.
First published January 29, 2002;10.1152/ajprenal.00258.2001
Received 16 August 2001; accepted in final form 16 January 2002.
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