Regulation of the voltage-gated K+ channel KCNA10 by KCNA4B, a novel beta -subunit

Shulan Tian1, Weimin Liu1, Yanling Wu1, Hamid Rafi1, Alan S. Segal2, and Gary V. Desir1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Voltage-gated K+ (Kv) channels are heteromultimeric complexes consisting of pore-forming alpha -subunits and accessory beta -subunits. Several beta -subunits have been identified and shown to interact with specific alpha -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 alpha -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 beta -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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -subunits very rarely match currents known to exist in native tissues (7). Most often, it is because the alpha -subunits interact with accessory proteins that modify their kinetic and pharmacological properties. Furthermore, accessory proteins often modulate the levels of expression of the alpha -subunits.

Several beta -subunits have been characterized. The Kvbeta genes encode soluble proteins (367-404 aa), related to the NAD(P)H-dependent oxidoreductase superfamily, that interact with many Kv alpha -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 beta -subunits, that binds to the COOH terminus of KCNA10 and modifies the channel's kinetic properties.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-beta -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 beta -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 beta -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.

Stage V-VI oocytes from Xenopus laevis were harvested from ovarian lobes and repeatedly washed in Ca2+-free OR2 (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES, pH 7.5). Oocytes were defolliculated by incubation with type IA collagenase (2 mg/ml) in Ca2+-free OR2 for 90-120 min at room temperature. After a second wash with Ca2+-free OR2, the oocytes were stored in ND96 (88 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, pH 7.5) with penicillin (100 U/ml)-streptomycin (100 µg/ml) and sodium pyruvate (550 mg/l) at 19°C. The vegetal poles of selected oocytes were injected with various combinations of 5'-capped KCNA10, c-Myc-KCNA10, and KCNA4B cRNAs (50 ng total) or RNase-free water as a control.

Whole cell currents were recorded using the standard two-microelectrode voltage-clamp method. Current was measured after 3-5 days of incubation, and expressed K+ currents were compared with those from water-injected control oocytes. For voltage clamping, oocytes were impaled with two microelectrodes filled with 1 M KCl (resistance 0.5-2 MOmega ). Oocyte current-voltage relationships were obtained by applying command voltage ramp or step protocols and measuring the resulting membrane current with a current-to-voltage amplifier (model OC-725, Warner Instruments, Hamden, CT). Data acquisition was controlled by PULSE 8.1 (Heka Elektronik) or pClamp 5.5 (Axon Instruments, Foster City, CA). Currents were filtered at 1-2 kHz and digitized to hard disk at 2.5-5 ksamples/s. Data were analyzed using PULSE-Fit (Heka Elektronik), Origin 5.0 (Microcal Software, Northampton, MA), and Igor-Pro (WaveMetrics, Lake Oswego, OR). The extracellular recording solution was ND96 unless otherwise noted.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -subunits, but with a conserved aldoketoreductase motif (aa 76-92).


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 1.   Nucleotide and amino acid sequences, chromosomal localization, and intron-exon organization of KCNA4B. A: nucleotide and deduced amino acid sequences for KCNA4B. The 1.4-kb insert was isolated and sequenced as described in MATERIALS AND METHODS. A 600-bp region, containing the longest open reading frame, is shown (accession no. AF262975, GenBank). Deduced amino acid sequence for KCNA4B (141 aa) is shown in single-letter code. Single putative protein kinase A phosphorylation sites are underlined. *, Protein kinase C sites; **, ER localization signal. B: chromosomal localization and intron-exon organization. A genomic 101,830-bp clone (accession no. AL359454, GenBank) from chromosome 13 was identified by BLAST search using KCNA4B. KCNA4B spans a 16-kb region, and its coding region is made up of 5 small (61-310 bp) exons.

A search of the GenBank databases using BLAST revealed a cDNA sequence that encodes a protein of unknown function, with an amino acid sequence identical to that of KCNA4B. This clone had previously been isolated from D34+ hematopoietic stem cells (accession no. AF125097) and leukemic cells (accession no. AF275807). A genomic clone was also identified (accession no. AL359454), and its analysis indicates that KCNA4B is located on chromosome 13 at q12.3-13q14.2. The gene spans ~16 kb and contains five exons (Fig. 1B). There is also a related sequence at chromosome Xq13.2-21.1 (accession no. AL121882) that is intronless and contains numerous stop codons and small deletions. That sequence most likely represents a pseudogene.

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.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 2.   Expression pattern of KCNA4B. A multiple-tissue Northern blot containing poly(A)+ mRNA isolated from a variety of human tissues was hybridized at high stringency with a 32P-labeled (106 cpm/ml hybridization) 1.4-kb cDNA fragment containing the entire coding region of KCNA4B. Final wash was at 65°C for 20 min in 0.1× saline-sodium citrate and 0.1% SDS. The blot was then exposed to film at -70°C for 24 h. KCNA4B expression is highest in heart, muscle, kidney, small intestine, and placenta. Hybridization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is shown as control for amounts of RNA loaded in each lane.

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.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3.   KCNA4B interacts with full-length KCNA10 and its COOH terminus. A: interaction of KCNA4B with the COOH terminus of KCNA10. Lane 1, COOH-terminal construct c-Myc-KCNA10 was translated in vitro, labeled with 35S, and immunoprecipitated with monoclonal anti-hemagglutinin (anti-HA); there is no nonspecific binding to the column, and anti-HA does not cross-react with c-Myc-KCNA10. Lane 2, HA-KCNA4B was translated in vitro, labeled with 35S, and immunoprecipitated with monoclonal anti-c-Myc. There is no nonspecific binding to the column, and anti-c-Myc does not cross-react with hemagglutinin (HA)-KCNA. Lane 3, c-Myc-KCNA10 was translated in vitro, labeled with 35S, and immunoprecipitated using anti-c-Myc monoclonal antibody. Lane 4, HA-KCNA4B was translated in vitro, labeled with 35S, and immunoprecipitated using anti-HA monoclonal antibody. Lane 5, c-Myc-KCNA10 and HA-KCNA4B were translated in vitro and labeled with 35S. Both proteins can be immunoprecipitated with a c-Myc-specific monoclonal antibody, indicating that the proteins interact in vitro. B: interaction of KCNA4B with full-length KCNA10. Lane 1, HA-KCNA10 was translated in vitro, labeled with 35S, and immunoprecipitated using anti-c-Myc monoclonal antibody. There is no nonspecific binding to the column, and anti-c-Myc does not cross-react with HA-KCNA10. Lane 2, c-Myc-KCNA4B was translated in vitro, labeled with 35S, and immunoprecipitated using anti-HA monoclonal antibody. There is no nonspecific binding to the column, and anti-HA does not cross-react with c-Myc-KCNA4B. Lane 3, HA-KCNA10 was translated in vitro, labeled with 35S, and immunoprecipitated using anti-HA monoclonal antibody. Predicted molecular mass of KCNA10 is ~55 kDa. Identity of the higher molecular mass proteins is unclear; they may represent multimeric complexes containing KCNA10 or could be the result of posttranslational modifications. Lane 4, c-Myc-KCNA4B was translated in vitro, labeled with 35S, and immunoprecipitated using anti-c-Myc monoclonal antibody. Lane 5, c-Myc-KCNA10 and HA-KCNA4B were translated in separate reactions in vitro and labeled with 35S, and both proteins were then mixed and immunoprecipitated with a c-Myc-specific monoclonal antibody. KCNA4B also interacts with full-length KCNA10 in vitro. Lane 6, HA-KCNA10 and c-Myc-KCNA4B were translated in vitro, labeled with 35S, and applied to a protein A-Sepharose column. There was no significant nonspecific binding to column. Lane 7, same as lane 5, except c-Myc-KCNA10 and HA-KCNA4B cRNAs are mixed together before translation.

To test whether full-length KCNA10 also interacted with KCNA4B, an HA epitope tag was inserted at the NH2 terminus of KCNA10 (HA-KCNA10), while a c-Myc tag was placed at the NH2 terminus of KCNA4B (c-Myc-KCNA4B). We confirmed, in control experiments, that the epitope tag did not interfere with the expression and function of KCNA10. Indeed, HA-KCNA10 cRNA injected into oocytes yielded K+ currents that were identical to wild-type KCNA10: 2.5 ± 0.5 µA (n = 5) vs. 2.2 ± 0.1 µA (n = 5). These constructs were then used to examine the interaction of full-length KCNA10 with KCNA4B. As shown in Fig. 3B, in the absence of antibody, the proteins did not interact with the protein A-Sepahrose column (lanes 1 and 2). The HA antibody efficiently immunoprecipitated full-length KCNA10 (lane 3), while the c-Myc antibody immunoprecipitated KCNA4B (lane 4). Anti-KCNA10 also immunoprecipitates proteins of molecular mass greater than expected for KCNA10, and the identity of these complexes is unknown. If the two proteins are translated separately and then mixed together, the HA antibody can coimmunoprecipitate both proteins (lane 5). Finally, translating both proteins in the same reaction by mixing both cRNAs before translation did not appear to affect their interaction (lanes 6 and 7). Taken together, these data strongly support the notion that KCNA4B is closely associated with KCNA10 and that the association may be biologically significant.

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.


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4.   KCNA4B increases KCNA10 current expression. A: current expression by KCNA4B. Whole cell currents were recorded from Xenopus oocytes using the 2-electrode voltage-clamp method. Voltage protocol is shown at right. Current expression for oocytes injected with KCNA4B (0.2 ± 0.02 µA, n = 10) alone is the same as for water-injected oocytes (0.35 ± 0.02 µA, n = 10). B: effect of KCNA4B on KCNA10 expression. Whole cell currents were recorded from Xenopus oocytes expressing KCNA10 alone or KCNA10 and KCNA4B. Voltage protocol is the same as in A. Representative current traces are shown. KCNA4B increased current expression levels significantly. C: effect of KCNA4B on KCNA10 and Kv1.3 current expression. Whole cell currents were recorded from Xenopus oocytes expressing KCNA10 or Kv1.3 alone or in combination with KCNA4B. Voltage protocol is the same as in A. Peak currents measured at 40 mV are shown. Coexpression of KCNA10 and KCNA4B increases expressed K+ currents by 2.8-fold. KCNA4B did not affect expression of a related Kv channel, Kv1.3.


                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Comparison of inhibitor profiles for KCNA10 and KCNA10/KCNA4B

The COOH terminus of KCNA10 is unique among Kv1 channels by virtue of its putative CNB domain. Sensitivity to cyclic nucleotides was also tested in oocytes coexpressing KCNA10 and KCNA4B. cGMP (1 mM 8-bromo-cGMP) decreased expressed peak current slightly in oocytes expressing KCNA10 alone (12 ± 1.8%, n = 10) and in those expressing KCNA10 and KCNA4B (15.5 ± 1.7%, n = 10). In contrast to cGMP, cAMP regulation was differentially affected by coexpression of KCNA4B. Indeed, we found that although, as previously reported (10), cAMP does not affect KCNA10 current levels (2 ± 0.3% increase, n = 10), it does cause a significant increase in KCNA10/KCNA4B currents (Fig. 5).


View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5.   Activation of KCNA10/KCNA4B current by cAMP. Whole cell currents were recorded from Xenopus oocytes expressing KCNA10 alone or KCNA10 and KCNA4B. See Fig. 4A for voltage protocol. A: representative current traces obtained after addition of 1 mM 8-bromo-cAMP. Current levels increase within 5 min after addition of cAMP. B: effect of 8-bromo-cAMP (1 mM) and 8-bromo-cGMP (1 mM) on peak currents at 40 mV for KCNA10 alone (n = 6) and KCNA10/KCNA4B (n = 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -alpha -beta fold, characteristic of nucleotide-binding proteins. Interestingly, the first class of Kv beta -subunits cloned is homologous to members of the aldoketoreductase family (11). Although the significance of this observation is unknown, these beta -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 Kvbeta 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 beta -subunit interacts with a few specific alpha -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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abbott, GW, Butler MH, Bendahhou S, Dalakas MC, Ptacek LJ, and Goldstein SA. MiRP2 forms potassium channels in skeletal muscle with Kv3.4 and is associated with periodic paralysis. Cell 104: 217-231, 2001[ISI][Medline].

2.   Abbott, GW, Goldstein SA, and Sesti F. Do all voltage-gated potassium channels use MiRPs? Circ Res 88: 981-983, 2001[Free Full Text].

3.   Abbott, GW, Sesti F, Splawski I, Buck ME, Lehmann MH, Timothy KW, Keating MT, and Goldstein SA. MiRP1 forms IKr potassium channels with HERG and is associated with cardiac arrhythmia. Cell 97: 175-187, 1999[ISI][Medline].

4.   Archer, SL, Huang JM, Hampl V, Nelson DP, Schultz PJ, and Weir EK. Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci USA 91: 7583-7587, 1994[Abstract].

5.   Barhanin, J, Lesage F, Guillemare E, Fink M, Lazdunski M, and Romey G. KvLQT1 and IsK (minK) proteins associate to form the IKs cardiac potassium current. Nature 384: 78-80, 1996[ISI][Medline].

6.   Brüggemann, A, Pardo LA, Stuhmer W, and Pongs O. Ether-à-go-go encodes a voltage-gated channel permeable to K+ and Ca2+ and modulated by cAMP. Nature 365: 445-448, 1993[ISI][Medline].

7.   Coetzee, WA, Amarillo Y, Chiu J, Chow A, Lau D, McCormack T, Moreno H, Nadal MS, Ozaita A, Pountney D, Saganich M, Vega-Saenz de Miera E, and Rudy B. Molecular diversity of K+ channels. Ann NY Acad Sci 868: 233-285, 1999[Abstract/Free Full Text].

8.   Gomora, JC, and Enyeart JJ. Ca2+ depolarizes adrenal cortical cells through selective inhibition of an ATP-activated K+ current. Am J Physiol Cell Physiol 275: C1526-C1537, 1998[Abstract/Free Full Text].

9.   Kuryshev, YA, Gudz TI, Brown AM, and Wible BA. KChAP as a chaperone for specific K+ channels. Am J Physiol Cell Physiol 278: C931-C941, 2000[Abstract/Free Full Text].

10.   Lang, R, Lee G, Liu W, Tian S, Rafi H, Orias M, Segal AS, and Desir GV. KCNA10: a novel ion channel functionally related to both voltage-gated potassium and CNG cation channels. Am J Physiol Renal Physiol 278: F1013-F1021, 2000[Abstract/Free Full Text].

11.   McCormack, T, and McCormack K. Shaker K+ channel beta  subunits belong to an NAD(P)H-dependent oxidoreductase superfamily. Cell 79: 1133-1135, 1994[ISI][Medline].

12.   Perez-Garcia, MT, Lopez-Lopez JR, and Gonzalez C. Kvbeta 1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to Kv4.2 but not to Shaker channels. J Gen Physiol 113: 897-907, 1999[Abstract/Free Full Text].

13.   Pongs, O, Leicher T, Berger M, Roeper J, Bahring R, Wray D, Giese KP, Silva AJ, and Storm JF. Functional and molecular aspects of voltage-gated K+ channel beta -subunits. Ann NY Acad Sci 868: 344-355, 1999[Abstract/Free Full Text].

14.   Sanger, F, Nicklen S, and Coulson AR. DNA sequencing with chain-terminating inhibitors. Biotechnology 24: 104-108, 1977.

15.   Sanguinetti, MC, Curran ME, Spector PS, and Keating MT. Spectrum of HERG K+-channel dysfunction in an inherited cardiac arrhythmia. Proc Natl Acad Sci USA 93: 2208-2212, 1996[Abstract/Free Full Text].

16.   Sanguinetti, MC, Curran ME, Zou A, Shen J, Spector PS, Atkinson DL, and Keating MT. Coassembly of KvLQT1 and minK (IsK) proteins to form cardiac IKs potassium channel. Nature 384: 80-83, 1996[ISI][Medline].

17.   Santoro, B, Grant SGN, Bartsch D, and Kandel ER. Interactive cloning with the SH3 domain of N-src identifies a new brain-specific ion channel protein, with homology to Eag and cyclic nucleotide-gated channels. Proc Natl Acad Sci USA 94: 14815-14820, 1997[Abstract/Free Full Text].

18.   Schwartz, PJ, Priori SG, Locati EH, Napolitano C, Cantu F, Towbin JA, Keating MT, Hammoude H, Brown AM, and Chen LS. Long QT syndrome patients with mutations of the SCN5A and HERG genes have differential responses to Na+ channel blockade and to increases in heart rate---implications for gene-specific therapy. Circulation 92: 3381-3386, 1995[Abstract/Free Full Text].

19.   Sentenac, H, Bonneaud N, Minet M, Lacroute F, Salmon JM, Gaymard F, and Grignon C. Cloning and expression in yeast of a plant potassium ion transport system. Science 256: 663-665, 1992[ISI][Medline].

20.   Trudeau, MC, Warmke JW, Ganetzky B, and Robertson GA. HERG, a human inward rectifier in the voltage-gated potassium channel family. Science 269: 92-96, 1995[ISI][Medline].

21.   Warmke, JW, and Ganetzky B. A family of potassium channel genes related to eag in Drosophila and mammals. Proc Natl Acad Sci USA 91: 3438-3442, 1994[Abstract].

22.   Wible, BA, Yang Q, Kuryshev YA, Accili EA, and Brown AM. Cloning and expression of a novel K+ channel regulatory protein, KChAP. J Biol Chem 273: 11745-11751, 1998[Abstract/Free Full Text].

23.   Xu, L, and Enyeart JJ. Purine and pyrimidine nucleotides inhibit a noninactivating K+ current and depolarize adrenal cortical cells through a G protein-coupled receptor. Mol Pharmacol 55: 364-376, 1999[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 283(1):F142-F149