KChAP/Kvbeta 1.2 interactions and their effects on cardiac Kv channel expression

Y. A. Kuryshev1,2, B. A. Wible1,3, T. I. Gudz1, A. N. Ramirez1, and A. M. Brown1,2

1 The Rammelkamp Center for Education and Research, MetroHealth Campus, and Departments of 2 Physiology and Biophysics and 3 Biochemistry, Case Western Reserve University, Cleveland, Ohio 44109


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

KChAP and voltage-dependent K+ (Kv) beta -subunits are two different types of cytoplasmic proteins that interact with Kv channels. KChAP acts as a chaperone for Kv2.1 and Kv4.3 channels. It also binds to Kv1.x channels but, with the exception of Kv1.3, does not increase Kv1.x currents. Kvbeta -subunits are assembled with Kv1.x channels; they exhibit "chaperone-like" behavior and change gating properties. In addition, KChAP and Kvbeta -subunits interact with each other. Here we examine the consequences of this interaction on Kv currents in Xenopus oocytes injected with different combinations of cRNAs, including Kvbeta 1.2, KChAP, and either Kv1.4, Kv1.5, Kv2.1, or Kv4.3. We found that KChAP attenuated the depression of Kv1.5 currents produced by Kvbeta 1.2, and Kvbeta 1.2 eliminated the increase of Kv2.1 and Kv4.3 currents produced by KChAP. Both KChAP and Kvbeta 1.2 are expressed in cardiomyocytes, where Kv1.5 and Kv2.1 produce sustained outward currents and Kv4.3 and Kv1.4 generate transient outward currents. Because they interact, either KChAP or Kvbeta 1.2 may alter both sustained and transient cardiac Kv currents. The interaction of these two different classes of modulatory proteins may constitute a novel mechanism for regulating cardiac K+ currents.

chaperone; modulation; potassium channels; voltage-gated potassium 1.4 channel; voltage-gated potassium 1.5 channel; voltage-gated potassium 2.1 channel; voltage-gated potassium 4.3 channel


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

KCHAP (11, 24) and Kvbeta -subunits (5, 7, 27) are two types of cytoplasmic proteins known to interact with and alter processing of voltage-dependent K+ (Kv) channels. Kvbeta -subunits interact with members of the Kv1.x subfamily (10, 13, 14, 18), with Kv2.2 (11) and Kv4.x (16) channels, whereas KChAP in addition to Kv1.x interacts with Kv2.1 and Kv4.3 channels (12, 25). Kvbeta -subunits and KChAP differ in how they affect Kv currents, however. First, Kvbeta -subunits coassemble with Kvalpha -subunits to form stable Kvalpha /beta heterooligomeric channels (16, 20, 21), whereas KChAP interacts with Kvalpha -subunits only transiently (12, 25). Second, Kvbeta -subunits enhance the trafficking of Kv channels to the plasma membrane without affecting whole cell Kvalpha -subunit protein levels (17, 21), whereas KChAP increases Kvalpha -subunit protein levels (12, 25). Third, Kvbeta -subunits change Kv1.x channel gating by virtue of their tight association with mature channels (1, 10, 18), whereas KChAP has no effect on gating (12, 25). Thus KChAP manifests true chaperone behavior while Kvbeta -subunits exhibit pseudochaperone behavior that more closely resembles that of other coassembled, accessory K+ channel subunits such as minK with KvLQT1 (4, 19, 23) or the beta -subunit with the Ca2+-activated maxi-K+ channel (22).

Not only do KChAP and Kvbeta -subunits interact with Kv channels, they also interact with each other. In fact, KChAP was discovered in yeast two-hybrid experiments using Kvbeta 1.2 as bait (25). This interaction led us to propose that interactions between KChAP and Kvbeta -subunits might significantly alter membrane currents in cells expressing Kv1.x, Kv2.1, or Kv4.3, such as cardiomyocytes. Kv4.x and Kv1.4 contribute to the transient outward current (ITo) in heart, and the differential expression of these channels between endocardium and epicardium is thought to be critical for the timing and directionality of ventricular repolarization. In a previous paper, we showed that KChAP interacted with Kv2.1 and Kv4.3 in rat heart (12). Here we report that Kvbeta 1.2 is expressed in rat and human heart and that complexes between Kvbeta 1.2 and Kv1.2, Kv1.4, and Kv1.5 are detected. Having shown that all of the participants are present in heart, we tested our hypothesis in a model cell system using heterologous expression in Xenopus oocytes. First, we found that KChAP did not act as a chaperone for Kvbeta 1.2 in that levels were unaltered in the presence of KChAP. However, KChAP did prevent the effects of Kvbeta 1.2 on expression and gating of its target Kv1.x channels. Conversely, Kvbeta 1.2 prevented the chaperone effects of KChAP on Kv2.1 and Kv4.3. Thus KChAP may increase K+ currents directly by its chaperone effects on Kv2.1 and Kv4.3 and indirectly by suppressing the block of Kv1.x channels by Kvbeta 1.2. Conversely, Kvbeta -subunits may decrease K+ currents directly by block of Kv1.x channels or indirectly by suppressing the chaperone effect of KChAP on Kv2.1 and Kv4.3. Such mechanisms would provide an additional level of control over Kv currents and could be controlled by the relative levels of distinct but interacting modulatory proteins.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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mRNA Extraction and Northern Blot Analysis

mRNA from rat heart and brain was isolated using the Poly(A)Pure kit (Ambion) following the manufacturer's instructions. Poly(A)+ RNA (2 µg/lane) was electrophoresed on a 1.0% agarose/formaldehyde gel and transferred to Bright-Star membranes (Ambion). The blot was hybridized to a 32P-labeled riboprobe spanning the region encoding the NH2-terminal 79 residues of human Kvbeta 1.2. A T7 promoter sequence was engineered directly onto the end of the coding fragment using the Lig'nScribe kit from Ambion and the riboprobe synthesized with the Strip-EZ T7 kit (Ambion). The blot was hybridized with probe (106 counts · min-1 · ml-1) overnight at 44°C in ULTRAhyb buffer (Ambion), washed at 50°C in 0.1× saline-sodium citrate (SSC)-0.1% SDS, and exposed to BioMax MS film (Kodak) at -70°C with an intensifying screen.

PCR Amplification of Rat Heart Kv Channel beta -Subunit cDNA Fragments

Sets of specific primers were designed to amplify unique DNA fragments corresponding to regions of rat Kvbeta -subunits by RT-PCR. Primer sequences and locations in published cDNA sequences in GenBank of the National Center for Biotechnology Information were as follows: for Kvbeta 1.1 (accession number X70662), 5'-ATG CAA GTC TCC ATA GCC TGC ACA GAG C-3' (sense primer; nt 332-359) and 5'-GTC ATC AGC CGT TCA GCA ACC TCA TCT G-3' (antisense primer; nt 627-654); Kvbeta 1.3, 5'-GAC AAC AGC AAG TTT AGA AAG CAG TC-3' (sense primer) and 5'-GTC ATC AGC CGT TCA GCA ACC TCA TCT G-3' (antisense primer); Kvbeta 2 (accession number X76724), 5'-ATC TAC AGT ACT CGG TAT GGG AGT C-3' (sense primer; nt 662-686) and 5'-GCC TCC ATG ATC TCC ATG GAG C-3' (antisense primer; nt 1163-1185); Kvbeta 3 (accession number X76723), 5'-ATG CAG GTG TCT ATC GCG TGT AC-3' (sense primer; nt 388-410) and 5'-AAG CCT CGC TCA GTT TCT GCC-3' (antisense primer; nt 881-902).

RT-PCR. Poly(A)+ mRNA (100 ng) was denatured at 70°C for 5 min and reverse-transcribed into first-strand cDNA by priming with random hexamers (First-Strand cDNA Synthesis Kit; Perkin-Elmer). PCR was performed with the Advantage PCR System (Clontech) using the following cycling program: one cycle (2 min at 95°C); 35 cycles (15 s at 94°C, 15 s at 50°C, 1 min at 68°C); one cycle (10 min at 72°C). PCR products were resolved by electrophoresis on 1.2% agarose gels and transferred to BrightStar-Plus membranes (Ambion). Channel-specific PCR products were identified by hybridization to the following 32P-labeled internal oligonucleotides specific for either the Kvbeta 1 subfamily (5'-GTT TCG TGC TTG GGT CTT GGA ACA TGG-3'), Kvbeta 2 (5'-GAG GCC AGA TCA CAG ATG AG-3'; nt 774-793), or Kvbeta 3 (5'-GGC TCT CAG ATC TCA GAT GAG ACA G-3'; nt 691-715). The blots were washed with 0.1× SSC-0.1% SDS at 44°C and exposed to BioMax MS film (Kodak) at -70°C with an intensifying screen.

Antibodies

An anti-Kvbeta 1.2 antibody was raised to a fusion protein consisting of the unique NH2 terminus (residues 1-79) of human Kvbeta 1.2, which was PCR amplified and cloned in-frame into an expression vector (pET-15b) encoding six histidine tags on the NH2 terminus. The fusion protein was purified by metal chelation chromatography using the His-Bind purification kit (Novagen) and injected into rabbits (Research Genetics). Antisera was affinity purified on a fusion protein-Sepharose column. An antibody to human Kv1.5 (COOH-terminal residues D528-L613) was prepared by creating a maltose-binding fusion protein (New England Biolabs). Polyclonal antisera from rabbits was affinity purified on a fusion-protein Sepharose column. Monoclonal antibodies against Kv1.2 and Kv1.4 were obtained from Upstate Biotechnology. The monoclonal anti-green fluorescent protein used for Western blotting was from Clontech (no. 8362-1). The polyclonal anti-KChAP antibody was previously described (12).

Tissue Lysate and Membrane Preparation

Rat tissue lysates were prepared from freshly dissected organs. Heart, kidney, and skeletal muscle were minced and placed in ice-cold lysis or immunoprecipitation (IP) buffer (1:7 wt/vol) containing 150 mM NaCl, 50 mM Tris · HCl, 1 mM EDTA, 0.2% BSA, and 1% Triton X-100, pH 7.5, supplemented with a protease and phosphatase inhibitor cocktail (Complete; 50 mM sodium fluoride-0.2 mM sodium vanadate; Roche). Samples were homogenized with a Polytron at setting 6 for 5 s. All of the other tissue samples were homogenized in a Teflon-glass homogenizer (1:7 wt/vol) in the same lysis buffer. After 1 h of incubation on ice, the lysates were centrifuged at 3,000 g for 10 min to remove insoluble material. Human atrial appendage tissue was pooled from adult patients undergoing aortocoronary bypass surgery. A sample of human left ventricle was obtained from an explanted heart at transplant.

To isolate crude cellular membranes, rat tissues were homogenized in 0.3 M sucrose and 10 mM phosphate, pH 7.4, supplemented with a protease and phosphatase inhibitor cocktail as above. After nuclei and debris were removed by centrifugation at 3,000 g for 10 min, the supernatant was centrifuged at 50,000 g for 1 h to pellet membranes. Protein concentrations were determined by the bicinchoninic acid method (Pierce). IP and immunoblotting were performed as previously described (12).

Constructs and Yeast Two-Hybrid Assay

Full-length KChAP (residues 1-619) and the KChAP NH2-terminal fragment (KChAP-N; residues 46-354), the middle fragment of KChAP (KChAP-M; residues 355-452), and the COOH-terminal fragment (KChAP-C; residues 453-619) in the GAL4 yeast two-hybrid vector pGAD424 were used as described (11). Kvbeta 1.2 in pGBT9 was tested for interaction with individual KChAP fragments by cotransformation of host strain Y190 as previously described (25).

Transfection

COS-1 cells were plated to ~70% confluence in 60-mm dishes 1 day before transfection. Cotransfection of either enhanced green fluorescent protein (EGFP)-C2 or KChAP-M/EGFP-C2 with Kvbeta 1.2/pCR3 was achieved with FuGene (Roche) using 1 µg of each DNA plus 6 µl FuGene/60-mm dish. As a control, Kvbeta 1.2/pCR3 was substituted with pcDNA3 in some experiments. At 24 h posttransfection, cells were lysed in IP buffer (as described above). For IP, equal amounts of protein (150 µg/500 µl) were incubated overnight with or without anti-Kvbeta 1.2 antibody (1:200 dilution).

Expression in Xenopus Oocytes and Electrophysiology

cRNAs for expression in Xenopus oocytes were prepared using the mMESSAGE mMACHINE kit (Ambion). Plasmids encoding Kvbeta 1.2 and Kv1.5 were previously described (1) as were KChAP, Kv2.1, and Kv4.3 (12). cRNAs were injected as previously described (24).

Measurement of Xenopus oocyte whole cell current was performed using the standard two-microelectrode voltage-clamp technique. Bath solution contained (in mM) 5 KOH, 100 NaOH, 0.5 CaCl2, 2 MgCl2, 100 methanesulfonic acid, and 10 HEPES (pH 7.4). Electrodes were filled with 3 M KCl and had a resistance of 0.3-0.6 MOmega . All recordings were made at room temperature. Linear leakage and capacity transient currents were substracted (P/4 prepulse protocol) unless specified, and data were low-pass filtered at 1 kHz. pCLAMP software (Axon Instruments) was used for voltage-pulse protocols and data acquisition. Data are reported as means ± SE. Comparisons among multiple groups of oocytes were performed by one-way ANOVA, Student's t-test, and the Student-Newman-Keuls post hoc test. Means are considered to be significantly different at P < 0.05.


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The molecules of interest to us in rat cardiomyocytes are KChAP, Kv1.2, Kv1.4, Kv1.5, Kv2.1, and Kv4.3, and the particular Kvbeta s represented in these cells. We have already shown that KChAP interacts with Kv2.1 and Kv4.3 in rat heart (12). The expression of Kv1.2, Kv1.4, and Kv1.5, and Kv2.1 and Kv4.3 in rat heart has been reported elsewhere (8, 9, 26). Kvbeta 1.2 was cloned from human heart (10, 13), but the complement of Kvbeta s in rat heart is unknown.

Kvbeta -Subunit Expression in Rat Heart

To determine whether KChAP and Kvbeta -subunits interact in heart, we had to first identify which Kvbeta -subunits are expressed in rat heart. Kvbeta 1.2 was examined first, since it had been cloned previously from human heart (10, 13), and its expression was suggested by RT-PCR from rat heart (14). To confirm its expression in adult rat heart, we did Northern blot analysis and compared its expression with adult rat brain. As shown in Fig. 1A, both heart and brain exhibited a single band of ~5 kb that hybridized with a Kvbeta 1.2- specific riboprobe. Expression of the other Kvbeta -subunit mRNAs was examined by RT-PCR using Kvbeta 1.1-, Kvbeta 1.3-, Kvbeta 2-, or Kvbeta 3-specific primers and rat heart and brain mRNA. Of these four Kvbeta -subunits, only Kvbeta 1.1 mRNA was detected in rat heart and brain (Fig. 1B). Transcripts for Kvbeta 1.3, Kvbeta 2, and Kvbeta 3 were not found in rat heart, although strong signals were detected in brain.


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Fig. 1.   Expression of voltage-dependent K+ (Kv) beta -subunit in rat heart. A: Northern blot analysis of Kvbeta 1.2 expression in adult rat heart and brain. Brain and heart poly(A)+ RNA (2 µg/lane) was separated on a formaldehyde/agarose gel, blotted to BrightStar membrane (Ambion), and hybridized [overnight in ULTRAhyb (Ambion) at 44°C] with a 32P-labeled riboprobe prepared from a fragment of human Kvbeta 1.2 encoding the unique NH2-terminal 79 residues. Washes in 0.1× saline-sodium citrate and 0.1% SDS were done at 50°C, and autoradiography was done at -70°C with Kodak Biomax MS film and an intensifying screen. RNA size markers are indicated on left. B: RT-PCR analysis of the expression of other Kvbeta -subunits in rat heart and brain. Oligonucleotide primers designed to specifically amplify either Kvbeta 1.1, Kvbeta 1.3, Kvbeta 2, or Kvbeta 3 (see METHODS for details) were used in RT-PCR reactions from adult rat heart or brain poly(A)+ RNA. As a control, first-strand cDNA reactions were performed either with (+) or without (-) RT. PCR products were separated by electrophoresis, blotted to BrightStar membrane (Ambion), and Southern blotted with 32P-labeled oligonucleotides designed to detect specific Kvbeta PCR products. Note that, in all reactions, the lanes labeled -RT had no signal, confirming that the bands detected in the experiment were coming from mRNA and not DNA contamination. The blots were overexposed to confirm that no signal could be detected in rat heart for Kvbeta 1.3, Kvbeta 2, or Kvbeta 3 mRNA. C: anti-Kvbeta 1.2 antibody recognizes Kvbeta 1.2 overexpressed in Xenopus oocytes. Lysates prepared 2 days postinjection from oocytes injected with cRNA for Kvbeta 1.2 (lane 2) or from uninjected oocytes (lane 1) were examined by Western blotting with affinity-purified polyclonal anti-Kvbeta 1.2. Immunoreactive bands were visualized with ECL-Plus (Amersham Pharmacia Biotech). Molecular mass markers (kDa) are indicated on left, and the position of Kvbeta 1.2 is marked on right. No Kvbeta 1.2 is detected in uninjected oocytes. D: Western blot analysis of Kvbeta 1.2 expression in membrane-enriched fractions from rat tissues. Crude membrane fractions (40 µg/lane) from 14-day-old rat heart (lane 1), brain (lane 2), and skeletal muscle (lane 3) were probed with the anti-Kvbeta 1.2 antibody. E: Western blot analysis of expression of Kvbeta 1.2 in rat and human tissue lysates. Lysates prepared from rat brain (lane 1), rat lung (lane 2), rat liver (lane 3), rat skeletal muscle (lane 4), rat kidney (lane 5), rat atrium (lane 6), rat ventricle (lane 7), human atrial appendage (lane 8), and human ventricle (lane 9), all at 40 µg/lane, were examined.

Given the expression of Kvbeta 1.2 message in both rat and human heart, we raised a polyclonal antibody to the unique NH2 terminus of Kvbeta 1.2 to examine protein levels in native tissue. Affinity-purified anti-Kvbeta 1.2 antisera detected a single band of ~45 kDa in lysates of oocytes injected with Kvbeta 1.2 cRNA, consistent with the predicted molecular mass of Kvbeta 1.2 (Fig. 1C, lane 2). In adult rat tissue lysates (Fig. 1E), anti-Kvbeta 1.2 antibody detected a band of ~45 kDa in skeletal muscle (lane 4), atrium (lane 6), and ventricle (lane 7). In lung (lane 2) and kidney (lane 5) lysates, the antibody labeled a faint protein band of slightly lower mobility, ~47 kDa. No signal was obtained from rat liver (lane 3) or brain (lane 1) lysates. The lack of Kvbeta 1.2 reactivity in brain was surprising, since Kvbeta 1.2 message was detected by Northern blotting (Fig. 1A). Because Kvbeta -subunits are Kv1.x channel-associated proteins, we examined the expression of Kvbeta 1.2 in enriched membrane fractions from rat tissues (Fig. 1D). As shown in Fig. 1E, Kvbeta 1.2 was detected in membranes from rat brain (lane 2), but at a much lower level than in heart (lane 1) or skeletal muscle (lane 3). In human heart, Kvbeta 1.2 was detected at equivalent levels in the human atrial appendage (Fig. 1E, lane 8) and ventricle (lane 9).

Kvbeta 1.2 Coimmunoprecipitates with Kvalpha 1-Subunits in Rat Heart

To identify binding partners for Kvbeta 1.2, we performed coimmunoprecipitation experiments. In Fig. 2A, a membrane fraction from 3-day postnatal rat heart was solubilized with 1% Triton X-100 buffer, immunoprecipitated with anti-Kvbeta 1.2, and Western blotted with anti-Kv1.4. Kv1.4 was detected as a double band of 90 and 110 kDa in adult rat brain (Fig. 2A, left) and 3-day postnatal rat heart (Fig. 2A, right) membranes. Both bands were also seen in complexes immunoprecipitated with anti-Kvbeta 1.2. Figure 2B shows that Kv1.2 was also immunoprecipitated with Kvbeta 1.2, in this case from adult rat heart lysates. Anti-Kv1.2 detected a prominent band at ~76-kDa in both the lysate and immunocomplexes. Because the anti-Kv1.5 antibody that we raised recognizes the human channel much better than rat, we examined association of Kv1.5 with Kvbeta 1.2 from the human atrial appendage. In lysates from human atrial appendage, we observed a major band of ~70 kDa, which was also present in immunocomplexes with Kvbeta 1.2 (Fig. 2C). Thus Kvbeta 1.2 was found in association with all three Kv1.x subunits that have been shown to be expressed in either rat or human heart, Kv1.2, Kv1.4, and Kv1.5. By contrast, we were never able to identify two other cardiac Kv channels, Kv2.1 or Kv4.3, in immunocomplexes with Kvbeta 1.2 (data not shown).


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Fig. 2.   Kvbeta 1.2 associates with Kv1.4 (A), Kv1.2 (B), and Kv1.5 (C) in heart. A: membranes prepared from 2-day-old rat heart were solubilized in 1% Triton X-100 buffer and immunoprecipitated with anti-Kvbeta 1.2 antibody. Immunocomplexes were collected on secondary antibody-coated beads. As a control for nonspecific binding to the beads, primary antibody was omitted from one reaction. Right: solubilized membranes (40 µg) and material collected on the beads with (+) or without (-) primary antibody were examined by Western blotting with monoclonal anti-Kv1.4 (1:500 dilution; Upstate Biotechnology). Left: solubilized brain membranes from 2-day-old rat were blotted for comparison of molecular size. IP, immunoprecipitate. B: total protein lysates from 2-day-old rat heart were immunoprecipitated with Kvbeta 1.2 and blotted with monoclonal anti-Kv1.2 (1:500 dilution; Upstate Biotechnology). Immunoprecipitated material was compared with anti-Kv1.2 reactivity in lysates (40 µg/lane). C: total protein lysates of human atrial appendage were immunoprecipitated with anti-Kvbeta 1.2 and blotted with polyclonal anti-Kv1.5 (1:100). Immunoprecipitated material was compared with anti-Kv1.5 reactivity in lysates (40 µg/lane). Positions of molecular weight markers are indicated on left, and arrows at right mark the mobility of each Kvalpha 1 polypeptide.

Kvbeta 1.2 Binds to the KChAP-M Fragment

Having demonstrated Kvbeta 1.2 association with a subset of cardiac Kv channels, we examined the interaction of Kvbeta 1.2 with KChAP. In the yeast two-hybrid system, we showed that KChAP binds to the conserved Kvbeta 1 COOH-terminus of Kvbeta 1.2, the same fragment to which the Kvalpha 1 NH2-terminus binds (25). To determine whether Kvbeta 1.2 interacts with KChAP in myocardium, we performed coimmunoprecipitation experiments with anti-Kvbeta 1.2 and blotted with anti-KChAP. We were unable to detect KChAP reproducibly in these experiments, suggesting a weak or very transient interaction.

In a previous study, the Kvalpha -subunit binding fragment of KChAP was localized to a stretch of 98 amino acids in the middle of the protein, which we called KChAP-M (12). To determine if KChAP-M also interacted with Kvbeta 1.2, we used yeast two-hybrid assays. As shown in Fig. 3, A and B, we found that KChAP-M did interact with Kvbeta 1.2, but, just as was observed with Kvalpha NH2-termini, there was no evidence of interaction between the other KChAP fragments (N and C) and Kvbeta 1.2. This interaction was confirmed using coimmunoprecipitation experiments from cotransiently transfected COS-1 cells. EGFP-tagged KChAP-M was found in association with Kvbeta 1.2 (Fig. 3C, left). As a control for nonspecific binding of EGFP-KChAP-M to the secondary antibody-coated beads, we did the same experiments with lysates from cells transfected with only EGFP-KChAP-M and no Kvbeta 1.2 and did not detect any KChAP-M attached to the beads (Fig. 3C, middle). In a second control experiment, we saw that EGFP did not coimmunoprecipitate with Kvbeta 1.2 (Fig. 3C, right). Thus binding sites for Kvalpha NH2 termini and Kvbeta -subunits are located within the same 98-amino acid fragment of KChAP.


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Fig. 3.   Middle fragment of KChAP (KChAP-M) interacts with Kvbeta 1.2, but KChAP does not alter Kvbeta 1.2 protein levels in heterologous expression systems. A: KChAP fragments tested for interaction with Kvbeta 1.2 in the yeast two-hybrid (Y2H) assay. B: positive interaction in Y2H was obtained between full-length KChAP and between Kvbeta 1.2 and KChAP-M and Kvbeta 1.2. C: Kvbeta 1.2 and KChAP-M coimmunoprecipitate in lysates from transiently transfected COS-1 cells. Lysates from cells transfected with either Kvbeta 1.2/pCR3 plus EGFP-C2/ KChAP-M (left), pcDNA3 plus EGFP-C2/KChAP-M (middle), or Kvbeta 1.2/pCR3 plus EGFP-C2 (right) were immunoprecipitated with anti-Kvbeta 1.2. Parallel reactions omitted primary antibody to control for nonspecific binding to secondary antibody-coated beads. Immunoprecipitated material was electrophoresed next to aliquots of the lysate (10 µg/lane), and Western blots (WB) were probed with monoclonal anti-green fluorescent protein (GFP; Clontech). Note that EGFP/KChAP-M coimmunoprecipitated with Kvbeta 1.2 but EGFP did not. D: KChAP does not alter Kvbeta 1.2 protein levels in heterologous expression systems. Uninjected oocytes or oocytes injected with cRNAs for Kvbeta 1.2 alone, KChAP alone, or equivalent amounts of Kvbeta 1.2 plus KChAP were homogenized 1 day postinjection in 1% Triton X-100 buffer. Equal amounts of protein (10 µg/lane) were analyzed by Western blotting with anti-Kvbeta 1.2 (D) and anti-KChAP (E). Note that the presence of KChAP does not affect Kvbeta 1.2 expression, and the presence of Kvbeta 1.2 does not alter KChAP protein levels.

KChAP Does Not Increase Kvbeta 1.2 Protein Levels

We have shown previously that KChAP acts as a novel chaperone to increase protein levels and functional expression of the Kv channels Kv2.1 and Kv4.3 (12, 25). With the exception of Kv1.3, the levels of Kv1.x subunits do not change when coexpressed with KChAP, even though KChAP is able to bind to the NH2-termini of these subunits. To determine whether KChAP can affect the level of expression of Kvbeta 1.2, we examined steady-state protein levels after coexpression of the two proteins in Xenopus oocytes. As shown in Fig. 3D, the amount of Kvbeta 1.2 was not changed when Kvbeta 1.2 was either expressed alone (lane 2) or with an equimolar ratio of KChAP cRNA (lane 3). Conversely, Kvbeta 1.2 did not alter KChAP levels either (Fig. 3E). Note the equivalent KChAP protein levels in oocytes injected with KChAP alone (lane 2) or with an equimolar ratio of Kvbeta 1.2 cRNA (lane 3).

Functional Consequences of KChAP-Kvbeta 1.2 Interactions on Kv Channel Expression

Both KChAP and Kvbeta 1.2 bind to the NH2 termini of Kv1.2, Kv1.4, and Kv1.5; Kvbeta 1.2 alters processing and can introduce or accelerate open-channel block of Kv1.x subunits (10, 13, 21) while KChAP produces no obvious alteration in either parameter (12, 25). Because both proteins bind to the NH2 termini of Kv1.x subunits and to each other, we hypothesized that KChAP may indirectly affect Kv1.x channel expression through the modulation of Kvbeta 1.2/Kv1.x subunit interactions. We tested this hypothesis by heterologous expression of KChAP with the cardiac channel Kv1.5 and the COOH-terminus of Kvbeta 1.2 in Xenopus oocytes. We showed previously that the Kvbeta 1 COOH terminus (Kvbeta 1-C) produced a significant decrease in the expression of Kv1.5 currents (1) and have used this construct rather than full-length Kvbeta 1.2 to analyze effects in the absence of the fast inactivation introduced by the beta -ball. Kv1.5 cRNA alone, Kv1.5, and Kvbeta 1-C cRNAs or Kv1.5, Kvbeta 1-C and KChAP cRNAs were injected in Xenopus oocytes. As shown in Fig. 4, injection of Kvbeta 1-C produced dramatic suppression of Kv1.5 current. When KChAP cRNA was added to the injectate, we observed a significant recovery in Kv1.5 current. Five days after injection, the steady-state currents at +70 mV potential were 1.14 ± 0.03, 0.20 ± 0.03, and 0.61 ± 0.08 µA for Kv1.5 alone, Kv1.5 plus Kvbeta 1-C, and Kv1.5 plus Kvbeta 1-C and KChAP, respectively (n = 10 for each group). Averaged whole cell current traces are shown in Fig. 4A. The disinhibition by KChAP was concentration dependent (Fig. 4B). As we reported previously, coexpression of Kv1.5 with Kvbeta 1-C (1) or with KChAP (25) had no effects on kinetics and voltage dependence of the Kv1.5 current. Simultaneous coexpression of Kvbeta 1-C and KChAP with Kv1.5 also did not affect kinetics of the Kv1.5 current (Fig. 4C). Time constants of activation were 6.64 ± 0.29, 6.72 ± 0.38, and 6.92 ± 0.28 ms for Kv1.5 alone, Kv1.5 with Kvbeta 1-C, and Kv1.5 with Kvbeta 1-C plus KChAP, respectively, and current-voltage relationships were scaled.


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Fig. 4.   KChAP modulates the effect of the Kvbeta 1 COOH terminus (Kvbeta 1-C) on Kv1.5 expression. A: average whole cell currents from Xenopus oocytes injected with Kv1.5 cRNA (100 ng/µl) alone, oocytes coinjected with Kv1.5 and Kvbeta 1-C cRNAs (100 and 250 ng/µl, respectively), or oocytes coinjected with Kv1.5, Kvbeta 1-C, and KChAP cRNAs (100, 250, and 500 ng/µl, respectively). Recordings were performed 5 days after injection (1 injection series, n = 10 for each group of oocytes). Holding potential was -90 mV, and 175-ms pulses were from -80 to +80 mV with 10-mV steps; 5 mM K+ was in bath solution. B: bar graph of steady-state whole cell currents measured in Xenopus oocytes injected with Kv1.5 cRNA (100 ng/µl) alone or coinjected with a constant amount of Kvbeta 1-C cRNA and with increasing amounts of KChAP cRNA. cRNA concentrations (ng/µl) are indicated at top of bars. Currents were recorded 5 days postinjection at the end of 175-ms pulses to +70 mV (n = 10 for each group of oocytes). *Significant difference from the value of the currents measured in oocytes coinjected with Kv1.5 and Kvbeta 1-C. C: normalized and average steady-state currents (same injection series as in A) plotted as function of test potential recorded from oocytes injected with Kv1.5 cRNA (100 ng/ml) alone (n = 10; ) or coinjected with Kv1.5, Kvbeta 1-C, and KChAP cRNAs (100, 250, and 500 ng/µl, respectively; n = 10; triangle ). Inset: superimposition of averaged and normalized currents from oocytes injected with Kv1.5 cRNA alone and oocytes coinjected with Kv1.5, Kvbeta 1-C, and KChAP cRNAs at +70-mV test potential.

In contrast to Kv1.5, Kv1.4 current amplitude was increased by Kvbeta 1-C or Kvbeta 2 (1, 17). This increase was suppressed by coinjection with KChAP (Fig. 5). On the second day after injection, oocytes coinjected with Kv1.4 and Kvbeta 2 expressed current that was two to three times larger than in oocytes injected with Kv1.4 alone. Addition of KChAP cRNA to the injectate suppressed the stimulatory effect (Fig. 5A). At a test potential of +70 mV, averaged peak currents were 1.9 ± 0.2, 5.2 ± 0.4, and 2.8 ± 0.3 µA for oocytes injected with Kv1.4 alone, Kv1.4 plus Kvbeta 2, and Kv1.4 plus Kvbeta 2 and KChAP, respectively (n = 8-10 for each group). Additionally, KChAP attenuated the acceleration of inactivation of Kv1.4 current produced by Kvbeta 2. Time constants of the inactivation were 15.02 ± 0.73, 12.31 ± 0.53, and 14.11 ± 0.49 ms for oocytes injected with Kv1.4 alone, Kv1.4 plus Kvbeta 2, and Kv1.4 plus Kvbeta 2 and KChAP, respectively (Fig. 5B).


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Fig. 5.   KChAP modulates the effect of Kvbeta 2 on Kv1.4 channel expression. A: averaged whole cell currents from Xenopus oocytes injected with Kv1.4 cRNA (5 ng/µl) alone and oocytes coinjected with Kv1.4 and Kvbeta 2 cRNAs (5 and 125 ng/µl, respectively) or coinjected with Kv1.4, Kvbeta 2, and KChAP cRNAs (5, 125, and 250 ng/µl, respectively). Recordings were performed 2 days after injection (1 injection series, n = 8-10 for each group of oocytes). Holding potential was -90 mV, and 200-ms pulses were from -50 to +80 mV with 10-mV steps; 50 mM K+ was in bath solution. B: normalized and averaged steady-state currents (same injection series as in A) plotted as function of test potential recorded from oocytes injected with Kv1.4 cRNA (5 ng/ml) alone (n = 10), coinjected with Kv1.4 and Kvbeta 2 cRNAs (5 and 125 ng/µl, respectively; n = 8), coinjected with Kv1.4, Kvbeta 2, and KChAP cRNAs (5, 125, and 250 ng/µl, respectively; n = 8), and coinjected with Kv1.4 and KChAP cRNAs (5 and 250 ng/µl, respectively; n = 8) . Inset: superimposition of averaged and normalized currents from oocytes injected with Kv1.4 cRNA alone and oocytes coinjected with different combinations of Kv1.4, Kvbeta 2, and KChAP cRNAs at +70-mV test potential.

We hypothesized that Kvbeta 1.2, although it does not interact directly with Kv2.1 or Kv4.3, might suppress the stimulatory effect of KChAP Kv2.1 or Kv4.3 currents. As we have described previously (25) and as replicated in this experiment, KChAP produced a twofold increase of Kv2.1 current (Fig. 6 A and B). When increasing concentrations of Kvbeta 1.2 cRNA were added, a dose-dependent reduction in the effect of KChAP on Kv2.1 was seen. At a 2:1 ratio of Kvbeta 1.2 to KChAP cRNA, the expression enhancement of Kv2.1 currents by KChAP was eliminated. Coexpression of Kvbeta 1.2 cRNA alone or in combination with KChAP produces no changes in voltage dependence or kinetics of Kv2.1 currents (Fig. 6C). For Kv2.1 alone and Kv2.1 coinjected with KChAP and Kvbeta 1.2, time constants of activation were 17.1 ± 0.2 (n = 10) and 17.4 ± 0.2 (n = 10) ms, respectively.


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Fig. 6.   Kvbeta 1.2 blocks the KChAP-mediated enhancement of Kv2.1 channel expression. A: averaged whole cell currents from oocytes injected with Kv2.1 cRNA (0.62 ng/µl) alone, oocytes coinjected with Kv2.1 and KChAP cRNAs (0.62 and 125 ng/µl, respectively), or oocytes coinjected with Kv2.1, KChAP, and Kvbeta 1.2 cRNAs (0.62, 125, and 250 ng/µl, respectively). Recordings were performed 5 days after injection (1 injection series, n = 10 for each group of oocytes). Holding potential was -90 mV, and 200 ms pulses were from -70 to +80 mV with 10-mV steps; 5 mM K+ was in bath solution. B: bar graph of whole cell currents measured in oocytes injected with Kv2.1 cRNA (0.62 ng/µl) alone or coinjected with a constant amount of KChAP cRNA and an increasing amount of Kvbeta 1.2 cRNA. cRNA concentrations (ng/µl) are indicated at top of bars. Currents were recorded 5 days postinjection (n = 10 for each group of oocytes). Currents were measured at the ends of 200-ms pulses to +70 mV. **Significant difference from the value of the currents measured in oocytes coinjected with Kv2.1 and KChAP. C: normalized and averaged steady-state currents (same injection series as in A) plotted as a function of test potential recorded from oocytes injected with Kv2.1 cRNA (0.62 ng/ml) alone (n = 10; ) or coinjected with Kv2.1, KChAP, and Kvbeta 1.2 cRNAs (0.62, 125, and 250 ng/µl, respectively; n = 10; triangle ). Inset: superimposition of averaged and normalized currents from oocytes injected with Kv2.1 alone and oocytes coinjected with Kv2.1, KChAP, and Kvbeta 1.2 at +70-mV test potential.

Similar results were obtained when KChAP and Kv4.3 were coexpressed in the presence of Kvbeta 1.2 (Fig. 7). KChAP also produced an approximate twofold increase in Kv4.3 currents that was abolished when an excess of Kvbeta 1.2 cRNA was coinjected. By itself, Kvbeta 1.2 had no effect on Kv4.3 current amplitude or kinetics. On the second day after injection, average peak currents at +70 mV were 1.38 ± 0.11, 4.01 ± 0.24, 2.34 ± 0.16, and 1.50 ± 0.13 µA for oocytes injected with Kv4.3 cRNA alone, Kv4.3 plus KChAP cRNAs, Kv4.3 plus KChAP and Kvbeta 1.2 cRNAs, and Kv4.3 plus Kvbeta 1.2 cRNAs, respectively (n = 8-12). Time constants of inactivation for Kv4.3 alone, Kv4.3 coinjected with Kvbeta 1.2, and Kv4.3 coinjected with KChAP and Kvbeta 1.2 were 47.2 ± 1.0, 47.3 ± 0.7, and 48.3 ± 0.3 ms, respectively (n = 5). Because Kvbeta 1.2 does not bind to Kv2 or Kv4 channels, these results are consistent with the interpretation that Kvbeta 1.2 binds to KChAP and prevents its association with the channel.


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Fig. 7.   Kvbeta 1.2 blocks the KChAP-mediated enhancement of Kv4.3 channel expression. A: averaged whole cell currents from oocytes injected with Kv4.3 cRNA (10 ng/µl) alone, oocytes coinjected with Kv4.3 and KChAP cRNAs (10 and 125 ng/µl, respectively), or oocytes coinjected with Kv4.3, KChAP, and Kvbeta 1.2 cRNAs (10, 125, and 250 ng/µl, respectively). Recordings were performed 2 days after injection (1 injection series, n = 10 for each group of oocytes). Holding potential was -90 mV, and 200-ms pulses were from -70 to +80 mV with 10-mV steps; 5 mM K+ was in bath solution. B: bar graph of peak whole cell currents at +70 mV measured in oocytes injected with Kv4.3 cRNA alone or coinjected with different combinations of KChAP or Kvbeta 1.2 cRNA. Currents were recorded 2 days postinjection (n = 10 for each group). cRNA concentrations (ng/µl) are indicated at top of bars. C: normalized and averaged peak currents (same injection series as in A) plotted as function of test potential recorded from oocytes injected with Kv4.3 cRNA (10 ng/ml) alone (n = 10; ) or Kv4.3, KChAP, and Kvbeta 1.2 cRNAs (10, 125, and 250 ng/µl, respectively; n = 10; triangle ). Inset: superimposition of averaged and normalized currents from oocytes injected with Kv4.3 alone and oocytes coinjected with Kv4.3, KChAP, and Kvbeta 1.2 cRNAs at +70-mV test potential.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The modulation of Kv channels by accessory proteins is an expanding area of research. Here we report that two distinct modulatory subunits may interact with each other to regulate Kv channels. The interactions between KChAP and Kvbeta -subunits may have important consequences for the properties, i.e., expression level and gating, of Kv channels. Results from this model system predict that controlling the relative levels of interacting modulatory subunits within a single cell can constitute a novel mechanism for modulating Kv currents.

Kvbeta -subunits and KChAP are representative of the two functionally distinct classes of cytoplasmic Kv channel modulatory proteins that have been described to date. The first class includes those subunits that bind tightly to the NH2 termini of Kvalpha -subunits, exist as part of the mature cell-surface channel complexes, and alter channel gating and/or expression. These include the Kvbeta -subunits, which are specific for Kvalpha 1 channels (27), and the recently described KChIP family, which binds only to Kvalpha 4 subunits (3). KChAP belongs to a second class of modulators and acts as a chaperone to increase a subset of Kvalpha currents. KChAP differs from Kvbeta -subunits or KChIP in that it interacts only transiently with the NH2 termini of Kvalpha -subunits, thus behaving more as a true chaperone (12, 25). The functional relationship between KChAP and KChIPs is not known. It will be interesting to determine whether these two subunits can compete for binding sites on Kv4 channels to produce altered currents.

The present experiments show for the first time at the protein level that Kvbeta 1.2 is expressed in both rat and human heart and that it coimmunoprecipitates with cardiac Kv1.2, Kv1.4, and Kv1.5. The association of Kvbeta 1.2 with the very slowly inactivating Kv1.2 and Kv1.5 proteins in cardiomyocytes could result in NH2-type inactivation and raises the possibility that Ito in these cells is in part a result of these associations.

We were unable to demonstrate an association between Kvbeta 1.2 and KChAP in rat heart by coimmunoprecipitation. This result was consistent with a lack of a detectable interaction between KChAP and Kvbeta 1.2 overexpressed in Xenopus oocytes and may reflect the transient nature of the interaction. However, KChAP-M, the middle 98-residue fragment that contains the Kvalpha -NH2-terminal binding sites, was found to interact with Kvbeta 1.2 by both yeast two-hybrid assays and coimmunoprecipitation from transfected cells. Interestingly, this is the same fragment of KChAP that binds to the NH2 terminus of Kvalpha -subunits. Competition for the same binding site on KChAP could explain the interference of Kvbeta 1.2 with the enhancement of the functional expression of Kv2.1 and Kv4.3 by KChAP. There is no evidence that Kvbeta -subunits bind directly to Kv2 or Kv4 NH2 termini, so it is likely that Kvbeta 1.2 binding to KChAP would prevent its association with these channels. Because KChAP coimmunoprecipitates with Kv2.1 and Kv4.3 in rat cardiomyocytes, it is possible that KChAP modulates these currents directly via its chaperone effect and that Kvbeta 1.2 modulates them indirectly via interactions with KChAP.

The situation with KChAP, Kvalpha -subunit, and Kvbeta -subunit is more complex because Kvbeta -subunits can also bind to the NH2 terminus of Kvalpha 1 subunits. At this point, we are not able to distinguish whether the interference of KChAP on the modulation of Kv1.5 and Kv1.4 by Kvbeta -subunits is a result of 1) KChAP binding to Kvbeta and preventing its association with Kv1.x or 2) KChAP binding to the Kv1.x NH2 terminus and preventing Kvbeta binding. The latter explanation would give some functional significance to the interactions between Kvalpha 1 subunits and KChAP, since, with the exception of Kv1.3, KChAP does not act as a chaperone for Kv1.x subunits.

Both Kvbeta -subunits and KChAP are thought to interact with Kvalpha -subunits at early stages in channel synthesis or assembly. Kvbeta -subunits have been shown to assemble with Kvalpha 1 subunits while the channels are in the endoplasmic reticulum (15, 21). This complex promotes trafficking of the channels to the cell surface (17). We have evidence from in vitro translation experiments to suggest that KChAP interacts with Kvalpha -subunits cotranslationally (25). Furthermore, for KChAP to act as a chaperone for Kvalpha -subunits in oocytes, the two cRNAs must be injected in the same pipette (25). Because the timing of interaction of both types of subunits with Kvalpha -subunits during processing may be comparable, it is reasonable to assume that preventing the initial interactions of the channel with the modulatory subunit would eliminate the influence of this subunit entirely. It is likely then that the extent to which different modulatory subunits might affect Kv currents would be dependent on their levels relative to other binding partners. For KChAP, this is especially intriguing, since it belongs to a family of proteins known to interact with and modulate the activity of certain transcription factors such as STAT3 (6). Regulating the availability of auxiliary subunits for channel binding might constitute a system in which channel expression could be rapidly altered without changing channel message levels. In the future, experiments addressing these possibilities will be targeted directly in native cardiomyocytes that express all of the component molecules.


    ACKNOWLEDGEMENTS

We thank Dr. W. Dong for technical assistance with the oocytes.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-55404, HL-36930, and NS-23877 (to A. M. Brown), HL-60759 (to B. A. Wible), and HL-61436 (to D. L. Kunze for support of A. N. Ramirez).

Address for reprint requests and other correspondence: A. M. Brown, Rm. 301, Rammelkamp Center, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109-1998 (E-mail: abrown{at}metrohealth.org).

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.

Received 1 July 2000; accepted in final form 13 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Accili, EA, Kiehn J, Yang Q, Wang Z, Brown AM, and Wible BA. Separable Kvbeta subunit domains alter expression and gating of potassium channels. J Biol Chem 272: 25824-25831, 1997[Abstract/Free Full Text].

2.   Accili, EA, Kuryshev YA, Wible BA, and Brown AM. Separable effects of human Kvbeta1.2 N- and C-termini on inactivation and expression of human Kv14. J Physiol (Lond) 512: 325-336, 1998[Abstract/Free Full Text].

3.   An, WF, Bowlby MR, Betty M, Cao J, Ling HP, Mendoza G, Hinson JW, Mattsson KI, Strassle BW, Trimmer JS, and Rhodes KJ. Modulation of A-type potassium channels by a family of calcium sensors. Nature 403: 553-556, 2000[ISI][Medline].

4.   Barhanin, J, Lesage F, Guillemare E, Fink M, Lazdunski M, and Romey G. K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. Nature 384: 78-80, 1996[ISI][Medline].

5.   Barry, DM, and Nerbonne JM. Myocardial potassium channels: electrophysiological and molecular diversity. Annu Rev Physiol 58: 363-394, 1996[ISI][Medline].

6.   Chung, CD, Liao J, Liu B, Rao X, Jay P, Berta P, and Shuai K. Specific inhibition of Stat3 signal transduction by PIAS3. Science 278: 1803-1805, 1997[Abstract/Free Full Text].

7.   Deal, KK, England SK, and Tamkun MM. Molecular physiology of cardiac potassium channels. Physiol Rev 1: 49-67, 1996.

8.   Dixon, JE, and McKinnon D. Quantitative analysis of potassium channel mRNA expression in atrial and ventricular muscle of rats. Circ Res 75: 252-260, 1994[Abstract].

9.   Dixon, JE, Shi W, Wang HS, McDonald C, Yu H, Wymore RS, Cohen IS, and McKinnon D. Role of the Kv4.3 K+ channel in ventricular muscle A molecular correlate for the transient outward current. Circ Res 79: 659-668, 1996[Abstract/Free Full Text].

10.   England, SK, Uebele VN, Shear H, Kodali J, Bennett PB, and Tamkun MM. Characterization of a voltage-gated K+ channel beta subunit expressed in human heart. Proc Natl Acad Sci USA 92: 6309-6313, 1995[Abstract].

11.   Fink, M, Duprat F, Lesage F, Heurteaux C, Romey G, Barhanin J, and Lazduski M. A new K+ channel beta subunit to specifically enhance Kv2.2 (CDRK) expression. J Biol Chem 271: 26341-26348, 1996[Abstract/Free Full Text].

12.   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].

13.   Majumder, K, De Biasi M, Wang Z, and Wible BA. Molecular cloning and functional expression of a novel potassium channel beta -subunit from human atrium. FEBS Lett 361: 13-16, 1995[ISI][Medline].

14.   Morales, MJ, Castellino RC, Crews AL, Rasmusson RL, and Strauss HC. A novel beta  subunit increases rate of inactivation of specific voltage-gated potassium channel alpha  subunits. J Biol Chem 270: 6272-6277, 1995[Abstract/Free Full Text].

15.   Nagaya, N, and Papazian DM. Potassium channel alpha  and beta  subunits assemble in the endoplasmic reticulum. J Biol Chem 272: 3022-3027, 1997[Abstract/Free Full Text].

16.   Nakahira, K, Shi G, Rhodes KJ, and Trimmer JS. Voltage-gated K+ channel beta -subunits with alpha -subunits. J Biol Chem 271: 7084-7089, 1996[Abstract/Free Full Text].

17.   Peri, R, Wible BA, and Brown AM. Mutations in the Kvbeta 2 binding site for NADPH and their effects on Kv1.4. J Biol Chem 276: 738-741, 2001[Abstract/Free Full Text].

18.   Rettig, J, Heinemann SH, Wunder F, Lorra C, Parcej DN, Dolly JO, and Pongs O. Inactivation properties of voltage-gated K+ channels altered by presence of beta -subunit. Nature 369: 289-294, 1994[ISI][Medline].

19.   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].

20.   Scott, VES, Rettig J, Parcej DN, Keen JN, Findlay JBC, Pongs O, and Dolly JO. Primary structure of a beta  subunit of alpha -dendrotoxin-sensitive K+ channels from bovine brain. Proc Natl Acad Sci USA 91: 1637-1641, 1994[Abstract].

21.   Shi, G, Nakahira K, Hammond S, Rhodes KJ, Schechter LE, and Trimmer JS. beta Subunits promote K+ channel surface expression through effects early in biosynthesis. Neuron 16: 843-852, 1996[ISI][Medline].

22.   Wallner, M, Meera P, Ottolia M, Kaczorowki GJ, Latorre R, Garcia ML, Stefani E, and Toro L. Characterization and modulation by a beta -subunit of a human maxi KCa channel cloned from myometrium. Receptors Channels 3: 185-199, 1995[ISI][Medline].

23.   Wang, W, Xia J, and Kass RS. MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled IsK channel. J Biol Chem 273: 34069-34074, 1998[Abstract/Free Full Text].

24.   Wang, Z, Kiehn J, Yang Q, Brown AM, and Wible BA. Comparison of binding and block produced by alternatively spliced Kvbeta 1 subunits. J Biol Chem 271: 28311-28317, 1996[Abstract/Free Full Text].

25.   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].

26.   Xu, H, Dixon JE, Barry DM, Trimmer JS, Merlie JP, McKinnon D, and Nerbonne JM. Developmental analysis reveals mismatches in the expression of K+ channel alpha subunits and voltage-gated K+ channel currents in rat ventricular myocytes. J Gen Physiol 108: 405-419, 1996[Abstract].

27.   Xu, J, and Li M. Auxiliary subunits of Shaker-type potassium channels. Trends Cardiovasc Med 8: 229-234, 1998[ISI].


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