Cardiovascular Research

Cardiovascular Research 2001 52(2):255-264; doi:10.1016/S0008-6363(01)00374-1
© 2001 by European Society of Cardiology

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Decher, N. Articles by Wollnik, B. Search for Related Content PubMed PubMed Citation Articles by Decher, N. Articles by Wollnik, B. Social Bookmarking          What's this?

Copyright © 2001, European Society of Cardiology

hKChIP2 is a functional modifier of hKv4.3 potassium channels

Cloning and expression of a short hKChIP2 splice variant

Niels Decher^a, Oya Uyguner^b, Constanze R Scherer^a, Birsen Karaman^b, Memnune Yüksel-Apak^b, Andreas E Busch^a, Klaus Steinmeyer^a,* and Bernd Wollnik^b,*

^aAventis Pharma Deutschland GmbH, DG Cardiovascular Diseases, D-65926 Frankfurt am Main, Germany
^bCenter of Molecular Cardiac Arrhythmia, Division of Medical Genetics, Institute of Child Health, Istanbul University, Istanbul, Turkey

klaus.steinmeyer{at}aventis.com

^* Corresponding authors. Tel.: +49-69-305-3416; fax: +49-69-305-16393 wollnik{at}superonline.com

Received 4 April 2001; accepted 6 June 2001

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
Objective: The Ca²⁺ independent transient outward K⁺ current (I_to1) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K⁺ channel -subunit contributes to the I_to1 current in many regions of the human heart. Consistently, downregulation of hKv4.3 transcripts in heart failure and atrial fibrillation is linked to reduction in I_to1 conductance. The recently cloned KChIP family of calcium sensors has been shown to modulate A-type potassium channels of the Kv4 K⁺ channel subfamily. Methods and results: We describe the cloning and tissue distribution of hKChIP2, as well as its functional interaction with hKv4.3 after expression in Xenopus oocytes. Furthermore, we isolated a short splice variant of the hKChIP2 gene (hKCNIP2), which represents the major hKChIP2 transcript. Northern blot analyses revealed that hKChIP2 is expressed in the human heart and occurs in the adult atria and ventricles but not in the fetal heart. Upon coexpression with hKv4.3 both hKChIP2 isoforms increased the current amplitude, slowed the inactivation and increased the recovery from inactivation of hKv4.3 currents. For the first time we analyzed the influence of a KChIP protein on the voltage of half-maximal inactivation of Kv4 channels. We demonstrate that the hKChIP2 isoforms shifted the half-maximal inactivation to more positive potentials, but to a different extent. By elucidating the genomic structure, we provide important information for future analysis of the hKCNIP2 gene in candidate disorders. In the course of this work we mapped the hKCNIP2 gene to chromosome 10q24. Conclusions: Heteromeric hKv4.3/hKChIP2 currents more closely resemble native epicardial I_to1, suggesting that hKChIP2 is a true β-subunit of human cardiac I_to1. As a result hKChIP2 might play a role in cardiac diseases, where a contribution of I_to1 has been shown.

KEYWORDS Arrhythmia (mechanisms); K-channel; Long QT syndrome; Membrane currents

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The Ca²⁺ independent transient outward current I_to1 is an important repolarizing K⁺ current in human heart [1], which is especially important for the early phase of repolarization [2,3]. It controls the height of the early plateau of the action potential and thereby influences other currents that function in following phases of repolarization. The I_to1 current is not uniformly distributed within the mammalian [4–8] and the human heart [9,10]. Differences in the current density, biophysical properties and regulation of I_to1 exist in different heart regions and in the ventricular free wall [6,9,10].

The magnitude of I_to1 is reduced in various cardiac diseases, including ventricular failure [11,12], myocardial infarction [13] and atrial fibrillation [14,15]. Downregulation of I_to1 is accompanied by a significant prolongation of action potential duration (APD) [16]. Although this finding suggests a direct link between the magnitude of I_to1 and APD, this is difficult to prove experimentally, since in failing heart, for example, other repolarizing currents are altered as well [16,17].

Members of the Shal or Kv4 family of potassium channel proteins are thought to underlie native cardiac I_to1 currents. hKv4.3 in particular is believed to be responsible for the major part of human I_to1 current [17,18]. However, heterologously expressed hKv4.3 channels do not completely reproduce the properties of the native I_to1 currents. In particular, the considerably slower recovery from inactivation of hKv4.3 channels compared to native currents [2,3,9], indicates the presence of modulatory subunits within the native I_to1 channel complex.

KChIP proteins have recently been shown to associate with rat Kv4 proteins [19] and to reconstitute features of native brain A-type potassium channels [19]. This finding prompted us to investigate whether hKChIP2 is able to modulate hKv4.3 currents and to reproduce cardiac I_to1 channels. We show that hKChIP2 is expressed in human heart and that the majority of transcripts encode a short splice variant of hKChIP2. We demonstrate that both isoforms of hKChIP2 strongly changed the current amplitude and kinetics of hKv4.3 and notably enhanced the recovery from inactivation. Therefore, we conclude that hKChIP2 is a physiological regulatory subunit of hKv4.3 that may also contribute to the heterogeneity of I_to1 currents in the human heart. As a molecular constituent of I_to1 channels, hKChIP2 is possibly involved in the genesis or progression of human heart diseases like arrhythmias or heart failure. To allow a systematic mutational screening in candidate disorders, we determined the complete exon–intron organization and the chromosomal localization of the hKCNIP2 gene.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Cloning and tissue distribution of the hKChIP2 isoforms
Both hKChIP2 isoforms were cloned by PCR from a human heart Marathon-Ready cDNA (Clontech), using primers (forward: 5'-GCCATGCGGGGCCAGGGC-3' and reverse: 5'-GGGCTAGATGACATTGTCAAAG-3') derived from the published hKChIP2 cDNA sequence (GenBank accession number AF199598 [GenBank] ). Amplified products were automatically sequenced on both strands (ABI 310, Perkin Elmer) and cloned into pSGEM vector [20]. For Xenopus oocyte expression, capped cRNA was synthesized using the T7 mMessage mMachine kit (Ambion). For Northern blot analysis, a DIG-labeled riboprobe of 0.75 kb was generated with the DIG RNA Labeling kit (Roche) according to the manufacturers instructions and hybridized to a series of human RNA blots (Clontech). The riboprobe was designed from the full length hKChIP2 isoform-1. Membranes were exposed on a Lumi-Imager (Roche).

2.2 Construction of the N2–67 hKv4.3long mutant
Mutant channels with 67 amino-acid N-terminal deletion (N2–67) were constructed for coexpression studies. The N-terminal deletion was obtained using standard PCR techniques. Amino-terminal and carboxy-terminal primers carried a Kozak-site followed by a translation inititation codon and a termination codon.

2.3 Determination of the genomic structure
To identify the genomic organization of the hKCNIP2 gene, BLAST searches were performed against genomic databases (NCBI/EMBL), as well as unfinished chromosome specific databases (Sanger Center). Bioinformatic analysis of the partially sequenced BAC-clone RP11-190J1 (AC010789 [GenBank] ) predicted the presence of most of the exon–intron boundaries of the hKCNIP2 gene. Additionally, exon–intron boundaries were determined by long-range PCR (Expand^TM Long Template PCR System, Roche) on uncloned, adaptor-ligated human genomic DNA fragments (Genome Walker Kit, Clontech) using staggered oligonucleotide primers derived from the published cDNA. Intron sizes were determined by amplification between adjacent exons. Intronic primer pairs were designed using standard software to amplify each of the nine exons (GenBank accession numbers AY026329 [GenBank] , AY026330 [GenBank] , AY026331 [GenBank] ) of the hKCNIP2 gene and amplification products were sequenced to confirm the genomic organization and exon–intron boundaries of the hKCNIP2 gene.

2.4 Chromosomal mapping of the hKCNIP2 gene
Electronic PCR software (NCBI) was used to analyze the RP11-190J1 clone, which contains the hKCNIP2 gene, for the presence of sequence tagged sites (STS). Several STS were predicted, further analyzed and located on GeneMap99.

Fluorescence in situ hybridization (FISH) analysis was performed on metaphase spreads obtained from primary blood samples. Preparations were made according to standard protocols [21]. The probe was generated and labeled by PCR techniques. Exonic and intronic regions of the hKCNIP2 gene were amplified (200–500 bp each) and labeled by DIG-dUTP (Roche) in the PCR reaction mix. Hybridization was performed using 300–500 ng of labeled probe, 10 µg of human Cot-1 DNA (Roche), and hybridization buffer as described before [22]. The signal was visualized and amplified by standard procedures. Chromosomes were counterstained with 4',6-diamidino-2-phenylindol dihydrochloride (DAPI).

2.5 Electrophysiological studies
Xenopus laevis oocytes were obtained from tricaine anesthetized animals. Ovaries were collagenase treated (1 mg/1 ml, Worthington, type II) in OR2 solution (NaCl 82.5 mM, KCl 2 mM, MgCl₂ 1 mM, HEPES 5 mM, pH 7.4) for 120 min and subsequently stored in recording solution ND96 containing (in mmol/l) NaCl 96, KCl 2, CaCl₂ 1.8, MgCl₂ 1, HEPES 5, pH 7.4) with additional Na-Pyruvate (275 mg/l), theophylline (90 mg/l) and gentamycin (50 mg/l) at 18°C. At the beginning of our coexpression experiments, we tested, whether the effects of hKChIP2 on hKv4.3 currents are dose dependent. Therefore, the two proteins were coexpressed using different concentrations of hKChIP2 cRNA. After onset of the kinetic changes caused by hKChIP2, no additional alterations in kinetics were observed by increasing the amount of coinjected hKChIP2 cRNA. Subsequently oocytes were individually injected with 10 ng cRNA encoding hKv4.3long [23] and coexpressed with 3 ng of either hKChIP2 isoform. Standard two-electrode voltage-clamp recordings were performed at room temperature with a Turbo Tec 10CD (NPI) amplifier and an ITC-16 interface combined with Pulse software (Heka). Macroscopic currents were recorded 2–4 days after injection. The pipette solution contained 3 M KCl.

2.6 Statistical analysis
Pulse software (Heka) and Origin version 5.0 (Microcal Software) were used for data acquisition on Pentium II PC. All fitting procedures were based on the simplex algorithm. Results are reported as means±S.E.M. Statistical differences of electrophysiological data were evaluated by a two population Student’s unpaired t-test using Origin software. Significance was assumed if P<0.01 and indicated by an asterisk and/or plus sign.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
3.1 Cloning of hKChIP2 isoforms
PCR on human heart cDNA was used to amplify the recently published hKChIP2 cDNA [19]. Two PCR products were generated, a major fragment with a size of 663 bp and a minor band of 759 bp (Fig. 2C). Sequencing demonstrated, that the longer fragment corresponded to the published hKChIP2 sequence (AF199598 [GenBank] ) and that the shorter fragment encoded a hKChIP2 splice variant. This splice variant, named hKChIP2 isoform-2 (GenBank accession number AY026328 [GenBank] ), encodes a protein of 220 amino-acids and lacks amino-acid residues 25–56 (in frame deletion, N25–56) of the long isoform-1 (Fig. 1A). In addition, the asparagine at position 57 is changed to aspartic acid.

View larger version (35K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Tissue distribution of human KChIP2. (A), Northern blots of multiple human tissues and (B), subregions of human heart containing poly(A)+ RNA (Clontech), hybridized with a hKChIP2-specific DIG-labeled RNA probe. Control hybridizations with G3PDH or β-actin probes are shown below. (C), Semi-quantitative RT-PCR of the human heart showing the two hKChIP2 isoforms.

 

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Exon/intron structure and isoforms of the hKCNIP2 gene. (A), Exons are shown in black boxes and numbered above; the start ATG and stop signal for translation are indicated by arrows. Additionally, the composition of isoform-1 and 2 of the hKCNIP2 gene is shown. Note, that isoform-2 does not contain exon 2 of the gene. (B), Splice junctions of the hKCNIP2 gene and exon/intron sizes. Exonic sequences are shown in capital letters, intronic sequences in lower case letters and invariant AG/GT motifs are shown in bold letters. The donor splice junction of exon 2 contains a GC motif.

 
3.2 Tissue distribution of hKChIP2
Northern blot experiments indicate that hKChIP2 is expressed in the human heart (Fig. 2A). Within the heart, hKChIP2 transcripts are detected in adult atria and ventricles but not in fetal tissue (Fig. 2B). Coexpression of hKChIP2 with hKv4.3 in atria and ventricles [18,24], renders interactions between these two proteins a real possibility. Our RT-PCR experiments results further indicate that the shorter isoform-2 is the predominant KChIP2 isoform in the human heart (Fig. 2C).

3.3 Genomic organization and chromosomal mapping of the hKCNIP2 gene
Elucidation of the exon–intron boundaries of the hKCNIP2 gene was carried out by comparing the known hKChIP2 cDNA sequence with the genomic sequence included in the BAC-clone RP11-190J1 (AC010789 [GenBank] ), which contained most of the hKCNIP2 gene. In addition, long-range PCR methods on uncloned, adaptor-ligated human genomic DNA fragments were used to determine the exon–intron boundaries of exon 2. The hKCNIP2 gene consists of nine exons, and splice junctions contain the canonical dinucleotides GT and AG for donor and acceptor sites, except the donor splice site of exon 2, which contains a GC dinucleotide motif (Fig. 1B). It was reported that less than 0.6% of human splice sites contain non-canonical dinucleotides GC–AG splice site pairs [25]. If a non-canonical splice site has any influence on the efficiency of splicing is not yet known. Our computational splice site prediction could not identify a difference in the quality of splice site recognition between the GC and a hypothetically analyzed GT donor site. Interestingly, exon 2 is the exon, which is not spliced into the isoform-2 of hKChIP2. The exon and intron sizes of the hKCNIP2 gene are listed in Fig. 1B.

We were able to localize several STS markers on the clone RP11-190J1, which allowed us to map the hKCNIP2 gene to chromosome 10q24, into the interval defined by the markers D10S603 and D10S597 (Fig. 3A). This localization is in accordance with the result from the FISH analysis (Fig. 3B).

View larger version (51K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 The hKCNIP2 gene maps to chromosome 10q24. (A), Ideogram of chromsome 10 and localization of clone RP11-190J1 containing the hKCNIP2 gene and the indicated STS markers. The region marked ‘atrial fibrillation’ defines the locus of a familial form of atrial fibrillation described by Brugada et al. [39]. (B), FISH mapping of the hKCNIP2 gene on normal human metaphase chromosomes. Arrows mark the localization of the hKCNIP2 gene on chromosomes 10.

 
3.4 Characterization of hKv4.3long currents
Injection of hKv4.3 cRNA in Xenopus oocytes resulted in the expression of fast activating and inactivating A-type potassium currents (Fig. 4A). During depolarizing pulses the first 200 ms of inactivating currents were fitted with an equation for single exponential decay. The time constant of inactivation at +50 mV was =62.3±2.1 ms (n=43) (Table 1). The half-maximal activation voltage was fitted by a Boltzmann equation using the normalized conductance [23] of hKv4.3long currents with –9.6±1.2 mV (n=18) and a slope of 20.3±0.6 (Fig. 4D). Steady-state inactivation parameters were obtained with a double pulse protocol with 1 s prepulses to voltages from –100 to +5 mV in 5 mV increments from a holding potential of –80 mV as previously described [23]. Peak currents were normalized to the maximal transient outward current and fitted individually for each oocyte by a single Boltzmann equation. Half-maximal inactivation voltage was –51.0±1.3 mV (n=24) with a slope of 6.5±0.4 (Fig. 5C). Recovery from inactivation was analyzed by a two pulse protocol. Cells were depolarized to +50 mV for 200 ms and then allowed to recover for variable durations at –80 mV before a second depolarizing pulse to +50 mV. The time constant for recovery from inactivation was obtained by a monoexponential fit at –80 mV, with a value of 249.8±11.3 ms (n=18). Currents took over 1 s to fully recover from inactivation (Fig. 6A and C).

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Coexpression of hKChIP2 with hKv4.3long in Xenopus oocytes. (A), Expression of hKv4.3long generates rapidly activating and inactivating currents, with an activation threshold around –40 mV and a half-maximal activation V1/2 of – 9.6±1.2 mV, slope 20.3±0.6 (n=18). (B), Representative current traces of hKv4.3long coexpressed with hKChIP2 isoform-1. Current amplitude was increased, whereas activation threshold and half-maximal activation were not significantly altered. Similar results were obtained by coexpression with hKChIP2 isoform-2. (C), Currents were elicited from a holding potential of –80 mV by depolarizing voltage steps of 500 ms duration from –60 to +80 mV in 10 mV increments. Leak currents were not subtracted. (D), Representative steady-state activation curves of hKv4.3long (), hKv4.3long coexpressed with hKChIP2 isoform-1 () and hKv4.3long coexpressed with hKChIP2 isoform-2 ().

 

View this table: [in this window] [in a new window]   Table 1 Functional effects of the two hKChIP2 isoforms on Kv4.3long currentsa

 

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Steady-state inactivation of hKv4.3 in Xenopus oocytes coexpressing hKChIP2. (A), raw current traces of hKv4.3long obtained by a two-pulse protocol, with the first pulse of 1 s ranging from –100 mV to +5 mV in 5 mV steps and the second test pulse set to +50 mV. (B), raw current traces of an oocyte expressing hKv4.3long and hKChIP2 isoform-1. Similar results were obtained by coexpression with hKChIP2 isoform-2. (C), Representative steady-state inactivation curves of hKv4.3long (), hKv4.3long coexpressed with hKChIP2 isoform-1 () and hKv4.3long coexpressed with hKChIP2 isoform-2 ().

 

View larger version (19K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Recovery from inactivation was explored using two 200 ms depolarizing voltage steps to +50 mV with increasing interpulse time. Holding potential and potential during the recovery step were –80 mV. (A), Recovery from inactivation of hKv4.3long alone or (B), coexpressed with hKChIP2 isoform-1. Similar recovery current traces were obtained by coexpression with hKChIP2 isoform-2. (C), Percentage of recovery of currents from inactivation in dependence of the interpulse time. Representative single exponential fits to peak currents at various time points after an inactivating prepulse for hKv4.3long alone () and for coexpression with hKChIP2 isoform-1 or hKChIP2 isoform-2 ().

 
3.5 Coexpression of hKChIP2 isoforms with hKv4.3long
hKChIP2 alone did not generate currents in Xenopus oocytes. Coexpressing either hKChIP2 isoform with hKv4.3 significantly altered all the tested current properties, except for the half-maximal activation. The effects of both hKChIP2 isoforms on hKv4.3 currents were almost identical. Only the values for the half-maximal inactivation differed and reached statistical significance (Table 1).

Upon coexpression of hKv4.3long with either hKChIP2 isoform-1 or isoform-2, current amplitudes significantly increased from 12.2±1.0 µA (n=43) to 23.1±1.0 µA (n=93) and 24.2±1.7 µA (n=33), respectively (Fig. 4B). Moreover, the time constant of hKv4.3 inactivation increased from 62.3±2.1 ms (n=43) to 110.7±6.0 ms (n=92) for hKChIP2 isoform-1 and to 114.3±14.7 ms (n=19) for hKChIP2 isoform-2. Half-maximal activation of hKv4.3/hKChIP2 currents was not different from that of hKv4.3 alone, whereas half-maximal inactivation was shifted to more positive voltages. V_1/2 of inactivation was –38.9±0.6 mV with a slope of 4.7±0.1 (n=41) for hKChIP2 isoform-1 and –32.2±1.1 mV with a slope of 5.7±0.2 (n=26) for hKChIP2 isoform-2 (Fig. 5B). Thus, depending on the associated hKChIP2 isoform the resulting voltage shift in steady-state inactivation differentially changes the open window current for hKv4.3.

The most striking difference between native cardiac A-type currents and hKv4.3 currents in oocytes is the much faster rate of recovery from inactivation of I_to1 [9,10,23]. Interestingly, hKChIP2 drastically enhanced recovery from inactivation of hKv4.3. Time constants for recovery from inactivation decreased from 249.8±11.3 ms (n=18) to 36.7±2.2 ms (n=37) and to 39.8±4.3 ms (n=23) for Kv4.3/hKChIP2 isoform-1 and Kv4.3/hKChIP2 isoform-2, respectively. These values correspond well to those reported for I_to1 from human cardiac tissue [3,9,10]. After coexpression, currents are already fully recovered from inactivation in less than 200 ms (Fig. 6B and C).

To determine whether hKChIP2, in addition to altering current density and kinetic parameters, also changes pharmacological properties of hKv4.3, we tested the sensitivity to 4-aminopyridine (4-AP), a classical I_to blocker [26–28]. However, both splice forms of hKChIP2 did not affect hKv4.3 block by 4-AP (data not shown).

3.6 Coexpression of hKChIP2 with hKv4.3 N2-67
Yeast two hybrid analyses showed that hKChIP proteins interact with the N-terminus of Kv4 proteins [19]. Rapid inactivation of Shal-related Kv4 channel proteins, in contrast to Shaker-type Kv1 channels, is relatively insensitive to deletion of the N-terminal domain [29]. To determine whether binding of hKChIP2 to the N-terminus mediates modulation of hKv4.3 channels, we coexpressed it with an N-terminally truncated hKv4.3 mutant. Similar to the wild type, hKv4.3 N2–67 displayed rapidly activating and inactivating A-type currents. The voltage of half-maximal activation of the N2–67 hKv4.3 construct was –8.1±1.4 mV with a slope of 20.5±0.7 (n=16). Half-maximal inactivation voltage was –48.4±1.5 mV with a slope of 6.8±0.4 (n=16). The time constant of inactivation at +50 mV was =73.5±3.0 ms (n=16). The time constant of recovery from inactivation was =229.8±15.3 ms (n=16). However, coexpression of hKChIP2 no longer altered current amplitude nor changed the biophysical properties of the mutant currents (data not shown), thus supporting the physical binding studies. This result also shows that the binding site must be located within the first third of the N-terminal tail.

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 
In human heart, I_to1 is responsible for the initial phase of repolarization and determines the plateau voltage of the cardiac action potential. Thus, I_to1 indirectly affects other cardiac currents involved in AP shaping, like L-type Ca²⁺ channels [17]. Therefore, dysfunction of I_to1 may strongly alter the shape and duration of the cardiac action potential [17] and may predispose the heart to arrhythmias [30]. The human I_to1 shows regional differences in current density, kinetics and pharmacological properties, which are also present throughout the human ventricular free wall [3,9]. This functional heterogeneity is reflected by the contribution of several genes to the I_to1. The hKv1.4 channel was initially believed to be the molecular correlate of cardiac I_to1. However, its slow recovery from inactivation and its pharmacological properties (sensitivity to 4-AP and H₂O₂) differ drastically from those of native human I_to1 [28,31]. Subsequent work demonstrated the major contribution of Kv4.2 and Kv4.3 to the I_to1 in the mammalian heart [30,32–34]. Based on the tissue expression profile as well as biophysical and pharmacological properties, hKv4.3 is believed to encode the bulk of human transient outward current [18,24,32,35], whereas the hKv1.4 channel possibly represents the I_to1 of the endocardium [36]. Still, the native I_to1 current recovers much faster from its inactivation than hKv4.3, suggesting the presence of modulatory proteins within the native channel complex.

As recently shown, the KChIP proteins represent strong candidates for these modulating subunits. KChIP proteins belong to a family of neuronal calcium binding proteins and modify the properties of Kv4 -subunits [19]. From the three KChIP proteins, only KChIP2, in addition to its expression in the brain, is also expressed in the rat heart [19]. Interestingly, we could only detect hKChIP2 in the heart of humans and found that, similar to hKv4.3, it is expressed in the atrium and the ventricle. Human Kv4.3 exists in two isoforms that differ in the length of their C-terminal tails. We used the hKv4.3long variant for coexpression since this is the principal isoform in the heart [23]. We demonstrate that coexpression of human KChIP2 with hKv4.3 reconstitutes several features of native I_to1 by modulating the current density, inactivation kinetics and rate of recovery from inactivation in the heterologous Xenopus oocyte expression system. Moreover, we show that the first 67 amino-acids of the Kv4.3 -subunit are necessary for functional interaction with hKChIP2 [19].

While preparing our manuscript, Ohya et al. [37] reported the cloning of a novel short splice variant of hKChIP2. The authors only detected the short isoform in the human heart. This supports our observation that the new short splice variant of hKChIP2 is the major isoform in the human heart. Coexpression with both isoforms resulted in similar changes in current density and kinetics of hKv4.3 currents. This is the first analysis concerning KChIP protein influence on the voltage half-maximal inactivation of Kv4 channels. Interestingly, the shift in half-maximal inactivation of hKChIP2 isoform-2 was more pronounced than that of hKChIP2 isoform-1. Therefore, the open window current of the hKv4.3 encoded transient outward currents might differ depending on the associated hKChIP2 variant. Both hKChIP2 isoforms therefore may contribute to the heterogeneity of hKv4.3 generated cardiac I_to1 currents. Also the transmural heterogeneity of recovery from inactivation of human I_to1 might in part be due to a gradient of hKChIP2 expression in the ventricular free wall. Since it is possible that binding of KChIP proteins is selective for Kv4 potassium channels [19], hKChIP2 most probably does not affect the endocardial (H₂O₂ sensitive) I_to1 since this current is thought to be encoded by the Kv1.4 subunit.

The coexpression of rat Kv4.3 (rKv4.3) with low molecular weight (LMW) RNA from rat brain [26] increased the rate of inactivation, changed the sensitivity to 4-aminopyridine and caused a negative shift in the voltage of activation and inactivation. This is in contrast to our results where coexpressed hKChIP2 slowed inactivation and shifted the voltage of inactivation of hKv4.3 currents to more positive values.

Overall, hKv4.3/hKChIP2 currents more closely resemble native I_to1 of the human epicardium. In particular the change in half-maximal inactivation, together with the strongly enhanced recovery from inactivation, support the hypothesis that hKChIP2 is a physiological modifier of cardiac hKv4.3 channels, as only these rapid kinetics allow the native I_to1 to fully recover from inactivation within the time interval of the diastolic repolarization.

The reported half-maximal inactivation of human I_to1 ranges from –21 to –45 mV [3]. This variability might result from differences in experimental conditions, like the use of cadmium or nifedipine as L-type calcium channel blockers and use of different concentrations of calcium complexing agents [3]. However, the shift of half-maximal inactivation from –51 to –39 and –32 mV, as seen in our experiments, is clearly a shift towards the physiological voltage range of human epicardial I_to1.

Similar to the effects of rKChIP2 on rKv4.2 [19], hKChIP2 also slowed the inactivation of hKv4.3 currents, resulting in a time constant of inactivation of the heteromeric currents that is greater than that reported for the native cardiac I_to1. Interpretation of this apparent difference is difficult since a higher temperature, as well as the use of nifedipine for recordings of I_to1 currents in cardiomyocytes, can accelerate the rate of inactivation [3]. Thus, the remaining differences in kinetics between hKChIP2/hKv4.3 currents and native I_to1 might be caused by the different cellular environment of Xenopus oocytes and/or the presence of additional modulators of Kv4.3 in the human heart.

In contrast to factors encoded by rat brain LMW mRNA species, coexpressed hKChIP2 did not modify hKv4.3 sensitivity to 4-aminopyridine. Thus, there may be additional accessory proteins present in native channel complexes, which in contrast to KChIP proteins may interact with the C-terminal tails of Kv4 subunits [26,38].

Genes encoding components of the I_to1 channel conductance are possible candidates for inherited forms of excitability disorders. We determined the complete exon–intron structure and the chromosomal localization of the hKCNIP2 gene. This will allow a systematic screening for mutations in candidate disorders. We localized the hKCNIP2 gene to chromosome 10q24, a region where two human disorders have been mapped, namely an inherited form of atrial fibrillation [39] and a partial epilepsy with auditory features [40]. Both disease genes have not yet been identified. Since the critical region of the atrial fibrillation locus [39] does not overlap and is located more centromeric to the hKCNIP2 gene, it is unlikely that hKCNIP2 represents a positional candidate gene for atrial fibrillation in the three reported families. Although we could not observe hKChIP2 transcripts in the human brain, we cannot strictly exclude the hKCNIP2 gene as a possible candidate for the partial epilepsy, since hKChIP2 expression levels in the brain might be too low to be easily detected by Northern blot analysis.

The potential involvement of hKChIP2 in pathophysiological conditions of the human heart is one of the most interesting questions to be addressed in the future. As a regulatory subunit of the cardiac I_to1 channel complex, this possibility seems promising.

Time for primary review 20 days.

	Acknowledgements

 
The study was supported in parts by grants CRP/TUR98-05 from the International Centre for Gene Engineering and Biotechnology (ICGEB) and from the Research Fund of the University of Istanbul, project number 1518/28072000. We thank Stefan Müller, Susanne Siefert, Kader Yilmaz, Ilona Gutcher and Andrea Brüggemann for their help.

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion References

 

1. Escande D., Coulombe A., Faivre J.F., Deroubaix E., Coraboeuf E. Two types of transient outward currents in adult human atrial cells. Am. J. Physiol. (1987) 252:H142–H148.[Web of Science][Medline]
2. Wettwer E., Amos G., Gath J., Zerkowski H.R., Reidemeister J.C., Ravens U. Transient outward current in human and rat ventricular myocytes. Cardiovasc. Res. (1993) 27:1662–1669.[Abstract/Free Full Text]
3. Wettwer E., Amos G.J., Posival H., Ravens U. Transient outward current in human ventricular myocytes of subepicardial and subendocardial origin. Circ. Res. (1994) 75:473–482.[Abstract/Free Full Text]
4. Shimoni Y., Severson D., Giles W.R. Thyroid status and diabetes modulate regional differences in potassium currents in rat ventricle. J. Physiol. (1995) 488:673–688.[Abstract/Free Full Text]
5. Gomez A.M., Benitah J.P., Henzel D., Vinet A., Lorente P., Delgado C. Modulation of electrical heterogeneity by compensated hypertrophy in rat left ventricle. Am. J. Physiol. (1997) 272:H1078–H1086.[Web of Science][Medline]
6. Clark R.B., Bouchard R.A., Salinas-Stefanon E., Sanchez-Chapula J., Giles W.R. Heterogeneity of action potential waveforms and potassium currents in rat ventricle. Cardiovasc. Res. (1993) 27:1795–1799.[Abstract/Free Full Text]
7. Litovsky S.H., Antzelevitch C. Transient outward current prominent in canine ventricular epicardium but not endocardium. Circ. Res. (1988) 62:116–126.[Abstract/Free Full Text]
8. Litovsky S.H., Antzelevitch C. Rate dependence of action potential duration and refractoriness in canine ventricular endocardium differs from that of epicardium: role of the transient outward current. J. Am. Coll. Cardiol. (1989) 14:1053–1066.[Abstract]
9. Näbauer M., Beuckelmann D.J., Überfuhr P., Steinbeck G. Regional differences in current density and rate-dependent properties of the transient outward current in subepicardial and subendocardial myocytes of the human left ventricular. Circulation (1996) 93:168–177.[Abstract/Free Full Text]
10. Li G.-R., Feng J., Yue L., Carrier M. Transmural heterogeneity of action potentials and I_to1 in myocytes isolated from the human right ventricle. Am. J. Physiol. (1998) 275:H369–H377.[Web of Science][Medline]
11. Kääb S., Nuss H.B., Chiamvimonvat N., O’Rourke B., Pak P.H., Kass D.A., Marbán E., Tomaselli G.F. Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure. Circ. Res. (1996) 78:262–273.[Abstract/Free Full Text]
12. Näbauer M., Beuckelmann D.J., Erdmann E. Characteristics of transient outward current in human ventricular myocytes from patients with terminal heart failure. Circ. Res. (1993) 73:386–394.[Abstract/Free Full Text]
13. Jeck C., Pinto J., Boyden P. Transient outward currents in subendocardial Purkinje myocytes surviving in the infarcted heart. Circulation (1995) 92:465–473.[Abstract/Free Full Text]
14. Van Wagoner D.R., Pond A.L., McCarthy P.M., Trimmer J.S., Nerbonne J.M. Outward K⁺ current densities and Kv1.5 expression are reduced in chronic human atrial fibrillation. Circ. Res. (1997) 80:772–781.[Abstract/Free Full Text]
15. Yue L., Feng J., Gaspo R., Li G.R., Wang Z., Nattel S. Ionic remodeling underlying action potential changes in a canine model of atrial fibrillation. Circ. Res. (1997) 81:512–525.[Abstract/Free Full Text]
16. Beuckelmann D.J., Näbauer M., Erdmann E. Alterations of K⁺ currents in isolated human ventricular myocytes from patients with terminal heart failure. Circ. Res. (1993) 73:379–385.[Abstract/Free Full Text]
17. Greenstein J.L., Wu R., Po S., Tomaselli G.F., Winslow R.L. Role of the calcium-independent transient outward current I_(to1) in shaping action potential morphology and duration. Circ. Res. (2000) 87:1026–1033.[Abstract/Free Full Text]
18. Kääb S., Dixon J., Duc J., Ashen D., Näbauer M., Beuckelmann D.J., Steinbeck G., McKinnon D., Tomaselli G.F. Molecular basis of transient outward potassium current downregulation in human heart failure: a decrease in Kv4.3 mRNA correlates with a reduction in current density. Circulation (1998) 98:1383–1393.[Abstract/Free Full Text]
19. An W.F., Bowlby M.R., Betty M., Cao J., Ling H.-P., Mendoza G., Hinson J.W., Mattsson K.I., Strassle B.W., Trimmer J.S., Rhodes K.J. Modulation of A-type potassium channels by a family of calcium sensors. Nature (2000) 403:553–556.[CrossRef][Medline]
20. Villmann C., Bull L., Hollmann M. Kainate binding proteins possess functional ion channel domains. J. Neurosci. (1997) 17:7634–7643.[Abstract/Free Full Text]
21. Lichter P., Cremer T. Human cytogenetics: a practical approach. (1992) Oxford: I.R.L. Oxford University Press.
22. Ried T., Baldini A., Rand T.C., Ward D.C. Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. Proc. Natl. Acad. Sci. (1992) 89:1388–1392.[Abstract/Free Full Text]
23. Dilks D., Ling H.-P., Cockett M., Sokol P., Numann R. Cloning and expression of the human Kv4.3 potassium channel. J. Neurophysiol. (1999) 81:1974–1977.[Abstract/Free Full Text]
24. Grammer J.B., Bosch R.F., Kuhlkamp V., Seipel L. Molecular remodeling of Kv4.3 potassium channels in human atrial fibrillation. J. Cardiovasc. Electrophysiol. (2000) 11:626–633.[Web of Science][Medline]
25. Burset M., Seledtsov I.A., Solovyev V.V. Analysis of canonical and non-canonical splice sites in mammalian genomes. Nucleic Acids Res. (2000) 28:4364–4375.[Abstract/Free Full Text]
26. Serodio P., Vega-Saenz de Miera E., Rudy B. Cloning of a novel component of A-type K⁺ channels operating at subthreshold potentials with unique expression in heart and brain. J. Neurophysiol. (1996) 75:2174–2179.[Abstract/Free Full Text]
27. Kong W., Po S., Yamagishi T., Ashen M.D., Stetten G., Tomaselli G.F. Isolation and characterization of the human gene encoding Ito: further diversity by alternative mRNA splicing. Am. J. Physiol. (1998) 275:H1963–H1970.[Web of Science][Medline]
28. McKinnon D. Molecular identity of I_to: Kv1.4 redux. Circ. Res. (1999) 84:620–622.[Free Full Text]
29. Rasmusson R.L., Morales M.J., Wang S., Liu S., Campbell D.L., Brahmajothi M.V., Strauss H.C. Inactivation of voltage-gated cardiac K⁺ channels. Circ. Res. (1998) 82:739–750.[Abstract/Free Full Text]
30. Barry D.M., Xu H., Schuessler R.B., Nerbonne J.M. Functional knockout of the transient outward current, long-QT syndrome, and cardiac remodeling in mice expressing a dominant-negative Kv4 alpha subunit. Circ. Res. (1998) 83:560–567.[Abstract/Free Full Text]
31. Po S., Snyders D.J., Baker R., Tamkun M.M., Bennett P.B. Functional expression of an inactivating potassium channel cloned from human heart. Circ. Res. (1992) 71:732–736.[Abstract/Free Full Text]
32. Dixon J.E., Shi W., Wang H.S., McDonald C., Yu H., Wymore R.S., Cohen I.S., McKinnon D. Role of the Kv4.3 K⁺ channel in ventricular muscle. A molecular correlate for the transient outward current. Circ. Res. (1996) 79:659–668.[Abstract/Free Full Text]
33. Fiset C., Clark R.B., Shimoni Y., Giles W.R. Shal-type channels contribute to the Ca²⁺-independent transient outward K⁺ current in rat ventricle. J. Physiol. (1997) 500:51–64.[Abstract/Free Full Text]
34. Johns D.C., Nuss H.B., Marbán E. Suppression of neuronal and cardiac transient outward currents by viral gene transfer of dominant-negative Kv4.2 constructs. J. Biol. Chem. (1997) 272:31598–31603.[Abstract/Free Full Text]
35. Wang Z., Feng J., Shi H., Pond A., Nerbonne J.M., Nattel S. Potential molecular basis of different physiological properties of the transient outward K⁺ current in rabbit and human atrial myocytes. Circ. Res. (1999) 84:551–561.[Abstract/Free Full Text]
36. Kääb S Kartmann H Andrassy J Hinterseer M Barth A Näbauer M. Presence of Kv4.3 and Kv1.4 potassium channel mRNA in human left ventricular myocardium: transmural mRNA-gradients and correlation with current properties. Circulation (1998) 98(Suppl I):I-867. Abstract 4548.
37. Ohya S., Morohashi Y., Muraki K., Tomita T., Watanabe M., Iwatsubo T., Imaizumi Y. Molecular cloning and expression of the novel splice variants of K⁺ channel-interacting protein 2. Biochem. Biophys. Res. Commun. (2001) 282:96–102.[CrossRef][Web of Science][Medline]
38. Yang E.K., Alvira M.R., Levitan E.S., Takimoto K. Kvbeta subunits increase expression of Kv4.3 channels by interacting with their C-termini. J. Biol. Chem. (2001) 276:4839–4844.[Abstract/Free Full Text]
39. Brugada R., Tapscott T., Czernuszewicz G.Z., Marian A.J., Iglesias A., Mont L., Brugada J., Girona J., Domingo A., Bachinski L.L., Roberts R. Identification of a genetic locus for familial atrial fibrillation. N. Engl. J. Med. (1997) 336:905–911.[Abstract/Free Full Text]
40. Ottman R., Risch N., Hauser W.A., Pedley T.A., Lee J.H., Barker-Cummings C., Lustenberger A., Nagle K.J., Lee K.S., Scheuer M.L., Neystat M., Susser M., Wilhelmsen K.C. Localization of a gene for partial epilepsy to chromosome 10q. Nat. Genet. (1995) 10:56–60.[CrossRef][Web of Science][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				L. M. Boland, M. M. Drzewiecki, G. Timoney, and E. Casey Inhibitory effects of polyunsaturated fatty acids on Kv4/KChIP potassium channels Am J Physiol Cell Physiol, May 1, 2009; 296(5): C1003 - C1014. [Abstract] [Full Text] [PDF]

				G. C. L. Bett and R. L. Rasmusson Modification of K+ channel-drug interactions by ancillary subunits J. Physiol., February 15, 2008; 586(4): 929 - 950. [Abstract] [Full Text] [PDF]

				L. L. Shang, A. E. Pfahnl, S. Sanyal, Z. Jiao, J. Allen, K. Banach, J. Fahrenbach, D. Weiss, W. R. Taylor, A. M. Zafari, et al. Human Heart Failure Is Associated With Abnormal C-Terminal Splicing Variants in the Cardiac Sodium Channel Circ. Res., November 26, 2007; 101(11): 1146 - 1154. [Abstract] [Full Text] [PDF]

				D. Van Hoorick, A. Raes, and D. J. Snyders The aromatic cluster in KCHIP1b affects Kv4 inactivation gating J. Physiol., September 15, 2007; 583(3): 959 - 969. [Abstract] [Full Text] [PDF]

				H.-L. Li, Y.-J. Qu, Y. C. Lu, V. E. Bondarenko, S. Wang, I. M. Skerrett, and M. J. Morales DPP10 is an inactivation modulatory protein of Kv4.3 and Kv1.4 Am J Physiol Cell Physiol, November 1, 2006; 291(5): C966 - C976. [Abstract] [Full Text] [PDF]

				V. Salvador-Recatala, W. J. Gallin, J. Abbruzzese, P. C. Ruben, and A. N. Spencer A potassium channel (Kv4) cloned from the heart of the tunicate Ciona intestinalis and its modulation by a KChIP subunit J. Exp. Biol., February 15, 2006; 209(4): 731 - 747. [Abstract] [Full Text] [PDF]

				S. P. Patel and D. L. Campbell Transient outward potassium current, 'Ito', phenotypes in the mammalian left ventricle: underlying molecular, cellular and biophysical mechanisms J. Physiol., November 15, 2005; 569(1): 7 - 39. [Abstract] [Full Text] [PDF]

				N. Szentadrassy, T. Banyasz, T. Biro, G. Szabo, B. I. Toth, J. Magyar, J. Lazar, A. Varro, L. Kovacs, and P. P. Nanasi Apico-basal inhomogeneity in distribution of ion channels in canine and human ventricular myocardium Cardiovasc Res, March 1, 2005; 65(4): 851 - 860. [Abstract] [Full Text] [PDF]

				S. Zicha, L. Xiao, S. Stafford, T. J. Cha, W. Han, A. Varro, and S. Nattel Transmural expression of transient outward potassium current subunits in normal and failing canine and human hearts J. Physiol., December 15, 2004; 561(3): 735 - 748. [Abstract] [Full Text] [PDF]

				K. J. Rhodes, K. I. Carroll, M. A. Sung, L. C. Doliveira, M. M. Monaghan, S. L. Burke, B. W. Strassle, L. Buchwalder, M. Menegola, J. Cao, et al. KChIPs and Kv4 {alpha} Subunits as Integral Components of A-Type Potassium Channels in Mammalian Brain J. Neurosci., September 8, 2004; 24(36): 7903 - 7915. [Abstract] [Full Text] [PDF]

				N. Decher, A. S. Barth, T. Gonzalez, K. Steinmeyer, and M. C. Sanguinetti Novel KChIP2 isoforms increase functional diversity of transient outward potassium currents J. Physiol., June 15, 2004; 557(3): 761 - 772. [Abstract] [Full Text] [PDF]

				J. C. Choi, D. Park, and L. C. Griffith Electrophysiological and Morphological Characterization of Identified Motor Neurons in the Drosophila Third Instar Larva Central Nervous System J Neurophysiol, May 1, 2004; 91(5): 2353 - 2365. [Abstract] [Full Text] [PDF]

				N. Hatano, S. Ohya, K. Muraki, R. B. Clark, W. R. Giles, and Y. Imaizumi Two Arginines in the Cytoplasmic C-terminal Domain Are Essential for Voltage-dependent Regulation of A-type K+ Current in the Kv4 Channel Subfamily J. Biol. Chem., February 13, 2004; 279(7): 5450 - 5459. [Abstract] [Full Text] [PDF]

				F. G. Akar, R. C. Wu, I. Deschenes, A. A. Armoundas, V. Piacentino III, S. R. Houser, and G. F. Tomaselli Phenotypic differences in transient outward K+ current of human and canine ventricular myocytes: insights into molecular composition of ventricular Ito Am J Physiol Heart Circ Physiol, February 1, 2004; 286(2): H602 - H609. [Abstract] [Full Text] [PDF]

				X. Ren, S. H. Shand, and K. Takimoto Effective Association of Kv Channel-interacting Proteins with Kv4 Channel Is Mediated with Their Unique Core Peptide J. Biol. Chem., October 31, 2003; 278(44): 43564 - 43570. [Abstract] [Full Text] [PDF]

				L. M. Boland, M. Jiang, S. Y. Lee, S. C. Fahrenkrug, M. T. Harnett, and S. M. O'Grady Functional properties of a brain-specific NH2-terminally spliced modulator of Kv4 channels Am J Physiol Cell Physiol, July 1, 2003; 285(1): C161 - C170. [Abstract] [Full Text] [PDF]

				S. Y. Lee, P. J. Maniak, D. H. Ingbar, and S. M. O'Grady Adult alveolar epithelial cells express multiple subtypes of voltage-gated K+ channels that are located in apical membrane Am J Physiol Cell Physiol, June 1, 2003; 284(6): C1614 - C1624. [Abstract] [Full Text] [PDF]

				Y. Zhang, J. N. MacLean, W. F. An, C. C. Lanning, and R. M. Harris-Warrick KChIP1 and Frequenin Modify shal-Evoked Potassium Currents in Pyloric Neurons in the Lobster Stomatogastric Ganglion J Neurophysiol, April 1, 2003; 89(4): 1902 - 1909. [Abstract] [Full Text] [PDF]

				G. C. Amberg, S. D. Koh, Y. Imaizumi, S. Ohya, and K. M. Sanders A-type potassium currents in smooth muscle Am J Physiol Cell Physiol, March 1, 2003; 284(3): C583 - C595. [Abstract] [Full Text] [PDF]

				L. A. Schrader, A. E. Anderson, A. Mayne, P. J. Pfaffinger, and J. D. Sweatt PKA Modulation of Kv4.2-Encoded A-Type Potassium Channels Requires Formation of a Supramolecular Complex J. Neurosci., December 1, 2002; 22(23): 10123 - 10133. [Abstract] [Full Text] [PDF]

				G. C Amberg, S. D. Koh, W. J Hatton, K. J Murray, K. Monaghan, B. Horowitz, and K. M Sanders Contribution of Kv4 channels toward the A-type potassium current in murine colonic myocytes J. Physiol., October 15, 2002; 544(2): 403 - 415. [Abstract] [Full Text] [PDF]

				I. Deschenes, D. DiSilvestre, G. J. Juang, R. C. Wu, W. F. An, and G. F. Tomaselli Regulation of Kv4.3 Current by KChIP2 Splice Variants: A Component of Native Cardiac Ito? Circulation, July 23, 2002; 106(4): 423 - 429. [Abstract] [Full Text] [PDF]

				K. Takimoto, E.-K. Yang, and L. Conforti Palmitoylation of KChIP Splicing Variants Is Required for Efficient Cell Surface Expression of Kv4.3 Channels J. Biol. Chem., July 19, 2002; 277(30): 26904 - 26911. [Abstract] [Full Text] [PDF]

				S. P Patel, D. L Campbell, M. J Morales, and H. C Strauss Heterogeneous expression of KChIP2 isoforms in the ferret heart J. Physiol., March 15, 2002; 539(3): 649 - 656. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Disclaimer Google Scholar Articles by Decher, N. Articles by Wollnik, B. Search for Related Content PubMed PubMed Citation Articles by Decher, N. Articles by Wollnik, B. Social Bookmarking          What's this?

Online ISSN 1755-3245 - Print ISSN 0008-6363

Copyright © 2009 European Society of Cardiology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics