hKChIP2 is a functional modifier of hKv4.3 potassium channels: cloning and expression of a short hKChIP2 splice variant.

OBJECTIVE: The Ca(2+) independent transient outward K(+) current (I(to1)) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K(+) channel alpha-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 beta-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.

Identification of specific pore residues mediating KCNQ1 inactivation. A novel mechanism for long QT syndrome.

KCNQ1 inactivation bears electrophysiological characteristics different from classical N- and C-type inactivation in Shaker-like potassium channels. However, the molecular site of KCNQ1 inactivation has not yet been determined. KCNQ2 channels do not exert a fast inactivation in contrast to KCNQ1 channels. By expressing functional chimeras between KCNQ1 and KCNQ2 in Xenopus oocytes, we mapped the region of this inactivation to transmembrane domain S5 and the pore loop H5 and finally narrowed down the site to positions Gly(272) and Val(307) in KCNQ1. Exchanging these two amino acids individually with the analogous KCNQ2 residue abolished inactivation. Furthermore, a KCNQ1-like inactivation was introduced into KCNQ2 by mutagenesis in the corresponding region, confirming its relevance for the inactivation process. As KCNQ1 inactivation involves the regions S5 and H5, it exhibits a geography distinct from N- or C-type inactivation. Native cardiac I(Ks) channels comprising KCNQ1 and accessory MinK subunits do not inactivate because of the functional interaction of KCNQ1 with MinK. Mutations in KCNQ1 can lead to long QT1 syndrome, an inherited form of arrhythmia. The long QT1 mutant KCNQ1(L273F) displays a pronounced KCNQ1 inactivation. Here we show that when expressing mutant I(Ks) channels formed from KCNQ1(L273F) and MinK, MinK association no longer eliminates KCNQ1 inactivation. This results in smaller repolarizing currents in the heart and therefore represents a novel mechanism leading to long QT syndrome.

Gene expression profiles of CLC chloride channels in animal models with different cardiovascular diseases.

Here for the first time we investigated the potential involvement of the CLC chloride channel family at the transcriptional level in different cardiovascular diseases. Northern blot and semiquantitative RT-PCR analyses were used to study the gene expression profiles of all CLC genes present in the heart and kidney; namely, CLC-2, CLC-3, CLC-4, CLC-5, CLC-6, CLC-7, CLC-K1, and CLC-K2. Rat models with distinctive cardiovascular diseases were studied: These included spontaneously hypertensive rats, nutritionally- and surgically-induced hypertensive rats with cardiac hypertrophy, as well as rats suffering from chronic heart failure due to myocardial infarction. The present data show that it was not possible to detect apparent differences in the CLC mRNA expression between the hearts and kidneys of diseased and control animals. Our data strongly suggest that altered transcript regulation of CLC chloride channels does not contribute to the cardiac and renal pathology in the examined cardiovascular diseases.

Molecular cloning and functional expression of KCNQ5, a potassium channel subunit that may contribute to neuronal M-current diversity.

We have isolated KCNQ5, a novel human member of the KCNQ potassium channel gene family that is differentially expressed in subregions of the brain and in skeletal muscle. When expressed in Xenopus oocytes, KCNQ5 generated voltage-dependent, slowly activating K(+)-selective currents that displayed a marked inward rectification at positive membrane voltages. KCNQ5 currents were insensitive to the K(+) channel blocker tetraethylammonium but were strongly inhibited by the selective M-current blocker linopirdine. Upon coexpression with the structurally related KCNQ3 channel subunit, current amplitudes increased 4-5-fold. Compared with homomeric KCNQ5 currents, KCNQ3/KCNQ5 currents also displayed slower activation kinetics and less inward rectification, indicating that KCNQ5 combined with KCNQ3 to form functional heteromeric channel proteins. This functional interaction between KCNQ5 and KCNQ3, a component of the M-channel, suggests that KCNQ5 may contribute to a diversity of heteromeric channels underlying native neuronal M-currents.

Molecular impact of MinK on the enantiospecific block of I(Ks) by chromanols.

Slowly activating I:(Ks) (KCNQ1/MinK) channels were expressed in Xenopous: oocytes and their sensitivity to chromanols was compared to homomeric KCNQ1 channels. To elucidate the contribution of the ss-subunit MinK on chromanol block, a formerly described chromanol HMR 1556 and its enantiomer S5557 were tested for enantio-specificity in blocking I:(Ks) and KCNQ1 as shown for the single enantiomers of chromanol 293B. Both enantiomers blocked homomeric KCNQ1 channels to a lesser extent than heteromeric I:(Ks) channels. Furthermore, we expressed both WT and mutant MinK subunits to examine the involvement of particular MinK protein regions in channel block by chromanols. Through a broad variety of MinK deletion and point mutants, we could not identify amino acids or regions where sensitivity was abolished or strikingly diminished (>2.5 fold). This could indicate that MinK does not directly take part in chromanol binding but acts allosterically to facilitate drug binding to the principal subunit KCNQ1.