Abstract
For many model organisms traditionally in use for cardiac electrophysiological studies, characterization of ion channel genes is lacking. We focused here on two genes encoding the inward rectifier current, KCNJ2 and KCNJ12, in the dog heart. A combination of RT-PCR, 5′-RACE, and 3′-RACE demonstrated the status of KCNJ2 as a two exon gene. The complete open reading frame (ORF) was located on the second exon. One transcription initiation site was mapped. Four differential transcription termination sites were found downstream of two consensus polyadenylation signals. The canine KCNJ12 gene was found to consist of three exons, with its ORF located on the third exon. One transcription initiation and one termination site were found. No alternative splicing was observed in right ventricle or brain cortex. The gene structure of canine KCNJ2 and KCNJ12 was conserved amongst other vertebrates, while current GenBank gene annotation was determined as incomplete. In silico translation of KCN12 revealed a non-conserved glycine rich stretch located near the carboxy-terminus of the KIR2.2 protein. However, no differences were observed when comparing dog with human KIR2.2 protein upon ectopic expression in COS-7 or HEK293 cells with respect to subcellular localization or electrophysiological properties.
Highlights
In the mammalian heart, the resting membrane potential of cardiomyocytes is set and stabilized by the inward rectifier potassium current (I K1; Dhamoon and Jalife, 2005)
The main molecular determinants of cardiac I K1 are the KIR2.1 and KIR2.2 proteins expressed from the KCNJ2 and KCNJ12 genes respectively (De Boer et al, 2010a)
KCNJ2 loss of function mutation associated with Andersen–Tawil syndrome 1 regularly, but not in each case, displayed long repolarization times (LQT) and biventricular tachycardias
Summary
The resting membrane potential of cardiomyocytes is set and stabilized by the inward rectifier potassium current (I K1; Dhamoon and Jalife, 2005). The main molecular determinants of cardiac I K1 are the KIR2.1 and KIR2.2 proteins expressed from the KCNJ2 and KCNJ12 genes respectively (De Boer et al, 2010a). The model acquires its sensitivity and specificity from bradycardia associated volume overload and subsequent cardiac remodeling (Thomsen et al, 2007). The latter process translates into modified contractile, structural, and electrical function (Oros et al, 2008). Dog KCNJ2 and KCNJ12 genes stimulated molecular research in this model species (LindbladToh et al, 2005). To enable our future studies on KCNJ2 and KCNJ12 expression regulation in the dog, it is crucial to characterize the molecular structure of the respective genes. Canine KIR2.2 harbors a peculiar stretch of glycine residues in its carboxy-terminus that is not present in KIR2.2 proteins from other species, which did not affect its expression in cell systems neither its electrophysiological properties
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