Abstract
KIR2.1 potassium channels, producing inward rectifier potassium current (IK1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the “epestfind” webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical IK1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of IK1 channels.
Highlights
Since its cloning in the early 1990s (Kubo et al, 1993), many domains of the KIR2.1 primary protein sequence, encoded by KCNJ2, have been attributed to biological function and molecular structure, but not all
Since we noticed that this region was enriched in proline (P), glutamate (E), aspartate (D), serine (S), and threonine (T) residues, a hallmark of so-called KIR2.1 protein lacking the complete PEST domain (PEST) domains (Rechsteiner and Rogers, 1996), individual sequences were screened according to a PEST finding algorithm using the EMBOSS program epestfind
No PEST domains were found in human KIR2.2, KIR2.3 or KIR2.6 channel proteins, while KIR2.4 contains a PEST domain with a PEST score of 9.39 that starts upstream from the KIR2.1 PEST domain (Figure 1)
Summary
Since its cloning in the early 1990s (Kubo et al, 1993), many domains of the KIR2.1 primary protein sequence, encoded by KCNJ2, have been attributed to biological function and molecular structure, but not all. The resulting inward rectifying potassium current (IK1) is characterized by a larger inward than outward current from equal negative and positive deflections from the potassium equilibrium potential. This property allows for action potential formation in excitable cells, while providing a stable resting membrane potential in between action potentials (Van der Heyden and Jespersen, 2016). More understanding of the functions of different protein domains might provide new avenues for therapeutic approaches
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