Identification of a PEST Sequence in Vertebrate K _IR2.1 That Modifies Rectification

K _IR2.1 potassium channels, producing inward rectifier potassium current ( I _K1 ), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal K _IR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. K _IR2.1 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 K _IR2.1 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 K _IR2.1 sequences using the “epestfind” webtool. WT and ΔPEST K _IR2.1 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. K _IR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine K _IR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the K _IR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in K _IR2.2, K _IR2.3, and K _IR2.6 proteins. Deletion of the PEST domain in California kingsnake and human K _IR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST K _IR2.1 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 I _K1 , however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of K _IR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of I _K1 channels.