Structure/function analysis of Na⁺-K⁺-ATPase central isoform-specific region: involvement in PKC regulation

Abstract

Specific functions served by the various Na+-K+-ATPase α-isoforms are likely to originate in regions of structural divergence within their primary structures. The isoforms are nearly identical, with the exception of the NH2 terminus and a 10-residue region near the center of each molecule (isoform-specific region; ISR). Although the NH2 terminus has been clearly identified as a source of isoform functional diversity, other regions seem to be involved. We investigated whether the central ISR could also contribute to isoform variability. We constructed chimeric molecules in which the central ISRs of rat α1- and α2-isoforms were exchanged. After stable transfection into opossum kidney cells, the chimeras were characterized for two properties known to differ dramatically among the isoforms: their K+ deocclusion pattern and their response to PKC activation. Comparisons with rat full-length α1- and α2-isoforms expressed under the same conditions suggest an involvement of the central ISR in the response to PKC but not in K+ deocclusion.

The Isoform-Specific Region of the Na,K-ATPase Catalytic Subunit: Role in Enzyme Kinetics and Regulation by Protein Kinase C

“…Relative K + sensitivity of ouabain-sensitive Na + -ATPase activity in membranes from the different transfected OK cells are represented in Figure 4. The K + -induced inhibition pattern observed in this study for the wild-type R1 isoform ( Figure 4A) is comparable to the one obtained by Pierre et al (2). Exchange of the R1 ISR by either the H,K-or mycsequences increased K + inhibition to a notable extent in the initial phase of the curve as well as for the highest K + concentrations ( Figure 4A).…”

“…This is the result of pump translocation from intracellular pools via clathrin-coated vesicles, leading to increased pump abundance in the plasma membrane. This process requires the phosphorylation of both Ser 11 and Ser 18 of the R1 isoform by the PMA-inducible PKC-isoform (21) and seems to involve a dileucine motif present in the R1 ISR, which would act as a dynamic retention signal favoring R1 internalization (2). Thus, the increase in R1 abundance after PMA treatment in OK cells seems to reflect a balance between translocation of intracellular pools of pumps to the plasma membrane and endocytosis of these same pumps, with an overall increase in Na,K-ATPase cell surface expression.…”

“…Heterologous expression of the different constructs was achieved by transfection of opossum kidney (OK) cells using a cationic liposome preparation (2). Transfected OK cells expressing the introduced ouabain-resistant Na,K-ATPase constructs were selected for their ability to grow under 3 µM ouabain, a concentration sufficient to kill untransfected OK cells.…”

“…Earlier work by our laboratory using ISR exchanges among the isoforms suggests that this region contributes to the response of R1 to PKC-mediated phosphorylation (2). To evaluate further this contribution, we examined the effects of PKC stimulation on Na,KATPase-mediated transport in OK cells transfected with H,K- ATPase-and myc-substituted chimeras.…”

“…In a previous study, we have shown that the R1 ISR is essential in protein kinase C (PKC) regulation (2). The present study aims to investigate further the importance of the Na,K-ATPase R1 ISR and to describe the potential role of the R3 ISR to the kinetics and regulation of the enzyme, using a mutagenesis/heterologous expression strategy.…”