Critical role of the isoform-specific region in alpha1-Na,K-ATPase trafficking and protein Kinase C-dependent regulation

The isoform-specific region (ISR) is a region of structural heterogeneity among the four isoforms of the catalytic alpha-subunit of the Na,K-ATPase and an important structural determinant for isoform-specific functions. In the present study, we examined the role of a potential dileucine clathrin adaptor recognition motif [DE]XXXL[LI] embedded within the alpha1-ISR. To this end, a rat alpha1 construct where leucine 499 was replaced by a valine (as found in the alpha2 isoform sequence) was compared to wild-type rat alpha1 after stable expression in opossum kidney cells. Total Na,K-ATPase expression, activity, or in situ (86)Rb(+) transport was not affected by the L499V mutation. However, surface Na,K-ATPase expression was nearly doubled. This increase was associated with a reduced rate of internalization from the cell surface of about 50% after a 4 h chase and became undetectable if clathrin-coated pit-mediated trafficking was blocked with chlorpromazine. Further, PKC-induced stimulation of Na,K-ATPase-mediated (86)Rb(+) uptake was doubled in mutant-expressing cells, comparable to the chimera containing the intact alpha2-ISR. Similar results were observed when the potential motif was disrupted by means of an E495S mutation. These findings suggest that a dileucine motif embedded within the Na,K-ATPase alpha1-ISR plays a critical role in the surface expression of Na,K-ATPase alpha1 polypeptides at steady state and in the response to PKC activation.