Critical Role of the Isoform-Specific Region in α1-Na,K-ATPase Trafficking and Protein Kinase C-Dependent Regulation

Abstract

The Isoform Specific Region (ISR) is a region of structural heterogeneity among the 4 isoforms of the catalytic α-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 α1-ISR. To this end, a rat α1 construct where Leucine 499 was replaced by a Valine (as found in the α2 isoform sequence) was compared to wild-type rat α1 after stable expression in opossum kidney cells. Total Na,K-ATPase expression, activity, or in situ 86Rb+ transport were 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 86Rb+ uptake was doubled in mutant-expressing cells, comparable to the chimera containing the intact α2 ISR. Similar results were observed when the potential motif was disrupted by means of an E495S mutation. These findings suggest that a di-leucine motif embedded within the Na,K-ATPase α1-ISR plays a critical role in the surface expression of Na,K-ATPase α1 polypeptides at steady-state, and in the response to PKC activation.

Modulation of Na+-K+-ATPase cell surface abundance through structural determinants on the α1-subunit

“…The data presented in Fig. 3 confirmed our previously reported finding that basal surface expression of Na ϩ -K ϩ -ATPase ␣1-units is significantly higher in the mutant group without change in total expression (44). After 5 min of reperfusion, total expression of Na ϩ -K ϩ -ATPase ␣1-was unchanged (3B), but its surface expression was significantly decreased in both ␣1-and ␣1-L499V Na ϩ -K ϩ -ATPase-expressing cells compared with their respective controls.…”

“…Details on the experimental procedures related to expression vectors and site-directed mutagenesis, heterologous expression, and initial characterization of Na ϩ -K ϩ -ATPase enzyme properties in these cells can be found in Sottejeau et al (44).…”

“…Total abundance of the introduced rat ␣1 constructs was determined by electrophoresis and immunoblotting of proteins from cell lysates using anti-NASE antibody as described (44). For total expression, equal loading of the samples among the lanes of the gel was The conserved sequence of amino acid residues that form the dileucine motif appears in boldface.…”

“…Introduced ␣1 expressed at the cell surface was detected by biotinylation following the recommended procedures of Gottardi et al (22), as we have recently reported in detail (44). I-R-induced endocytosis of Na ϩ -K ϩ -ATPase ␣1 units was tested with a pulse-chase strategy as slightly modified from previous studies (27,44).…”

“…We have recently examined one of these molecular determinants, a dileucine-based motif for recognition by clathrin-coated vesicle (CCV) adaptor proteins of the structure n(p) [2][3][4] LL, where n is a negatively charged residue and p is a polar residue (26). The sequence is well conserved among all the known mammalian ␣1 sequences (Table 2), and our studies revealed that mutations targeting this motif such as L499V or E495S resulted in an increased abundance of Na ϩ -K ϩ -ATPase ␣1-units at the cell surface (44).…”