Nucleotide Binding Stabilizes Na,K‐ATPase Quaternary Structure and Facilitates Trafficking to Plasma Membrane

Abstract

Na,K‐ATPase (NKA) is an integral membrane protein that plays a central role in ionic homeostasis in animals by mediating transport of Na+ and K+ ions against their electrochemical gradients. NKA is a heterodimer of α and β subunits. It is not yet clear if the native structure of NKA consists of αβ‐protomers or higher order oligomeric states. α‐ α associations were earlier demonstrated using in vitro pull down assays with isolated ATP‐binding domain (ABD) and full‐length α (Costa et al., 2002). Our objective is to elucidate the oligomeric state of NKA throughout maturation and trafficking and the role it plays in protein‐protein interactions. Consistent with previous observations, results from our co‐immunoprecipitation experiments using lysates from HEK 293 cells stably expressing α‐subunit with v5‐ or myc‐tagged NKA α‐subunits demonstrate α‐α interaction. Moreover, this self‐association is stabilized by including 2mM Mg‐ATP in the assay. Our preliminary results from confocal imaging of HEK 293 cells co‐expressing GFP‐tagged ABD and mcherry‐tagged full length α‐subunit revealed GFP‐ABD in the plasma membrane (PM), consistent with the isolated ABD being “carried” to the PM with full‐length α‐subunits. We also found that the α‐subunit lacking the M4M5 cytoplasmic loop (αΔ45LL), and thus unable to bind ATP, was retained in ER. Studies are underway to see if expressing the isolated ABD will rescue αΔ45LL and facilitate its delivery to the PM. This work was supported by NIH grant GM061583.