Sodium Potassium Adenosine Triphosphatase (NaK)
Sodium-potassium ATPase (or NaK) is an ion pump responsible for establishing an electrochemical gradient across the cell membrane (Reeves and Yamanaka 1993). It is a P-type (or E1E2) ATPase ion co-transporter, meaning that it works via the phosphorylation of a covalent intermediate state (Emery et al. 1998). Like other P-type ATPases, NaK is sensitive to vanadate, alternates between an E1 and E2 conformational state, and has a large highly conserved polypeptide chain usually greater than 100 kDa, designated the alpha-subunit (Fagan and Saier Jr 1993). Found in the plasma membrane (Emery et al. 1998), it pumps 3 Na+ ions out of the cell and 2 K+ ions into the cell (Emery et al. 1998), and the subsequent electrochemical gradient that is established powers several other vital transport processes and cell signaling functions across the cell membrane (Reeves and Yamanaka 1993, Emery et al. 1998).
NaK is a heterodimer composed of the catalytically active alpha-subunit (containing approximately 1000 amino acids residues) and a glycosylated beta-subunit, the function of which is unknown (Emery et al. 1998, Reeves and Yamanaka 1993). The highly conserved alpha-subunit has been historically divided into three segments: Segment 1 consists of the N-terminus, four putative transmembrane helices, and some cytoplasmic and extracellular loops, including the ouabain-binding site. Segment 2 includes the large cytoplasmic region that contains the phosphorylation domain and the ATP binding site. Segment 3 contains the remaining C-terminal region (Fagan and Saier Jr 1993).
In invertebrates, NaK is a single copy nuclear protein-coding gene that can undergo post-transcriptional modifications to produce different transcripts (Reeves and Yamanaka 1993). Vertebrates, however, have evolved multiple copies of both the alpha and beta subunits, which allow for greater tissue specificity (Emery et al. 1998).
The NaK pump is easily disabled by the addition of ouabain, a cardiac glycoside (Labeyrie and Dobler 2004) and several studies have been carried out looking at the evolution of ouabain resistance in species of insects that feed on milkweed plants (Labeyrie and Dobler 2004, Holzinger and Wink 1996, Moore and Scudder 1986, Vaughan and Jungreis 1977). Tissue-specific -expression of NaK ATPase varies widely among species. Blood-feeding insects, for example, will express NaK in higher concentration within their midgut, where it acts to remove Na+ and water from the intestinal lumen (Emery et al. 1998).
Our primers were developed by examining an alignment of the ouabain-binding site of different insects as presented in Labeyrie and Dobler (2004), and by blasting the subsequent consensus against the Apis whole genome. The fragment amplified by our primers is an intronless region, about 1.5kb long, that stretches across the extracellular ouabain-binding site of the alpha-subunit. It is found near the N-terminus of Segment 1 of the NaK alpha-subunit, and studies of mutant NaKs indicate that the ouabain-binding site is on the first extracellular loop, between the first and second transmembrane helices (Fagan and Saier Jr 1993).
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