The Na+, K+-ATPase functions by actively transporting 3 Na+ ions out of and 2 K+ ions into the cell, thereby creating ion gradients crucial for many physiological processes. Recently, a combined structural and functional study of the closely related Ca2+-ATPase indicated the presence of a regulatory K+-binding site in the P-domain of the enzyme, identifying E732 as being of particular importance (Sorensen, Clausen et al. 2004). In addition, P709 is thought to play a significant role in the structural organization of this site. Both E732 and P709 are highly conserved among P-type ATPases (E732 is present as either glutamic acid or aspartic acid), which supports their importance and additionally raises the question whether this site may play a general role among P-type ATPases. In Na+, K+-ATPase, K+ functions directly as a substrate for membrane binding sites, however, an additional regulatory effect on the enzyme is yet-unknown. Here, we have investigated the presence of a potential regulatory K+-site in Na+, K+-ATPase. We have focused our attention on D742, corresponding to E732 of the Ca2+-ATPase, by investigating the functional alterations caused by specific substitutions of this residue. Interestingly, all mutations investigated display a reduced level of K+-occluded enzyme intermediate E2[K2] and increased the rate of K+ deocclusion. In addition, preliminary results suggest that the dephosphorylation reaction E2P→E2[K2] is affected. Our data obtained so far thus indicate that D742 indeed is critical to regulation of the enzyme by K+.
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PhD day 2008, Faculty of Health Sciences, University of Aarhus