1. The Na +,K + -ATPase Gail Virgin

2. Introduction Membrane Protein – Consists minimally of 2 subunits Uses ATP to transport 3 Na + ions into cell and 2 K + ions out of cell Converts 20 – 30 % of current ATP production in resting mammals to Na + and K + transport P 2 -Type ATPase – Alkali metal cations – Pump gets phosphorylated during cycle D-K-T-G-T-L-T

3. The Catalytic Cycle

4. Secondary Structures of the  -Subunit

5. Mutational Anaylsis D376A  -subunit mutant – Enzymatically inactive – Assembled with  -subunit and delivered to PM properly Na pump does not need to be active in order to reach PM All 3 glycosylation Asn replaced with Glu on  -subunit – Proper assembly and trafficking to PM with wild-type  -subunit – Catalytically active, but increased susceptibility to degradation Mutants with one or more S-S bridge reduced on  -subunit – Enzymatically inactive –  - and  -subunits assemble properly – Heterodimer is retained in ER Bridges provide stability  -subunit lacking cytoplasmic domain – Properly trafficked to PM but inactive

6. The Subunits  - subunit – 1028 amino acids – 110 kDa – 10 transmembrane segments – 3 domains A domain – Regulatory P domain – Phospohorylated N domain – ATP binding – 45% helical – 14%  -sheets  - subunit – Unique to counter K + transporting pumps – 305 amino acids – 55 kDa – 3 S-S bonds and 3 glycosylation sites – 16% helical – 16%  -sheets  – domain – FXYD protein – 74 amino acids – Regulates pump in tissue- and isoform-specific manner – 35% helical

7. Secondary Structures of the  - subunit and  -subunit

10. Ouabain Binding Pocket and K + Binding Site

11. Magnesium and Asp376

12. The  – and  – subunits

15. Cardiac Glycosides Used to treat congestive heart failure Inhibit the Na +,K + - ATPase – Causes increase in intracellular Na + levels – Slows down the Na + /Ca 2+ exchanger – Increases intracellular Ca 2+ levels – Results in stronger muscle contractions

16. References Kaplan JH. Biochemistry of the Na,K-ATPase. Annu. Rev. Biochem. 71:511-535, 2002 Laughery MD, Todd ML, Kaplan JH. Mutational analysis of a-b subunit interactions in the delivery of Na,K-ATPase heterodimers to the plasma membrane. J. Biol. Chem. 278:34794-34803, 2003. Ogawa H, Shinoda T, Cornelius F, Toyoshima C. Crystal structure of the sodium-potassium pump (Na +,K + -ATPase) with bound potassium and ouabain. PNAS. 106:13742-13747, 2009.. Shinoda T, Ogawa H, Cornelius F, Toyoshima C. Crystal structure of the sodium-potassium pump at 2.4Å resolution. Nature. 459:446-451, 2009.