The Na+,K+-ATPase

Gail Virgin

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

• P2-Type ATPase– Alkali metal cations– Pump gets phosphorylated during cycle

• D-K-T-G-T-L-T

The Catalytic Cycle

Secondary Structures of the -Subunit

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

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

Secondary Structures of the -subunit and -subunit

Ouabain Binding Pocket and K+ Binding Site

Magnesium and Asp376

The – and – subunits

Cardiac Glycosides

• Used to treat congestive heart failure

• Inhibit the Na+,K+-ATPase– Causes increase in

intracellular Na+ levels

– Slows down the Na+/Ca2+ exchanger

– Increases intracellular Ca2+

levels– Results in stronger muscle

contractions

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.