the na +,k + -atpase gail virgin. introduction membrane protein – consists minimally of 2 subunits...
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The Na+,K+-ATPase
Gail Virgin
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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
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The Catalytic Cycle
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Secondary Structures of the -Subunit
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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
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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
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Secondary Structures of the -subunit and -subunit
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Ouabain Binding Pocket and K+ Binding Site
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Magnesium and Asp376
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The – and – subunits
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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
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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.