Transcript Gail`s powerpoint

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 a-Subunit
Mutational Anaylsis
• D376A a-subunit mutant
– Enzymatically inactive
– Assembled with b-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 b-subunit
– Proper assembly and trafficking to PM with wild-type a-subunit
– Catalytically active, but increased susceptibility to degradation
• Mutants with one or more S-S bridge reduced on b-subunit
– Enzymatically inactive
– a- and b-subunits assemble properly
– Heterodimer is retained in ER
• Bridges provide stability
• b-subunit lacking cytoplasmic domain
– Properly trafficked to PM but inactive
The Subunits
• a - subunit
– 1028 amino acids
– 110 kDa
– 10 transmembrane
segments
– 3 domains
• A domain
– Regulatory
• P domain
– Phospohorylated
• N domain
– ATP binding
– 45% helical
– 14% b-sheets
• b - subunit
– Unique to counter K+
transporting pumps
– 305 amino acids
– 55 kDa
– 3 S-S bonds and 3
glycosylation sites
– 16% helical
– 16% b-sheets
• g – domain
– FXYD protein
– 74 amino acids
– Regulates pump in tissue- and
isoform-specific manner
– 35% helical
Secondary Structures of the bsubunit and g-subunit
Ouabain Binding Pocket and K+ Binding Site
Magnesium and Asp376
The b – and g – 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.