The Na

+

,K

+

-ATPase

Gail Virgin

Introduction

• • • • Membrane Protein – Consists minimally of 2 subunits Uses ATP to transport 3 Na + 2 K + ions out of cell ions into cell and 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

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 – g – 16% helical 16% b -sheets – domain – FXYD protein – 74 amino acids – Regulates pump in tissue- and isoform-specific manner – 35% helical

Secondary Structures of the

b

subunit 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 + /Ca 2+ exchanger – Increases intracellular Ca 2+ 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.