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Neurological disease

mutations

compromise a C-

terminal ion pathway

in the Na+/K+ -

ATPaseManipol, Ryel Katherine

Saldajeno, Maria Angela


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Introduction


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Introduction The Na+/K+-ATPase pumps three sodium ions out

of and two potassium ions into the cell for each

ATP molecule that is split, thereby generating

the chemical and electrical gradients across the

plasma membrane that are essential insignalling, secondary transport and volume

regulation in animal cells.


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Introduction Crystal structures of the potassium-bound form o

the pump revealed an intimate docking of the

subunit carboxy terminus at the transmembrane

domain. The structure shows that this element is a

key regulator of a previously unrecognized ion

pathway.

Current models of P-type ATPases operate with a

single ion conduit through the pump, but our data

suggest an additional pathway in the Na+/K+

ATPase between the ion-binding sites and thecytoplasm which is the C-terminal pathway.


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Introduction The C-terminal pathway allows a cytoplasmicproton to enter and stabilize site III when empty inthe potassium-bound state, and when potassium isreleased the proton will also return to the

cytoplasm, thus allowing an overall asymmetricstoichiometry of the transported ions.

The C terminus controls the gate to the pathway.Its structure is crucial for pump function, asdemonstrated by at least eight mutations in the

region that cause severe neurological diseases.


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Introduction This novel model for ion transport by the

Na+/K+-ATPase is established byelectrophysiological studies of C-terminal

mutations in familial hemiplegic migraine 2(FHM2) and is further substantiated bymolecular dynamics simulations. A similarion regulation is likely to apply to the H+/K+-ATPase and the Ca2+-ATPase.


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History 1957- Nobel Laureate Jens Christian Skou

was the first to describe the sodium-potassium pump

2007- atomic structure of sodium-potassium pump was determined


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Objectives


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Objectives To assess if the C-terminal region is directly

involved in ion transport.

To determine the role of C-terminal ion pathway to

the Na+/K+-ATPase conformation. To determine the effects of C-terminal truncations

or mutations.

To understand the involvement of C-terminalpathway in neurological disease.


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Procedure


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Procedure

Electrophysiology

Molecular dynamics


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ElectrophysiologyPlasmids

encodinghuman a2 andB1 subunits ofthe Na+/K+ -ATPase were

subcloned intothe pXOON

vector

MutationsQ116R andN127D were

introduced intoa2 by PCR

yielding thewild type

RNAstranscribed


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ElectrophysiologyB1 and a2were co-

injected intooocytes

fromXenopus

laevis

Oocyteswere

loaded

Twoelectrodevoltage

clampingperformed


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Electrophysiology

Steady-statecurrents weredetermined

Charge

movementwas

determined


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Electrophysiology study of the electrical properties of biological cells

and tissues

involves measurements of voltage change orelectric current on a wide variety of scales fromsingle ion channel proteins to whole organs likethe heart

it pertains to the flow of ions in biological tissuesand, in particular, to the electrical recordingtechniques that enable the measurement of thisflow


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Molecular DynamicsSimulations

implementedwith pig renal

aB dimer

Asp 930 andGlu 958 wereprotonated

Wild-typesystem wasequilibrated


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Molecular dynamics computer simulation of physical

movements by atoms and molecules

frequently used in the study of proteins

and biomolecules

based on statistical mechanics, statisticalensemble averages are equal to timeaverages of the system


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Results and Discussion


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Results and DiscussionMutated residues in C-terminal result to neurological

disease:

The crystal structures indicate that the two C-terminal tyrosines (Tyr 1019 and Tyr 1020) are

coordinated by two arginines (Arg 937 and Arg1002) through hydrogen bonds and cationpinteractions Naturally occurring mutations of eitherarginine, R937P14 or R1002Q15 in the a2 encodinggene ATP1A2 cause FHM2, a severe dominantform of migraine.


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Figure 1. Side view of theproposed alternative ionpathway with charged andpolar residues shown assticks. The color codingis as in a; yellow indicates

residues causing FHM2when mutated.



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Results and Discussion A weak but measurable current is generated

from the differences in the numbers of sodium

and potassium ions pumped in and out

frog eggs were used as model cells since ithad specific sodium- potassium pumps and

mutations and the pump currents were

measured by inserting small electrodes into

the eggs


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Results and Discussion pumps without the c-terminal ion

pathway as well as pumps with mutationswere measured

the currents showed that the c- terminalion pathway is important for binding andreleasing the sodium ions while it has noinfluence to potassium ions


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Results and Discussion it was conceptualized a protons from the

cell acts as a piston that pushes thesodium ions out and fills one of three "ion

containers" in the pump so that only twocontainers are left for the potassium

mutations of the c-terminal ion pathwaydisturb the piston function, thus causing

neurological diseases.


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model for the sodium-potassium pump


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Sodium Potassium Pump Left: the pump binds three sodium ions (green) from the

cell, locks them in, and when they are released on theother side, the piston (i.e. a proton from the cell) (black)helps to push them out.

Right: the proton remains in the pump, so there is onlyroom for two potassium ions (red) from the outside, thepotassium ions and the proton are occluded in thepump, and they are all subsequently released inside thecell. The pump thus forms large differences in both ionconcentrations and the number of charges on either

side of the membrane.


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Model for ion transport by the Na1/K1-ATPaseResults and Discussion


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Model for ion transport by the Na/K-ATPase

in a simplified PostAlbers scheme Formulated by Robert Lickely Post

designed to accommodate the observed kineticsof enzyme phosphorylation anddephosphorylation catalyzed by Na+ and K+,respectively

explaining the biochemical behavior of theisolated enzyme

describes the sequence of reaction steps of theNa,K-ATPase by which the charge translocationstake place


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Model for ion transport by the Na/K-ATPase

in a simplified PostAlbers scheme Enzyme is phosphorylated by ATP (in the presence

of Na+ and Mg2+) in one conformation yielding ahigh-energy intermediate E1P capable ofphosphorylating ADP, and then undergoes a

conformational change to a low-energy (ADP-resistant) E2P form that is rapidly dephosphorylatedin the presence of K+.


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Conclusion


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Conclusion neurons depend on power for fast and efficient

communication. this power comes from the sodiumpotassium pump which ensures that the sodium isoutside and potassium is inside and the cell is negative

so when a channel for sodium opens sodium flows intothe cell

if enough channels open, the cell becomes activatedand sends a message to others cells in the network

communication between neurons in the brainconstantly uses most of its energy (ATP) to operate the

sodium- potassium pumps.


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Conclusion if a mutation in the sodium potassium

pump affects a region that is essential formechanics, it will probably lead to either

cell death or diseaseA large number of the disease mutations

are located near the pumpstail


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Conclusion sodium-potassium pump is controlled by a

tiny pistonconsisting of just a singleprotonand genetic changes affecting

the piston can cause migraine or dystonicparkinsonism.

Different mutations in the sodiumpotassium pumps cause different

consequences


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Conclusion The sodium-potassium pump is essential for all

animal life and is the target of some of the oldestknown drugs.

The body generally uses about a quarter of its

available energy to operate the sodium-potassiumpump (and in the brain, this amounts to almostthree quarters of the energy), since it mustmaintain the vital intracellular and extracellularsalt concentrations to drive numerous processes,

e.g. electrical nerve impulses.


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Related Research The structure of the potassium channel:molecular basis of K+ conduction and selScience Apr 20, 1998... long, whereas the remainder of the pore iswider and lined withhydrophobic aminoacids. A large water-filledcavity and helix dipoles are positioned so asto overcome electrostatic destabilization ofan ion in the pore at ...


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Related ResearchOne way for the gastric proton pump

Poul NissenMolecular Biology, University of Aarhus,Gustav Wids Vej, Aarhus C, Denmark

mutational studies, where the subunitof the H+,K+-ATPase was truncated from theN-terminus by 4, 8 and 13 residues


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References1. Morth, J. P. et al. Crystal structure of the sodiumpotassium pump. Nature 450,10431049 (2007).

2. Shinoda, T., Ogawa, H., Cornelius, F. & Toyoshima, C. Crystal structure of thesodiumpotassium pump at 2.4 A

resolution. Nature 459, 446450 (2009).

3. Olesen, C. et al. The structural basis of calcium transport by the calcium pump.Nature 450, 10361042 (2007).

4. Takeuchi, A., Reyes, N., Artigas, P. & Gadsby, D. C. The ion pathway through the

opened Na1,K1-ATPase pump. Nature 456, 413416 (2008).

5. Toyoshima, C. & Nomura, H. Structural changes in the calcium pumpaccompanying the dissociation of calcium. Nature 418, 605611 (2002).

6. Morth, J. P. et al. The structure of the Na1,K1-ATPase and mapping of isoform

differences and disease-related mutations. Phil. Trans. R. Soc. B 364, 217227

(2009).7. Blanco-Arias, P. et al. A C-terminal mutation of ATP1A3 underscores the crucial

role of sodium affinity in the pathophysiology of rapid-onset dystoniaparkinsonism.

Hum. Mol. Genet. 18, 23702377 (2009).


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References8. Toyoshima, C., Nakasako, M., Nomura, H. & Ogawa, H. Crystal structure of thecalcium pump of sarcoplasmic reticulum at 2.6 A resolution. Nature 405,

647655 (2000).9. Li, C., Geering, K. & Horisberger, J. D. The third sodium binding site of Na,KATPase

is functionally linked to acidic pH-activated inward current. J. Membr. Biol.

213, 19 (2006).10. Ogawa, H. & Toyoshima, C. Homology modeling of the cation binding sites of

Na1K1-ATPase. Proc. Natl Acad. Sci. USA 99, 1597715982 (2002).

11. Meier, S., Tavraz, N. N., Durr, K. L. & Friedrich, T. Hyperpolarization-activated

inward leakage currents caused by deletion or mutation of carboxy-terminaltyrosines of the Na1/K1-ATPase a subunit. J. Gen. Physiol. 135, 115134 (2010).

12. Toustrup-Jensen, M. S. et al. The C-terminus of Na1,K1-ATPase controls Na1

affinity on both sides of the membrane through Arg935. J. Biol. Chem. 284,

1871518725 (2009).13. Yaragatupalli, S., Olivera, J. F., Gatto, C. & Artigas, P. Altered Na1 transport after

an intracellular alpha-subunit deletion reveals strict external sequential release of

Na1 from the Na/K pump. Proc. Natl Acad. Sci. USA 106, 1550715512 (2009).


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References14. Riant, F. et al. ATP1A2 mutations in 11 families with familial hemiplegic migraine.Hum. Mutat. 26, 281 (2005).

15. Jen, J. C. et al. Prolonged hemiplegic episodes in children due to mutations inATP1A2. J. Neurol. Neurosurg. Psychiatry 78, 523526 (2007).

16. Holmgren, M. et al. Three distinct and sequential steps in the release of sodium

ions by the Na1/K1-ATPase. Nature 403, 898901 (2000).17. Vasilyev, A., Khater, K. & Rakowski, R. F. Effect of extracellular pH on presteadystate

and steady-state current mediated by the Na1/K1 pump. J. Membr. Biol.

198, 6576 (2004).

18. Anselm, I. A., Sweadner, K. J., Gollamudi, S., Ozelius, L. J. & Darras, B. T. Rapidonsetdystonia-parkinsonism in a child with a novel ATP1A3 gene mutation.

Neurology 73, 400401 (2009).

19. Zanotti-Fregonara, P. et al. [123I]-FP-CIT and [99mTc]-HMPAO single photon

emission computed tomography in a new sporadic case of rapid-onsetdystoniaparkinsonism. J. Neurol. Sci. 273, 148151 (2008).

20. Bas, D. C., Rogers, D. M. & Jensen, J. H. Very fast prediction and rationalization of

pKa values for protein-ligand complexes. Proteins 73, 765783 (2008).


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References21. Skou, J. C. & Esmann, M. Effects of ATP and protons on the Na : K selectivity of the(Na1 1 K1)-ATPase studied by ligand effects on intrinsic and extrinsic

fluorescence. Biochim. Biophys. Acta 601, 386402 (1980).22. Jewell-Motz, E. A. & Lingrel, J. B. Site-directed mutagenesis of the Na,K-ATPase:

consequences of substitutions of negatively-charged amino acids localized in the

transmembrane domains. Biochemistry 32, 1352313530 (1993).23. Yamamoto, S., Kuntzweiler, T. A., Wallick, E. T., Sperelakis, N. & Yatani, A. Amino

acid substitutions in the rat Na1, K(1)-ATPase alpha 2-subunit alter the cation

regulation of pump current expressed in HeLa cells. J. Physiol. (Lond.) 495,

733742 (1996).24. Burnay, M., Crambert, G., Kharoubi-Hess, S., Geering, K. & Horisberger, J. D.

Electrogenicity of Na,K- and H,K-ATPase activity and presence of a positively

charged amino acid in the fifth transmembrane segment. J. Biol. Chem. 278,

1923719244 (2003).25. Jespersen, T., Grunnet, M., Angelo, K., Klaerke, D. A. & Olesen, S. P. Dual-function

vector for protein expression in both mammalian cells and Xenopus laevis oocytes.

Biotechniques 32, 536538 540 (2002).


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References26. Price, E. M. & Lingrel, J. B. Structurefunction relationships in the Na,K-ATPase asubunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-

122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry 27,84008408 (1988).

27. Berendsen, H. J. C., Postma, J. P. M., Vangunsteren, W. F., Dinola, A. & Haak, J. R.

Molecular dynamics with coupling to an external bath. J. Chem. Phys. 81,36843690 (1984).

28. Berger, O., Edholm, O. & Jahnig, F. Molecular dynamics simulations of a fluid

bilayer of dipalmitoylphosphatidylcholine at full hydration, constant pressure, and

constant temperature. Biophys. J. 72, 20022013 (1997).


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