Neurological disease mutations compromise a Cterminal ion pathway in the Na+/K+ ATPase

Manipol, Ryel Katherine Saldajeno, Maria Angela

Introduction

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 in signalling, secondary transport and volume regulation in animal cells.

Introduction

Crystal structures of the potassium-bound form of 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 the cytoplasm which is the C-terminal pathway.

Introduction

The C-terminal pathway allows a cytoplasmic proton to enter and stabilize site III when empty in the potassium-bound state, and when potassium is released the proton will also return to the cytoplasm, thus allowing an overall asymmetric stoichiometry of the transported ions. The C terminus controls the gate to the pathway. Its structure is crucial for pump function, as demonstrated by at least eight mutations in the region that cause severe neurological diseases.

Introduction
This

novel model for ion transport by the Na+/K+-ATPase is established by electrophysiological studies of C-terminal mutations in familial hemiplegic migraine 2 (FHM2) and is further substantiated by molecular dynamics simulations. A similar ion regulation is likely to apply to the H+/K+ATPase and the Ca2+-ATPase.

History
1957-

Nobel Laureate Jens Christian Skou was the first to describe the sodiumpotassium pump 2007atomic structure of sodiumpotassium pump was determined

Objectives

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-terminal pathway in neurological disease.

Procedure

Procedure

Electrophysiology
Molecular dynamics

Electrophysiology
Plasmids encoding human a2 and B1 subunits of the Na+/K+ ATPase were subcloned into the pXOON vector Mutations Q116R and N127D were introduced into a2 by PCR yielding the wild type

RNAs transcribed

Electrophysiology
B1 and a2 were coinjected into oocytes from Xenopus laevis Oocytes were loaded Two electrode voltage clamping performed

Electrophysiology
Steady-state currents were determined Charge movement was determined

Electrophysiology

study of the electrical properties of biological cells and tissues involves measurements of voltage change or electric current on a wide variety of scales from single ion channel proteins to whole organs like the heart it pertains to the flow of ions in biological tissues and, in particular, to the electrical recording techniques that enable the measurement of this flow

Molecular Dynamics
Simulations implemented with pig renal aB dimer Asp 930 and Glu 958 were protonated Wild-type system was equilibrated

Molecular dynamics
computer

simulation of physical movements by atoms and molecules frequently used in the study of proteins and biomolecules based on statistical mechanics, statistical ensemble averages are equal to time averages of the system

Results and Discussion

Results and Discussion

Mutated residues in C-terminal result to neurological disease: The crystal structures indicate that the two Cterminal tyrosines (Tyr 1019 and Tyr 1020) are coordinated by two arginines (Arg 937 and Arg 1002) through hydrogen bonds and cationp interactions Naturally occurring mutations of either arginine, R937P14 or R1002Q15 in the a2 encoding gene ATP1A2 cause FHM2, a severe dominant form of migraine.

Results and Discussion

Figure 1. Side view of the proposed alternative ion pathway with charged and polar residues shown as sticks. The color coding is as in a; yellow indicates residues causing FHM2 when mutated.

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 it had specific sodium- potassium pumps and mutations and the pump currents were measured by inserting small electrodes into the eggs

Results and Discussion

pumps

without the c-terminal ion pathway as well as pumps with mutations were measured the currents showed that the c- terminal ion pathway is important for binding and releasing the sodium ions while it has no influence to potassium ions

Results and Discussion

it

was conceptualized a protons from the cell acts as a piston that pushes the sodium ions out and fills one of three "ion containers" in the pump so that only two containers are left for the potassium mutations of the c-terminal ion pathway disturb the piston function, thus causing neurological diseases.

Results and Discussion

model for the sodium-potassium pump

Sodium Potassium Pump

Left: the pump binds three sodium ions (green) from the cell, locks them in, and when they are released on the other 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 only room for two potassium ions (red) from the outside, the potassium ions and the proton are occluded in the pump, and they are all subsequently released inside the cell. The pump thus forms large differences in both ion concentrations and the number of charges on either side of the membrane.

Results and Discussion

Model for ion transport by the Na1/K1-ATPase

Model for ion transport by the Na/K-ATPase in a simplified PostAlbers scheme

Formulated by Robert Lickely Post designed to accommodate the observed kinetics of enzyme phosphorylation and dephosphorylation catalyzed by Na+ and K+, respectively explaining the biochemical behavior of the isolated enzyme describes the sequence of reaction steps of the Na,K-ATPase by which the charge translocations take place

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 a high-energy intermediate E1P capable of phosphorylating ADP, and then undergoes a conformational change to a low-energy (ADPresistant) E2P form that is rapidly dephosphorylated in the presence of K+.

Conclusion

Conclusion

neurons depend on power for fast and efficient communication. this power comes from the sodium potassium pump which ensures that the sodium is outside and potassium is inside and the cell is negative so when a channel for sodium opens sodium flows into the cell if enough channels open, the cell becomes activated and sends a message to others cells in the network communication between neurons in the brain constantly uses most of its energy (ATP) to operate the sodium- potassium pumps.

Conclusion
if

a mutation in the sodium potassium pump affects a region that is essential for mechanics, it will probably lead to either cell death or disease A large number of the disease mutations are located near the pumps tail

Conclusion
sodium-potassium

pump is controlled by a tiny piston consisting of just a single proton and genetic changes affecting the piston can cause migraine or dystonic parkinsonism. Different mutations in the sodium potassium pumps cause different consequences

Conclusion

The sodium-potassium pump is essential for all animal life and is the target of some of the oldest known drugs. The body generally uses about a quarter of its available energy to operate the sodium-potassium pump (and in the brain, this amounts to almost three quarters of the energy), since it must maintain the vital intracellular and extracellular salt concentrations to drive numerous processes, e.g. electrical nerve impulses.

Related Research

The structure of the potassium channel: molecular basis of K+ conduction and sel Science Apr 20, 1998 ... long, whereas the remainder of the pore is wider and lined with hydrophobic amino acids. A large water-filled cavity and helix dipoles are positioned so as to overcome electrostatic destabilization of an ion in the pore at ...

Related Research
One

way for the gastric proton pump Poul Nissen

Molecular Biology, University of Aarhus, Gustav Wids Vej, Aarhus C, Denmark mutational studies, where the subunit of the H+,K+-ATPase was truncated from the N-terminus by 4, 8 and 13 residues

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