BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 36. MEMBRANE TRANSPORT

36.2. Discovery of the Sodium-Potassium Active Transport System

Most animal Cells maintain a high intracellular concentration of K⁺ and a low concentration of Na⁺ relative to their external environment. These ion gradients are generated by a specific transport system known as the (Na⁺ + K⁺) pump, as the movements of these two ions are coupled. The Active Transport of Na⁺ and K⁺ is of profound physiological significance. In fact, in animal organisms, more than one-third of the ATP consumed at rest is expended on this process. The concentration gradients of Na⁺ and K⁺ regulate Cell volume, provide the electrical excitability of nerve and Muscle cells, and serve as the driving force for the active transport of sugars and Amino Acids (Sec. 36.10).

In 1957, Jens Skou discovered an enzyme that hydrolyzes ATP only in the presence of both Na⁺ and K⁺ within a Mg²⁺-containing medium. This enzyme was named (Na⁺ + K⁺)-ATPase:

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Skou hypothesized that the (Na⁺ + K⁺)-ATPase is an integral component of the (Na⁺ + K⁺) pump and that ATP Cleavage provides the energy for the active transport of Na⁺ and K⁺. Since then, a wealth of evidence has been accumulated demonstrating that the (Na⁺ + K⁺)-ATPase is indeed an intrinsic part of the (Na⁺ + K⁺) pump.

1. The (Na⁺ + K⁺)-ATPase is found wherever active transport of Na⁺ and K⁺ takes place. The level of enzymatic activity correlates with the quantity of ions transported. For instance, Nerve Cells exhibit high activity of both the (Na⁺ + K⁺)-ATPase and the (Na⁺ + K⁺) pump, whereas in erythrocytes both parameters are low.

2. Both the (Na⁺ + K⁺)-ATPase and the pump are firmly bound to The Plasma Membrane.

3. The (Na⁺ + K⁺)-ATPase and the pump have the same orientation within the plasma membrane.

4. Changes in the concentrations of Na⁺ and K⁺ exert identical effects on the ATPase activity and The rate of ion transport.

5. Cardiotonic Steroids are specific inhibitors of both the (Na⁺ + K⁺)-ATPase and the (Na⁺ + K⁺) pump. The inhibitor concentration producing a half-maximal effect is identical for both processes.

Fig. 36.3. The (Na⁺ + K⁺)-ATPase [a component of the (Na⁺ + K⁺) pump] hydrolyzes ATP only when Na⁺ and K⁺ are simultaneously present in a medium containing Mg²⁺

6. When the operation of the pump is reversed under specific ionic conditions, ATP is synthesized from ADP and P_i.

36.3. Both the Enzyme and the Pump Show a Definite Membrane Orientation

Investigations of the (Na⁺ + K⁺) pump in erythrocyte ghosts have made it possible to determine the orientation of the (Na⁺ + K⁺)-ATPase and the (Na⁺ + K⁺) pump. In a hypotonic salt solution, an erythrocyte swells, and pores form in its membrane. Hemoglobin leaks out, leaving behind a clear membrane (ghost). The interior of the swollen erythrocyte can be equilibrated with the external medium. When the external solution is made isotonic, the membrane regains its permeability barrier. Consequently, the molecular and ionic COMPOSITION OF THE interior of the ghosts can be regulated by resealing them in an appropriate solution. Studies of transport processes and enzymatic activity in erythrocyte ghosts have demonstrated that the (Na⁺ + K⁺) pump is oriented as follows (Fig. 36.4).

Fig. 36.4. The (Na⁺ + K⁺) pump has a strict orientation in the plasma membrane

1. To activate the ATPase and achieve transmembrane transport, Na⁺ ions must be on the inside, and K⁺ ions must be on the outside.

2. Only intracellular ATP serves as an effective substrate for the ATPase and drives the pump.

3. Cardiotonic steroids inhibit the pump and the ATPase only when they are located on the extracellular side (outside The Cell).

4. Vanadate ions inhibit the pump and the ATPase only when they are present inside the cell.

36.4. ATP Transiently Phosphorylates the Sodium-Potassium Pump

How does ATP drive the active transport of Na⁺ and K⁺? The key to this puzzle came from the observation that, in the presence of Na⁺ and Mg²⁺, ATP phosphorylates the (Na⁺+ K⁺)-ATPase. The site of phosphorylation is the side chain of a specific aspartate residue. Subsequently, in the presence of K⁺, the phosphorylated intermediate (E—P) undergoes Hydrolysis. The phosphorylation reaction does not require K⁺, whereas the dephosphorylation reaction requires neither Na⁺ nor Mg²⁺:

Na⁺-dependent phosphorylation and K⁺-dependent dephosphorylation are not the only critical reactions involved. During its operation, the pump adopts at least two distinct Conformations, designated as E₁ and E₂. In fact, at least four conformational states of the enzyme participate in Na⁺ and K⁺ transport coupled with ATP hydrolysis: E₁, E₁—P, E₂—P, and E₂ (Fig. 36.5). The hydrolysis of a single ATP molecule drives the export of three Na⁺ ions and the import of two K⁺ ions. Consequently, the operation of the pump generates an electrical current across the membrane. In other words, the (Na⁺ + K⁺)-ATPase pump is electrogenic. The maximum turnover number of the ATPase is approximately 100 s^-1.

Fig. 36.5. Cyclic conformational changes of the (Na+ + K+)-ATPase during catalysis

Nanomolar concentrations of vanadate ions (V⁵⁺) inhibit the (Na⁺ + K⁺)-ATPase. This pentavalent ion locks the protein in the E₂ conformation. Vanadate acts as a transition-state analog for the hydrolytic Cleavage of the phosphoryl group because it readily adopts a bipyramidal geometry similar to that of phosphate (Fig. 36.6).

Fig. 36.6. Structure OF THE vanadate ion (V⁵+). The ligands surrounding the ion form a bipyramidal arrangement, identical to that around the phosphorus atom during the hydrolytic cleavage of a phosphoryl group.

36.5. Ion Transport and ATP Hydrolysis Are Closely Coupled

A key feature of the pump is that in the absence of Na⁺ and K⁺ transport, ATP hydrolysis does not occur. In other words, the system is coupled in such a way that ATP energy is not wasted. Tight coupling is a common characteristic of biological systems that mediate energy conversion. Recall that in Mitochondria, a normal electron flow through the Respiratory Chain is contingent upon the simultaneous generation of ATP (Section 14.13). Another example of this principle is the obligatory coupling between ATP hydrolysis and Muscle contraction.

The action of the (Na⁺ + K⁺)-pump can be reversed to drive ATP synthesis. Net synthesis of ATP from ADP and P_i takes place under conditions of steeply increased ion gradients. This is achieved by incubating erythrocytes in a medium with an abnormally high Na⁺ concentration and an extremely low K⁺ concentration.