BIOCHEMISTRY - L. Stryer - 1984

VOLUME 3

PART V. MOLECULAR PHYSIOLOGY

CHAPTER 36. MEMBRANE TRANSPORT

36.6. The Sodium-Potassium Pump Is an Oligomeric Transmembrane Protein

The (Nа⁺ + К⁺)-ATPase is an α₂β₂ tetramer with a molecular mass of 270 kDa. The large α-subunit (95 kDa) contains the site that catalyzes ATP Hydrolysis and the binding site for cardiotonic steroid inhibitors. The smaller β-subunit (40 kDa) contains carbohydrate moieties. Cross-links are readily formed either between the two α-subunits or between an α and a β subunit (but not between the two β-subunits). This observation suggests that the α-subunits are in contact with each other, whereas the β-subunits are spatially separated. As already mentioned, ATP hydrolysis takes place on the cytosolic side of the membrane, whereas the steroid inhibitor binding site is located on the extracellular face. Consequently, each α-subunit spans the entire membrane (Fig. 36.7). The carbohydrate chains of the β-subunits are located on the outside of Cell/30.html">The Plasma Membrane, as is typical for membrane Glycoproteins in general (Section 10.12).

Class="center">Fig. 36.7. Schematic diagram of the subunit Structure and membrane topology of the (Nа⁺ + К⁺)-pump

It is intriguing to note that this enzyme complex possesses a single steroid inhibitor binding site, a single phosphorylation site, and three Na⁺-binding sites. How can an α₂β₂ tetrameric structure contain an odd number of binding sites? One possibility is that the binding sites are located at the interfaces between subunits. As we recall, the α₂β₂ tetramer of Hemoglobin contains a single binding site for bisphosphoglycerate, situated in a central cavity within the molecule (Section 4.14). However, another possibility exists, namely, a cooperative interaction between the two enzyme halves such that binding at one site precludes binding at the other. Indeed, A number of oligomeric Enzymes exhibit this type of "half-of-the-sites" reactivity.

36.7. A Model for the Mechanism of the Sodium-Potassium Pump

Why do the phosphorylation and dephosphorylation of the ATPase result in The transport of Na⁺ and K⁺ across the membrane? The structure of the pump is not yet known in sufficient detail to allow a comprehensive description of its mechanism. Nevertheless, it is useful to consider a simple working model proposed by Oleg Jardetzky. According to this model, the structure of a protein functioning as a pump must satisfy three conditions.

1. The protein must contain a cavity of appropriate size to accommodate a small molecule or ion.

2. The protein must exist in two Conformations, with the cavity being open to the inner side in one conformation and to the outer side in the other.

3. These conformations must exhibit different affinities for the transported components.

Let us examine this model as applied to the transport of Nа⁺ and K⁺ (Fig. 36.8). The two protein conformations are the E1 and E₂ forms described previously. It is postulated that 1) the ion-binding cavity on E1 faces the inside of The Cell, whereas on E₂ it faces the outside, and 2) E1 has a high affinity for Nа+, while E₂ has a high affinity for K⁺. The model is also based on two established facts, namely, 1) Nа+ triggers phosphorylation, whereas K⁺ triggers dephosphorylation, and 2) phosphorylation stabilizes the E₂ form, while dephosphorylation stabilizes the E1 form. In Fig. 36.8, E1 and E₂ are depicted as having vastly different conformations. It should be emphasized, however, that the structural differences between these two forms do not necessarily have to be large. A shift of a few atoms by a distance of 2 Å may be sufficient to alter the orientation of the cavity and the affinity for Nа+ or K⁺. There is ample precedent to suggest that phosphorylation can induce changes of this magnitude. Recall The Effect of phosphorylation on The properties of Glycogen phosphorylase and glycogen synthase, or The change in the oxygen affinity of hemoglobin upon the non-covalent binding of bisphosphoglycerate.

Fig. 36.8. Schematic representation of the putative MECHANISM OF ACTION of the (Na⁺ + K⁺)-pump. The upper half of the figure shows The sequence of reactions directed toward the extrusion of three Nа⁺ ions; the lower half shows the sequence of reactions providing for the influx of two K⁺ ions. The E₁ (yellow) and E₂ (blue) forms differ markedly in conformation in the diagram. In reality, the conformational differences may be very subtle.

36.8. Cardiotonic steroids: specific inhibitors of (Na⁺ + K⁺)-ATPase and the (Nа⁺ + K⁺)-pump

Certain plant-derived Steroids are potent inhibitors of (Nа⁺ + K⁺)-ATPase and the pump. Half-maximal inhibition of both processes is observed at an inhibitor concentration on the order of 10^-8 M. Members of this class of inhibitors, notably digitoxigenin and ouabain, are called cardiotonic steroids due to their pronounced effect on Cardiac Activity (Fig. 36.9). The activity of cardiotonic steroids is determined by the presence in their structure of a 5- or 6-membered unsaturated lactone ring with a β-configuration at C-17. The hydroxyl group at C-14 and the cis-configuration of the C and D ring junction are also essential. In the molecules of ouabain and several other cardiotonic steroids, a sugar residue is attached at C-3; however, this sugar is not required for ATPase inhibition.

Fig. 36.9. Cardiotonic steroids, such as digitoxigenin and ouabain, inhibit the (Na⁺ + K⁺)-pump.

As previously mentioned, cardiotonic steroids inhibit the dephosphorylation reaction of (Na⁺ + K⁺)-ATPase. This inhibition occurs only when the cardiotonic steroids are located on the outer surface of the membrane. Thus, the suppression of dephosphorylation by these steroids is spatially as asymmetric as the activation of dephosphorylation by potassium ions.

Cardiotonic steroids, such as digitalis, are of paramount importance in medicine. Digitalis increases the force of myocardial contraction and is therefore a primary Treatment for acute Heart Failure. The inhibition of the (Na⁺ + K⁺)-pump by digitalis leads to an elevation of Na+ levels in cardiac Muscle Cells. This is accompanied by an increase in intracellular Ca²⁺ concentration, which in turn enhances myocardial contractility. Interestingly, digitalis (a foxglove alkaloid) was successfully used long before the discovery of (Na⁺ + K⁺)-ATPase. In 1785, the physician and botanist William Withering published *An Account of the Foxglove and Some of Its Medical Uses*, in which he described how he first learned about The Use of digitalis for treating acute heart failure.

“In the year 1775, my opinion was asked concerning a family receipt for the cure of the dropsy. I was told that it was a secret kept by an old woman in Shropshire, who had sometimes cured people whom the regular physicians had failed to benefit... Her remedy consisted of twenty or more different herbs, but it was not difficult for anyone knowledgeable in the subject to perceive that the active ingredient could be nothing other than the foxglove... The foxglove has a power over the pulse to an extent that no other medicine possesses, and this power may be converted to the cure of the disease.”