A K ATPase

Na, K -ATPase, a K ATPase Ca-+-ATPase, SR Ca" -ATPase, plasma membrane H -ATPase, yeast H" -ATPase, Neurospora... 

Active Transport. Maintenance of the appropriate concentrations of K" and Na" in the intra- and extracellular fluids involves active transport, ie, a process requiring energy (53). Sodium ion in the extracellular fluid (0.136—0.145 AfNa" ) diffuses passively and continuously into the intracellular fluid (<0.01 M Na" ) and must be removed. This sodium ion is pumped from the intracellular to the extracellular fluid, while K" is pumped from the extracellular (ca 0.004 M K" ) to the intracellular fluid (ca 0.14 M K" ) (53—55). The energy for these processes is provided by hydrolysis of adenosine triphosphate (ATP) and requires the enzyme Na" -K" ATPase, a membrane-bound enzyme which is widely distributed in the body. In some cells, eg, brain and kidney, 60—70 wt % of the ATP is used to maintain the required Na" -K" distribution. 

FIGURE 10.8 A schematic diagram of the Na, K -ATPase in mammalian plasma membrane. ATP hydrolysis occurs on the cytoplasmic side of the membrane, Na ions are transported out of the cell, and ions are transported in. The transport stoichiometry is 3 Na out and 2 in per ATP hydrolyzed. The specific inhibitor ouabain (Figure 7.12) and other cardiac glycosides inhibit Na, K -ATPase by binding on the extracellular surface of the pump protein. 

A minimal mechanism for Na, K -ATPase postulates that the enzyme cycles between two principal conformations, denoted Ej and Eg (Figure 10.11). El has a high affinity for Na and ATP and is rapidly phosphorylated in the presence of Mg to form Ei-P, a state which contains three oeeluded Na ions (occluded in the sense that they are tightly bound and not easily dissociated from the enzyme in this conformation). A conformation change yields Eg-P, a form of the enzyme with relatively low affinity for Na, but a high affinity for K. This state presumably releases 3 Na ions and binds 2 ions on the outside of the cell. Dephosphorylation leaves EgKg, a form of the enzyme with two... 

FIGURE 10.10 The reaction of tridated sodium borohydride with the aspartyl phosphate at the active site of Na, K -ATPase. Acid hydrolysis of the enzyme following phosphorylation and sodium borohydride treatment yields a tripeptide containing serine, homoserine (derived from the aspartyl-phosphate), and lysine as shown. The site of phosphorylation is Asp" in the large cytoplasmic domain of the ATPase. 

FIGURE 10.11 A mechanism for Na, K -ATPase. The model assumes two principal conformations, Ei and E9. Binding of Na ions to Ei is followed by phosphorylation and release of ADP. Na ions are transported and released and ions are bound before dephosphorylation of the enzyme. Transport and release of ions complete the cycle. 

FIGURE 10.15 A mechanism for Ca -ATPase from sarcoplasmic reticulum. Note the similarity to the mechanism of Na, K -ATPase (see also Figure 10.11). ( Out here represents the cytosol In represents the lumen of the SR.)... 

Inhibition of the Na+/K+-ATPase leads to a loss of potassium and an increase of sodium within the cell. Secondary intracellular calcium is increased via the Na VCa -exchanger. This results in a positive inotropic effect in the myocardium, with an increase of peak force and a decrease in time to peak tension. Besides this, cardiac glycosides increase vagal activity by effects on the central vagal nuclei, the nodose ganglion and increase in sensitivity of the sinus node to acetylcholine. 

Tlie Na+/K+-ATPase belongs to the P-type ATPases, a family of more than 50 enzymes that also includes the Ca2+-ATPase of the sarcoplasmic reticulum or the gastric H+/K+-ATPase. P-Type ATPases have in common that during ion transport an aspartyl phos-phointermediate is formed by transfer of the y-phosphate group of ATP to the highly conserved sequence DKTGS/T [1]. 

The catalytic cycle of the Na+/K+-ATPase can be described by juxtaposition of distinct reaction sequences that are associated with two different conformational states termed Ei and E2 [1]. In the first step, the Ei conformation is that the enzyme binds Na+ and ATP with very high affinity (KD values of 0.19-0.26 mM and 0.1-0.2 pM, respectively) (Fig. 1A, Step 1). After autophosphorylation by ATP at the aspartic acid within the sequence DKTGS/T the enzyme occludes the 3 Na+ ions (Ei-P(3Na+) Fig. la, Step 2) and releases them into the extracellular space after attaining the E2-P 3Na+ conformation characterized by low affinity for Na+ (Kq5 = 14 mM) (Fig. la, Step 3). The following E2-P conformation binds 2 K+ ions with high affinity (KD approx. 0.1 mM Fig. la, Step 4). The binding of K+ to the enzyme induces a spontaneous dephosphorylation of the E2-P conformation and leads to the occlusion of 2 K+ ions (E2(2K+) Fig. la, Step 5). Intracellular ATP increases the extent of the release of K+ from the E2(2K+) conformation (Fig. la, Step 6) and thereby also the return of the E2(2K+) conformation to the EiATPNa conformation. The affinity ofthe E2(2K+) conformation for ATP, with a K0.5 value of 0.45 mM, is very low. 

Na+/K+-ATPase. Figure 2 Specific Inhibitors of Na+/K+-ATPase. (a) Endogenous cardiac glycosides identified in mammals. Substances with a 5-membered lactone at position C17 of the steroid moiety are referred to as cardenolides, those with a 6-membered lactone as bufadienolides. (b) Palytoxin (C P NsO ) produced by corals of the genus Palythoa. 

If 50% of Europeans with essential hypertension are affected by this disease because of an elevated secretion of endogenous ouabain, then there might be a chance to block its interaction at the cardiac glycoside binding site of Na+/K+-ATPase and thus lower blood pressure. This therapeutic approach seems to be successfiil. Recent studies provide evidence that the cardenolide analogue Rostafuroxin (PST 2238 Fig. 4) at very low concentrations can overcome the ouabain-induced tise of hypertension in experimental animals [6]. This compound has recently entered the phase I of clinical trials and is certainly a prototype of a new class of antihypertensive drugs. 

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