A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes

I. The Molecular Design of Life
13. Membrane Channels and Pumps
13.1. The Transport of Molecules Across a Membrane May Be Active or Passive
Figure 13.2. Free Energy and Transport. The free-energy change in transporting (A) an uncharged solute from a
compartment at concentration c 1 to one at c 2 and (B) a singly charged species across a membrane to the side having the
same charge as that of the transported ion. Note that the free-energy change imposed by a membrane potential of 59 mV
is equivalent to that imposed by a concentration ratio of 10 for a singly charged ion at 25°C.
I. The Molecular Design of Life
13. Membrane Channels and Pumps
13.2. A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across
Membranes
The extracellular fluid of animal cells has a salt concentration similar to that of sea water. However, cells must control
their intracellular salt concentrations to prevent unfavorable interactions with high concentrations of ions such as calcium
and to facilitate specific processes. For instance, most animal cells contain a high concentration of K+ and a low
concentration of Na+ relative to the external medium. These ionic gradients are generated by a specific transport system,
+
+
+
+
an enzyme that is called the Na -K pump or the Na -K ATPase. The hydrolysis of ATP by the pump provides the
energy needed for the active transport of Na+ out of the cell and K+ into the cell, generating the gradient. The pump is
called the Na+-K+ ATPase because the hydrolysis of ATP occurs only when Na+ and K+ are bound to the pump.
Moreover, this ATPase, like all such enzymes, requires Mg2+ (Section 9.4.2). The active transport of Na+ and K+ is of
great physiological significance. Indeed, more than a third of the ATP consumed by a resting animal is used to pump
these ions. The Na+-K+ gradient in animal cells controls cell volume, renders neurons and muscle cells electrically
excitable, and drives the active transport of sugars and amino acids.
The subsequent purification of other ion pumps has revealed a large family of evolutionarily related ion pumps
including proteins from bacteria, archaea, and all eukaryotes. These pumps are specific for an array of ions. Of
particular interest are the Ca2+ ATPase, the enzyme that transports Ca2+ out of the cytoplasm and into the sarcoplasmic
reticulum of muscle cells, and the gastric H+-K+ ATPase, the enzyme responsible for pumping sufficient protons into the
stomach to lower the pH below 1.0. These enzymes and the hundreds of known homologs, including the Na+-K+
ATPase, are referred to as P-type ATPases because they form a key phosphorylated intermediate. In the formation of this
intermediate, a phosphoryl group obtained from the hydrolysis of ATP is linked to the side chain of a specific conserved
aspartate residue in the ATPase (Figure 13.3).
13.2.1. The Sarcoplasmic Reticulum Ca2+ ATPase Is an Integral Membrane Protein
We will consider the structural and mechanistic features of these enzymes by examining the Ca2+ ATPase found in the
sarcoplasmic reticulum (SR Ca2+ ATPase) of muscle cells. This enzyme, which constitutes 80% of the sarcoplasmic
reticulum membrane protein, plays an important role in muscle contraction, which is triggered by an abrupt rise in the
cytosolic calcium level. Muscle relaxation depends on the rapid removal of Ca2+ from the cytosol into the sarcoplasmic
reticulum, a specialized compartment for calcium storage, by the SR Ca2+ ATPase. This pump maintains a Ca2+
concentration of approximately 0.1 µM in the cytosol compared with 1.5 mM in the sarcoplasmic reticulum.
The SR Ca2+ ATPase is a single 110-kd polypeptide with a transmembrane domain consisting of 10 α helices. A large
cytoplasmic head piece constitutes nearly half the molecular weight of the protein and consists of three distinct domains
(Figure 13.4). The three cytoplasmic domains of the SR Ca2+ ATPase have distinct functions. One domain (N) binds the
ATP nucleotide, another (P) accepts the phosphoryl group on its conserved aspartate residue, and the third (A) may serve
as an actuator for the N domain. The relation between these three domains changes significantly on ATP hydrolysis. The
crystal structure in the absence of ATP shows the likely nucleotide-binding site separated by more than 25 Å from the
phosphorylation site, suggesting that the N and P domains move toward one another during the catalytic cycle. This
closure is facilitated by ATP binding and by the binding of Ca2+ to the membrane-spanning helices.
The results of mechanistic studies of the SR Ca2+ ATPase and other P-type ATPases have revealed two common
features. First, as we have seen, each protein can be phosphorylated on a specific aspartate residue. For the SR Ca2+
ATPase, this reaction takes place at Asp 351 only in the presence of relatively high cytosolic concentrations of Ca2+.
Second, each pump can interconvert between at least two different conformations, denoted by E1 and E2. Thus, at least
four conformational states E1, E1-P, E2-P, and E2 participate in the transport process. From these four states, it is
possible to construct a plausible mechanism of action for these enzymes, although further studies are necessary to
confirm the mechanism and provide more details (Figure 13.5):
1. The postulated reaction cycle begins with the binding of ATP and two Ca2+ ions to the E1 state.
2. The enzyme cleaves ATP, transferring the γ-phosphoryl group to the key aspartate residue. Calcium must be bound to
the enzyme for the phosphorylation to take place. Phosphorylation shifts the conformational equilibrium of the ATPase
toward E2.
3. The transition from the E1 to the E2 state causes the ion-binding sites to "evert" so that the ions can dissociate only to
the luminal side of the membrane.
4. In the E2-P state, the enzyme has low affinity for the Ca2+ ions, so they are released.
5. With the release of Ca2+, the phosphoaspartate residue is hydrolyzed, and the phosphate group is released.
6. The enzyme, devoid of a covalently attached phosphoryl group, is not stable in the E2 form. It everts back to the E1
form, completing the cycle.
Essentially the same mechanism is employed by the Na+-K+ ATPase. The E1 state binds three Na+ ions and transports
them across the membrane and out of the cell as a result of the protein's phosphorylation and transition to the E2 state.
The three Na+ ions are released into the extracellular medium. The E2 state of this enzyme also binds ions
K+
K+
namely, two
ions. These
ions are carried across the membrane in the opposite direction by eversion driven by the hydrolysis of
the phosphoaspartate residue and are released into the cytosol.
The change in free energy accompanying the transport of Na+ and K+ can be calculated (Section 13.1.1). Suppose that
the concentration of Na+ outside and inside the cell is 143 and 14 mM, respectively, and that of K+ is 4 and 157 mM. At
a membrane potential of -50 mV and a temperature of 37 °C, the free-energy change in transporting 3 moles of Na+ out
of and 2 moles of K+ into the cell is +10.0 kcal (+41.8 kJ mol-1). The hydrolysis of a single ATP per transport cycle
provides sufficient free energy, about -12 kcal mol-1 (-50 kJ mol-1) under cellular conditions, to drive the uphill transport
of these ions.
13.2.2. P-Type ATPases Are Evolutionarily Conserved and Play a Wide Range of Roles
Analysis of the complete yeast genome revealed the presence of 16 proteins that clearly belong to the P-type
ATPase family. More detailed sequence analysis suggests that 2 of these proteins transport H+ ions, 2 transport Ca2
+, 3 transport Na+, and 2 transport metals such as Cu2+. In addition, 5 members of this family appear to participate in the
transport of phospholipids with amino acid head groups. These latter proteins assist in the maintenance of membrane
asymmetry by transporting lipids such as phosphatidyl serine from the outer to the inner leaflet of the bilayer membrane
(Figure 13.6). Such enzymes have been termed "flippases."
All members of this protein family employ the same fundamental mechanism. The free energy of ATP hydrolysis drives
membrane transport by effecting conformational changes associated with the addition and removal of a phosphoryl
group at an analogous aspartate site in each protein.
13.2.3. Digitalis Specifically Inhibits the Na+-K+ Pump by Blocking Its
Dephosphorylation
Certain steroids derived from plants are potent inhibitors (Ki 10 nM) of the Na+-K+ pump. Digitoxigenin and ouabain
are members of this class of inhibitors, which are known as cardiotonic steroids because of their strong effects on the
heart (Figure 13.7). These compounds inhibit the dephosphorylation of the E2-P form of the ATPase when applied on the
extracellular face of the membrane.
Digitalis, a mixture of cardiotonic steroids derived from the dried leaf of the foxglove plant (Digitalis purpurea),
is of great clinical significance. Digitalis increases the force of contraction of heart muscle, which makes it a
choice drug in the treatment of congestive heart failure. Inhibition of the Na+-K+ pump by digitalis leads to a higher
level of Na+ inside the cell. The diminished Na+ gradient results in slower extrusion of Ca2+ by the sodium calcium
exchanger (Section 13.4). The subsequent increase in the intracellular level of Ca2+ enhances the contractility of cardiac
muscle. It is interesting to note that digitalis was effectively used long before the discovery of the Na+-K+ ATPase. In
1785, William Withering, a British physician, heard tales of an elderly woman, known as "the old woman of
Shropshire," who cured people of "dropsy" (which today would be recognized as congestive heart failure) with an extract
of foxglove. Withering conducted the first scientific study of the effects of foxglove on congestive heart failure and
documented its effectiveness.
I. The Molecular Design of Life
13. Membrane Channels and Pumps
13.2. A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes
Figure 13.3. Phosphoaspartate. Phosphoaspartate (also referred to as β-aspartyl phosphate) is a key intermediate in the
reaction cycles of P-type ATPases.
I. The Molecular Design of Life
13. Membrane Channels and Pumps
13.2. A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes
Figure 13.4. Structure of SR CA2+ ATPase. This enzyme, the calcium pump of the sarcoplasmic reticulum, comprises
a membrane-spanning domain of 10 α helices and a cytoplasmic headpiece consisting of three domains (N, P, and
A). Two calcium ions (green) bind within the membrane-spanning region. The aspartate residue characteristic of
this protein family is indicated.
I. The Molecular Design of Life
13. Membrane Channels and Pumps
13.2. A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes
Figure 13.5. Mechanism of P-Type ATPase Action. The binding of Ca2+ and the phosphorylation of the ATPase (steps
1 and 2), illustrated here for the Ca2+ ATPase, lead to the eversion of the binding sites (step 3) and the release of Ca2+ to
the luminal side of the membrane (step 4). Hydrolysis of phosphoaspartate (step 5) and eversion (step 6) reset the
enzyme to its initial state.
I. The Molecular Design of Life
13. Membrane Channels and Pumps
13.2. A Family of Membrane Proteins Uses ATP Hydrolysis to Pump Ions Across Membranes
Figure 13.6. P-Type ATPases Can Transport Lipids. Flippases are enzymes that maintain membrane asymmetry by
"flipping" phospholipids (displayed with a red head group) from the outer to the inner layer of the membrane.