Biological Membranes - A. N. Ogurtsov 2012

Structure and Functions of Biomembranes
Active Transport
Mechanism of Action of Ca2+-ATPase

Let us examine the Basic principles of ion pumps using the example of the sarcoplasmic reticulum (SR) Ca²⁺-ATPase from Muscle Cells (SR Ca²⁺-ATPase) (Figure 64). This enzyme constitutes 80% of the Proteins in the sarcoplasmic reticulum and plays a pivotal role in muscle Cell contraction, which is stimulated by an increase in cytosolic calcium ion concentration.

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Figure 64 - Structure OF THE SR Ca²⁺-ATPase

Muscle relaxation occurs As a result of a decrease in cytosolic calcium concentration and its transport back into the sarcoplasmic reticulum (a specialized compartment of the smooth Endoplasmic reticulum for calcium storage), a process carried out by the SR Ca²⁺-ATPase.

The concentration of Ca²⁺ ions in the Cytosol of muscle cells ranges from 10^-7 M (in the relaxed state) to over 10^-6 M (during contraction), whereas within the sarcoplasmic reticulum, the Ca²⁺ concentration can reach 10^-2 M.

The Ca²⁺-ATPase is a 110 kDa polypeptide whose transmembrane domain consists of ten a-helices. (1 Dalton (Da) = 1 a.m.u. = 1.66·10^-24 g). The cytoplasmic portion of the SR Ca²⁺-ATPase, which accounts for approximately half of the protein's molecular mass, consists of three domains: A, N, and P (Figure 64).

The three cytoplasmic domains of the SR Ca²⁺-ATPase perform distinct Functions. The nucleotide-binding domain N binds ATP, the phosphorylation domain P accepts a phosphate group onto the aspartate residue Asp 351, and the actuator (regulatory) domain A activates the N domain.

Ten transmembrane a-helices form a channel for the passage of Ca²⁺ across the membrane. Two of these helices extend only halfway across the membrane, and the regions where the protein strand transitions into a-helices serve as the calcium ion binding sites.

The spatial orientation of domain A regulates the affinity of Ca²⁺ ions for these binding sites, thereby controlling the subsequent release of Calcium Ions from the cytosol to The Cell exterior or into the sarcoplasmic reticulum.

In the initial state, the distance between the phosphorylation site and the Ca²⁺ binding sites is relatively large. During a single transport cycle, domain N tilts by 20° to the left (Figure 61), bringing the ATP-binding center into proximity with the aspartate residue Asp 351 (aspartic acid), while domain A rotates by 90° about the normal to the membrane.

These Conformational Changes in the molecule lead to the translocation of the Ca²⁺-binding sites, first to one side of the membrane and then to the other, thereby altering the affinity of these sites for Ca²⁺ ions from high on the cytoplasmic side of the membrane to low on the sarcoplasmic side.

Common to the operation of all pumps are:

1) the phosphorylation of a specific aspartate residue—Asp 351 in the case of the SR Ca²⁺-ATPase;

2) the existence of at least two distinct Conformations, which we designate as E₁ and E₂.

Taking phosphorylation into account, there are consequently at least four conformational states—E₁, E₁-P, E₂-P, and E₂—on The basis of which a General scheme of pump operation can be constructed (Figure 65). The reaction cycle of the pump consists of six stages.

Figure 65 - MECHANISM OF ACTION of the SR Ca²⁺-ATPase: binding of Ca²⁺ (1) and phosphorylation of the ATPase (2) lead to the translocation (3) of the binding sites from the cytosolic to the luminal side of the membrane and the release of Ca²⁺ (4); Hydrolysis of phosphoaspartate (5) and a conformational transition that returns the binding sites back to the inner side of the membrane (6) restore the ATPase to its initial state

1. The cycle begins with the binding of ATP and two Ca²⁺ ions to the E₁ conformation of the ATPase.

2. The ATPase transfers a phosphate group to the target aspartate. Calcium must already be bound by the enzyme for phosphorylation to proceed. Phosphorylation shifts the conformational equilibrium of the ATPase toward the E₂ conformation.

3. The transition from the E₁ to the E₂ conformation causes the ion-binding sites to "flip" across the membrane such that calcium ion dissociation occurs on the extracellular (or luminal) side of the membrane.

4. In the E₂ conformation, the ATPase exhibits a low affinity for Ca²⁺ ions, resulting in their release.

5. The release of Ca²⁺ ions stimulates the hydrolysis of phosphoaspartate (dephosphorylation) and the dissociation of the phosphate group.

6. Deprived of its covalently bound phosphate group, the ATPase is conformationally unstable in the E₂ state. It "flips back" into the E₁ conformation, completing the reaction cycle.

All P-type ion pumps, regardless of the specific ions they Transport Across the membrane, share a similar architecture. In all such pumps, aspartate phosphorylation occurs, and the transmembrane a-domains of all P-type pumps have approximately the same molecular weight and a conserved structural design composed of a-helices. All of this indicates that all P-type pumps share a common evolutionary ancestor, although over time these pumps have adapted to transport various ions.

Changes in cytosolic Ca²⁺ concentration play a critical role in cellular signaling mechanisms. To respond rapidly to a signal—which intracellular systems detect via a sharp surge in cytosolic calcium concentration—it is necessary to continuously maintain a low cytosolic Ca²⁺ concentration (below 0.1–0.2 µM) in the intervals between signals.

Ca²⁺ ions are removed from the cytosol by Plasma Membrane Ca²⁺-ATPases, The structure of which is similar to the SR Ca²⁺-ATPase. The activity of plasma membrane Ca²⁺-ATPases is regulated by calcium-binding cytosolic proteins called calmodulins.

At high cytosolic calcium concentrations, calmodulins undergo a conformational change upon binding to calcium ions and "wrap around" the Ca²⁺-ATPases. This, in turn, induces an allosteric activation of the ATPases, causing the pumps to rapidly extrude the ions from the cytosol.