Calcium pump in living cells
Yu.A.Vladimirov
Abstract
Calcium transporting ATPase (Ca-ATPase) - is a comparatively small enzyme composed of a single polypeptide chain. It performs the active transport of calcium ions across the membranes of the cell and intracellular vesicles thereby allowing the cell to maintain low calcium concentration in the (10-7 M) in spite of comparatively high concentration (310-3 M) of these ions outside the cell and inside intracellular vesicular depots. Though the exact spatial structure of the enzyme has not yet been determined, the main steps of the enzyme functioning are revealed and now it is clear how the energy of ATP hydrolysis is being spend to transport Ca2+ from diluted to concentrated solutions of these ions.
Introduction
Calcium concentration in cell cytoplasm is only 50–100нМ (5.10-8–1.10-7М) whereas in the environmental medium it is about 3нМ (310-3М). This concentration difference (by four orders of magnitude!) is maintained by the system of active Ca2+ transport, the main role in which is played by a Ca-pump - the enzyme Ca-ATPase. To be more precise, this is not a single enzyme but a group of Ca-ATPases differing by their localization in cell, structure and way of regulation. Though all these enzymes transfer calcium ions from cell juice to extracellular liquid or to intracellular calcium depots - endoplasmic reticulum vesicles at the expense of ATP hydrolysis energy, thus maintaining a low Ca2+ concentration in cytoplasm.
The maintenance of low Ca2+ concentration in the cytoplasm of resting cells creates the possibility of regulation of cell functions by way of enhancement of membrane permeability for Ca2+: entering a cell, these ions activate a great variety of different intracellular processes. A brilliant example is muscle contraction that begins from Ca2+ coming out from sarcoplasmic reticulum (SR) and interaction with contraction proteins. Under the action of an electric impulse spreading in plasmalemma calcium ions leave SR vesicles and cause .... contraction. The removal of calcium by SR results in muscle relaxation. The following removal of calcium from cytoplasm and its accumulation in SR reservoirs is carried out by Ca-ATPase and results in muscle relaxation (See Fig.1).
/Fig.1. Calcium ions as regulators of the contraction-relaxation cycle in striped muscles:
TT - terminal tanks of SR, LT - lateral tubes, IS - internal synapse, TS - T-system, C -cytoplasmic membrane (plasmalemma), A - myosin filaments, I - actin, Z - Z-plate, M - M-stripe/band.In other cells, calcium ions entering passively via opening channels connected with different receptors also play the role of “messengers» giving orders to switch on this or that intracellular system. After executing the orders, the messengers are showed our from cytoplasm by Ca-ATPases and Na+-Ca2+ exchangers. Ca-ATPases of cytoplasmic and intracellular membranes differ by some properties. All Ca-ATPases represent monomeric proteins, i.e. they consist of a single polypeptide chain, but they are somewhat different in their molecular weight. Thus, SR ATPase has a molecular weight of 108kD and plasma ATPase - 120kD. Ca-ATPase of the SR of striped muscles is investigated best of all, and its structure and functioning will be considered in detail in this paper.
Extraction and purification of Ca-ATPase
Studies on the mechanism of Ca2+ transport on Ca-ATPase functioning was mainly performed in isolated SR vesicles obtained after tissue homogenization by consecutive centrifugations. SR vesicles under electron microscope look the same like other membrane structures. On the spallings of frozen suspensions of isolated SR vesicles one can see intramembrane particles with a diameter of 9 nm. These globular particles on the surface of the spalling are formed due to the intrusion of the parts of Ca-ATPase polypeptide chain into the hydrophobic zone. The analysis of the protein composition of the vesicles shows that Ca-ATPase is the basic protein in reticulum (70-80% of all proteins). Using different methods, it is possible to purify Ca-ATPase from other proteins, though it is true that during purification membrane is usually damaged, and thus it is impossibly to study the transport function. Though, adding phospholipids, it is possible to restore the integrity of vesicles and to obtain a fine object for investigation of Ca-ATPase function: phospholipid vesicles with built in working enzyme.
Energetics of Ca2+ transport
Using different methods, it was revealed that during the hydrolysis of one ATP molecule SR ATPase transfers two calcium ions from the environment into vesicles, as it is shown in Fig.2.
Fig.2. SR vesicle with built-in Ca-ATPase molecule (scheme)
The enzyme's head (D=9nm) is directed into the external medium (cytoplasm). It is bound with ATP and calcium ions. Membrane is pierced by a channel by which calcium, as it is assumed, is carried during ATP hydrolysis. For details see Fig.4.
Ca2+ transfer is accompanied by the transfer of electric charges but the difference of potentials across membrane does not remain since SR membrane is well permeable for other ions.
In order to transfer across the membrane 2 gram-equivalent of calcium ions from cell juice (where its concentration Ci=110-7Ì) to the interior cavity of SR (where calcium concentration is close to 1 mM (C0=110–3)) the following energy should be spent:
G = 2=2[o + RTln(Ci/Co) + zF](1)
Since inside SR the potential is equal to the intracellular one (=0), and the value is approximately the same for calcium ions in water solutions (=0), the changes of free energy on the transfer of two moles of Ca2+ at 37C (310К) can be considered equal to 47.5 kJ/mole according to the following equation:
G=2RTln(Ci/C0)=47,5kJ/mole(2)
It is equal approximately to the energy of hydrolysis of ATP macroergic bond/link at physiological concentrations of ATP, ADP and orthophosphate. Thus, Ca2+ transport across SR membrane is carried out with a high efficiency, without energy losses. Besides, this assumes the reversibility of Ca-ATPase functioning. Indeed, it was shown that the synthesis of ATP from ADP and phosphate can be obtained if isolated SR vesicles are loaded with calcium and then calcium is removed from the environment adding a complexon - a Ca2+-binding compound. It should be noted that other transport ATPases can also function reversibly, such as Na/K-ATPase of cytoplasmic membranes and H+ATPase of mitochondria.
Mechanism of Ca2+ transfer
Though transport ATPases are called ion pumps, they differ from water pumps by the fact that they do not pump liquid by transfer discrete particles - ions. Each transfer cycle includes at least three stages: (1) particle must be caught by one side of the membrane, (2) transferred through the membrane (translocated), and (3) released by the other side of the membrane. Realization of these stages is attended by energy consumption, thus something must happen to ATP. The ATP molecule itself must be captured (1) and hydrolyzed with energy storing and its consumption on calcium transfer (2), while products (ADP and phosphate) must go from the bound-to-enzyme state to water solution (3). In each cycle, the enzyme uses simultaneously two substrates instead of one - intracellular calcium and ATP, and creates three products: calcium accumulated inside the vesicles of SR, ADP and orthophosphate.
Fig.3. Sequence of stages of ATPase functioning
1 - binding of Ca2+, 2 - binding of ATP, 3 - formation of enzyme-phosphate, 4 - splitting of calcium ions, 5 - hydrolysis of enzyme-phosphate, 6 - return of enzyme in to initial state. For other explanations see text
Due to the efforts of many researches, the sequence of stages in Ca-ATPases functioning was generally interpreted (See Fig.3); it includes all stages mentioned above and besides the stages of ATP “processing» alternate with the stages of Ca2+ transfer. Here are these stages:
1. Binding of two calcium ions on the surface of ATPase turned to cytoplasm (or outwards in isolated SR vesicles).
2. Binding of ATP molecule on the same surface.
3. Protein phosphorylation (formation of phosphoenzyme) and release of ADP.
4. Release of calcium ions from the surface of ATPase turned inwards to SR vesicles.
5. Transition of the enzyme molecule to the initial state (calcium-binding sites again appear to be on the surface of SR vesicles).
These stages will be considered in detail in sections below.
Ca2+-binding (Stage 1)
In order to study quantitatively the ability of SR vesicles to bind ions, different amounts of ions under study are added to a vesicle suspension with a known ATP concentration (Cа), and concentrations of bound ion (Cb) and ion remained in the solution (Cf)are measured by this or that method. On the basis of the obtained data, two basic binding parameters are calculated - binding constant (Kb) and number of binding sites (n). Binding constant is the constant of the following equilibrium:
free ions + vacant binding sitesbound ions.
According to determination, binding constant is:
(3)
It is seen from the equation that the binding constant Kb is equal to the inverse concentration of free ions at such their concentration Cf when the concentration of bound ions Cb is equal to the concentration of free binding sites nf , i.e. when half of all binding sites on the enzyme surface is occupied. The higher binding constant (i.e. the affinity of sites to ion), the lower is ion concentration at which protein is still possible to bind them.
Studies on binding of different ions by SR membrane vesicles showed that only for Ca2+ there are sites with high binding constant (2106М-1); there are two such sites for one ATPase molecule.
Table 1. Constants of binding of Ca2+ and ATP with Ca-ATPase.
Using Eq. 2, it can be calculated what is the part of all Ca-binding sites will be occupied by ions at a concentration of free ions in solution of 110-7 M.
Since the total quantity of binding sites n=Cb+nf , then it is not difficult to find out a part of occupied binding sites, according to Eq. 3:
(4)
Putting values Cf and Kb to Eq.3, find:
(5)
Thus, every sixth binding site on the surfaces of ATPase molecules is occupied by calcium ions at such very low (100 nM) their concentration which is typical for intracellular contents.
At once the question certainly arises whether the binding of calcium is really the first stage of its transfer, or the transfer and adsorption of calcium are different and independent processes. Much data have been obtained testifying to the fact that it is the binding of calcium with high-affinity sites that is the first stage of transfer. One of the evidence consists in the same dependence of binding and transfer on calcium concentration. On the other hand, every reduction of binding under the action of competing ions simultaneously reduced calcium transfer through membranes in each case. It should be noted that the presence of magnesium does not influence much the binding of calcium by ATPase since the affinity for Ca2+ of binding sites is 30000 times as much as that for Mg2+.
Binding of ATP (Stage 2)
Studies on the binding of ATP with calcium ATPase built in to membrane vesicles showed that ATP binds in a complex with Mg2+ (or Mn2+), the binding of Mg•АТФ complex occurring irrespective of Са2+. This means that there are two different sites for Mg•АТФ and for Са2+. The Mg•АТФ binding constant is 2105М-1, i.e. the affinity of active enzyme center for substrate is quite high - half ATP molecules bind at its concentration of 5 mcM. Besides one binding site possessing high affinity for ATP there is one more binding site, with low affinity, on the surface of ATPase molecule which takes no part in the process of ATP hydrolysis and calcium transfer, though it is probably important for regulation of the enzyme activity.
ATP hydrolysis
Thus, on the surface of ATPase there are binding sites for two calcium ions and one ATP molecule possessing high affinity for substrate. They interact (between each other) since Ca2+ binding «launches» the hydrolysis of ATP attached, together with Mg2+, to its site. It was also shown that ATP hydrolysis begins only after both calcium ions attach to their binding sites. This corresponds to the stoichiometry of Ca2+ transfer and ATP hydrolysis equal to 2 about which it has been already said above.
Protein phosphorylation (stage 3)
ATP hydrolysis is realized by Ca-ATPase in three stages: (1) ATP binding, (2) protein phosphorylation and splitting out of ADP, and (3) hydrolytic splitting of rotein-phosphate bond and release of orthophosphate. Phosphorylation is realized on the carboxyl group of asparagine acid residue. This stage is reversible - adding ADP in the presence of 1 mM Ca2+ to isolated endoplasmic reticulum vesicles containing enzyme-phosphate (Е~Р) one can observe almost complete transfer of phosphate from protein on to ADP with formation of ATP. Thus, in SR membranes there exists the following equilibrium
,
where K0 - constant of binding of calcium ions on ATPase at the outer surface of SR membrane vesicles, Kрh - constant of equilibrium of phosphorylation reaction. The latter value is close to 1.
As it is known, a large amount of energy is released during ATP hydrolysis due to which the linkage between phosphate and ADP in an ATP molecule is called macroergic (rich in energy). The reversibility of the process of protein phosphorylation means that the linkage of phosphate with asparagine residue in phosphorylated protein (designated as «~» in Fig.3) is also rich in energy which is released during its hydrolysis. It will be seen below that it is this energy which is spent on the active transfer of calcium ions.
Hydrolysis of enzyme-phosphate complex (stage 4)
A high-energetic (i.e. able to transfer phosphoric acid residue on too ADP) form of phosphorylated ATPase is stable only in the presence of millimolar (i.e. comparably high) Ca2+ concentrations. At lower Ca2+ concentrations, transfer of Ca2+ from Ca-binding sites of phosphoenzyme by Mg2+ (that are present in the medium and without which Ca-ATPase does not function) takes place; calcium ions pass to the surrounding solution during this process.
This stage of ATPase functioning (hydrolysis of EP) is the most important in the cycle of calcium transfer and is worth considering in more detail.
As it was discovered, the displacement of Ca2+ from Ca-binding sites of high-energetic phosphoderivative protein by Mg2+ occurs in two stages: (1) calcium outsplitting, and (2) hydrolysis of phosphate bond (outsplitting of inorganic phosphate):
(E~P)Ca2 + 2Mg2+ (E~P)Mg2 + 2Ca2+E + Pi
Calcium and magnesium complexes of enzyme-phosphate differ in principle by their capability of reacting with ADP to form ATP. As it has already been mentioned, the calcium-enzyme-phosphate complex (existing at high calcium concentrations) can transform into the initial Ca-ATPase with ATP synthesis (the so-called ionic phosphorylation). This is why it is said that phosphate is linked with protein by macroergic linkage, and the complex is designated as E ~ P. The complex of enzyme-phosphate with magnesium turned to possess no capability to transfer phosphate on to ADP: no enough energy is released at hydrolylic outsplitting of phosphate, the linkage of phosphate with protein is not macroergic (the complex is thus designated as EP).
What was the energy of phosphate bond spent on? It turned out to have been spent on the change of the constant of calcium binding with ATPase. Studies on the influence of Ca2+ on decay and formation of phosphoenzyme showed that on phosphorylation Ca2+-binding constant reduces more than 1000 times s much, i.e. it becomes < 2103М–1. In a 0,5 mM Ca2+ solution half of all earlier bound calcium ions leave ATPase after protein phosphorylation and ions pass to the solution. At a 1mM calcium concentration in the medium, two thirds of ions are in a bound state, others pass to the solution (See Eq.3). The existence of magnesium ions in medium additionally reduces the amount of bound magnesium since the difference in affinity for Ca2+ and Mg2+ of phosphorylated ATPase is not very large.
Before phosphorylation, ATPase bound calcium at low concentrations (10-7M) and after phosphorylation only at high ones (10-3M). The energy of ATP was spent on ATPase's «pushing out» to a concentrated solution of calcium ions which the enzyme «caught out» of their diluted solution. Moreover, ATPase binds Ca2+ at one membrane side and splits out at the other side.
Calcium transfer through membrane (translocation)
Ca-ATPase molecules in SR vesicles are oriented in a strictly definite way so that binding of Ca2+ and ATP takes place on the outer surface of vesicles, and calcium release on the inner one. Studies on Ca2+ binding at different stages of Ca-ATPase functioning showed that in non-phosphorylated state Ca-binding sites of ATPase are accessible for Ca2+ only at the outer surface of vesicles and are not on the inner surface. After the enzyme's phosphorylation, Ca-binding sites become accessible on the inner surface and inaccessible on the outer one. Thus, phosphorylation leads to transfer of Ca-binding sites across the membrane (translocaton). Since ion transfer is carried out by a protein molecule, it is obvious that some of its parts must be transferred or, as it is said, changes in the conformation of the protein molecule must take lace. At the same time, as it has already been said, the affinity of binding sites for Ca2+ suffer changes. Mechanics become integrated with energetics.
Final stages of the cycle - hydrolysis of phosphoenzyme (stages 5 and 6)
The magnesium complex of enzyme-phosphate is quickly hydrolyzed and the enzyme acquires its initial properties. At the same time, Ca-binding sites with high affinity appear on the outer surface of the enzyme again. It is obvious that the EP hydrolysis (stage 5 in Fig.3) results in (1) removal of magnesium from binding sites, and (2) their reverse translocation (stage 6). On the outer enzyme surface, calcium binding sites again acquire a high affinity for these ions. Thus, EP dephosphorylation results in space translocations of part oof protein molecule and in changes in ion-binding energy in the same way as ATPase phosphorylation though in the opposite/reverse direction. The cycle of the enzyme work is thus closed (see Fig.3).
Direct evidence of enzyme's «stirring/moving» when working
Though the idea about changes in protein conformation during/due to enzyme working was suggested long ago, direct evidence were obtained only for few known enzymatic reactions. In this respect, transport ATPases are very good objects since these mechanochemical machines transfer ions from one side/surface of the membrane on to the other. It is clear that such actions are associated with displacement in space of definite protein molecule sections. Changes in Ca-ATPase conformation during the enzyme's work were shown by the methods of differential spectrophotometry, intrinsic protein fluorescence, and spin probes. Nevertheless, it is more interesting to know what are the stages of enzyme's work on which the most significant changes in protein conformation occur. During the experiments carried out in our laboratory in collaboration with V.B.Ritov by the method of spin probes we discovered a great enhancement of the mobility of the protein section containing SH-groups during Mg•АТФ addition to the enzyme. (Spin probe is a chemical group that contains a stable free radical iminoxyl (>NO) whose EPR signal depends on the physical properties of the medium that surrounds iminoxyl (>NO) group. If the mobility of the polypeptide chain to which iminoxyl radical is added/attached enhances, at the same time the EPR signal changes correspondigly). We also managed to show changes in the enzyme's conformation (the mobility of spin probe added to SH-group) during the formation as well as splitting of phosphoenzyme. Thus, the mechanical shifts of enzyme sections, which had been predicted on the basis of the studies on individual stages of ATP working (Fig.3), were confirmed by direct experiments.
Physical state of lipids and Ca-ATPase working