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Transport of Ions: Mechanisms and Models

21. Transport of Ions: Mechanisms and Models
21. Transport of Ions:
Mechanisms and Models
I.  Coupling Between ATP Hydrolysis and Transport
II.  Synthesis of ATP by Transport ATPases
III.  Models of Ion Transport and Structure
Suggested Reading
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Examining simple models and possible alternatives sometimes can provide insights into biological
processes. This approach has proved very useful in sorting out the data on ion transport and their possible
interpretation, and it provides the perspective of this chapter.
Section I examines data obtained in a study of Na+, K+-ATPase and, for discussion, uses the model
represented in Fig. 1 based on the experiments presented in Chapter 20. This model undoubtedly will
require extensive modification and elaboration, but it is a useful summary. Very similar data are
available from studies of the Ca2+-ATPase and a similar model could also be drawn for the transport of
Ca2+. Section II examines some of the characteristics of the phosphorylation of ADP by inorganic
phosphate, catalyzed by transport ATPases in the absence of ionic gradients. These phenomena may
reveal some new features of the ATPases and perhaps have some bearing on our understanding of the
synthesis of ATP by the ATP synthase of mitochondria, chloroplasts and bacteria. Section III
concentrates on possible molecular mechanisms of ion transport and discusses the information gained
from knowledge of the amino acid sequences and the reconstruction of the structure of the Ca2+-ATPase.
The evidence reviewed in Chapter 20 unmistakably demonstrates that the active efflux of Na+ and the
influx of K+ are coupled to the hydrolysis of ATP. As suggested in step 1 of Fig. 1, the coupling between
the translocation of the ions and the hydrolysis of ATP may result from the required phosphorylation of
the transporter molecule. The incubation of membrane preparations with ATP labeled with [32P] in its
terminal position, labels the membranes. The phosphate, and not the whole ATP molecule, is
incorporated since [14C]ATP does not label the membranes. Table 1 (Post et al., 1965) summarizes the
incorporation of [32P] into kidney plasma membranes as a function of the cation present in the medium.
When Na+ is present, the incorporation is highest, 97 pmoles/mg protein, compared to the incorporation
in its absence (between 14 and 29 pmoles). Even in the presence of Na+, the incorporation may not seem
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21. Transport of Ions: Mechanisms and Models
very large. This is because the Na+,K+-ATPase is a minor component of the cell membrane (see below).
Much higher values can be obtained for membrane fragments containing the Ca2+-ATPase, which
represents a very large proportion of the total protein of the sarcoplasmic reticulum. Actually, in both
cases the amount of [32P] incorporated corresponds to one per ATPase molecule. In step 1 of the model
of Fig. 1, the phosphorylation of X produces Y~P. Different letters, X and Y, are used to denote the two
forms because they have very different properties. Y is able to bind Na+ (step 2) and transfer it to the
external membrane interface (step 3), from which it is released (step 4). X-P is generated from Y~P (step
5) and it binds K+ (step 6), transfers it to the internal membrane interface (step 7), and releases it to the
cell’s interior (step 8) with hydrolysis of X-P.
Fig. 1 Early model of the functioning of the Na+,K+-ATPase. The step corresponds to the following: step
1, the phosphorylation of the ATPase indicated as Y; step 2, the binding of Na+ to Y~P; step 3, the
movement of the binding group from the cytoplasmic side of the membrane to the outside; step 4, the
release of Na+; step 5, the hydrolysis of Y~P to form a different form of Y, X; step 6, the binding of K+;
step 7, its displacement to the cytoplasmic side of the membrane; and step 8, its release into the
Table 1 Effect of Monovalent Cations on Labeling of Kidney Membranes After Incubation with
Mg2+ and [32P]ATP
Addition Labelling
(pmol 32P/mg protein)
None 26
Li+ 20
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21. Transport of Ions: Mechanisms and Models
Na+ 97
K+ 16
NH4+ 14
Rb+ 18
Cs+ 14
Tris+ 19
Reproduced with permission from R.L. Post et al., J. Biol. Chem. 240:1437-1445. Copyright ©1965 The American Society
for Biochemistry and Molecular Biology.
This scheme suggests that the formation of Y~P requires the presence of Na+, as shown by the results in
Table 1. Other univalent cations cannot substitute. Fig. 2 shows the dependence of the phosphorylation
expressed as % of the maximum (ordinate) on the concentration of Na+ (abscissa). The phosphorylation
is related to transport, as shown by the inhibition of a large portion of the Na+-dependent
phosphorylation by ouabain (Post et al., 1965) which blocks the transport of Na+ and K+. In Chapter 20
we saw that ouabain is an inhibitor of the Na+,K+-ATPase. The scheme also predicts that K+ would favor
the hydrolysis of Y~P, as shown by the experiment represented in Fig. 3 (Post et al., 1965). In this
experiment the membranes were first labeled with radioactive [32P]ATP. Then after the addition of
unlabelled ATP, they were incubated in the presence of K+. Although the [32P] is released even in the
absence of K+, the release is sharply accelerated when K+ is present. The rates of phosphorylation and
dephosphorylation are comparable to those of the ATPase activity (e.g., Kyte, 1974) which, in turn,
correspond very closely to the moles of ions being transported, as shown in Table 6 of Chapter 20.
The nature of the phosphorylated ATPase has also been examined in relation to its sensitivity to ADP.
The increased hydrolysis favored by K+ also appears in the results of the experiment of Fig. 4 (curve 1),
carried out with the same protocol as in Fig. 3, but with the addition of K+ or ATP after a 5 min
incubation (Post et al., 1965). In this experiment, K+ decreases the radioactivity as expected (curve 1),
whereas the addition of ADP (curve 2) has no effect, suggesting that the phosphorylated ATPase is no
longer in a high-energy form. In its high energy form, the phosphorylation of the enzyme would be
The results are different when the ATPase has first been treated with N-ethylmaleimide (NEM), which
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21. Transport of Ions: Mechanisms and Models
reacts with sulfhydryl groups. Treatment of the ATPase with NEM, blocks the ATPase activity but not
the phosphorylation. As shown in Fig. 5 (Post et al., 1965), the NEM-treated ATPase is not sensitive to
K+ but is sensitive to ADP. These results suggest that the ATPase may be present in two distinct forms: a
form with a high and another with a low phosphate group transfer potential, the latter corresponding to a
K+-sensitive form. NEM blocks the conversion of the high energy form to the low-energy form. This
scheme is consistent with the following reactions:
Nai+ + E1 + ATP ↔ E1~P.Na+ (1)
E1~P.Na+↔ E2-P + Nao+ (2)
E2-P + Ko+ ↔ E2-P.K+ (3)
E2-P.K+↔ E2 + K+i + Pi (4)
where E represents the transporter molecule. The subscripts are used to distinguish the various molecular
configurations of the enzyme; E1 and E2 correspond to the Y and X of Fig. 1, respectively.
Fig. 2 Effect of ouabain on the sensitivity of the [32P]-labeled intermediate to the concentration of sodium
ion. The concentration of ouabain was 2.5 x 10-4 M, and that of Mg-ATP was 0.1 mM. Incubation was for
12 s at 23C. The results are the average of two experiments. Reproduced with permission from R. L. Post,
et al., Journal of Biological Chemistry, 240:1437-1445. Copyright ©1965 The American Society for
Biochemistry and Molecular Biology.
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21. Transport of Ions: Mechanisms and Models
Fig. 3 Influence of K+ on the rate of breakdown of the [32P]-labeled intermediate. Kidney membranes
were stirred with 0.04 mM Mg-ATP labeled with [32P] for 2 min at 8.5C in the presence of 16 mM Na+ in
a volume of 1.0 ml. ( ) K+ absent; ( ) K+ present at 0.04 mM. Then 0.1 ml of 20 mM unlabeled (Tris)
ATP was added to reduce the specific activity of the labeled ATP to 2% of its initial value. After the time
intervals on the horizontal axis the reaction was stopped with acid. The solid line indicates exponential
disappearance with a time constant of 21 s. The dashed line is similar, with a time constant of 4 s.
Reproduced with permission from R. L. Post, et al., Journal of Biological Chemistry, 240:1437-1445.
Copyright ©1965 The American Society for Biochemistry and Molecular Biology.
The estimates of size of the molecule, together with estimates of the turnover number of the transport
ATPase [i.e., moles of product x (moles of enzyme x minutes)-1], permit a number of interesting
approximations. The turnover number was calculated to be about 12,000, based on the phosphate
hydrolyzed. 1 mmol of Pi per hour is hydrolyzed from the ATP by 1 liter of cells. If we assume that there
are 1.1 x 1013 cells per liter, there must be 1.3 x 10-22 moles of enzyme per cell. Multiplied by
Avogadro’s number (the number of molecules in one mole) this value corresponds to about 80 transporter
molecules per cell. Assuming that the volume of each transporter molecule is 3.2 x 10-19 cm3, the total
volume of transporter per cell is 80 X (3.2 x 10-19) = 2.6 x 10-17 cm3. The red blood cell surface area is
about 1.55 X l0-6 cm2 and its thickness is approximately 5 nm, therefore, the volume of the membrane is
about 0.78 x 10-12 cm3. Thus, the transport ATPase occupies about 0.0003% of the membrane volume,
an extremely tiny portion of the cell membrane. In the red cell ghost, the Na+,K+-ATPase has been found
by a cytochemical electron microscopic method (Charnock et al., 1972) to be distributed evenly over the
membrane surface. However, other membranes are quite different. The Ca2+-ATPase is the major protein
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21. Transport of Ions: Mechanisms and Models
present in the sarcoplasmic reticulum. In addition, the distribution may not be even, the Na+, K+-ATPase
of polar cells such as epithelial cells, is present only on one surface, the apical surface.
Fig. 4 Sensitivity of the phosphorylated intermediate of the native enzyme to ADP and K+. The ATPase
was labeled with [32P]ATP. At zero time the radioactivity of the ATP was chased using a 100-fold excess
of unlabeled ATP. From Post et al. (1969). Reproduced from The Journal of General Physiology, by
copyright © permission of the Rockefeller University Press.
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Fig. 5 Sensitivity of the phosphorylated intermediate of the Na+,K+-ATPase to ADP and K+ after
treatment with N-ethylmaleimide. From Post et al. Reproduced from The Journal of General Physiology,
by copyright permission ©1969 of the Rockefeller University Press.
As we saw in Chapter 10, when ion pumps are run in reverse, ATP can be synthesized from ADP and Pi.
These findings have certain implications related to the model of Fig. 1. ATP can be synthesized only if
the phosphorylated form of Y(Y~P) is a high-energy form (high phosphate group transfer potential); i.e.,
the G for its hydrolysis is sufficiently low to support the synthesis of ATP from ADP. However, as
described above, the evidence indicates that the usual phosphorylated form of the ATPase is hydrolyzed
with the addition of K+, but not ADP. As already noted, a possible explanation is that there are two
phosphorylated forms of the transporter molecule: a high-energy form involved in the transport of Na+
and a low-energy form that interacts with K+ (X-P). Furthermore, since X-P is a low-energy form, it
should be possible to phosphorylate the molecule with Pi in the absence of Na+, and this was found to be
the case (Post et al., 1965; Schoot et al., 1977; Sen et al., 1969). In the formulation of Fig. 1, Y~P would
then be the precursor of X-P. The Na+,K+-ATPase is present in two forms, E1 and E2, which differ in
conformation, as shown by a variety of techniques means such as exposure of regions of the molecule at
the membrane surface to tryptic digestion (see below). The Y~P and X-P would then correspond,
respectively, to the phosphorylated forms of E1 and E2 (Jorgensen and Petersen, 1979). Digestion of the
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21. Transport of Ions: Mechanisms and Models
enzyme phosphorylated with either ATP or Pi, produces identical electrophoretic patterns (Bontig et al.,
1979; Siegel et al., 1969).
However, it is not necessary to have an ion gradient to synthesize ATP in the case of either the Na+,K+-ATPase (Post et al., 1974) or the Ca2+-ATPase (Knowles and Racker, 1975). The in vitro synthesis is
carried out in two steps. First, the ATPase is phosphorylated; we saw that this can be done in the case of
the Na+,K+-ATPase by incubation with Pi. Then ATP is synthesized when ADP is added in the presence
of a high concentration of Na+. Obviously, this proceeds only for a single turnover.
The sequence of events perhaps can be understood best by examining the reactions in some detail. If K+
is ignored, the reactions would be as shown in Eqs. (5) to (7):
E2 + Pi↔ E2-P (5)
E2-P + Na+ ↔ E1P.Na+ (6)
E1P.Na+ + ADP ↔ E1 + ATP + Na+ (7)
These reactions represent the reverse of the normal sequence of active transport. The passage from Eq.
(5) to Eq. (7) would be highly improbable unless the Na+ concentration was raised sufficiently, which is
predictable from the law of mass action. However, as discussed more fully in Section III, the
phosphorylation of the ATPase by ATP, presumably reaction (7) run from right to left, decreases the
binding constant of the cation — and the effect is reversible. When the binding of one component (e.g.,
the phosphorylation) to a protein capable of undergoing conformational change affects the binding of
another (e.g., the cation), the inverse will be true. The nature of the enzyme-phosphate bond will thereby
be affected by the binding of the cation (Weber, 1972, Weber, 1974). Presumably, the binding of Na+
would then convert the low-energy bond into a high-energy bond.
The possibility of obtaining ATP from the reverse of ion transport can be explained by the considerations
discussed in this section. The high Na+ present on the outside of the cell will permit the formation of the
high-energy phosphate [reaction of Eq. (6)]. The phosphorylation of ADP removes the phosphate and the
enzyme can be used again for another cycle of phosphorylation.
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21. Transport of Ions: Mechanisms and Models
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The simplest model for transport of an ion would include the following steps: (1) binding of the ion by
specific binding groups on the transporter molecule at the loading site; (2) movement of the complex
from one interface to the other; and, (3) release of the ion at the discharge site of the membrane. This
model ignores a membrane potential to avoid complications unnecessary for our present discussion. The
Ca2+ pump of the sarcoplasmic reticulum imports 2 Ca2+ and exports 1 H+per ATP hydrolyzed; the
Na+/K+ pump exports from the cell 3 Na+ and simultaneously imports 2 K+ per ATP hydrolyzed.
The model would carry out the net transport of the ion. Active transport, i.e., transport against an
electrochemical gradient, could take place in this same model when two other conditions are met: (1) the
affinity of the binding group changes from high at the loading interface to low at the discharge interface
and (2) the free energy of the sequence of reactions decreases. In a transport ATPase the energy is
provided by the coupled hydrolysis of ATP.
Models capable of carrying active transport can be constructed without postulating a change in binding
constants. However, all transport systems known have been shown to have this feature (see Table 2). For
simplicity, in the present discussion we assume that the transport of all ions occurs by the same basic
process. This approach is not unreasonable because, as we saw in Chapter 20, there is considerable
evidence that the transport functions are analogous for the Na+,K+-ATPase, Ca2+-ATPase, the H+,K+-ATPase and the H+-ATPase of plants and Neurospora. Furthermore, the properties of these molecules
are very similar.
A mechanism of active transport, involving the phosphorylation of the transporter and changes in binding
constants, is supported by a variety of observations. The experiments discussed here (Ikemoto, 1976)
were carried out with a stop-flow apparatus (Fig. 6), which delivers reactants and enzyme (from syringes
shown at A) into the same chamber (B) with very rapid mixing in relation to the time course of the
reaction. Then the flow is stopped, also very rapidly. The light absorption of the contents of the chamber
can be recorded (E). An oscillosope (D) is required to record very rapid reactions. These experiments
used a purified preparation of Ca2+-ATPase from the sarcoplasmic reticulum and the Ca2+ indicator
Arsenazo III, which changes color when it binds Ca2+. The record of Fig. 7 represents the light
absorption with time. The two sets of panels differ in the time scale: set I shows fast changes (intervals
correspond to 50 ms) and set II shows slower changes (intervals represent 5 s). The downward
deflections reflect increases in the concentration of Ca2+. The concentration of ATP added is shown at
the left in the records. In the control (IA and IA), no ATP was added and no Ca2+ was released. The Ca2+
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released increases with the concentration of ATP added (compare B and D) until the system appears
saturated (compare D and E), as would be expected because the amount of ATPase is finite. As shown by
the longer time scale in set II, the release is temporary; eventually the Ca2+ is bound again, presumably
when all the ATP is hydrolyzed. The results show that the ATPase binds Ca2+ and that activation by ATP
reversibly decreases the binding. Fig. 8 shows the level of phosphorylation of the enzyme (determined
after rapid filtration, curve 1) compared to the Ca2+ release (curve 2) calculated from Fig. 7. The two
panels represent identical results plotted on two different time scales. The changes in phosphorylation of
the ATPase precede the release of the Ca2+, suggesting that phosphorylation is responsible for the change
in binding constants. These results indicate the Ca2+ is bound more tightly (larger binding constant)
before activation. Similar data are available for other transport systems, such as Na+,K+-ATPase (Masui
and Homareda, 1982; Yamaguchi and Tonomura, 1980). The binding constants on the two sides of the
membrane for different transport systems are shown in Table 2 (Tanford, 1983).

Fig. 6 Stop-flow apparatus.
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Fig. 7 ATPase-coupled changes in Ca2+ binding to purified Ca2+-ATPase of the sarcoplasmic reticulum.
From Ikemoto (1976), with permission.
The transport process seems to involve precise stoichiometry. As mentioned, 2 Ca2+ are transported per
ATP hydrolyzed. Furthermore, 2 Ca2+ are bound per phosphorylated transporter molecule (Inesi et al.,
1980). In the case of active transport, the affinity of the binding groups of the transporter for the ligand,
decreases when the transporter molecule is phosphorylated and this lower affinity should represent the
state of the transporter on the side with the higher concentration at steady state. A model of active
transport involving ion binding sites and shuttling of ions across the plasma membrane is consistent with
the data.
However, these considerations do not resolve how the binding sites can move from one interface to the
other without a major movement of the transporter. Integral proteins have distinct domains corresponding
to the two different membrane surfaces. It follows that the transporter molecule does not flip or rotate.
Furthermore, the Na+,K+-ATPase continues to function even when anchored at one interface with an
antibody (Kyte, 1974). These difficulties could be resolved by proposing that the binding sites do not
traverse the whole membrane thickness, but rather move over much shorter distances. This would be
possible if the binding sites were inside a channel traversing the membrane.
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Fig. 8 Relationship between Ca2+ release and rebinding and the formation and decay of the
phosphorylated intermediate. ( ) Ca2+ release; ( ) P in enzyme. Reproduced with permission from N.
Ikemoto, Journal of Biological Chemistry, 251:7275-7277. Copyright &copy1976 The American Society
of Biological Chemistry and Molecular Biology.
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Table 2 Binding Constants for Transported Ions
Binding constant, Keq(M-1)
Protein  Ion  Uptake side  Discharge side
SR Ca2+ pump Ca2+  107-108  300
Na+ pump Na+  4 X 103  <20
Chloroplast FoF1 H+  >108  <106
Na+/ Ca2+ exchange Ca2+  2 x 105-106  400
Tanford (1983), reproduced with permission from the Annual Review of Biochemistry Vol. 52, copyright &copy1983 by
Annual Reviews Inc.
There are, in fact, many indications that the transporters can act as channels, at least when reconstituted
in artificial membrane systems. Addition of purified Ca2+-ATPase to a bilayer (in this case a bilayer
made of oxidized cholesterol) changes its conductivity (Shamoo and MacLennan, 1975). Under some
conditions, the Na+, K+-ATPase incorporated into planar bilayers of phospholipid shows electrical
conductance transitions typical of channels (Last et al., 1983). The anion transporter (Giebel and Passow,
1960) also behaves like a channel. In this case, the selectivity of the transport system appears to depend
on the size of the molecule, suggesting a channel 0.8 to 0.9 nm in diameter. The channel behavior is
related to the transport process of the native systems and not some irrelevant coincidence. This is shown
by the sensitivity of the channel behavior to inhibitors of transport. HgCl2 inhibits both the Ca2+-ATPase
activity and the Ca2+ conductivity in parallel. Ouabain and vanadate, both inhibitors of the Na+/K+
transport, inhibit the Na+, K+-ATPase channel behavior.
Evidence of conformational rearrangements comes from many kinds of experiments. The sensitivity of
the transporter molecule to proteolytic digestion differs at different stages of transport (e.g., see Chapter
4). Gresalfi and Wallace (1984) have examined the circular dichroism (CD) spectra of purified and
membrane-attached Na+,K+-ATPase in its E1 and E2 forms, obtained by introducing either Na+ or K+.
The spectra for the peptide backbone (190-240 nm) were consistent with extensive conformational
differences between E1 and E2. The changes appear to be reversible when the ion composition is altered.
Phosphorylation of the ATPase is accompanied by fluorescence changes of its tryptophan residues
(Nakamura et al., 1994), indicating a conformational change.
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The involvement in transport of rearrangements within the ATPase is also shown by x-ray diffraction
studies of packed membranes from the sarcoplasmic reticulum containing Ca2+-ATPase (Blasie et al.,
1985). A significant portion of the ATPase juts into the cytoplasmic phase, as also shown by three-dimensional reconstruction of negatively stained crystals in sarcoplasmic reticulum membranes (see
below). Activation of the ATPase produces a conformational change with a displacement of the structure
into the bilayer. Both the binding of Ca2+ (DeLong and Blasie, 1993; Cheong et al., 1996) and the
phosphorylation of the enzyme (Blasie et al., 1985, Pascolini et al., 1988) were found to change the
conformation of the enzyme as seen using X-ray diffraction. The experiments correlating specific steps
in the transport used flash photolysis of caged ATP, a compound which releases ATP in response to a
flash of light, assuring rapid and synchronous activation.
Important details have been provided by other studies, some of them more recent. Ten transmembrane
helices (M1 to M10, shown by the numbers in Fig. 9) have been proposed based on the amino acid
sequence (MacLennan et al., 1985) and confirmed by high resolution EM (8-Å, Zhang et al., 1998). The
functional portions of the cytosolic domains of the head region of the molecule are distinct. The P
domain is involved in the phosphorylation, the N domain contains the nucleotide binding site and the A
or actuator domain (also called the transducer domain) is thought to have a special role in the
transduction (see Toyoshima et al., 2000; Toyoshima and Nomura, 2002). These are represented in the
diagram of Fig. 9 and discussed below. Fig. 9 was drawn from models derived from X-ray diffraction
data (Toyoshima et al., 2000).
The more recent crystallographic study (Toyoshima and Nomura, 2002) with a resolution of 3.1 Å has
provided data on the Ca2+-ATPase in its E2-state (with no Ca2+, but protonated with 2 H+).The
conformation of E2 was found to differ from that of E1 (with 2 bound Ca2+) as follows. In E1 the three
domains (P, N and A domains) are widely separated. In E2 they form a compact structure as shown in
Fig. 9. In addition, six out of the ten transmembrane segments also undergo conformational changes
when assuming the E2 conformation. These changes might be required for the release of the Ca2+ into the
SR lumen by opening a channel for the passage of Ca2+ and the permitting the counter-transport of 2 H+
to cytoplasm in exchange for 1 Ca2+ per 1 ATP hydrolyzed .(see fig. 2 of Green and MacLennan, 2002)
as indicated in the model of Fig. 16 below.
The structure deduced for the Ca2+-ATPase allowed the construction of an atomic homology model of
the H+-ATPase of Neurospora by comparing it to an 8 Å map of the Neurospora proton pump derived
from electron microscopy (Kühlbrandt et al., 2002). The model, shows the probable path of the proton
through the membrane and indicates that the nucleotide-binding domain rotates by approximately 70o to
deliver ATP to the phosphorylation site of the ATPase. This model differs somewhat from that proposed
for the Ca2+-ATPase.
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Fig. 9 Schematic representations of the Ca2+-ATPase in the Ca2+-binding configuration, based on the
reconstruction of Toyoshima et al. (2000). The red P in the P-domain represents the phosphorylated site.
The yellow oval represent the site of nucleotide binding in the N-domain. The ten helices traversing the
membrane are represented by cylinders and the two red dots represent the bound Ca2+.
In summary, it appears that the transport of ions proceeds by binding the ions to specific sites. These sites
are probably present in a channel of the transporter that traverses the membrane. The translocation is
associated with some movement of the binding sites, so that the sites are exposed first to one, and then to
the other side of the membrane and major rearrangments of the large cytoplasmic domains of the
How can this information be put together in a single model? The presence of a conventional channel
would only allow passive flow in the direction of the gradient and could not carry out transport against an
electrochemical gradient. For this reason, the models generally considered propose alternating access
(see Fig. 10), in which a small conformational change (in this case a rotation) exposes the binding sites
first to the water phase on one side of the membrane, and then to the water phase on the other side
(Tanford, 1983). The channel would remain closed at all times, but would alternate using two different
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“gates”, comparable to the gates in a lock connecting two bodies of water of different heights. The
movement of the binding groups could increase the distance between them, as represented in the diagram
and, therefore, could also account for the change in the affinity for the transported ion. During the
working cycle of the alternating access pump, when both “gates” are closed, the ion is unavailable for
exchange. The ion in the transporter molecule is said to be occluded. Occlusion suggests the presence of
an intermediate position of the binding sites, apart from their location at either the uptake or the
discharge site (see Glynn and Karlish, 1990). An alternating access model of the Ca2+-ATPase is shown
in Fig.16.

Fig. 10 Representation of the alternate-access model of transport. The structures represent polypeptide
chains traversing the phospholipid bilayer of the plasma membrane. The circles indicate the binding sites
of the transported ion. The closeness of the binding groups on the left accounts for the high-affinity
binding, the separation on the right for the decrease in affinity. The slight rotation of the polypeptides
accounts for the access of the binding sites from either the uptake site (left) or the discharge site (right).
Mutational studies have identified amino acids critical for transport (see MacLennan et al., 1997). Site-specific mutagenesis substitutes amino acids at defined locations in the molecule and delineates the
functional role of amino acids or amino acid clusters in the transport. In some of these experiments,
mutant DNA was incorporated into COS cells, a transformed simian cell line, using a vector and then
assayed for function (see MacLennan, 1990). These studies have identified amino acids critical for
transport (see MacLennan et al., 1997). Negatively charged residues in M4, M5, M6 and M8 are thought
to constitute high affinity Ca2+-binding site (Clarke et al., 1989). The two Ca2+-binding sites are formed
by the juxtaposition of acidic and oxygen containing amino acids next to each other in the middle of the
four transmembrane helices (Clarke et al., 1989; see Andersen, 1995 and MacLennan et al., 1997) as
represented in Fig. 15. Small changes in the position of the helices forming this cluster would disrupt
these binding sites. The study of (Toyoshima et al., 2000) suggests the pathway lined by oxygen atoms,
allowing for the in-and-out passage of Ca2+ and shows the disruption of structure of the M4 and M6
helices to provide a Ca2+-binding cavity. In addition, they identified mutation-sensitive carbonyl groups
in the M4 helix.
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The properties common to at least some of the transport systems are summarized in Table 3. Some of
these examples correspond to active transport, others do not. For all transport systems, the transporter
binds the transported substrate. Furthermore, the transporters have been shown to undergo a
conformational change. Channel behavior has been shown, at least under some conditions, for some of
the transporters.
Table 3 Summary of the Properties of Some Transport Systemsa
Ion or Solute Active
change of
No  Yesb  Yesc  Yesd
Na+, K+
Yese  Yes
(Table 2)
Yesf  Yesg
Yes  Yes
(Table 2)
Yesh  Yesi
(ATP synthase)
Yes  Yes
(Table 2)
Yesj  Yes
(see Chapter 17)
Yes  —  —  Yesk
Na+-amino acid
Yes  —  —  Yesl
a Yes indicates that the phenomenon has been observed for the solute or the transporter b Falke et al. (1984b); c Giebel and
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21. Transport of Ions: Mechanisms and Models
Passow (1960); d Falke et al (1984b);e Yamaguchi and Tonomura (1980);f Last et al. (1983); gJorgensen (1975); Karlish
and Yates (1978); Koepsell (1972) h Shamoo and MacLennan (1975) i Imamura et al. (1984); j Tanford (1983); k Peerce
and Wright (1984);l Wright and Peerce, 1984.
Many years of collected evidence support the alternate access model represented in Fig. 10. This model,
adapted to reflect the various experimental findings for the Ca2+-ATPase, is shown in Fig. 11. In this
figure, 2 Ca2+ are shown to be bound sequentially. One of these is not readily accessible from either side
of the ATPase-channel (occlusion). ATP phosphorylates the ATPase so that the two Ca2+ are released
sequentially. In this figure the stripes indicate the Ca2+ which is bound to the ATPase first (reaction 1-2)
and the dotted circles represent the second Ca2+ bound in reaction 3. As shown, the first Ca2+ is not
readily available from the outside or from inside the vesicle. It will equilibrate slowly with Ca2+ in the
medium. However, phosphorylation of the enzyme (reaction 4) allows the sequential discharge of Ca2+
to the inside of the vesicles: the first Ca2+ to be bound is released into the vesicles first (reaction 5); the
second Ca2+ to be bound is released second (reaction 7) and corresponds to the Ca2+ which is readily
exchangeable with 40[Ca2+] before phosphorylation.
An alternate access model for the Ca2+-ATPase highlighting the structural aspects is shown in Fig. 16
(Inesi, 1987).

Fig. 11 Diagram representing the sequential mechanism of calcium binding and translocation upon ATP
hydrolysis by SR ATPase. From Inesi, 1987. Reproduced by permission. Copyright &copy1987 The
American Society for Biochemistry and Molecular Biology.
Inesi (1987) explored details of the Ca2+-ATPase mediated transport of the SR with a pulse chase
technique. In one experiment, SR vesicles containing Ca2+-ATPase were first equilibrated with the
radioactive isotope [45Ca]2+. This incubation was then followed by a chase with nonradioactive [40Ca]2+.
The time course of the release of the labelled Ca2+ at low temperature is shown in Fig. 12 (Inesi, 1987).
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21. Transport of Ions: Mechanisms and Models
In this figure, bound radioactive Ca2+ in the ordinate is shown as a function of the time after addition of
the nonradioactive Ca2+. The total initial binding corresponds to 2 Ca2+ per enzyme molecule. A rapid
initial release, over in about 0.2 s, is followed by a much slower one, which cannot be seen with the time
scale used. The vastly different rates of release are in agreement with the sequential model of Fig. 11.
The faster release corresponds to the Ca2+ which is bound second and readily accessible from the outside
La3+ displaces all bound Ca2+, so that in the presence of ATP, any Ca2+ not displaced by La3+ represents
Ca2+ which has been occluded or transported into the vesicle. The relationship between translocation and
binding was examined in an experiment whose results are represented in Fig. 13. The experimental
design is shown diagrammatically on the left side of the figure. Curve A represents results obtained
without a chase. [45Ca]2+ was first bound to the ATPase of the vesicles and ATP added subsequently.
La3+ was added at the various times indicated in the abscissa. The Ca2+ translocated into the vesicle is
first very rapid, corresponding to the translocation of Ca2+ initially bound to the ATPase. This is
followed by a slower transport that represents the Ca2+ subsequently transported into the vesicle. When
ATP is added simultaneously to a chase with [40Ca]2+, the amount transported (curve B) corresponds
exactly to that bound (2 Ca2+/enzyme); no additional translocation of the radioactive Ca2+ can take place
because of the chase. As indicated by Fig. 12, a chase of 0.2 s with [40Ca]2+ removes the molecule of
Ca2+ that was bound second by the ATPase. The transport of the remaining Ca2+ ion (the first to be taken
up) can, therefore, be followed by introducing ATP after a 0.2 s chase with the non-radioactive Ca2+
(curve C). After the 0.2 seconds chase, only half of the radioactive Ca2+ was transported into the vesicle
(curve C). This indicates that the Ca2+ which occupies the position closer to the outside, is transported
first into the vesicle, as predicted from a sequential model of Fig. 11.

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21. Transport of Ions: Mechanisms and Models
Fig. 12 Isotopic exchanges of bound Ca2+. From Inesi, 1987. Reproduced by permission. Copyright
&copy1987 The American Society for Biochemistry and Molecular Biology.

Fig. 13 Quench-flow measurements of ATP-dependent calcium uptake. From Inesi, 1987. Reproduced by
permission. Copyright &copy1987 The American Society for Biochemistry and Molecular Biology.
The Ca2+ uptake of the initial burst (Fig. 13A) may include Ca2+ that is not exchangeable and is trapped
in the ATPase, i.e. occluded. A different experimental design can differentiate between bound Ca2+ and
occluded Ca2+. When ADP is added in the presence of a Ca2+-chelator [ethylene glycol-bis-(β-aminoethyl ether) N,N,N’,N’-tetraacetic acid (EGTA)], the phosphorylation of the transporter is reversed.
ADP is phosphorylated and the occluded Ca2+ is released into the medium. Only one single cycle of the
enzyme is possible because there is no Pi present. In contrast, the Ca2+ transported into the vesicles
would be retained (and would not be released by La3+). The results of this experiment are shown in Fig.
14, which shows the radioactive Ca2+ uptake in the ordinate. The time shown in the abscissa represents
the time of addition of ADP + EGTA which is then followed by the addition of La2+. In curve A, the
preparation is preincubated in [45Ca]2+. Then [40Ca]2+ and ATP are added simultaneously. In this case,
the Ca2+ uptake after the ADP+EGTA addition represents the transported Ca2+ (amount taken up +
amount occluded). At the earlier times of addition of ADP + EGTA, 4 to 5 nanomoles of Ca2+ are taken
up per mg, compared to 9 to 10 without the ADP + EGTA treatment (Fig. 14A). Therefore,
approximately half of the original Ca2+ taken up is in the occluded form. When ATP is added after the
0.2 s of [40Ca]2+ chase (which removes the more external Ca2+) (Fig. 14B), half of the Ca2+ uptake has
already become ADP + EGTA insensitive. This shows that the insensitive Ca2+ (released into the
vesicles) is the one that was bound first (see Fig. 11). These results elaborate and support the alternating-site model and indicate a sequential release of the Ca2+.
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21. Transport of Ions: Mechanisms and Models
Much the same information is available for the Na+, K+-ATPase from entirely different experiments. As
we have seen, 3 Na+ and 2 K+ bind to separate sites of the protein. First they become occluded (i.e.,
trapped inside the transporter) and then are released to the other side (see Post et al., 1972; Beaugé and
Glynn, 1979). In the absence of K+, Na+ is still translocated (Garrahan and Glynn, 1967) and the
translocation is electrogenic (Fendler et al., 1985; Nakao and Gadsby, 1986). The electrical signal during
the ion pumping corresponds to the movement of the ions across the channel that traverses the membrane
(e.g., Hilgemann, 1994) and is associated with charge movements. The rate of these electrogenic
reactions is dependent on the membrane potential, so that enzymes conformations can be shifted. High
speed voltage jumps can be used to initiate this redistribution. Three phases are apparent (Holmgren et
al., 2000), reflecting the de-occlusion of the three ions. The results indicate that three are released one at
a time, in order.

Fig. 14 ADP reversal of ATP-induced calcium translocation. From Inesi, 1987, reproduced by permission.
Analyses of the phosphorylating reactions were also carried out. Either ATP or Pi can phosphorylate the
enzyme. The ATP phosphorylation depends on high affinity Ca2+ binding. In contrast, the
phosphorylation by Pi is blocked by Ca2+.
The Ca2+ occlusion was studied on detergent solubilized SR vesicles in the presence of CrATP. CrATP
allows occlusion without the hydrolysis of ATP and it also stabilizes the Ca2+-enzyme complex. A
HPLC-molecular sieve procedure was used (see Chapter 1) to separate the proteins from free Ca2+.
Mutations at the sites thought to bind Ca2+, prevented occlusion (Vilsen and Andersen, 1992, Andersen
and Vilsen, 1994).
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21. Transport of Ions: Mechanisms and Models

Summary of amino acid substitution introduced into the predicted Ca2+-binding domain. Glu309, Glu771,
Asn796, Thr799, Asp800, and Glu908 are thought to be in the transmembrane segments M4, M5, M6 and
M8 respectively. From Clarke et al., 1990b. Reproduced by permission.
As already discussed, there is considerable evidence that the ATPase pumps require a channel-like
structural arrangement. Modeling of the four helices thought to be involved in Ca2+ binding and which
are amphiphilic, show that polar and charged residues are predominantly in one face of each helix with
the hydrophobic residues in the opposite face. The hydrophilic components could therefore form
hydrophilic clusters in the internal surfaces, thereby forming a channel. The hydrophobic residues, on the
other hand, could interact with the bilayers providing the transmembrane arrangement.
Present information (e.g., Inesi et al., 1992; Toyoshima et al. 2000) indicates that the Ca2+-binding
domain and the catalytic domain are separated by 50 Å. This spatial arrangement would require that any
interaction would be indirect, via a conformational change. We have seen that conformational changes
have been demonstrated (see Fig. 9). A possible mechanism for the transport of Ca2+ involving the
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21. Transport of Ions: Mechanisms and Models
transmembrane helices is indicated in Fig. 16 (MacLennan, 1990). The shift from E1 to the E2 form
accompanying phosphorylation, would shift the negatively charged binding groups from the outer to the
inner interface. Furthermore, the conformational shift would disrupt the arrangement of the high affinity
binding groups to produce low affinity binding sites.

Fig. 16 Model illustrating the possible mechanism of Ca2+ transport by the Ca2+-ATPase. In the E1
configuration, high affinity Ca2+-binding sites are accessible to the cytoplasmic Ca2+. ATP hydrolysis
induces the E2 configuration, in which the access of the binding groups from the cytoplasmic side is
blocked and their configuration of the binding groups is disrupted. The disruption results in a low affinity
binding. From MacLennan, 1990, reproduced by permission.
Inesi, G., Zhang, Z., Sagara, Y. and Kirtley, M.E. (1994) Intracellular signaling through long-range
linked functions in Ca2+ ATPase, Biophys. Chem. 50:129-138. (Medline)
MacLennan, D.H., Rice, W.J. and Green, N.M. (1997) The mechanism of Ca2+ transport by
sarco(endo)plasmic reticulum Ca2+-ATPases, J. Biol. Chem. 272:28815-28818. (MedLine)
Stein, W.D. and Lieb, W.R. (1986) Transport and Diffusion Across Cell Membranes, Chapter 6, pp. 475-http://www.albany.edu/~abio304/text/21part2.html (15 of 16) [3/5/2003 8:24:10 PM]
21. Transport of Ions: Mechanisms and Models
612. Academic Press, New York.
Tanford, C. (1984) The sarcoplasmic reticulum calcium pump. Localization of free energy transfer to
discrete steps of the reaction cycle, FEBS Lett. 166:1-7. (Medline)
General Reviews
Inesi, G.(1994) Teaching active transport at the turn of the twenty first century: recent discoveries and
conceptual changes, Biophys. J. 66:554-560. http://www.biosci.umn.edu/biophys/OLTB/BJ/Inesi.pdf
Adobe Acrobat from http://www.adobe.com is required for reading pdf files.
Lauger, P. (1984) Channels and multiple conformational states: interrelations with carriers and pumps,
Curr. Top. Membr. Transport 21:309-326.
Search the textbook
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Chapter 21: References
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