(Received for publication, November 27, 1995; and in revised form, January 31, 1996)
From the
Treatment of purified canine renal Na,K-ATPase with a range of
photoactivatable amiloride derivatives results in inhibition of ATPase
activity prior to illumination. Inhibition by amiloride derivatives
substituted on a guanidium N could not be prevented by the presence of
either K or Na; however, these cations could protect the enzyme against
inhibition by derivatives substituted on the 5-position of the pyrazine
ring. In the case of
5-(N-ethyl-[2`-methoxy-4`-nitrobenzyl])amiloride
(NENMBA), the presence of monovalent cations (Na, K, and Rb) protected
the enzyme effectively against inhibition, with concentrations in the
millimolar range. ATP did not prevent inhibition; furthermore, native
and NENMBA-treated enzyme exhibited normal levels of high affinity
[H]ADP (and hence ATP) binding. The rate of
inhibition increased with increasing concentrations of NENMBA.
Extensive washing of NENMBA-inhibited enzyme did not restore ATPase
activity, showing that NENMBA has an extremely slow off-rate for
dissociation from its inhibitory site. Partially inhibited enzyme could
be rapidly pelleted and resuspended in NENMBA-free buffer and
inhibition was observed to continue, albeit at a somewhat diminished
rate, suggesting that NENMBA gains access to its inhibitory site after
partitioning into the lipid phase rather than directly from the aqueous
solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent
incorporation of the reagent into the
-subunit of the Na,K-ATPase,
as observed by separation of labeled protein on a Laemmli gel and
Western analysis using a polyclonal amiloride antibody. Almost all of
the covalent labeling could be prevented by the presence of Rb in the
incubation and labeling medium. These results suggest that NENMBA
inhibits the Na,K-ATPase by disruption of the cation transport domain
rather than the catalytic domain of the enzyme and that it promises to
be a useful tool for cation site localization.
The sodium pump or Na,K-ATPase (EC 3.6.1.37) is the plasma
membrane protein responsible for maintaining the resting concentrations
of sodium and potassium in animal cells. It is composed of an
-subunit and a
-subunit. The
-subunit consists of about
1020 amino acid residues and has been cloned from several
sources(1, 2, 3, 4, 5, 6) .
The
-subunit is composed of about 300 amino
acids(7, 8, 9) . The enzyme undergoes a
series of conformational changes through its catalytic cycle, which
couple binding of ATP and enzyme phosphorylation to the active
transport of the monovalent cations against their electrochemical
potential gradients (for reviews see (10, 11, 12) ). In its normal transport mode,
the enzyme is phosphorylated by ATP when cation binding residues which
selectively bind sodium are exposed to the cytosol; the enzyme then
undergoes a major conformational transition and transports three
sodiums across the cell membrane, where they dissociate to the external
medium. The (phospho-) enzyme cation ligating residues are now exposed
to the external medium and are more potassium-selective; two potassiums
(or cogeners for potassium such as rubidium, cesium, etc.) bind, and
the phospho-enzyme bond is hydrolyzed. The potassiums are then occluded
within the protein, and the enzyme undergoes the second major
conformational transition of its catalytic cycle when ATP binds with
low affinity (but does not phosphorylate the protein) and so
facilitates the release of potassium to the cytosol. The binding of
sodium ions then reinitiates the cycle.
A detailed molecular
description of these transport events includes the localization of
which amino acid residues are involved in binding and transport of
sodium and potassium across the cell membrane. One method that has been
used to address this problem is the utilization of residue-selective
chemical probes to modify purified sodium pump protein (for review, see (13) ). It has been assumed that negatively charged amino acids
are involved in cation coordination. This hypothesis has been tested by
using aspartate and glutamate-specific carbodiimides which inactivate
the sodium pump in a cation-preventable
manner(14, 15, 16, 17, 18, 19, 20, 21) .
There are inherent ambiguities in the use of such reagents, as they can
also induce the formation of endo-peptide bonds; such crossing linking
is also cation-preventable(16, 19) . In order to
circumvent these problems, 4-(diazomethyl)-7-(diethylamino)-coumarin
(DEAC), ()a stable diazomethane analogue, which reacts
specifically and unambiguously with carboxylates to form stable ester
derivatives of the protein, has been recently shown to modify Glu-779
in a cation-preventable manner(22, 23) . However, by
analogy with the naturally occurring ionophores, we would infer that
not only glutamate or aspartate side chains are involved but the
electron lone pairs of other, neutral residues might also be involved
in monovalent cation transport.
In this report we have examined the
effects of four photoactivatable amiloride derivatives on the
Na,K-ATPase. Amiloride derivatives were chosen because they are known
to inhibit other sodium transporting proteins such as epithelial Na
channels, the Na/H antiporter, and the Na/Ca
exchanger(24, 25) . Furthermore, there are well
defined structure-activity relationships for amiloride derivatives;
guanidium-substituted probes show high affinity for epithelial Na
channels, whereas pyrazine ring derivatives evidence a higher affinity
for exchangers than channels(25) . The four compounds we tested
are representative of the two classes of inhibitors. All four reagents
inhibit the Na pump, but only the pyrazine ring-substituted derivatives
in a cation-preventable manner. The most effective of these probes,
NENMBA (Fig. 1), was shown to be covalently incorporated into
the -subunit of the Na pump upon irradiation, as shown by Western
analysis using a polyclonal antibody. A preliminary report of some of
these results has appeared(26) .
Figure 1: Chemical structures of the four photoactivatable, amiloride derivatives.
Figure 2: Summary of inhibition of purified Na,K-ATPase by the amiloride derivatives. The enzyme (0.05 mg/ml) was incubated with each compound (100 µM) at 37 °C for 2 min ± Rb, K (10 mM) or Na (60 mM, where applicable) at pH 7.5 (40 mM Hepes) and activity was assayed as described under ``Materials and Methods.''
Figure 3:
Effect of varying [NENMBA] on
the time course of Na,K-ATPase activity. The enzyme (0.05 mg/ml) was
incubated with 20 µM (), 40 µM (
), 60 µM (
), 80 µM (
), and 100 µM (
) NENMBA at 37 °C at
pH 7.5 (40 mM Hepes), and aliquots were assayed at the time
points indicated.
Figure 4: Protective effect of Rb (A) and Na (B) on the inhibition of purified Na,K-ATPase by NENMBA (Rb is used as a cogener for K in these experiments). The enzyme (0.05 mg/ml) was incubated with NENMBA (100 µM with Rb, 40 µM with Na) at 37 °C at pH 7.5 (40 mM Hepes) for 2 min with the concentrations of cations indicated and activity was assayed as described under ``Materials and Methods.''
Figure 5: Effect of varying pH on the inhibition of purified Na,K-ATPase by NENMBA. The enzyme (0.05 mg/ml) was incubated with NENMBA (100 µM) at 37 °C for 2 min, and activity was assayed as described under ``Materials and Methods.'' The buffers used were: Mes (6.5), Hepes (7.0, 7.5), Taps (8.0, 8.5), and Ches (9.0).
Figure 6:
NENMBA access to inhibitory site. The
enzyme (0.05 mg/ml) was incubated with NENMBA (100 µM) at
37 °C for 2 min at pH 7.5 (40 mM Hepes), and then half the
sample was pelleted rapidly (450,000 g, 4 °C, 2
min) and resuspended in NENMBA-free buffer. Aliquots were taken at the
time points indicated, and activity was assayed as described under
``Materials and Methods.''
Fig. 3shows that increasing [NENMBA] increases the
rate of inhibition of the Na pump. If fully inhibited enzyme (<2%
residual activity) is pelleted by centrifugation, washed in
inhibitor-free buffer, and resuspended in buffer, no recovery of enzyme
activity is observed. Thus, the dissociation of the inhibitor appears
to be an extremely slow process. The lack of recovery is independent of
the composition of the resuspension media (± Rb, ± ATP,
± BSA, ± Na). The presence of monovalent cations (Na, K,
or Rb) in the incubation medium can prevent the inhibitory binding of
NENMBA to the Na pump. These cations protect the enzyme with
concentrations in the mM range: K for
Rb (which is recognized as a cogener for K by the Na pump) is about 1
mM (see Fig. 4A), and for Na is about 7 mM (Fig. 4B), when a concentration of 100 µM NENMBA was used to inhibit the enzyme at 37 °C for 10 min.
However, since NENMBA has an extremely slow off-rate, eventually the
enzyme is fully inhibited even in the presence of 100 mM monovalent cations.
Figure 7:
Photoincorporation of NENMBA into
Na,K-ATPase -subunit. The enzyme (0.05 mg/ml) was incubated with
NENMBA (100 µM) at 37 °C for 5 min ± Rb (20
mM) at pH 7.5 (40 mM Hepes) and irradiated with a
high pressure Hg arc lamp for 2 min through a 313 nm narrow band-pass
filter, separated using a Laemmli gel (10%), and transferred to
nitrocellulose. Covalent incorporation of NENMBA was tested using a
polyclonal amiloride antibody. No signal was observed in the Western
analysis from NENMBA (lane 1), Na pump (lane 2), Na
pump and NENMBA without irradiation (lane 4). Irradiation
produces a stable chemical bond between NENMBA and Na pump
-subunit (lane 3); the presence of Rb (lane 5)
prevents most of this incorporation.
In the present work we have shown that NENMBA, a pyrazine-substituted amiloride derivative, is a potent inhibitor of the Na,K-ATPase. The inhibition is prevented by the simultaneous presence of monovalent cations, which are transported by the Na pump, and the inhibitor is covalently coupled to the protein upon irradiation at 313 nm.
It has been known for some time that amiloride itself inhibits the Na pump, albeit with low affinity, and that pyrazine-ring substituted amiloride derivatives exhibit an enhanced affinity for the Na pump compared to the parent compound(34, 35) . However, these studies were performed either on various cell lines or on Na pump preparations which had 1.5% the specific activity of the enzyme used in this report. The intent of the present studies was to exploit the binding of photoactivatable amiloride derivatives to the Na pump, so as to localize non-acidic amino acid residues in the cation transport domain of the enzyme. The fundamental requirement for the success of this strategy is that the cation photoaffinity probes bind with high affinity to Na pump prior to photolysis, in a cation-preventable manner, so that reasonably specific covalent incorporation of the probe into the protein can subsequently be achieved.
The tight binding or
``occlusion'' of both Na and K
have been well characterized kinetically and it is thought that
these intermediates do in fact form part of the normal catalytic cycle
of the Na pump(10, 38, 39, 40) . An
important characteristic of the cation occlusion phenomenon is the slow
association and dissociation of the cations. It is tempting to
speculate that since NENMBA does not seem to dissociate from the enzyme
it also becomes occluded by the Na pump; however, since other
substituted amines(41) , including guanidines(42) , do
not seem to be occluded by the Na,K-ATPase, we deem it unlikely that
NENMBA is exceptional in this regard.
Fig. 6shows that when
partially inhibited Na pump is pelleted and resuspended in NENMBA-free
buffer, the observed rate of inhibition is reduced 2-3-fold.
Several conclusions can be drawn from this observation. First, as
observed previously and mentioned above, inhibition is not relieved by
resuspension in inhibitor-free medium, i.e. there is a very
slow off-rate from the inhibitory site. Second, it seems highly likely
that inhibitor in the membrane phase is associated with the slow
development of inhibition. NENMBA has been found to be highly
hydrophobic, having a partition coefficient of 200:1 between chloroform
and 0.1 M phosphate buffer at pH 7.4 (24) . We
estimate that the volume ratio of the pellet/resuspension medium is
about 1:100. We would expect the partition equilibrium to be
re-established very rapidly on resuspension, and thus the concentration
of NENMBA in the membrane following resuspension would be about
one-half of its value prior to resuspension, and the rate of
development of inhibition would fall by about 2-fold, which is what is
observed (Fig. 6). Therefore, the probe gains access to its
inhibitory site after it has partitioned into the lipid
environment. If NENMBA bound to the Na pump directly from the aqueous
solution, pelleting and resuspension of the partially inhibited protein
should effectively stop any further inhibition; such is not the case.
NENMBA has a pK of about 8.3(24) ; thus,
as the pH is raised from 6.5 to 9.0 (Fig. 5), the reagent
deprotonates and partitions more completely into the membrane. Entry to
the inhibitory site seems to occur from the membrane phase, so that the
rate of inhibition thus increases as the pH is raised.
Second, NENMBA promises to be a useful cation binding site photo-activated probe since the nitroanisole chromophore reacts by displacement of the methoxy group through a photoaromatic nucleophilic substitution reaction(43) . These types of chromophores are stable upon irradiation in the absence of nucleophiles(44) . This property makes them rather different from the more widely used aromatic azides, which produce reactive nitrenes upon illumination. If these nitrenes do not react with the target protein, they decompose to inert side products; for this reason, observed yields of covalent incorporation of such photoaffinity probes are often very low. In principle, the efficiency of covalent modification of the target enzyme can be much higher with the chromophore used in NENMBA, since it returns to ground state starting materials, if it does not encounter a nearby nucleophile, and so can be re-excited repeatedly during irradiation until a productive encounter occurs.
Two other types of cation binding site chemical probes have been used with the Na pump. The most extensively used are carbodiimides, which react with carboxyls (i.e. glutamates and aspartates) to produce an activated ester, which can react with a nucleophile to produce an amide; thus, intramolecular cross-linking is possible with these probes(16, 19) . This disadvantage has been recently circumvented by use of a cationic substituted diazomethane (DEAC), which also reacts chemo-selectively with carboxylates to form stable ester derivatives of amino acids directly(22, 23) . These reagents have been used because it has been thought for some time that negatively charged amino acid residues might be involved in cation binding and transport. However, inspection of the structures of naturally occurring monovalent cation ionophores suggests that serines, threonines, and tyrosines could also be involved in cation co-ordination of Na and K by the Na pump. Furthermore, the first atomic-resolution crystal structure of a Na/K binding enzyme (the dialkylglycine decarboxylase) shows no Asp or Glu involved in Na/K binding at one of its two cation co-ordination centers(45) .
In summary, we found that four
photoactivatable amiloride analogues inhibit the Na,K-ATPase and
monovalent cations protect the enzyme only when the photolabile
substituent is on the pyrazine ring. Upon illumination, NENMBA is
covalently incorporated into the -subunit as shown by Western
analysis using an amiloride specific antibody. Further studies are
under way to localize the amino acid residue labeled by NENMBA.