(Received for publication, July 18, 1995; and in revised form, September 19, 1995)
From the
This paper describes properties of a novel family of aromatic
isothiouronium derivatives, which act as Na-like
competitive antagonists on renal Na/K-ATPase. The derivatives are
reversible competitors of Rb
and Na
occlusion. K
values of the most
potent compounds, 1bromo-2,4,6-tris(methylisothiouronium)benzene
(Br-TITU) and
1,3-dibromo-2,4,6-tris(methylisothiouronium)benzene(Br
-TITU),
0.65 and 0.32 µM, respectively, are 15-30-fold lower
than K
values of the bis-guanidinium
derivatives described previously (David, P., Mayan, H., Cohen, H., Tal,
D. M., and Karlish, S. J. D.(1992) J. Biol. Chem. 267,
1141-1149), and represent the lowest reported values for cation
antagonists. Using fluorescein-labeled Na/K-ATPase, all derivatives
have been shown to stabilize the E
conformation when bound
at high affinity sites (i.e. they are sodium-like). In
addition, in one condition (10 mM Tris-HCl, pH 8.1), high
concentrations of Br-TITU (K
10
µM) appear to stabilize an E
conformation. We
propose a model which allows for simultaneous binding of the
antagonists to high affinity cytoplasmic sites and low affinity sites,
which may be at the extracellular surface. Blockage of cation occlusion
by the isothiouronium derivatives at the cytoplasmic surface probably
occurs at the entrance to the occlusion sites, which is recognized both
by Na
antagonists and by Na
or
K
ions. Unlike the alkali metal cations, the
Na
antagonists are not occluded or transported (see
also Or, E., David, P., Shainskaya, A., Tal, D. M., and Karlish, S. J.
D.(1993) J. Biol. Chem. 268, 16929-16937). The
isothiouronium derivatives appear to be promising candidates for
further development as affinity labels of cation binding domains, for
kinetic analysis of isoforms or mutated Na/K pumps, or as probes of
other cation transport proteins.
Knowledge of the structure and organization of cation occlusion sites is central to an understanding of the mechanism of active transport by the Na/K pump (Glynn and Karlish, 1990). In the absence of detailed molecular structure, a variety of techniques is being applied to obtain information on the sites, including chemical modification with carboxyl-specific labels (Goldshleger et al., 1992; Argüello and Kaplan, 1994) and site-directed mutagenesis (Van Huysse et al., 1993; Jewell-Motz and Lingrel, 1993; Vilsen, 1993). Another approach is the use of extensive proteolysis in order to define the minimal peptide components capable of cation occlusion. In a specifically trypsinized preparation of renal Na/K-ATPase, so-called ``19-kDa membranes,'' trans-membrane segments and extracellular loops remain, while cytoplasmic loops are removed. Cation occlusion capacity and ouabain binding are intact but ATP binding is destroyed (Karlish et al., 1990; Capasso et al., 1992; Shainskaya and Karlish, 1994; Schwappach et al., 1994). The concept that emerges from these approaches is that a cation occlusion ``cage,'' is located within the membrane domain, and consists of carboxyl and neutral ligating groups contributed by several trans-membrane segments. Nevertheless each technique is subject to inherent limitations and there is no firm evidence as to which residues or segments are directly involved in cation binding.
Affinity labeling of cation sites is, potentially,
an important approach. The objective would be to use the reactive
derivative to label and map the region of polypeptide chain in the
vicinity of the cation sites. In addition an affinity label of cation
sites with a known sidedness of action could help to distinguish
between different topological models. An affinity or photoaffinity
label could be based on an organic analogue of the alkali metal cations
and should bind with a K about equal to
the concentration of the sites, in practice, 0.1-1
µM.
Cation analogues which act on the Na/K-ATPase with
dissociation constants of 0.1-1 µM have not been
described. By contrast, the pyridineimidazole class of
potassium-competitive inhibitors of gastric H/K-ATPase and gastric acid
secretion represents an example of high affinity cation antagonists
(Wallmark et al., 1987), derivatives of which have been used
to successfully chemically modify the related H/K-ATPase (Munson et
al., 1991). Competition with alkali metal cations of different
alkyl or aryl amines and diamines on the Na/K-ATPase have been
described (Kropp and Sachs, 1977; Schuurmans-Stekhoven et al.,
1988; Forbush, 1988), but the dissociation constant of ethylenediamine,
the compound with the highest affinity, is only about 25
µM (Forbush, 1988). A photoaffinity probe based on a
precursor with such a low affinity would probably be unselective, due
to rapid dissociation of the active species and labeling of regions of
the protein other than the cation sites. Recently, we have described
synthesis and characterized functional effects of alkyl and aryl
bis-guanidinium derivatives (m- or p-xylylene
bis-guanidinium, mXBG ()or pXBG) (David et al.,
1992). The guanidinium derivatives competitively inhibit Na
or Rb
occlusion, stabilize the E
conformation of the enzyme, and block Na/K-ATPase activity. Thus
they are competitive Na
antagonists. pXBG or mXBG
compete with a K
8 µM.
Systematic structure-activity studies on the guanidinium derivatives
(David et al., 1992) and amiloride analogues (David et
al., 1993) led to a number of provisional conclusions concerning
expected properties of a putative high affinity Na antagonist. These conclusions form the basis for a search and
design of a new set of compounds of higher affinity. In order to
simplify syntheses, the positively charged guanidinium has been
replaced by the isosteric positively charged isothiouronium moiety.
This paper describes the interactions of the new high affinity
antagonists with renal Na/K-ATPase. Detailed syntheses of the compounds
are described elsewhere (Tal and Karlish, 1995).
In both cases, 0.5 ml of ice-cold 200 mM sucrose was added to dilute and cool the mixture, which was
immediately transferred to ice-cold Dowex columns. The enzyme with
occluded Rb or Na
was eluted with 1.0
ml of the ice-cold 200 mM sucrose.
Rb
was measured as its Cerenkov radiation. For
Na
, 3 ml of scintillation liquid
(Lumax:xylene = 1:3) was added and radioactivity was measured in
a scintillation counter.
The structures, IUPAC names, and abbreviations, of the new isothiouronium derivatives are shown in Fig. 1. The benzyl, xylyl, or mesityl derivatives contain either one, two, or three isothiouronium groups, respectively, without or with methyl substituents, and none, one, or two bromine atoms in the aromatic ring.
Figure 1: Structures of isothiouronium derivatives.
Figure 2:
A, inhibition by Br-TITU and
Br-TITU of Rb
occlusion. Na/K-ATPase (25
µg) was incubated in 50 µl of reaction medium containing 100
mM choline chloride, 10 mM Tris-HCl, pH 8.0, 50
µM
RbCl, and the indicated concentrations of
the inhibitors. B, competitive inhibition of Rb
occlusion by Br-TITU. Na/K-ATPase (25 µg) was incubated in 50
µl of reaction medium containing 100 mM choline chloride,
10 mM Tris-HCl, pH 8.0, and 0.05-1.2 mM Rb
in the absence (
) and presence (
)
of 3.13 µM Br-TITU.
Fig. 3shows the effect of Br-TITU on
FITC-labeled Na/K-ATPase. In the high ionic strength medium at pH 8.1,
the enzyme is in the E conformation. If Rb
(1 mM) is added first to induce the quench, subsequent
addition of Br-TITU (25 µM) largely reverses the signal (Fig. 3A). (The lack of complete reversal of the
fluorescence signal by Br-TITU (25 µM) is due to a small
contribution of nonspecific fluorescent quenching, observed when high
concentrations of Br-TITU are added after RbCl.) At lower concentration
of RbCl (300 µM) and Br-TITU (8 µM), reversal
of fluorescence quenching was complete (not shown). Thus, Br-TITU
behaves essentially like Na
ions in stabilizing the
E
form. Accordingly, if Br-TITU is added first, it does not
affect the fluorescence and subsequent addition of Rb
has no effect (Fig. 3B). Stabilization of the
E
conformation was observed for all the isothiouronium
derivatives, except Me
-Br-TITU, due apparently to
precipitation of the FITC-labeled enzyme. At very low ionic strength
the enzyme is mainly in the E
state (Skou and Esmann,
1980). Addition of Br-TITU in the absence of Rb
resulted in a strong enhancement, which can be reversed by
Rb
(Fig. 3C). When Rb
is added first, almost no change was observed, showing that the
enzyme is indeed mainly in the E
conformation. Subsequent
addition of Br-TITU did not reverse the signal at the low concentration
used, due to competition with the Rb
(Fig. 3D). Experiments with other derivatives
confirm that all are competitive Na
analogues which
stabilize the E
state. In fluorescence experiments,
observed K
values are significantly lower than in
occlusion experiments. This result is attributable to the higher enzyme
concentration in occlusion experiments, for the derivatives are bound
to the membranes, reducing the free concentration in the medium (see
David et al.(1992)).
Figure 3:
Effects of isothiouronium derivatives on
conformational changes. A, FITC-labeled Na/K-ATPase (10
µg) was suspended in 1.5 ml of 100 mM choline chloride, 10
mM Tris-HCl, pH 8.1, with addition of 1 mM Rb
and subsequent addition of 25 µM Br-TITU. B, same as in A, but Br-TITU was added
first. C, FITC-labeled Na/K-ATPase (
10 µg) was
suspended in 1.5 ml of 1 mM Tris-HCl, pH 8.5, with addition of
5 µM Br-TITU and subsequent addition of 0.5 mM Rb
. D as in C, but
Rb
was added first. E, FITC-labeled
Na/K-ATPase (
10 µg) was suspended in 10 mM Tris-HCl,
pH 8.1, and additions were made as
indicated.
In the course of systematic testing of
effects of conditions on fluorescence responses, an unexpected
phenomenon was observed for a medium of intermediate ionic strength (10
mM Tris-HCl, pH 8.1, Fig. 3E). The K of Br-TITU for reversing the
Rb
-induced quenching was lower than in the other
media, (K
0.13 µM in Fig. 3E), and addition of higher concentrations of
Br-TITU caused the fluorescence to be quenched again. Subsequent
addition of Na
ions, 20 mM, largely reversed
this quench. The apparent affinity for this E
-stabilizing
effect of Br-TITU effect is about 10 µM, i.e. about 80-fold lower than that of the high affinity site. The same
effect was observed for the mono- and dimethyl-Br-TITU derivatives,
although they are much less potent inhibitors of occlusion than Br-TITU (Table 1). In a medium of high ionic strength (100 mM choline chloride, 10 mM Tris-HCl, pH 8.1) addition of a
very high concentration of Br-TITU caused quenching of fluorescence,
but this effect appears to be nonspecific since it was not reversed by
Na
(data not shown).
Figure 4:
Br-TITU does not protect a 19-kDa fragment
of the subunit against tryptic digestion. Na/K-ATPase, was
digested as described by Capasso et al.(1992) in the presence
of 10 mM RbCl or 10 mM choline chloride plus 200
µM Br-TITU. 25 µg of protein was applied to a 10%
Tricine Mini-gel as indicated.
Most
of these features are similar to those of the aromatic bis-guanidinium
derivatives, pXBG and mXBG (David et al., 1992; Or et
al., 1993). pXBG and mXBG act as competitive Na antagonists, with selectivity for the cytoplasmic surface, but
have much lower affinities than the Br-TITU and Br
-TITU.
The previous work led to a proposal that cation occlusion from the
cytoplasmic surface occurs in two steps (Or et al., 1993). In
an initial recognition step, either transported cations or
Na
antagonists interact with carboxyl groups, while a
second step is selective for transported cations, and involves
occlusion of either K
or Na
ions and
a conformational change to a compact structure, which is resistant to
proteolysis and thermal inactivation. The isothiouronium derivatives,
like the lower affinity Na
antagonists, are presumed
to be sterically hindered from becoming occluded, and so block cation
occlusion and Na/K-ATPase activity (see David et al., 1992; Or et al., 1993). In another recent study we have looked at
effects of diffusion potentials on inhibition by Na
antagonists, using reconstituted proteoliposomes (Or et al.,
1994). The data are interpreted on the basis of a model which assumes
that two of the three Na
transport sites are
negatively charged and serve also as the K
transport
sites while the third Na
transport site is
electrically neutral and lies inside a cytoplasmic access pathway or
``ion well'' (Goldshleger et al., 1987; Glynn and
Karlish, 1990; Laüger, 1991). The
Na
antagonists do not appear to enter the narrow
Na
access pathway, or become occluded, but block
transport by interacting with the negatively charged sites located
nearer the surface. Evidently, these studies provide fairly clear
structural implications for affinity labels based on the competitive
Na
antagonists. One can expect that such affinity
labels would modify the protein at the entrance to the cytoplasmic
cation sites.
One feature of tris-isothiouronium derivatives, not
found previously for the bis-guanidinium derivatives, was observed in
experiments with FITC-labeled Na/K-ATPase (Fig. 3). At high
ionic strength, Br-TITU and other isothiouronium derivatives, like the
pXBG and mXBG, behave like Na ions (Fig. 3, A-D). However, at a lower ionic strength (10
mM Tris-HCl, pH 8.1), whereas the
``Na
-like'' effect was observed at a low
concentration (K
0.13 µM),
higher concentrations of Br-TITU (K
10
µM), and its methylated derivatives, appear to be
``potassium-like'' (Fig. 3E). The model in Fig. 5can explain the findings in Fig. 3E. There
are two sites for Br-TITU, high affinity, cytoplasmic
(K
K
) and low affinity, (K
K
), respectively. In this model K
1 (K
=
[E
L]/[E
L]), while K
1, (K
=
[E
L
]/[E
L
]).
At low concentrations, Br-TITU occupies a high affinity cytoplasmic
site and stabilizes the E
conformation, but at higher
concentrations it occupies simultaneously both high and low affinity
sites and stabilizes the E
conformation. Stabilization of
the E
state is reminiscent of effects of ouabain on the
Na/K pump (see Forbush, 1983) or the substituted pyridyl(1,2a)imidazole
K
competitor, SCH28080, on the gastric H/K pump
(Wallmark et al., 1987; Munson and Sachs, 1988). Possibly,
Br-TITU at high concentrations, like ouabain or SCH28080, binds to the
extracellular surface. Simultaneous binding of Br-TITU at opposite
surfaces might appear paradoxical in view of the accepted view that
Na
and K
are transported in a
consecutive fashion (Glynn, 1985; Sachs, 1991). Note, however, that we
are not dealing with a transported cation but with a cation antagonist.
It is conceivable that elements of the cation path are fixed and
co-exist at two surfaces of the membrane (see also Karlish and
Stein(1985)).
Figure 5:
Model with simultaneous binding of two
molecules of Na antagonists. See text for
explanation.
(b) Br-TITU and Br-TITU might be very
useful for kinetic analysis of functionally expressed Na/K pump
isoforms, and mutated or other structurally modified pumps. Significant
differences have been observed in monovalent cation activation of
Na/K-ATPase of
1,
2, or
3 isoforms (Jewell and Lingrel,
1991; Munzer et al., 1994), or between wild type and mutated
subunits (Jewell-Motz and Lingrel, 1993; Vilsen, 1993, 1995). In
some cases mutated pumps show little or no difference from wild type
(Van Huysse et al., 1993; Van Huysse and Lingrel, 1994). A
problem for the interpretation is that it is difficult to distinguish
effects on intrinsic cation binding constants from indirect effects on
rate constants of the catalytic cycle. By contrast, the derived
inhibition constant of a competitive sodium antagonist, should equal
the intrinsic binding affinity K
, provided that
the predominant form of the enzyme (E
) is that to which the
inhibitor binds (see Stein, 1986). In media of high ionic strength the
E
form predominates (Skou and Esmann, 1980).
(c) Preliminary experiments ()show that Br-TITU
acts as a potassium-competitive inhibitor of gastric H/K-ATPase, and in
addition it is an effective blocker of the epithelial Na
channel. (
)Thus, although the Br-TITU and
Br
-TITU inhibit Na/K-ATPase with high affinity, the
isothiouronium moiety does not appear to be highly selective for
Na
binding sites on the pump. This conclusion is
compatible with the concept that the isothiouronium moiety interacts at
the entrance of the cation sites on the Na/K-ATPase. The isothiouronium
derivatives may become useful probes for other cation pumps,
co-transporters, exchange diffusion systems, or cation channels.