(Received for publication, November 17, 1995; and in revised form, February 16, 1996)
From the
This paper demonstrates that specific chymotryptic digestion of
the cytoplasmic domain of the subunit of Na/K-ATPase leads to
changes in the kinetics of occlusion of Rb
ions. The
experiments utilize extensively trypsinized Na/K-ATPase, ``19-kDa
membranes,'' which lack cytoplasmic loops of the
subunit,
whereas membrane-embedded fragments (a COOH-terminal 19 kDa and three
fragments of 8.1-11.7 kDa) containing transmembrane segments and
extracellular loops are intact. The
subunit is partially split
into NH
- and COOH-terminal fragments of 16 and
50 kDa,
respectively. Cation occlusion and ouabain binding are preserved. The
19-kDa membranes were incubated, at 37 °C, with a selection of
proteases, in the presence of Rb
ions. In these
conditions, only
-chymotrypsin destroyed the ability to occlude
Rb
ions. This process was associated with truncation
of the 16-kDa fragment of the
subunit in two stages. In the first
stage, chymotrypsin removed 10 residues from the 16-kDa fragment to
form a 15-kDa fragment (NH
-terminal Ile
) and 4
or 6 residues from the NH
terminus of the
subunit
fragment beginning at Asp
. In these membranes
Rb
occlusion was still intact at 37 °C.
Strikingly, however, deocclusion of two Rb
ions, which
is characteristically biphasic in 19-kDa membranes, displayed
deocclusion kinetic with mainly one fast phase. These membranes also
showed a much lower affinity for Rb
ions compared with
19-kDa membranes; and, consistent with the lower Rb
affinity, Rb
ions, at nonsaturating
concentrations, protected less well against thermal inactivation of
Rb
occlusion. In the second stage, the 15-kDa fragment
was truncated further to a 14-kDa fragment (NH
-terminal
Leu
), followed by thermal destabilization of Rb
occlusion even at high concentrations of Rb
ions. Eventually, the thermally inactivated complex of fragments
of
and
subunits was digested to the limit peptides. The
results suggest that the cytoplasmic domain of the
subunit
interacts with that of the
subunit, possibly with residues
leading into the first transmembrane segment, and controls access of
Rb
ions into or out of the occlusion sites.
Na/K-ATPase represents one of a family of active cation
transport systems, the P-type pumps. Na/K-ATPase consists of (112
kDa) and
(33 kDa) subunits in an equimolar ratio and also a
subunit of unknown function (6.5 kDa). The
subunit of gastric
H/K-ATPase shows the highest degree of homology with the
subunit
of Na/K-ATPase, and H/K-ATPase is the only other P-type pump known to
have a
subunit. The
subunits of Na/K- and H/K-ATPase are
thought to contain 10 transmembrane segments (for recent evidence, see
Goldshleger et al.(1995)) and the functional sites for ATP and
cations and sites for the cardiac glycosides on Na/K-ATPase or
K
-competitive inhibitors of H/K-ATPase (Glynn and
Karlish, 1990; Lingrel and Kuntzweiler, 1994; Sachs et al.,
1995).
The subunit with a single transmembrane segment, three
conserved S-S bridges, and three glycosylation sites (seven for
H/K-ATPase
), is an essential component of the functional pump
unit, as shown by expression of
and
subunits in oocytes,
yeast, insect, and animal cells (Noguchi et al., 1987;
Horowitz et al., 1990; McDonough et al., 1990; Blanco et al., 1994; for a recent review see Chow and Forte(1995)).
One major role of the
subunit is stabilization of the
subunit in a functional conformation and facilitation of transfer from
the endoplasmic reticulum to the plasma membrane (Geering, 1991). An
important functional role has been inferred from observations that
different isoforms of the
subunit (
1,
2,
3), or
the H/K-ATPase
subunit, are partially or wholly interchangeable
and have significant effects on the apparent affinity of extracellular
potassium or cytoplasmic sodium for activating ATPase activity (Jaisser et al., 1992, 1994; Blanco et al., 1994, 1995) or
modulating ouabain binding (Eakle et al., 1992, 1994, 1995).
Further evidence comes from the finding that DTT (
)inactivates cation occlusion of renal Na/K-ATPase
presumably due to reduction of the extracellular S-S bridge(s)
(Lutsenko and Kaplan, 1993).
Segments involved in subunit assembly
and functional effects include 26 or fewer residues in the
extracellular loop between M7 and M8 of the subunit (Lemas et
al., 1992, 1994; Fambrough et al., 1994; Shin and Sachs,
1994). In the extracellular domain of the
subunit the region near
the first S-S bridge is thought to interact with the
subunit
(Renaud et al., 1991; Hamrick et al., 1993; Fambrough et al., 1994; Noguchi et al., 1994; Lutsenko and
Kaplan, 1994; Goldshleger et al., 1995), but hydrophobic
residues at the COOH-terminal domain of the
subunit (Beggah et al., 1993) or a conserved proline residue in the loop of
the third S-S bridge (Geering et al., 1993) are also
important, possibly via indirect conformational effects. Two studies
show clearly that the cytoplasmic domain and/or the transmembrane
segment of the
subunit is important for assembly and stability of
functional complexes (Eakle et al., 1994; Jaunin et
al., 1993). Obviously, several regions of subunit interactions are
likely to exist and remain to be identified.
This paper describes an
application of proteolytic digestion to investigate a role of the
subunit in modifying the kinetics of cation occlusion. The experiments
utilize ``19-kDa membranes'' produced by extensive tryptic
digestion of pig kidney Na/K-ATPase, in the presence of Rb
or Na
ions and absence of Ca
ions (Karlish et al., 1990; Capasso et al.,
1992). 19-kDa membranes lack most of the cytoplasmic domain of the
protein, whereas the transmembrane segments and extracellular loops are
intact (Karlish et al., 1990; Capasso et al., 1992).
19-kDa membranes consist of a 19-kDa fragment,
Asn
-Tyr
, corresponding to
transmembrane segments M7-M10, and five smaller peptides
containing pairs of the other transmembrane segments of the
subunit (11.7, 11.3, and 10.9 kDa, NH
-terminal
Asp
, M1/M2; 8.5 kDa, NH
-terminal
Ile
, M3/M4; 8.1 kDa, NH
-terminal
Gln
, M5/M6). The
subunit is intact or is partially
cut to a fragment of 16 kDa (NH
-terminal Ala
),
extending from the cytoplasmic domain via the transmembrane segment to
the loop between the first extracellular S-S bridge, and a glycosylated
fragment of about 50 kDa (NH
-terminal Gly
)
corresponding to the remaining COOH-terminal domain. In 19-kDa
membranes, Rb
and Na
occlusion and
ouabain binding are fully preserved, but ATP-dependent functions are
lost (Karlish et al., 1990; Capasso et al., 1992;
Schwappach et al., 1994).
Direct evidence for a role of the
subunit in cation occlusion would be obtained if it could be
demonstrated that a defined proteolytic split in the
subunit
causes a defined change in kinetics of cation occlusion. However, such
evidence is hard to obtain because 19-kDa membranes are highly
protected against proteases in the presence of Rb
ions
(Capasso et al., 1992), whereas the occlusion is rapidly
thermally inactivated in the absence of occluded Rb
(Or et al., 1993; Shainskaya and Karlish, 1994).
Previous, indirect evidence, includes the finding that potassium or
sodium ions protect the 16-kDa fragment of the
subunit against
proteases. This result led Capasso et al.,(1992) to discuss a
possible role of the
subunit in cation occlusion, probably via
conformational interactions with the
subunit. More recently, we
have demonstrated that dissociation of occluded Rb
ions leads to digestion of all fragments of
and
subunits to limit membrane-embedded peptides (Shainskaya and Karlish,
1994). The cytoplasmic sequence Ala
-Arg
and the extracellular sequence
Asp
-Arg
, removed upon further digestion of
the 16-kDa fragment of the
subunit, were suggested to be
candidates for interactions with the
subunit. We now report that
in the presence of Rb
ions,
-chymotrypsin digests
19-kDa membranes and destroys Rb
occlusion, as a
result of selective splits of the 16-kDa fragment of the
subunit.
The experiments suggest that interactions of the cytoplasmic domains of
the
and
subunit control access of Rb
ions
into and out of the sites and so affect the observed kinetics of cation
occlusion and deocclusion.
Na/K-ATPase was prepared from fresh pig kidney red outer medulla by the rapid procedure described by Jørgensen(1974). Protein, by the method of Lowry, and ATPase activity were determined as described by Jørgensen(1974). Specific activity was in the range of 13-20 units/mg of protein. Before use, the enzyme was dialyzed at 4 °C against 1,000 volumes of a solution containing 25 mM histidine, pH 7.0, and 1 mM EDTA (Tris). Standard conditions for preparation of tryptic 19-kDa membranes were described by Capasso et al.(1992). After digestion, membranes were washed, suspended, and stored in a standard medium containing 25 mM imidazole, pH 7.5, 1 mM EDTA, to which 2 mM RbCl was added.
Prior to SDS-polyacrylamide gel electrophoresis, pellets were resuspended in standard medium, dissolved with 4% SDS, and protein was precipitated by the addition of ice-cold methanol (4:1 v/v) and stored overnight at -20 °C. The delipidated protein was collected by centrifugation for 30 min at 10,000 rpm in a Sorvall centrifuge, dried in a stream of nitrogen, and dissolved in 10% SDS or the gel buffer.
Figure 1:
SDS-polyacrylamide gel electrophoresis
of 19-kDa membranes treated with different proteases. 19-kDa membranes
were suspended in the standard medium at pH 8.0 with 10 mM RbCl added. The membranes were incubated for 1 h at 37 °C with
different proteases (1:5 w/w). The mixture of inhibitors was added, and
samples were withdrawn for measurement of Rb occlusion. The membranes were washed as described under
``Experimental Procedures.'' Equivalent amounts of
delipidated protein (
100 µg) were applied per lane of a 16.5%
Tricine gel. Lane 1, control 19-kDa membranes; lane
2, clostripain; lane 3, trypsin; lane 4,
thermolysin; lane 5, carboxypeptidase B; lane 6,
chymotrypsin; lane M, marker
peptides.
Figure 2:
Time course of inactivation of
Rb occlusion on 19-kDa membranes treated with
chymotrypsin. 19-kDa membranes (2 mg/ml) were incubated at 37 °C in
the standard medium, pH 8.0, containing also 10 mM RbCl +
Rb
, and chymotrypsin 1:1 (
) or 1:5
(
) (w/w). At times from 1 min to 1.5 h samples were transferred to
the Dowex 50 columns.
A characteristic time course of the fragmentation pattern upon
chymotryptic digestion is presented in Fig. 3A.
Eventually the 19-kDa fragment and other smaller fragments are
digested, and Rb occlusion disappears (Fig. 3A, 90 min; see also Fig. 1, lane
6, and Fig. 6A). The chymotryptic destruction of
occlusion is correlated with disappearance of the 19-kDa fragment, as
was found previously for destruction of occlusion by trypsin in the
presence of Ca
(Karlish et al., 1990).
However, the fragments appearing prior to loss of occlusion provided
more information on the nature of events leading to destruction of
occlusion. Identification of intermediate fragments by
NH
-terminal sequencing is presented in Table 1, and
these are also depicted visually in Fig. 9A. The
striking finding in Fig. 3A is that the 16-kDa fragment
of the
subunit (NH
-terminal Ala
; Capasso et al.(1992)) was progressively digested to a fragment a, of
15 kDa with NH
-terminal Ile
(Table 1) i.e. by removal of 10 residues from the NH
terminus. At the end of the lag period (Fig. 3A,
20 min), when Rb
occlusion was still intact, most of
the 16-kDa fragment had been truncated to the 15-kDa fragment. Upon
further digestion, and loss of Rb
occlusion (30 and 90
min), the 15-kDa fragment was truncated further to a fragment of 14 kDa
(NH
-terminal Leu
), and then the 19-kDa and all
other fragments of the
subunit were digested. Note that the band
at 11.7 kDa was also progressively digested to the smaller fragments of
11.3 and 10.9 kDa, fragment f, in parallel with truncation of the 16-
to the 15-kDa fragment of the
subunit. The 11.7-kDa fragment has
NH
-terminal Asp
and contains the M1/M2
transmembrane segments (see Capasso et al.(1992)), whereas
fragment f (seen also in Fig. 3B) was found to be a
mixture of two peptides, in roughly equal amounts, having
NH
-terminal Ala
and Thr
(see Table 1). Of the other smaller peptides (8-12 kDa), the
fragment of 8.1 kDa disappeared within 1 min, and two fragments, marked
d and e, of 7.8 and 7.3 kDa, respectively, appeared. Fragment d,
NH
-terminal Ala
, is a truncated form of the
8.1-kDa fragment (NH
-terminal Gln
) containing
the M5/M6 transmembrane segments of the
subunit (Capasso et
al., 1992). Fragment e is a fragment of a water channel protein
from erythrocytes and proximal tubule (Preston and Agre, 1991) and
represents a contaminant in the preparation. However, these very early
splits are unrelated to inactivation of occlusion (see
``Discussion''). The fragments of 9.5 kDa (
subunit) and
8.7 kDa (M3/M4) were not cut. Fragment c of 13.5 kDa is a contaminant
fragment of chymotrypsin and was not seen in all experiments (see Fig. 6).
Figure 3:
Electrophoretic profile of digestion of
19-kDa membranes with chymotrypsin. Panel A, 37 °C; panel B, 20 °C. 19-kDa membranes (2 mg/ml) were incubated
with chymotrypsin (1:5 w/w) in the standard medium, pH 8.0, and 10
mM RbCl. At times from 1 min to 1.5 h, as indicated, aliquots
were withdrawn, the reaction was stopped, and membranes were washed
twice with 150 mM KCl as described under ``Experimental
Procedures.'' Equal amounts of delipidated protein (100
µg) were applied per lane of a 16.5% Tricine gel. Incubation times
with chymotrypsin and Rb
occlusion (% of control) were
as follows. In panel A: first lane, control 19-kDa
membranes; second lane, 1 min/100%; third lane, 5
min/100%; fourth lane, 10 min/100%; fifth lane, 20
min/100%; sixth lane, 30 min/51%; seventh lane, 90
min/14%. In panel B: first lane, control 19-kDa
membranes; second lane, 1 h/100%. M, molecular mass
markers.
Figure 6:
Panel A, detailed time course of digestion
of chymotryptic intermediate with chymotrypsin at 37 °C. Panel
B, levels of 15- and 14-kDa fragments compared with inactivation
of Rb occlusion. Chymotryptic intermediate (1.25
mg/ml) membranes were incubated with chymotrypsin (1:1, w/w) in the
standard medium, pH 8.0, with 20 mM RbCl. At times from 1 min
to 1.5 h aliquots were withdrawn, membranes were treated as in Fig. 3, and
100-µg aliquots of delipidated protein were
applied per lane of a 16.5% Tricine gel.
Figure 9:
Models depicting chymotryptic splits in
fragments of 19-kDa membranes and accompanying effects on Rb deocclusion. Panel A, arrows depict positions
of the splits in stages 1 and 2. Panel B, loss of ordered
deocclusion of Rb
ions accompanying stages 1 and 2,
depicted for simplicity as a widening of a
pocket.
Fig. 3B shows that digestion at 20 °C with a high ratio of chymotrypsin to 19-kDa membranes (1:5 or even 1:1 w/w) led to quantitative conversion of the 16-kDa to the 15-kDa fragment and also conversion of the M1/M2-containing 11.7-kDa fragment to the smaller 10.9-kDa fragment, peptide f; i.e. the electrophoretic profile is similar to that at the end of the lag phase at 37 °C (Fig. 3A, 20 min). In this condition there was no loss of occlusion, and at 20 °C the 15-kDa fragment was stable for hours upon application of chymotrypsin, and occlusion was maintained (data not shown). A similar pattern was obtained at 37 °C using a low ratio of chymotrypsin to 19-kDa membranes (1:40 w/w) for 1 h (not shown).
The availability of a stable form (Fig. 3B), referred to as the chymotryptic
intermediate, has allowed us to compare some properties of the
Rb occlusion in these membranes with those of the
19-kDa membranes. Since digestion experiments indicated a difference in
behavior at 20 and 37 °C, it was of interest to investigate
possible temperature-dependent differences in properties of
Rb
occlusion.
Measurements of Rb occlusion with varying Rb
concentrations at 37
°C showed a large difference in apparent K
between 19-kDa membranes (24-35 µM in
different experiments) and the chymotryptic intermediate (211.1
± 62.9 µM), whereas no difference was observed at
20 °C (31.6 ± 3.9 and 35.0 ± 4.3 µM)
(experiments not shown). A possible complication with such experiments
at 37 °C is that occlusion is partially thermally inactivated, at
nonsaturating concentrations of Rb
(Or et
al., 1993 and see below). An alternative approach, which is free
of this complication, was to compare dissociation of occluded
Rb
ions, at 37 and 20 °C, into media
containing nonradioactive Rb
ions (Fig. 4, A and B). At 20 °C (Fig. 4A)
deocclusion is markedly biphasic as reported previously (Karlish et
al., 1990), and again, there was essentially no difference between
19-kDa membranes and the chymotryptic intermediate. At 37 °C (Fig. 4B), dissociation from 19-kDa membranes remains
strongly biphasic, with fitted rates of 0.52 s
and
0.017 s
and equal amplitudes of the two phases (0.52
and 0.48) respectively. For the chymotryptic intermediate, at 37
°C, dissociation of
Rb
was fitted to
a biphasic curve with a rate constant of 1.14 s
and
0.014 s
and amplitudes of 0.86 and 0.14
respectively. Thus,
Rb
deocclusion from
the chymotryptic intermediate is characterized by a large increase in
the amplitude and rate of the first phase, so that the slow phase is
much reduced in amplitude (see ``Discussion'' and Fig. 9A). This phenomenon is consistent with a large
decrease in Rb
affinity at 37 °C for the
chymotryptic intermediate (see ``Discussion'').
Figure 4:
Rate of Rb
deocclusion from 19-kDa membranes and the chymotryptic intermediate. Panel A, 20 °C; panel B, 37 °C. 19-kDa
membranes (
) or the chymotryptic intermediate (
) (20
µg of protein) were used; see ``Experimental
Procedures.'' In panel B, the lines represent
the theoretical curves for double exponential decays, using the best
fit parameters quoted under
``Results.''
Another
way of demonstrating the decrease in Rb affinity of
the chymotryptic intermediate utilized a phenomenon of thermal
inactivation of Rb
occlusion in 19-kDa membranes and
protection by occluded cations such as Rb
or
Na
ions (Or et al., 1993; Shainskaya and
Karlish, 1994). The experiment in Fig. 5compared the thermal
stability at 37 °C of Rb
occlusion on 19-kDa
membranes and the chymotryptic intermediate, in the absence of
Rb
ions, at a very high Rb
concentration (30 mM) or at a nonsaturating
Rb
concentration (0.125 mM). The result is
that in the absence of Rb
ions, 19-kDa membranes and
the chymotryptic intermediate are inactivated at the same rate. At the
high Rb
concentration, 30 mM, both
preparations are fully protected. At the intermediate Rb
concentration (0.125 mM) the chymotryptic intermediate
is significantly more sensitive to thermal inactivation.
Figure 5:
Thermal stability at 37 °C of 19-kDa
membranes and chymotryptic intermediate, with different concentrations
of Rb ions. 19-kDa membranes (
,
,
)
or chymotryptic intermediate (
,
,
) (1 mg/ml) were
incubated at 37 °C in the standard medium, pH 7.5, without
Rb
, with 0.125 mM RbCl, or with 30 mM RbCl. At a time from 1 min to 1 h, 10-µl samples were
withdrawn and mixed with 10 µl of an ice-cold standard reaction
medium containing 5 mM RbCl +
Rb
. After 1 h at 0 °C, samples were
transferred to room temperature for 30 min and were then transferred to
Dowex columns for measurement of occluded
Rb
.
A more detailed time course of the profile of chymotryptic fragments identified later steps in the digestion (Fig. 6). Since the 15-kDa fragment is stable at 20 °C but not at 37 °C membranes digested at 20 °C as in Fig. 3B were subjected to a second chymotryptic digestion at 37 °C (Fig. 6A). Note that although eventually, the 15-kDa fragment was digested to the 14-kDa fragment, and occlusion was destroyed (Fig. 6A, 60 min), after 1 and 2 min the 15-kDa fragment appeared to undergo partial conversion to the 14-kDa fragment, while occlusion was fully intact. Quantification of the ratios of 15- to 14-kDa fragments by scanning the gel (Fig. 6B) showed that conversion of the 15- to the 14-kDa fragment preceded destruction of occlusion, about 30% of the 15-kDa fragment being converted to the 14-kDa fragment before occlusion began to drop.
A strong hint as to order and
mechanism of later steps in the destruction of occlusion was obtained
in the experiment of Fig. 7. We compared thermal stability, at 5
mM RbCl and 37 °C, of the chymotryptic intermediate with
that of a preparation redigested for 2 min (as in Fig. 6A), so that about 30% of the 15-kDa fragment is
converted to the 14-kDa fragment, but Rb occlusion is
still 100% intact or redigested for 10 min when 58% of occlusion
remained. In the latter two preparations, a fraction of the occlusion
is distinctly more thermolabile than that of the chymotryptic
intermediate. The remaining fraction was inactivated at about the same
rate as the control. The result in Fig. 7could be expected if
the more labile fraction represented those molecules in which the
15-kDa fragment has been truncated further to the 14-kDa fragment (see
``Discussion'' and Fig. 9B).
Figure 7:
Comparison of thermal stability of
chymotryptic intermediate and membranes redigested with chymotrypsin.
Chymotryptic intermediate () membranes (1 mg/ml) were
preequilibrated at room temperature for 30 min in the standard medium,
pH 7.5, containing 20 mM RbCl. Aliquots were transferred to 37
°C, and chymotrypsin (1:5, w/w) was added. After 2 (
) and 10
(
) min the reaction was stopped by the addition of ice-cold
reaction medium, containing 1.5 mM RbCl +
Rb
, 100 mM Tris
HCl, pH
7.5, 1 mM PMSF, and 0.2 mM TPCK. After 1 h at 0
°C, samples were incubated at room temperature for 20 min and then
transferred to 37 °C. At a time from 5 min to 1 h, 20-µl
aliquots were transferred to Dowex 50 columns for measurement of
Rb
occlusion.
An additional indication for interactions between
the subunit and the
subunit has come from an observation of
effects of ouabain. Ouabain is known to decrease greatly the rate of
deocclusion of Rb
ions from native Na/K-ATPase
(Forbush, 1983) and as shown recently also from 19-kDa membranes. (
)Fig. 8A demonstrates a similar effect of
ouabain on Rb
deocclusion from the chymotryptic
intermediate at 37 °C. Deocclusion in the presence of ouabain is a
double exponential with rate constants of 0.23 and 0.025
s
, with equal amplitudes for the two phases compared
with the fit without ouabain to rate constants of 0.81 s
and 0.016 s
and amplitudes 0.76 and 0.2,
respectively (seen also in Fig. 4B). An alternative way
of demonstrating that ouabain greatly decreased the rate of
Rb
deocclusion comes from a finding that 2 mM ouabain stabilized the chymotryptic intermediate to thermal
inactivation of Rb
occlusion, at 2 mM Rb
(Fig. 8B). Thus, ouabain,
which is thought to be bound to the
subunit, appears to be able
largely to reverse the functional changes characteristic of the
chymotryptic intermediate.
Figure 8:
Effects of ouabain on the chymotryptic
intermediate. Panel A, Rb
deocclusion. Panel B, thermal stability of Rb
occlusion. Panel A, 20 µl of chymotryptic
intermediate (20 µg of protein) was preequilibrated for 40 min at
20 °C in a standard medium, containing 50 µM of
Rb
, with (
) and without (
)
1 mM ouabain. 20 µl of the medium, containing 3 mM RbCl at 37 °C, was added. At times from 0 to 10 min samples
were transferred to Dowex 50 columns for measurement of occluded
Rb
. The lines represent the theoretical
curves for double exponential decays, using the best fit parameters
quoted under ``Results.'' Panel B, chymotryptic
intermediate membranes (1 mg/ml) were preequilibrated at 20 °C for
40 min in the standard medium, pH 7.5, and 2 mM RbCl +
Rb
with (
) and without (
) 2
mM ouabain. Tubes were then incubated at 37 °C. At times
from 3 min to 3 h samples were transferred to Dowex 50
columns.
The
earliest event is truncation of the 8.1-kDa fragment
(NH-terminal Gln
, containing M5/M6) to 7.8
kDa (NH
-terminal Ala
) (Fig. 3, A and B, 1 min; see Fig. 9A). However, this
split is not correlated with loss of occlusion or the end of the lag
period. Furthermore, a control experiment showed that after 1 min of
chymotryptic digestion, the thermal stability of Rb
occlusion at 37 °C and nonsaturating Rb
is
unchanged (result not shown). The 7.8-kDa fragment is similar to a
fragment (NH
-terminal Ser
) described earlier
(Karlish et al., 1991), which was produced by digestion of
renal Na/K-ATPase with trypsin untreated with TPCK, i.e. it
was generated by traces of chymotrypsin in the trypsin. This truncation
also had no effect on Rb
occlusion (Karlish et
al., 1990).
The first stage, prior to loss of occlusion, is
associated with truncation of the 16-kDa fragment of the subunit
to the 15-kDa fragment by removal of 10 residues, AKEEGSWKKF, and also
by truncation of the 11.9- and 11.3-kDa fragments of the
subunit
(M1/M2) by removal of 4 or 6 residues, DGPN or DGPNAL, to produce
overlapping 10.9-kDa fragments (Fig. 9A). Which of
these splits is necessary for inactivation of occlusion, and are the
two splits related? The specific chymotryptic split of the
subunit between Phe
and Ile
appears to be an
essential first step, for there is prior evidence that a proteolytic
split at the Asn
-Ala
bond is not a
prerequisite for inactivation of Rb
occlusion. In the
presence of magnesium, P
, and ouabain, trypsin truncates
the 11.7-kDa fragment to a fragment with NH
-terminal
Ala
, without inactivating Rb
occlusion
(Lutsenko and Kaplan, 1994). In Rb
-containing media
with added Ca
ions, trypsin gains access and splits
the Asn
-Ala
bond only when
Rb
occlusion is already thermally inactivated
(Shainskaya and Karlish, 1994). There are two hints that the
chymotryptic splits of the 16- and 11.7-kDa fragments are connected.
First, these two fragments are digested with a similar time course (Fig. 3A). Second, a comparison of the tryptic and
chymotryptic digestion is suggestive of the connection. As seen in Fig. 1, trypsin truncated the 11.7- and 11.3-fragments to a
10.9-kDa fragment, with the same NH
-terminal residue,
Asp
, i.e. it cut at the COOH-terminal side, in
the presence of Rb
ions. By contrast to the result
with chymotrypsin, the 16-kDa fragment of the
subunit was not cut
by trypsin, and Rb
occlusion was not affected. The
specific chymotryptic split between Phe
and Ile
of the
subunit may facilitate the truncation of the M1/M2
fragment between Asn
and Ala
, which is a
nonclassical chymotryptic split site as well as between Leu
and Thr
. (The converse hypothesis, that chymotrypsin
first truncates M1/M2 and as a result the
Phe
-Ile
bond of the
subunit
becomes accessible, is less likely because the split between Asn
and Ala
is unspecific and could be expected to occur
with trypsin or other proteases in the same conditions.)
The
chymotryptic intermediate showed a decrease in apparent affinity for
Rb and faster deocclusion rate, particularly from the
second site, at 37 °C but not at 20 °C (Fig. 4).
Biphasic deocclusion kinetics reflects an ordered release of
Rb
from the two sites in a narrow pocket
(Glynn et al., 1985) or one that is intermittently open
because of the presence of a flickering gate (Forbush, 1987). The
result is that the nonradioactive Rb
in the medium is
able to block dissociation of the deeper of the two
Rb
ions. For the chymotryptic
intermediate, at 37 °C, the medium Rb
is largely
unable to block dissociation of the inner Rb
ion, and
thus both Rb
ions dissociate rapidly and randomly.
This phenomenon is depicted in Fig. 9B, step 1, as a
result of a wider pocket, but it might also be depicted as the result
of more frequent openings of a gate. The lower Rb
affinity of the chymotryptic intermediate is also reflected in
the faster rate of thermal inactivation of occlusion at nonsaturating
concentrations of Rb
ions (Fig. 5). Thus,
Rb
ions protect the chymotryptic intermediate less
well than 19-kDa membranes against thermal inactivation at 37 °C,
even though the intrinsic thermal stability of the two membranes in the
absence of Rb
is quite similar (Fig. 5).
Protection by ouabain against thermal inactivation, associated with a
reduced dissociation of occluded Rb
(Fig. 8A), provides strong support for this
mechanism. At a high Rb
concentration (30 mM in Fig. 5and also 10 mM) the chymotryptic
intermediate is stable at 37 °C, i.e. thermal instability
is not the immediate cause of destruction of occlusion. At least one
additional chymotryptic split is required to destroy Rb
occlusion at 37 °C.
The second stage of digestion appears
to be further truncation of the 15-kDa to the 14-kDa fragment. The
Phe-Leu
bond may become accessible to
chymotrypsin at 37 °C but not at 20 °C, because of loss of a
stabilizing interaction between the 15-kDa fragment and other domains
of the protein. The key observation is that at 37 °C, the membranes
with the 15-kDa fragment partially truncated to 14 kDa, but with intact
Rb
occlusion, display a fraction of thermally unstable
molecules compared with the chymotryptic intermediate, even at a high
Rb
concentration (Fig. 7). Presumably, if it
were possible to produce an intermediate consisting of only 14-kDa
fragments, a much higher fraction of thermally unstable occlusion might
be observed. The Rb
affinity in those molecules may be
even lower than in the chymotryptic intermediate (Fig. 9B, step 2).
A third and final stage appears
to involve irreversible thermal inactivation of Rb occlusion and then digestion of all fragments in the membranes.
The process is similar to that we have described for digestion of
19-kDa membranes by trypsin following displacement of occluded
Rb
by Ca
ions (Shainskaya and
Karlish, 1994). Displacement of occluded Rb
ions leads
to thermal inactivation of Rb
occlusion, loss of
Rb
-dependent stabilizing interactions, and
disaggregation of the complex of fragments, and exposure of all the
extramembrane tails and loops of
and
subunits to trypsin.
The peptide products of the chymotryptic digestion were not analyzed in
detail, but the overall picture (Fig. 3A, 90 min, or Fig. 6A, 60 min) is quite similar to that observed for
the Ca
/trypsin digestion. Eventually the 19-kDa
fragment is digested to small peptides of 3-4 kDa containing
single transmembrane segments, while the peptides of 8-11 kDa
containing M1/M2, M3/M4, and M5/M6 are truncated at either or both
NH
- and COOH-terminal tails to peptides of 6-8 kDa,
still containing the pairs of transmembrane segments. The present
results are in line with our previous conclusions that maintenance of
occlusion requires an intact 19-kDa fragment, and the fragments in
19-kDa membranes exist in a Rb
-stabilized complex
(Karlish et al., 1990; Shainskaya and Karlish, 1994).
The
proposed mechanism whereby a change in Rb affinity
causes dissociation of Rb
ions and then thermal
inactivation due to loss of stabilizing interactions may be applicable
to other structural modifications. A case in point is inactivation of
Rb
occlusion in 19-kDa membranes by DTT (Lutsenko and
Kaplan, 1993). In the experiments of Lutsenko and Kaplan(1993)
inactivation by DTT occurred in the presence of 2 mM Rb
, whereas 25 mM Rb
ions protected partially. (The chymotryptic intermediate displays
a similar sensitivity to DTT, and Rb
ions partially
protect (experiment not shown).) Rb
occlusion in
19-kDa membranes is saturated at 2 mM Rb
(K
40 µM; Karlish et
al.(1990)), and hence the requirement for 25 mM Rb
for protection implies that reduction of the
S-S bridge(s) decreased the affinity for Rb
but did
not itself inactivate occlusion. However, dissociation of Rb
ions at 2 mM should then lead to irreversible thermal
inactivation. This mechanism is consistent with the proposal that
reduction of S-S bonds in the
subunit inactivates the enzyme by
causing delocalized denaturation of the
subunit (Kirley, 1990).
It is less easily reconciled with Lutsenko and Kaplan's
proposal(1993) that the extracellular portion of the
subunit
directly gates cation occlusion, closing access to the sites by
interacting with the intramembrane portion.
How might a
structural interaction between the cytoplasmic domains of the and
subunits affect gating of the occlusion domain, so that
relatively minor structural modification associated with the
chymotryptic digestion produces the subtle functional changes seen in Fig. 4, 5, 7? One possibility is that the cytoplasmic domains of
the
and
subunits interact directly within the occlusion
domain at the flickering gate (Forbush, 1987), i.e. the
NH
-terminal domain of the
subunit constitutes a
component of the gate. This is reminiscent of the ball-and-chain
mechanism suggested for inactivation of potassium channels (see Jan and
Jan(1992)). Alternatively, the gate may lie deeper within the
transmembrane segments, so that disruption of interactions of
cytoplasmic domains of the
and
subunits affects deocclusion
less directly, by loosening the interactions between transmembrane
segments. The indirect mechanism appears to be somewhat favored by the
finding that ouabain decreases the rate of Rb
deocclusion on the chymotryptic intermediate and restores the
ordered release phenomenon (Fig. 8). Ouabain is bound in the
subunit and is thought to interact with residues in several
extracellular loops of the
subunit (Price and Lingrel, 1988;
Lingrel et al., 1991; Canessa et al., 1993; Shultheis et al., 1993; Blostein et al., 1993) and also with
residues within the M1 transmembrane segment (Arystarkhova et
al., 1992; Canessa et al., 1992; Shultheis et
al., 1993; Antolovic et al., 1995).
Independent
evidence for a functional role of the cytoplasmic domain of the
subunit has been obtained recently (Jaisser et al., 1996). Xenopus
1 subunits truncated at the NH
terminus were expressed with Bufo
1 subunit in Xenopus oocytes. Truncation of 34 residues, at a position equivalent to
Trp
in the pig
sequence, induced a large decrease of
the apparent affinity for extracellular potassium. Truncation at
positions equivalent to Gly
or Asn
in the pig
sequence had no effect. Chow and Forte (1993) have reported that
an inhibitory monoclonal antibody, recognizing epitopes on the
cytoplasmic surface of the
subunit of H/K-ATPase, affects
potassium activation of p-nitrophenol phosphatase, suggesting
again a role for this domain in modulating potassium activation of
H/K-ATPase.