From the
The roles of 1) inactivation of Na-Ca+K exchange and 2)
Ca
Ca
Most of our
knowledge on Na-Ca or Na-Ca+K exchangers is derived from studies
of macroscopic Ca
All experimental procedures employed in this study have been
described in detail elsewhere and are only briefly outlined here.
Low doses of A23187 (<200
nM) selectively permeabilize the disc membranes and release
intradiscal Ca
In darkness, a kinetic equilibrium between Ca
At least two factors other
than Ca
I gratefully acknowledge the expert technical
assistance of Robert T. Szerencsei.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
release from discs in regulation of cytosolic free
Ca
were examined in intact rod outer segments (ROS)
purified from bovine retinas. Measurements of cytosolic free
Ca
(with fluo-3) were combined with Ca
flux measurements (
Ca) in ROS that contained about
600 µM total Ca
.
Na
-induced Ca
extrusion was measured
in a Ca
-free medium and did not lower cytosolic free
Ca
to below 1 nM as expected from a coupling
stoichiometry of
4Na
:(1Ca
+1K
).
Instead, cytosolic free Ca
was rapidly (20 s) lowered
from about 1300 nM to 100-150 nM, while at the
same time about 35% of total ROS Ca
was removed.
During the next 40 min cytosolic free Ca
remained
virtually steady, but total ROS Ca
was reduced by a
further 50% at a 100-fold lower rate than that observed for the initial
fast phase. The steady cytosolic Ca
concentration
resulted from Ca
release from discs and subsequent
removal across the plasma membrane by Na-Ca+K exchange operating
at a greatly reduced rate. Addition of the alkali cation channel
ionophore gramicidin led to a persistent increase in cytosolic free
Ca
concentration to about 400 nM, presumably
caused by an increase in intracellular Na
. It is
suggested that cytosolic free Ca
is not determined by
the Na
:Ca
coupling ratio of the
exchanger, but rather by a sensor on its cytoplasmic domain that
controls inactivation of the Ca
extrusion mode and is
sensitive to intracellular Ca
, Na
,
and K
.
homeostasis in retinal rod outer segments
(ROS)
(
)
is thought to be dominated by dynamic
fluxes across the plasma membrane: Ca
influx is
mediated by the light-sensitive cGMP-gated channels, and Ca
efflux is carried out by the Na-Ca+K exchanger
(1) .
In darkness, a kinetic equilibrium between the above two Ca
fluxes keeps cytosolic relatively high. Light causes a decrease
in Ca
influx, while continued Ca
efflux via the exchanger lowers cytosolic Ca
,
and this is thought to mediate, at least in part, the process of light
adaptation in both retinal rods and cones
(2, 3) . The
coupling stoichiometry of the ROS Na-Ca+K exchanger has been shown
to be 4Na:(1Ca+1K)
(4, 5) , from which, under
physiological conditions, a thermodynamic equilibrium free
Ca
concentration of below 1 nM can be
calculated
(6) . Therefore, the Na-Ca+K exchanger is
expected to lower cytosolic free Ca
below 1
nM when Ca
influx via the cGMP-gated
channels is interrupted for a prolonged time, e.g. when rods
are saturated under bright daylight illumination.
fluxes or exchange currents when
these exchangers operate far from thermodynamic equilibrium. In the
absence of cGMP and cGMP-gated Ca
fluxes, the
Na-Ca+K exchanger appears to be the only cation transporter
operating in the ROS plasma membrane
(7, 8, 9) .
In three previous studies we have loaded the cytosol of purified bovine
ROS with the fluorescent Ca
-indicating dye fluo-3 and
combined measurements of changes in cytosolic free Ca
with
Ca flux
measurements
(10, 11, 12) . Unexpectedly, it
proved difficult to lower free cytosolic Ca
below
50-100 nM upon stimulation of the Ca
efflux mode of the exchanger, even if thermodynamic
considerations would predict a free Ca
concentration
of less than 1 nM, and despite the ability of the
Ca
influx mode of the exchanger to operate at very
low external free Ca
concentrations of 10-20
nM. We concluded that the Ca
extrusion mode
of the exchanger operates in a brief burst (10-30 s) of high
velocity activity followed by a nearly complete inactivation, a novel
feature for Na-Ca(+K) exchangers. As this may present a mechanism
to prevent lowering of cytosolic free Ca
to
undesirably low values when rods are saturated with light for prolonged
periods of time, this study examines 1) the activity of the plasma
membrane Na-Ca+K exchanger on time scales between a few seconds
and 40 min, 2) the contribution of internal Ca
stores
to Ca
homeostasis under those conditions, and 3)
factors that determine at which free Ca
concentration
the Ca
efflux mode of the exchanger inactivates. The
significance of our findings for the mechanism(s) employed by ROS to
set intracellular free Ca
is discussed.
ROS Preparation
Bovine retinas were dissected from fresh
eyeballs obtained at a local abattoir and kept in a light-tight
container. Intact Ca-depleted bovine ROS were
purified on mixed sucrose-Ficoll gradients containing Na
and EDTA and washed with sucrose-EDTA solutions
(13) . ROS
were stored in concentrated suspensions (200-300 µM)
at 4 °C in 600 mM sucrose, 5% w/w Ficoll 400, 20
mM Hepes (pH 7.4), and 0.1 mM EDTA (pH in this and
all other solutions was adjusted to 7.4 with arginine). Up to this
point, all procedures were carried out under dim red illumination.
Fluorescence Measurements of Free Cytosolic
Ca
A suspension of bleached intact and
Ca-depleted bovine ROS (final opsin concentration 100
µM) was incubated for 45 min at room temperature with 50
µM fluo-3AM in a medium containing 400 mM
sucrose, 20 mM Hepes (pH 7.4), 10 mM KCl, 1
mM HEDTA, 0.25 mM CaHEDTA, and
Ca (for
the flux experiments only). The unhydrolyzed fluo ester and
unincorporated
Ca was removed by sedimentation of ROS
through a cushion of 600 mM sucrose, 20 mM Hepes (pH
7.4). Further experimental procedures and calibration procedures were
carried out as described before
(10, 12) . Fluorescence
measurements were carried out in an SLM-Aminco series 2 luminescence
spectrometer (SLM Instruments, Urbana, IL).
Ca fluxes in
ROS were measured with a rapid filtration technique over borosilicate
glass fiber filters as previously described
(8) . Calculations of
total Ca
fluxes assume that bovine ROS contain an
overall rhodopsin concentration of 3 mM or 2.9
10
rhodopsin molecules.
Accessibility of ROS Ca
The objective of the experiments
reported in this study was to analyze compartmentalization of
Ca to Removal by
Na-Ca or Sr-Ca Exchange
fluxes in isolated intact bovine ROS and its
relationship to the observed inability of the Ca
extrusion mode of the ROS Na-Ca+K exchanger to lower free
cytosolic Ca
to the low values expected from the
coupling stoichiometry of
4Na:(1Ca+1K)
(11, 12, 14) . Analysis of both
total internal Ca
, cytosolic free Ca
and internal Ca
sequestration with sufficient
analytical precision necessitated to prepare first ROS that contained
no measurable Ca
(Na
-enriched and
Ca
-depleted ROS were prepared by isolating and
purifying ROS in Ca
-free and
Na
-containing media). Subsequently,
Ca
-depleted ROS were loaded with
Ca
,
Ca and fluo-3, and stored in a
Na
- and Ca
-free medium; the
Ca
extrusion mode of the Na-Ca+K exchanger was
initiated by addition of Na
. A
Ca
-free medium was used to eliminate Ca
influx from the external medium. The above experimental paradigm
is nonphysiological, but it enables us to analyze compartmentalization
of Ca
and Ca
fluxes without
ambiguity. In the first two figures experiments are illustrated that
were designed to establish localization of total ROS Ca
to the cytosol and to internal stores, respectively.
Ca
-depleted ROS were loaded with Ca
(at an external free Ca
concentration of 350
nM) and
Ca to yield moderate Ca
loads of 0.1-0.3 mol Ca
/mol rhodopsin,
equivalent to a total ROS Ca
content of 300-900
µM. Ca
-loaded ROS were stored in a
Na
-free buffered sucrose solution and both cytosolic
free Ca
and total Ca
levels were
stable for 24 h when ROS were stored as a concentrated suspension
(200-300 µM rhodopsin) at 4 °C. Different
aliquots of the same ROS suspension were diluted to a final rhodopsin
concentration of 1-2 µM, NaCl was added to the
external medium to initiate Ca
extrusion via the
Na-Ca+K exchanger and either fluo-3 fluorescence was measured to
monitor cytosolic free Ca
or
Ca
radioactivity was measured to monitor total ROS Ca
.
Addition of NaCl caused a rapid Ca
efflux phase
lasting about 20 s, during which most of the cytosolic Ca
was removed from ROS (free Ca
dropped from 1300
nM to 150 nM), and during which about 40% of total
ROS Ca
was removed (Fig. 1, left
panels). The ROS Na-Ca+K exchanger also executes Ca-Ca
self-exchange fluxes, in which Sr
can replace
Ca
(8, 15) . Sr
can
also replace Ca
in binding to fluo-3, but at a
reduced fluorescence yield. When 0.5 mM SrCl
was
added to Ca
-loaded ROS, a fast Sr-Ca exchange phase
replaced cytosolic Ca
by Sr
and
reduced the fluo-3 fluorescence (Fig. 1). The half-time for both
Na
- and Sr
-induced lowering of
Ca
fluo-3 fluorescence in the ROS cytosol was
4-7 s. Consistent with a rapid removal of cytosolic
Ca
via both Sr-Ca and Na-Ca exchange, both
Sr
and Na
caused a rapid release of
about 40% of the total Ca
present in ROS. Similar
experiments in over 50 Ca
-loaded ROS preparations
with total Ca
loads ranging from 300 µM
to 4 mM showed that consistently 40-50% of total
internal Ca
could be rapidly exchanged against either
Sr
or Na
and was probably localized
to the cytosol.
Figure 1:
Comparison between changes in cytosolic
and total Ca in bovine ROS induced by Na-Ca and Sr-Ca
exchange. Ca
-,
Ca-, and fluo-loaded
intact bovine ROS were prepared as described under ``Materials and
Methods'' and were incubated in 600 mM sucrose, 20
mM Hepes (pH 7.4), 2 mM KCl, 500 nM FCCP,
0.1 mM EDTA at a final opsin concentration of 1.5
µM. Sr
- or
Na
-stimulated Ca
release was
initiated at time 0 by addition of 500 µM SrCl
(open circles) or 50 mM NaCl (inverted
triangles); controls represent changes in
Ca content
(filled circles in top panels) or in free cytosolic
Ca
(bottom panels) without additions to the
incubation medium. Incorporation of
Ca in ROS in this and
subsequent figures is expressed as the percentage of total
Ca present. Temperature, 25
°C.
Subsequent to the first 20 s of rapid Ca release, Sr-Ca exchange caused little further change in
fluorescence during the next 20 min, while Na-Ca exchange gradually
lowered cytosolic Ca
from a value of 150 nM
to 100 nM after 20 min (Fig. 1, bottom right-hand
panel). Therefore, the Na-Ca+K exchanger mediated very little
net Ca
removal from the cytosol in the time interval
between 20 s and 20 min after addition of Sr
and
Na
, respectively. In contrast, slow components were
observed for total Ca
efflux from ROS (measured with
Ca, Fig. 1, top right-hand panel): both
Na-Ca and Sr-Ca exchange removed about 35% of the initial ROS
Ca
content (
Ca at time 0) in the period
between 20 s and 10 min after addition of Na
and
Sr
, respectively, whereas 1 h after addition of
Na
or Sr
still 10-15% of the
initial
Ca content remained (not illustrated).
Localization of Slowly Exchanging Ca
The experiment in
Fig. 1
illustrates that the slow component of both Na-Ca exchange
and Sr-Ca exchange could change the total to
an Internal Membranous Store
Ca content of
ROS without a concomitant change in cytoplasmic Ca
,
suggesting that this slow component represents Ca
removal from some internal compartment, i.e. the
intradiscal space. Two approaches were used to substantiate the above
conclusion, one employing selective permeabilization of disc membranes
by A23187, and the second by selective permeabilization of the plasma
membrane with streptolysin S.
into the cytosol resulting in a rise
in cytosolic free Ca
(12) . Here, the amplitude
of the A23187-induced increase in cytosolic Ca
was
used as an indicator of intradiscal Ca
content as
illustrated in Fig. 2. Superimposed fluo-3 fluorescence traces
are shown of Sr-Ca exchange; these traces were obtained with a series
of separate cuvettes to which 100 nM A23187 was added at
different time points (as indicated by the upward pointing
arrows) after initiation of Sr-Ca exchange at time 0 (just prior
to addition of A23187, 2 mM EDTA was added to chelate all
external Sr
and Ca
and stop Sr-Ca
exchange across the plasma membrane). Initially, addition of A23187
caused a large increase in fluorescence to indicate Ca
release from internal stores that competed with cytosolic
Sr
for binding to fluo-3. The amplitude of the
A23187-induced rise in fluorescence gradually decreased as the interval
between Sr
addition and addition of EDTA/A23187 was
increased indicating progressive loss of Ca
from the
internal store. The time course with which the amplitude of the
A23187-induced fluorescence signals decreased was quite similar to the
time course of the slow phase of Sr
-induced
Ca efflux (Fig. 2, inset), and both are
consistent with removal of Ca
originally localized
within a membranous internal compartment, i.e. the intradiscal
space.
Figure 2:
Sr-Ca
exchange removes both cytosolic Ca and intradiscal
Ca
. Experimental conditions were as described in the
legend of Fig. 1. Seven superimposed traces are illustrated that
represent seven different cuvettes. In all cases Sr-Ca exchange was
initiated at time 0 by addition of 500 µM
SrCl
. The upward pointing arrows (as well as at 20 and 60
min) indicate addition of 2 mM EDTA to chelate external
Sr
and addition of 100 nM A23187 to release
internally sequestered Ca
. The insert in the top
right corner compares the amplitude of the A23187 signals (filled
circles) with the remaining
Ca content of ROS (open
circles); in the latter case, 100% represents the
Ca
content after the fast Sr-Ca exchange phase had removed cytosolic
Ca. Temperature, 25 °C.
Further support for the above localization of internal
Ca to cytosolic and intradiscal pools, respectively,
was obtained by selective permeabilization of the ROS plasma membrane
with streptolysin S. Streptolysin S caused the complete release of
fluo-3 from ROS, whereas only a partial release of
Ca was
observed that matched the amount released by the rapid component of
Na-Ca exchange (not illustrated).
Internal Ca
The experiment illustrated in Fig. 1implies that
internal Ca Release from ROS
Discs
stores in isolated ROS lost
Ca
after the initial rapid component of Na-Ca or
Sr-Ca exchange had removed most cytosolic Ca
. To
demonstrate Ca
release from discs into the cytosol,
Na
-stimulated Ca
release was allowed
to proceed for 30 s (which extruded most cytosolic
Ca
) and then stopped by a 40-fold dilution into a
Na
-free medium (dilution medium contained 5
mM Mg
to inhibit residual Na-Ca exchange at
1 mM external Na
and 1 mM EGTA to
prevent Ca
uptake from the external medium). When
Ca
removal across the plasma membrane via the
Na-Ca+K exchanger was minimized this way, a large rise in
cytosolic free Ca
from about 150 nM to about
600 nM was observed, whereas in the continued presence of 50
mM Na
a further drop to about 80 nM
was observed (Fig. 3, left-hand panel). Measurements of
the
Ca content showed that dilution into the
Na
-free, Mg
-containing medium
greatly reduced net Ca
loss from ROS compared with
the loss observed in the continued presence of 50 mM
Na
(Fig. 3, right-hand panel, compare
filled triangles with open squares). Free cytosolic
Ca
was lowered during the first 5 s following
addition of 50 mM Na
at an average rate of
200 nM/s. In contrast, the subsequent slow phase of
Na
-induced Ca
release took 10 min to
lower free cytosolic Ca
from 150 nM to 100
nM at an average net rate of Ca
efflux of
0.08 nM/s. The initial rate of Ca
release
from discs was between 2 and 4 nM/s (as judged from the
dilution experiment) and it took 10 min to raise cytosolic free
Ca
from 150 nM to 500 nM (average
rate over 10 min of 0.6 nM/s).
Figure 3:
Ca release from ROS
discs. Ca
- and fluo-loaded intact bovine ROS were
prepared as described under Methods and were incubated in 600
mM sucrose, 20 mM Hepes (pH 7.4), 500 nM
FCCP, 1 mM EGTA, and 2 mM KCl (final opsin
concentration 1.5 µM). Ca
release was
initiated at time 0 by addition of 50 mM NaCl (open
squares), 1 mM NaCl (open triangles), 5
mM MgCl
(filled inverted triangles) or no
addition (filled circles or labeled no
Na
). In one case 50 mM NaCl was added to
ROS at an opsin concentration of 75 µM; after 30 s ROS
were diluted 50-fold with the above incubation medium containing 5
mM MgCl
(filled triangles, trace labeled
50 mM Na
for 30 s).
Left panel, fluo traces representing free cytosolic
Ca
; right panel,
Ca content
representing total ROS Ca
. Temperature, 25
°C.
Fig. 3
also
illustrates two cases in which Ca release from the
discs could occur without first lowering cytosolic free Ca
to <200 nM. First,
Ca release in
Na
- and Ca
-free medium was greatly
reduced by addition of 5 mM Mg
to the
external medium (compare filled triangles with filled
circles), perhaps by stabilizing the ROS structure. A steady rise
in cytosolic free Ca
was observed in the fluo-3
traces under this condition indicating Ca
release
from discs. Second, Ca
release was measured at a low
external Na
concentration of 1 mM resulting
in a modest Ca
extrusion rate due to low occupancy of
the Na
binding sites (Na
dissociation
constant is 35 mM)
(13, 16) . At 1 mM
Na
, a concurrent Ca
release was
observed in cytosolic free Ca
measurements and total
Ca measurements, i.e. no separation in time
existed between net Ca
efflux from the cytosol and
net intradiscal Ca
loss as was observed at high
Na
.
Intracellular Cation Dependence of Cytosolic Free
Ca
Gramicidin is an alkali cation-selective channel
ionophore that greatly facilitates Na Set by the Na-Ca+K
Exchanger
and K
entry across the ROS plasma membrane (16). Gramicidin caused a
large rise in cytosolic Ca
(from 153 nM to a
maximal value of about 450 nM) when added 15 s after
initiation of Na
-induced Ca
efflux
(Fig. 4, left panel). In contrast, the rate of net
Ca efflux from ROS was little affected by addition of
gramicidin and indicated a continued net efflux of Ca
across the plasma membrane (Fig. 4, right panel).
While the
Ca efflux rate gradually declined in the 40 min
following Na
addition, the gramicidin-induced rise in
Ca
persisted. The gramicidin-induced rise in
cytosolic free Ca
was dependent on Ca
release from discs, since it was greatly reduced by pretreatment
of ROS with 100 nM A23187 (not illustrated). Pretreatment with
100 nM A23187 caused selective Ca
permeabilization of the disc membrane and enabled subsequent
Na
-dependent Ca
extrusion to remove
intradiscal Ca
released by A23187 before inactivation
occurred
(12) .
Figure 4:
Ca release from discs
and long term inactivation of Na-Ca exchange. Experimental conditions
were as described in the legend of Fig. 3. Four different experimental
protocols are represented and the fluo traces in the left panel are labeled accordingly: 1) 50 mM NaCl was added at time
0 (open triangles); 2) 50 mM NaCl was added at time
0, 100 nM gramicidin was added at 15 s (filled
triangles); 3) 50 mM NaCl was added at time 0, 100
nM gramicidin was added at 15 s, 50 mM KCl was added
at 2 min (open squares); 4) no addition (open
circles). The
Ca panel contains the additional
condition where Na
-stimulated Ca
release is stopped after 30 s by 50-fold dilution in incubation
medium containing 5 mM MgCl
(filled
squares). Left panel, fluo traces representing free
cytosolic Ca
; right panel,
Ca
content representing total ROS Ca
. Temperature, 25
°C.
High external K did not
prevent the gramicidin-induced and Na
-dependent rise
in Ca
per se (not shown), but it abolished
the persistent nature of the gramicidin-induced rise in
Ca
. When K
in the medium was raised
from 5 to 55 mM at the peak of the gramicidin-induced rise in
Ca
, cytosolic free Ca
was lowered
to a new steady value of about 100 nM, while
Ca
efflux across the plasma membrane was slightly increased
(Fig. 4). The above experimental protocol was repeated by
increasing the external K
concentration to different
values, and this resulted in different cytosolic free Ca
concentrations (Fig. 5; Li
could not
replace K
). The data illustrated in Fig. 3and
Fig. 4
were obtained with the same ROS preparation, but very
similar results were obtained in at least six other experiments with
different ROS preparations.
Figure 5:
Effect of cations on cytosolic free
Ca. Experimental conditions were as described in the
legend of Fig. 3. Seven different traces are illustrated representing
different cuvettes. In all cases Na
-stimulated
Ca
release was initiated at time 0 by addition of 50
mM NaCl (indicated by the first arrow). In one
cuvette, changes in cytosolic free Ca
were followed
for 4 min. To the other six cuvettes, NaCl addition was followed after
4 s by addition of 100 nM gramicidin (indicated by the
second arrow), and after 30 s by addition of the indicated
concentration of KCl or LiCl (indicated by the third arrow).
Temperature, 25 °C.
From the above results it might be
anticipated that the gramicidin-induced rise in cytosolic
Ca would mirror that observed for A23187 and would
gradually decrease as the slow component of Na-Ca+K exchange
removed Ca
from discs (as judged from the amplitude
of A23187-induced Ca
release, e.g.Fig. 2
and Fig. 7). This was not observed, the
gramicidin-induced rise in Ca
decreased much more
rapidly (Fig. 6) suggesting that the rate of Ca
release from discs and its potentiation by gramicidin was not a
simple function of the intradiscal Ca
content.
Figure 7:
Gramicidin-induced Ca
release does not reflect the intradiscal Ca
content.
Experimental conditions were as described in the legend of Fig. 3.
Seven different traces are illustrated representing different cuvettes.
In all cases Na
-stimulated Ca
release was initiated at time 0 by addition of 100 mM
NaCl (indicated by the first arrow). In one cuvette, changes
in cytosolic free Ca
were followed for 520 s. To the
other six cuvettes, NaCl addition was followed after different time
intervals by addition of 100 nM gramicidin or 100 nM
A23187 (as indicated). Temperature, 25
°C
Figure 6:
Time course of gramicidin-induced
Ca release from discs. Experimental conditions were
as described in the legend of Fig. 3. Seven different traces are
illustrated representing different cuvettes. In all cases
Na
-stimulated Ca
release was
initiated at time 0 by addition of 50 mM NaCl (indicated by
the first arrow). In one cuvette, changes in cytosolic free
Ca
were followed for 4 min. To the other six
cuvettes, NaCl addition was followed after different time intervals by
addition of 100 nM gramicidin (indicated by the small
arrows) which resulted in a rise in cytosolic
Ca
. Temperature, 25
°C.
Comparison between A23187- and Gramicidin-induced
Intracellular Ca
Both A23187 and
gramicidin were able to cause a rise in cytosolic free Ca Release
by increasing the rate of Ca
release from
discs. In the case of A23187, this was due to selective
permeabilization of the disc membrane by A23187 and Ca
release was most likely carried by A23187 as the release rate
depended on the A23187 concentration
(12) . In contrast,
gramicidin-induced Ca
release did not appear to be
mediated by gramicidin itself as the Ca
release rate
displayed little dependence on the gramicidin concentration in the
range of 1 nM to 1 µM (data not illustrated). Two
further differences could be noted. 1) Application of A23187 during the
rapid phase of Na
-stimulated Ca
release resulted in a delayed rise in cytosolic Ca
and the final Ca
level reached was less than
that observed when A23187 was added during the plateau phase
(Fig. 7). As pointed out before, application of A23187 before or
during the fast phase of Na
-stimulated Ca
release, resulted in partial or complete removal across the
plasma membrane of Ca
that was released from discs by
A23187. Clearly, this mechanism did not apply to gramicidin-induced
Ca
release. 2) Gramicidin resulted in a persistent
rise in cytosolic free Ca
without any sign of
reactivation of the exchanger. In contrast, the rise in Ca
caused by addition of A23187 resulted in partial reactivation of
the exchanger and in complete removal of Ca
released
from discs
(12) .
influx via the cGMP-gated channels and Ca
extrusion via the Na-Ca+K exchanger is thought to maintain
cytosolic free Ca
at a relatively high level and an
average dark value of 550 nM (range 320-880) was
observed in tiger salamander rods
(14) . In the latter study,
bright light closed all cGMP-gated channels and Ca
influx associated with it, but Ca
extrusion
failed to lower free cytosolic Ca
below 50
nM, well above a value of 300 pM expected from
thermodynamic considerations. Likewise, in our earlier studies on
Na-Ca+K exchange in isolated bovine ROS we observed that
Na
-dependent Ca
extrusion did not
lower free cytosolic Ca
to values below 50-100
nM, both in Ca
-containing
(11) and
Ca
-free media
(12) . In the latter study it was
shown that inactivation of the Ca
extrusion mode of
the exchanger accounted for these observations. The above cited studies
examined lowering of cytosolic free Ca
on relatively
short time scales (seconds to 10s of seconds) that are relevant for
visual excitation. Inactivation of Na-Ca+K exchange may play an
important role when rod photoreceptors are saturated for prolonged
periods of time during daylight illumination, during which time the
Na-Ca+K exchanger could lower cytosolic free Ca
toward the thermodynamic equilibrium value of <1 nM.
The Na-Ca+K exchanger is thought to be the only Ca
removal mechanism in the ROS plasma membrane, while internal
Ca
sequestration and release are thought not to occur
in ROS (reviewed in Refs. 9 and 17). This study had two objectives: 1)
the contribution of Na-Ca+K exchange to regulation of free
cytosolic Ca
was measured on time scales ranging from
1 s to 40 min, and 2) the issue of Ca
fluxes
originating from internal compartments (i.e. ROS discs) was
reexamined and their role in Ca
homeostasis in ROS
considered. Our main observation is that a combination of inactivation
of the Ca
extrusion mode of the Na-Ca+K
exchanger coupled with Ca
release from discs is
responsible for a persistent (up to 40 min) and steady cytosolic free
Ca
concentration of about 100 nM, even if
thermodynamic considerations would predict a free cytosolic
Ca
concentration of less than 1 nM. The
coupling stoichiometry of the Na-Ca+K exchanger dictates that
under any reasonable physiological condition the direction of
Ca
transport mediated by the exchanger is
Ca
extrusion; however, the coupling stoichiometry
does not appear to be involved in setting the actual free cytosolic
Ca
concentration. Below I will discuss the
observations that led to this conclusion, and I will discuss the
factors that control the free cytosolic Ca
concentration set by fluxes via the exchanger.
Localization of Total ROS Ca
In this study, Ca to Cytoplasm and
Internal Stores
-depleted ROS
were loaded with fluo-3, Ca
, and
Ca to
modest total Ca
loads of 0.1-0.3 mol
Ca
/mol rhodopsin (300-900 µM total
Ca
). The ``in vivo''
Ca
content of either bovine or amphibian ROS is not
known with certainty (reviewed in Ref. 9). In view of the potential for
rapid adjustments in total internal Ca
via the
Na-Ca+K exchanger, any isolation procedure (e.g. cooling,
altering external Na
or Ca
) could
conceivably result in unphysiologically low or high Ca
loads. Frog ROS with part of the inner segments attached can be
purified on Percoll gradients in Ringer's solution: they contain
about 1 mol of Ca
/mol of rhodopsin and produce
physiological responses to light
(18, 19) . Therefore,
the ROS Ca
loads used in this study are not
unreasonably high. The experiments illustrated in Fig. 1and
Fig. 2
demonstrate the protocol for localization of total internal
ROS Ca
to the cytoplasmic and intradiscal pool,
respectively: a rapid (half-time of 4-8 s) exchange of cytosolic
Ca
was observed against either external Na
(net Ca
efflux) or Sr
(indicative of self-exchange). A subsequent slow phase of net
Ca
removal from ROS (as judged from
Ca
loss) did not represent changes in cytosolic Ca
(as
indicated by the steady fluo-3 fluorescence), but led to a steady
reduction of intradiscal Ca
as judged by the
amplitude of Ca
release from discs into the cytosol
stimulated by low doses of the Ca
ionophore A23187
(Fig. 2).
Ca loaded into ROS did not to any
significant amount become tightly bound to high affinity binding sites
as high concentrations of A23187 caused a rapid release of all
Ca taken up
(12) .
Inactivation of Ca
Ca Extrusion via
Na-Ca+K Exchange Represents at Least a 20-Fold Reduction in
Maximal Rate
fluxes observed in the
experiments illustrated in Fig. 3and Fig. 4(which were
done with the same ROS preparation) were calculated from the fluo
experiments on the basis of a cytosolic Ca
buffer
capacity of 200 µM total
Ca
/µM free Ca
(12) as well as from changes in total ROS Ca
content (similar numbers were obtained in other experiments with
different ROS preparations). The rapid phase of
Na
-stimulated Ca
release caused a
change in free cytosolic Ca
at an average rate over
the first 5 s of 188 nM/s, equivalent to a net efflux of 3.6
10
cytosolic Ca
/ROS/s (fluo
experiment), while the total Ca
loss from ROS
occurred at an efflux rate of 4.9
10
Ca
/ROS/s (
Ca experiment). For
comparison, Na-stimulated Ca
release from
Ca
-enriched bovine ROS occurs at a rate of 5
10
Ca
/ROS/s
(13, 16) .
After 30 s of high velocity Ca
extrusion, the total
Ca
release rate dropped by a 100-fold to a rate of
4.6
10
Ca
/ROS/s with a negligible
rate of change in cytosolic Ca
(186
Ca
/ROS/s). The average cytosolic free Ca
concentration during the first 5 s of
Na
-stimulated Ca
extrusion was 800
nM, while the free Ca
concentration during
the first 5 min of the subsequent slow phase of Ca
extrusion was 130 nM. The apparent internal
Ca
dissociation constant of the Na-Ca+K
exchanger in bovine ROS is 0.9 µM(16) , from which
a 3.7-fold drop in Ca
extrusion rate can be
calculated when the internal Ca
concentration is
lowered from 800 to 130 nM (this calculation is based on a
simple Michaelis-Menten occupancy of the Ca
transport
site). Even if the apparent Ca
dissociation constant
of the exchanger would have been 10 µM (for example due to
competition with internal Na
), the calculated drop in
rate is only 5.8-fold. The above calculations suggest that inactivation
of the Ca
extrusion mode of the exchanger reflects a
drop in maximal velocity by at least 20-fold.
Ca
The first 5 min of the
slow phase of Na Release from Discs and
Ca
Homeostasis in ROS
-dependent Ca
extrusion illustrated in Fig. 3occurred at a rate of 4.6
10
Ca
/ROS/s. Subsequently, the
Ca
extrusion rate dropped to low values of 706
Ca
/ROS/s and 187 Ca
/ROS/s after 15
and 35 min, respectively. In the time interval between 30 s and 40 min
after initiation of Na
-dependent Ca
extrusion, free cytosolic Ca
had dropped from
139 to 83 nM, during which period net Ca
loss from the cytosol was less than 5% of the total
Ca
loss. Ca
release from discs was
responsible for the steady cytosolic Ca
concentration
and the initial release rate from discs was 2-4 nM/s or
3.8-7.6
10
Ca
/ROS/s
(Fig. 3), very similar to the rate calculated above for the first
5 min of the slow Ca
release phase. As cytosolic free
Ca
did not change very much over the next 35 min, the
dramatic drop in total Ca
release rate from ROS must
have reflected an equally large drop in Ca
release
rate from discs. We previously reported that inactivation of the
Ca
extrusion mode of the Na-Ca+K exchanger was
abolished when intradiscal Ca
was removed by
selective Ca
permeabilization of the disc membranes
with low doses of A23187 and we proposed a role for intradiscal
Ca
in inactivation
(12) . This suggestion was
based on the absence of a long lived steady cytosolic free
Ca
concentration after pretreatment with A23187 and
subsequent Na
-dependent
Ca
-extrusion. Either intradiscal Ca
is somehow directly involved in inactivation or an equilibrium
between Ca
release from discs and inactivated
Na
-dependent Ca
extrusion determines
the steady state cytosolic free Ca
concentration and
removal of intradiscal Ca
by A23187 pretreatment
disturbs this equilibrium. Such a scheme would also explain the
observation that the free Ca
concentration at which
the exchanger inactivated increased when the intradiscal Ca
content was increased (not illustrated; see also in
Ref. 11).
Does Cytosolic Free Ca
Above it is suggested that the long lived steady
cytosolic free Ca Control
Inactivation of Ca
Extrusion via Na-Ca+K
Exchange?
concentration reflects an apparent
equilibrium between Ca
release from discs and its
subsequent removal across the plasma membrane by an inactivated
Na-Ca+K exchanger. However, the low velocity Ca
extrusion mode operated over a more than 20-fold range (from 4.6
10
Ca
/ROS/s initially to a final
value of 187 Ca
/ROS/s), while free cytosolic
Ca
changed very little from 139 nM to 83
nM (Fig. 3). This suggests an alternative interpretation
in which a sensor controls cytosolic free Ca
either
by adjusting the Ca
extrusion rate or the
Ca
release rate from discs. Perhaps a Ca
sensor mediates inactivation of the Ca
efflux
mode when the appropriate free cytosolic Ca
concentration, far above that expected from the coupling
stoichiometry, is reached. Two properties of this putative sensor can
be noted. 1) It must cause prolonged inactivation of the Ca
extrusion mode. When cytosolic free Ca
was
raised to 500 nM by A23187-induced Ca
release from discs, it took seconds for the Ca
extrusion rate to reactivate and only a partial reactivation was
observed
(12) (Fig. 7). 2) The Ca
release rate inactivated within a very narrow range of free
Ca
concentration (e.g.Fig. 1
), which
suggests a highly cooperative mechanism. The cytosolic domain of the
Na-Ca+K exchanger contains multiple repeats of the highly acidic
decamer AQIEGEDEDG in which the underlined residues are
conserved
(20) . These repeats may represent allosteric
Ca
binding sites that are involved in the
inactivation process. Inactivation of Na-Ca+K exchange is limited
to the Ca
extrusion mode and no internal
Ca
dependence of Ca
influx via
reverse exchange is apparent
(10) .
appear to influence the putative
intracellular Ca
sensor, intracellular Na
and intracellular K
(Fig. 4). Addition of
the alkali cation ionophore gramicidin in the presence of external
Na
caused a persistent increase in cytosolic free
Ca
suggesting a shift in the Ca
sensor controlling inactivation to a higher free Ca
concentration (Fig. 4-6). Gramicidin equilibrates
internal and external alkali cation concentrations when applied to
bovine ROS
(16, 21) and this suggests that an increase
in intracellular Na
caused the above-noted shift in
the Ca
sensor. K
reversed the action
of Na
and resulted in lower values of cytosolic free
Ca
(Fig. 4-6). The gramicidin-induced and
Na
-dependent rise in cytosolic Ca
is
useful to analyze the mechanisms involved in regulating cytosolic free
Ca
. First of all, the Na-Ca+K exchanger
``allowed'' the free cytosolic Ca
to
increase to a new set value despite the fact that it still mediated net
Ca
efflux from ROS (Fig. 4). This is perhaps
the most compelling evidence that the exchanger possesses a sensor to
set free cytosolic Ca
at levels far above those
predicted from the coupling stoichiometry. In our experiments no
Ca
was present in the external medium and, therefore,
the exchanger can only dictate free cytosolic Ca
by
regulating its Ca
extrusion rate. Moreover,
exchanger-mediated Ca
influx to raise cytosolic
Ca
would violate the thermodynamic consequences of
the coupling stoichiometry. Ca
release from discs was
required as a source of Ca
to raise cytosolic
Ca
and this suggests that Ca
efflux
from discs is important in keeping cytosolic free Ca
well above values predicted from the coupling stoichiometry.
Further experiments are under way to delineate further 1) the role of
Ca
release from discs in Ca
homeostasis in ROS, 2) the mechanism(s) responsible for
Ca
uptake in and Ca
release from
discs, and 3) the secondary cation binding sites that are proposed to
constitute a sensor that controls the Ca
extrusion
rate of the Na-Ca+K exchanger.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.