(Received for publication, June 29, 1995; and in revised form, August 7, 1995)
From the
An increase in cytoplasmic calcium is an early event in hormone
(cytokinin)-induced vegetative bud formation in the moss Physcomitrella patens. Whole cell and calcium transport
studies have implicated 1,4-dihydropyridine-sensitive calcium channels
in this increase in cellular calcium. To understand the molecular
nature of the dihydropyridine-sensitive calcium channel, we have
established conditions for the binding of the arylazide
1,4-dihydropyridine, [H]azidopine, to its
receptor in moss plasma membranes. [
H]Azidopine
bound specifically in a saturable and reversible manner. The K
for [
H]azidopine
binding was 5.2 nM and the B
was 35.6
pmol/mg of protein. Association and dissociation of the receptor and
[
H]azidopine were temperature-dependent, and
association varied as a function of pH. Binding was inhibited by
dihydropyridine, phenylalkylamine, and benzothiazepine calcium channel
blockers, bepridil, lanthanum, and N-ethylmaleimide.
[
H]Azidopine binding was stimulated by cations
including calcium, strontium, manganese, and barium.
[
H]Azidopine binding was also stimulated by
cytokinin with a K
value for kinetin of
0.13 nM. These studies utilize a simple plant system to
provide a biochemical framework for understanding calcium regulation
during development and have implications for understanding mechanisms
of signal transduction in plants.
Controlled changes in cellular calcium concentrations have been identified as important components of signal transduction pathways in plants. Cytoplasmic calcium concentrations are highly regulated; levels are modulated by coordinating passive fluxes and active transport across organellar and plasma membranes(1, 2, 3, 4) . Cytoplasmic calcium levels have been shown to increase in response to a variety of stimuli including light (5, 6) and hormones(7, 8) , and small fluctuations in cellular calcium may modulate processes as diverse as secretory activity in the barley aleurone(7) , pollen tube growth(9) , and phase transition in mitosis(10) . Studies examining the effect of calcium channel inhibitors on physiological processes (11, 12, 13, 14) have suggested a role for calcium channels in stimulus-induced increases in cytoplasmic calcium levels; however, little is known about the biochemical or molecular properties of these transport systems.
Calcium acts as an
intracellular messenger in hormone (cytokinin)-induced vegetative bud
formation during the development of the filamentous protonemata (the
young gametophore) in the moss Physcomitrella
patens(15, 16, 17, 18) .
Formation of vegetative buds is an integral part of the moss life cycle
leading to the development of the mature gametophore which is essential
for subsequent sexual reproduction. Cytokinin applied to moss cells
causes profuse premature bud formation(19) . Localized
increases in calcium take place after addition of cytokinin but precede
the cytokinin-induced cell division (15, 18) . In moss
cells not stimulated by cytokinin, cytoplasmic calcium levels (250
nM) are three orders of magnitude lower than levels in the
external medium (0.1-1.0 mM)(18) . After
addition of cytokinin, cytoplasmic calcium levels increase to 750
nM(18) . Whole plant studies indicate that
cytokinin-modulated calcium entry takes place via dihydropyridine
(DHP)-sensitive channels(20) . In moss protonemata,
application of DHP calcium channel agonists in the absence of cytokinin
stimulates bud initial formation, whereas DHP calcium channel
antagonists block cytokinin-induced bud formation(20) . We have
previously characterized calcium influx into isolated moss protoplasts
and have established that the transport activity of the moss calcium
channel shares common characteristics with L-type calcium channels in
animal cells. Calcium transport in moss is voltage-dependent,
stimulated by DHP agonists, and inhibited by DHP antagonists,
phenylalkylamines, and benzothiazepines(21) . A novel feature
of the transport activity of this channel is hormonal modulation by
cytokinin(21) .
Cytokinin-induced bud formation in moss is a simple, highly ordered developmental process and is one of few plant responses that allows direct study of stimulus-response coupling at the biochemical and molecular levels. In view of the importance of this DHP-sensitive, hormone-stimulated calcium channel in early events in bud formation, it is of considerable interest to determine the molecular properties of this channel. In this study, conditions are established to investigate the interaction of DHPs with moss membranes. We demonstrate the presence of a DHP binding activity, determine its localization in purified plasma membranes, show an absolute requirement for physiological concentrations of calcium for binding, and also provide evidence for regulation of DHP binding by cytokinin.
To fractionate microsomal
membranes, the following modifications to the above procedures were
used. The crude microsomal pellet was resuspended, and 12 ml were
layered over a three-step (4, 6, and 12%) dextran gradient (8 ml of
each). After centrifugation for 2 h at 70,000 g (Beckman SW 28 rotor, r
), membranes at the
0-4, 4-6, and 6-12% interfaces were collected and
used for localization studies.
UDPase activity (Golgi membrane) was determined in the presence of
Triton X-100(25) . The final concentrations of reaction
components were 30 mM Hepes-BTP (pH 7.0), 3 mM MgSO, 3 mM UDP-Na
, 0.02% Triton
in a volume of 0.5 ml. Reactions were initiated by addition of UDP and
incubated at 35 °C for 30-40 min. Reactions were terminated
with 1% ammonium molybdate
((NH
)
Mo
)
4H
O)
in 2 N H
SO
plus 1% SDS at room
temperature.
Antimycin A-insensitive NADH cytochrome c reductase activity (endoplasmic reticulum) was assayed by monitoring the reduction of cytochrome c spectrophotometrically at 550 nm(26) .
Protein was determined by the method of Lowry with bovine serum albumin as the standard(27) .
For
determination of association rate constants (k),
the amount of labeled ligand-receptor complex in aliquots taken at
various times between initiation of the reaction and the time to
equilibrium was determined, and the data were analyzed as previously
described(28) . Dissociation rate constants (k
) were determined by measuring the amount
of labeled ligand-receptor complex remaining at various times after the
addition of excess (100 µM) unlabeled ligand.
Figure 1:
Equilibrium binding of
[H]azidopine to moss membranes. Binding was
measured in reaction mixtures as described under ``Experimental
Procedures'' with increasing concentrations of
[
H]azidopine (0-100 nM) at 20
°C for 60 min. A, binding to moss membranes in the absence
(
, total binding) or presence (
, nonspecific binding) of
100 µM unlabeled nifedipine. Nonspecific binding was
subtracted from total binding to give specific binding (
). Points
represent the means ± S.E. of three experiments. B,
Scatchard plot of specific binding (y = 6.871 -
0.193x, R
= 0.992). The specific
binding component resulted in an equilibrium dissociation constant (K
) of 5.2 nM and a maximum
binding capacity (B
) of 35.6 pmol/mg of
protein.
Figure 2:
Kinetics of formation and dissociation of
the [H]azidopine-moss receptor complex. A, the concentration of the
[
H]azidopine-receptor complex formed was measured
by sampling the reaction mixture (containing 10 nM [
H]azidopine) after the indicated times of
incubation at 20 °C. Data are the means ± S.E. of three
experiments. B, pseudo-first order representation of the data (y = 4.049 - 0.025x, R
= 0.876). B
represents the
equilibrium specific binding of [
H]azidopine and B
is specifically bound
[
H]azidopine at assay time t. The
association rate constant (k
) was 9.6
10
M
s
. C, after equilibrium was reached
(60 min), the rate of dissociation of the complex was monitored
following addition of 100 µM nifedipine. Points represent
the means ± S.E. of three experiments. D, first order
representation of specifically bound [
H]azidopine (y = 4.503 - 0.132x, R
= 0.823). The dissociation rate constant (k
) was 5.1
10
s
.
The dissociation of
[H]azidopine from its receptor in moss plasma
membranes was examined by incubating [
H]azidopine
with membranes for 60 min, adding 100 µM nifedipine, and
examining the residual binding at different time points (Fig. 2C). The time course of dissociation followed
first-order kinetics (Fig. 2D) producing a rate
constant of dissociation (k
) of 5.1
10
s
(n = 3) and
a half-life of dissociation (t) of 2.26 min. The K
value determined from the ratio k
/k
is 5.3 nM which agrees well with results from equilibrium experiments (Fig. 1B).
Figure 3:
pH dependence of
[H]azidopine binding to moss plasma membrane
receptors. Hepes buffer (20 mM final concentration) was
adjusted with NaOH to the desired pH at 20 °C. Points represent the
means ± S.E. of at least three
experiments.
Figure 4:
Inhibition of
[H]azidopine binding to moss plasma membrane
receptors by dihydropyridines. Binding of
[
H]azidopine was measured with the addition of
increasing concentrations of nifedipine (A) or Bay K8644 (B). Control [
H]azidopine binding was 38
pmol/mg of protein. Points represent the means ± S.E. of three
independent experiments. Mean I
values (µM)
and the maximal inhibition (%) observed at the highest drug
concentrations are 5.0 and 86 (nifedipine) and 0.01 and 78 (Bay K8644),
respectively.
Figure 5:
Inhibition of
[H]azidopine binding to moss plasma membrane
receptors by bepridil, diltiazem, and verapamil. Binding of
[
H]azidopine was measured with the addition of
increasing concentrations of bepridil (A), diltiazem (B), or verapamil (C). Points represent the means
± S.E. of three independent experiments. Mean I
values (µM) and the maximal inhibition (%) observed
at the highest drug concentration are 6.3 and 60 (bepridil), 20.0 and
71 (diltiazem), and 2.0 and 80 (verapamil),
respectively.
[H]Azidopine binding was sensitive to
sulfhydryl alkylating agents and reducing agents (Fig. 6). The
sulfhydryl blocking reagent, N-ethylmaleimide, inhibited
binding, suggesting that free thiol groups are essential for
receptor-ligand interaction. The moss azidopine receptor showed reduced
sensitivity to iodoacetamide (Fig. 6). This lack of inhibition
is similar to the effect of iodoacetamide on skeletal muscle T-tubule
azidopine receptors and suggests that the essential thiol group may be
located in a hydrophobic domain(43) . Reducing agents like
dithiothreitol (Fig. 6) and
-mercaptoethanol (data not
shown) inhibited [
H]azidopine binding at high
concentrations implying that intact, but not easily cleaved, disulfide
bridges are required for the channel to bind
[
H]azidopine with high affinity(30) .
Figure 6:
Effect of sulfhydryl reagents and reducing
agents on [H]azidopine binding to moss plasma
membrane receptors. Membranes (0.066 mg of protein/ml) were
preincubated with N-ethylmaleimide (
), dithiothreitol
(
) or iodoacetamide (
) at the indicated concentrations
for 30 min at 37 °C. Membranes were then added to binding
reactions, total and nonspecific binding was determined, and specific
binding was normalized with respect to controls. Points represent the
means ± S.E. of three experiments. Mean I
values
(µM) and the maximal inhibition (%) observed at the
highest drug concentration are 38.9 and 66 (N-ethylmaleimide)
and 603.0 and 52 (dithiothreitol),
respectively.
Figure 7:
Effect of calcium on
[H]azidopine binding to moss plasma membrane
receptors. A, calcium regulation of
[
H]azidopine binding to moss plasma membranes.
Total and nonspecific [
H]azidopine binding were
determined without KCl and with calcium concentrations varying from
0-10 mM. Points represent the means ± S.E. of
three experiments. B, Eadie-Hofstee plot of the data (y = 340.8655 - (0.0161)x, R
= 0.901) resulted in a K
for
calcium of 16.1 µM.
The specific binding of
[H]azidopine was modulated by a number of
divalent metal ions (Table 2). Some stimulation of binding was
observed with low concentrations of most of the cations examined;
however, the patterns varied considerably. Calcium, strontium, and
manganese provided the greatest stimulation of
[
H]azidopine binding; however, maximal
stimulation by strontium and manganese were only two-thirds and
one-half as great as with calcium, respectively (Table 2). Cobalt
had both agonist and antagonist effects as low levels of cobalt
stimulated [
H]azidopine binding while inhibitory
effects were seen at higher concentrations. Ions such as lanthanum,
which block the transport of calcium, inhibited
[
H]azidopine binding at all concentrations
tested.
Figure 8:
Effect of hormones on
[H]azidopine binding to moss plasma membrane
receptors. A, kinetin regulation of
[
H]azidopine binding to moss plasma membranes.
Total and nonspecific [
H]azidopine binding were
determined without KCl or CaCl
and with kinetin
concentrations varying from 0-1 µM. Points represent
the means ± S.E. of three experiments. B, Eadie-Hofstee
plot of the data (y = 132.8804 -
(0.1329)x, R
= 0.639) resulted in
a K
for kinetin of 0.13
nM.
Plasma membranes from the moss P. patens contain a
single class of binding sites for the calcium channel blocker
[H]azidopine (Fig. 1).
[
H]Azidopine binding is saturable (Fig. 1A) and reversible (Fig. 2C and Fig. 4). The maximum binding capacity is high (Fig. 1B), similar to values reported for DHP receptor
densities found in T-tubule membranes from rabbit muscle (31, 32) and significantly higher than values for
receptors in brain, heart, and smooth muscle microsomes (0.1-1
pmol/mg of protein)(33, 34) . The equilibrium
dissociation constant (K
) for the
azidopine-membrane complex suggests that
[
H]azidopine binds with high affinity to
membranes in this plant. Specific binding of azidopine was eliminated
by pretreatment of the membranes with trypsin or chymotrypsin at
concentrations of 0.1 mg/ml (data not shown). This protease sensitivity
suggests that the interaction of azidopine with moss membranes is
protein-mediated, and we have used the term receptor to describe this
protease-sensitive binding activity. Prior to the present study, only
preliminary evidence existed for binding of DHPs to plant membranes;
Hetherington and Trewavas (35) showed that microsomal membranes
isolated from etoliated pea shoots exhibited a low level of binding of
the DHP antagonist, nitrendipine.
Kinetic parameters indicate that
association of [H]azidopine with its receptor is
slower than would be expected for a diffusion controlled process
(10
M
s
< k
< 10
M
s
). This rate of association suggests that a
conformational change of the ligand (L)-receptor (R) complex occurs
following the association of [
H]azidopine, as has
been observed in inhibitor binding to skeletal muscle T-tubule
membranes(36) . The interaction to form the complex (L + R
&lrhar2; LR* &lrhar2; LR) likely takes place in at least two steps: via
a rapid second order absorption of the ligand to the receptor (L +
R &lrhar2; LR*) followed by a slow first order rearrangement of the
complex (LR* &lrhar2; LR)(37) .
Membrane fractionation
studies indicate that [H]azidopine binding sites
are located in the plasma membrane. Maximum receptor densities were
found in membranes isolated from the 6-12% dextran interface, the
fraction that was highly enriched in the plasma membrane marker,
vanadate-sensitive ATPase activity (Table 1). Similar levels of
vanadate-sensitive ATPase activity were seen when plasma membranes were
isolated using aqueous two-phase partitioning (38) (data not
shown); however, yields of purified plasma membranes were so low as to
make this method impractical for subsequent binding studies. Recent
electrophysiological studies have identified calcium channels on the
vacuolar membranes of broad bean guard cells (39) and sugar
beet cell suspension cultures (40, 41) that share a
number of properties with animal L-type channels. No information is
available, however, about the abundance of these vacuolar channels or
their binding affinity for DHPs. While
[
H]azidopine binding to moss membranes isolated
from the 0-4% dextran interface may represent binding to
receptors in the vacuolar membrane, two lines of evidence suggest that
binding to this fraction (and to membranes isolated from the 4-6%
interface) represents binding to contaminating plasma membranes: (i)
The K
values calculated for binding in these
fractions were very similar to the K
calculated
for binding in the 6-12% interface, and (ii) there was a low
level of vanadate-sensitive ATPase activity in these fractions.
There is strong evidence for three calcium antagonist receptor sites
on the L-type voltage-dependent calcium channels in animal cells: one
for DHPs, one for phenylalkylamines, and one for
benzothiazepines(42) . We investigated the ability of these
molecules to compete for [H]azidopine binding
sites in moss plasma membranes. All molecules reduced
[
H]azidopine binding in a manner qualitatively
similar to their ability to inhibit calcium influx into moss
protoplasts(21) . Inhibition is most likely due to a direct
competition for the azidopine-binding site by the DHPs and due to an
allosteric inhibition of azidopine binding by verapamil and
diltiazem(30, 31, 32, 33) . While it
has been possible to show involvement of phenylalkylamine-sensitive
calcium channels in processes in higher (vascular)
plants(43, 44, 45, 46, 47) ,
it has been difficult to show a role for DHP-sensitive calcium
channels. Recent experiments examining the ability of nifedipine to
inhibit hormone-induced tracheary element differentiation in Zinnia
elegans suggest that DHP-sensitive channels are present in higher
plants but may be in low abundance, and that their expression may be
temporal and highly tissue specific(48) . The DHP effect seen
in the Zinnia studies required the use of a simplified culture
system (suspension cultures) rather than the whole plant. The ability
to demonstrate DHP-sensitivity in moss membranes may be due to the high
density of high affinity DHP binding sites and suggests that the
simplicity of the moss system makes it particularly well suited for
understanding calcium channel involvement in plant processes.
In the
present study, increasing concentrations of KCl added to the binding
buffer stimulated [H]azidopine binding to moss
membranes. Maximum stimulation of binding (250%) was seen with 5 mM KCl (data not shown). This K
stimulation of
binding may reflect a direct stimulation of azidopine binding to its
receptor. Alternatively, because the membranes used in this study were
isolated using dextran gradients that enrich for sealed
vesicles(49) , K
stimulation of binding may be
due to an altered conformation of the channel caused by a change in the
potential across the vesicle membrane. The membranes used likely
represent a mixture of right-side out and inside-out vesicles.
K
depolarization of the plasma membrane in inside-out
vesicles, leading to increased [
H]azidopine
binding, would be equivalent to a hyperpolarization of the plasma
membrane potential in the cell. However, we have shown previously that
conditions that depolarize the plasma membrane in moss protoplasts
(K
gradients, K
>
K
) stimulate calcium influx(21) .
This depolarization-induced calcium influx suggests that we are most
likely measuring [
H]azidopine binding to
right-side out membrane vesicles and that the binding activity of the
channel may also be stimulated by a depolarization of the plasma
membrane.
[H]Azidopine binding to moss plasma
membranes has an absolute requirement for the presence of calcium (Fig. 7). [
H]Azidopine binding in the
absence of calcium was very low (0.18 pmol/mg of protein, Fig. 7, Table 2); additions of calcium as low as 10 nM significantly stimulated binding. Similar calcium-dependence has
been seen with DHP binding to calcium channels in a number of animal
cells(33, 50, 51, 52, 53) .
While the site of DHP binding is still
uncertain(54, 55, 56) , a conserved region in
the cytoplasmic domain adjacent to segment IVS6 has been shown to bind
DHPs in the purified rabbit skeletal muscle calcium
channel(54) . This conserved DHP binding site contains a
putative calcium binding site (an EF-hand domain). Binding of calcium
to this region may be necessary to induce correct folding of the DHP
binding domain and may explain the calcium stimulation of azidopine
binding. The ability of various cations to stimulate or inhibit
[
H]azidopine binding correlates with their known
agonist or antagonist activities at calcium channels in animal
cells(34) . Strontium and barium are known animal calcium
channel agonists and mimic the effects of calcium in stimulating
[
H]azidopine binding to receptors in moss plasma
membranes (Table 2). Lanthanum and cobalt, classical animal
calcium channel antagonists, reduce [
H]azidopine
binding to moss receptors (Table 2). Inorganic calcium
antagonists (manganese and cobalt) can also stimulate
[
H]azidopine binding at low concentrations,
suggesting that these ions possess agonist as well as antagonist
properties at the calcium channels depending on their concentration.
Similar effects were seen with binding of the DHP-antagonist
[
H]nitrendipine to brain membranes(34) .
The relative potencies of these ions in stimulating or inhibiting
[
H]azidopine binding may be related to their
ionic crystal radii. Maximal stimulation occurs at a diameter
corresponding to that of strontium with substantially lesser effects
for ions with smaller or larger diameters (Table 2). These
results suggest that it will be important to link
[
H]azidopine binding data with studies
correlating ionic crystal radius and action at the moss calcium
channel.
Previous studies showed that cytokinins stimulate calcium
influx into moss protoplasts(21) . These effects were seen
without prior incubation of the protoplasts with the hormone,
suggesting a primary effect. To understand the interaction of
cytokinins with the channel, we examined the action of hormones on
[H]azidopine binding to moss plasma membranes.
Low levels of cytokinins consistently caused a stimulation of
[
H]azidopine binding in a manner qualitatively
similar to their ability to stimulate calcium influx into moss
protoplasts. These cytokinin effects on binding were also seen without
prior incubation of the membranes with the hormone (Table 3),
providing additional evidence for a primary effect. The stimulation of
binding to moss plasma membrane receptors by cytokinins appears to be
specific as adenine did not stimulate azidopine binding (Table 3). As with studies of cytokinin effects on calcium influx
into moss protoplasts, the less active cytokinin, cis-zeatin,
was less effective in stimulating azidopine binding (Table 3).
Cytokinin may stimulate [
H]azidopine binding by
interacting with the channel and altering its conformation,
facilitating inhibitor binding.
Whole cell studies of the effect of
DHPs on bud formation in moss suggested that cytokinin-stimulated bud
formation is modulated by calcium entry through calcium
channels(20) . Subsequent studies characterizing calcium influx
into moss protoplasts established that calcium influx is stimulated by
DHP agonists and inhibited by DHP antagonists(21) . The current
study establishes the presence of abundant, high affinity sites for DHP
binding in the moss plasma membrane. The ability to monitor the
[H]azidopine receptor activity demonstrated in
this study should allow the identification of the protein responsible
for this ligand interaction and ultimately lead to an understanding of
the expression and regulation of the calcium channel during moss
development. In addition, information learned about the moss
DHP-sensitive calcium channel may serve as a model to allow
characterization and identification of the channel homolog in higher
plants.