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(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, [
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) 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
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 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
Figure 1:
Equilibrium binding of
[
Figure 2:
Kinetics of formation and dissociation of
the [
The dissociation of
[
Figure 3:
pH dependence of
[
Figure 4:
Inhibition of
[
Figure 5:
Inhibition of
[
[
Figure 6:
Effect of sulfhydryl reagents and reducing
agents on [
Figure 7:
Effect of calcium on
[
The specific binding of
[
Figure 8:
Effect of hormones on
[
Plasma membranes from the moss P. patens contain a
single class of binding sites for the calcium channel blocker
[ Kinetic parameters indicate that
association of [ Membrane fractionation
studies indicate that [ 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 [ In the
present study, increasing concentrations of KCl added to the binding
buffer stimulated [ [ 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
[ 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
[
Volume 270,
Number 40,
Issue of October 06, pp. 23461-23467, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
PROPERTIES OF RECEPTORS FOR A CALCIUM CHANNEL ANTAGONIST (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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 Kvalue 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.
-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) .
Plant Material
P. patens (Hedw.) Br.
Eur. was cultured and grown aseptically using a modified Knop's
medium solidified with 1.5% (w/v) agar (basal medium)(22) .
Plants were grown at 25 °C under continuous white light
(45-50 µE/m
s) supplied by
fluorescent tubes. Petri dishes containing appropriately supplemented
basal medium overlaid with sterile cellophane were inoculated with
spore suspensions. To prepare spore suspensions, mature sporophytes
were harvested, and 50 capsules were sterilized by soaking in 70%
ethanol (5 ml) for 2 min followed by 5.25% (w/v) sodium hypochlorite,
0.1% Triton X-100 (10 ml) for 10 min with occasional swirling. The
capsules were washed four times with sterile distilled water (10 ml
each) and resuspended in 10 ml of sterile distilled water. Capsules
were opened with sterile forceps, and plates were inoculated with 1 ml
of spore suspension each (approximately 4 
10 viable
spores). After incubation at room temperature for 2 weeks, protonemal
tissue was harvested and ground in sterile water (1 g/6 ml) with a
tissue homogenizer (Tissue Tearor, Biospec Products Inc., Bartlesville,
OK) for 2 min at 11,000 rpm. Ground tissue (0.25 g/1.5 ml) was
transferred to sterile flasks containing 50 ml of Gottwald's
medium(23) . Flasks were incubated at room temperature under
fluorescent lights (as above) on a rotary shaker at 100 rpm for 3
weeks. The tissue was then subcultured by transferring 9-ml aliquots of
culture into flasks containing 50 ml of Gottwald's medium.
Cultures were incubated for an additional 2 weeks, and the tissue was
harvested, weighed (yield: approximately 1 g tissue/flask), and used
for membrane isolation.Plasma Membrane Isolation
All procedures were
conducted at 4 °C. Moss vegetative tissue (35-60 g) was
homogenized by mortar and pestle in a medium containing 250 mM
sorbitol, 3 mM EGTA, 25 mM Hepes-BTP (pH 7.4), 1
mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 1 mM iodoacetamide, 0.01 mM pepstatin A,
0.2% bovine serum albumin (fatty acid free), and 0.25 g/g of fresh
weight polyvinylpolypyrrolidone at a medium-to-tissue ratio of 1.5 ml/g
of fresh weight. After filtration through cheesecloth, the debris was
homogenized again in 1 ml of homogenization medium/g of the original
tissue weight, washed in 0.5 ml/g of the same medium, and filtered. The
homogenate was centrifuged for 15 min at 13,000 g; the
resulting supernatant was centrifuged for 30 min at 60,000 g (Beckman SW 28 rotor, r). The resulting pellet
(crude microsomal fraction) was resuspended in 250 mM sorbitol, 2.5 mM Hepes-BTP (pH 7.2), and 1 mM dithiothreitol (resuspension buffer). The suspension (1.1 ml) was
layered over a two-step (6 and 12%, w/w) dextran gradient (5 ml each)
prepared in resuspension buffer. After centrifugation for 2 h at 70,000
g (Beckman SW 28.1 rotor, r), a turbid
band at the 6-12% dextran interface was collected and is referred
to as plasma membrane-enriched vesicles. g (Beckman SW 28 rotor, r), membranes at the
0-4, 4-6, and 6-12% interfaces were collected and
used for localization studies.Protein Determination and Enzyme Assays
For all
ATPase assays, reaction mixtures (0.5 ml) contained 10-30 µg
of protein, 4.5 units of pyruvate kinase, 5 mM
MgSO
, 5 mM phosphoenol pyruvate, 0.012% Triton
X-100, with 30 mM Hepes-BTP and 5 mM ATP-BTP buffered
to the appropriate pH. Reactions were initiated by addition of ATP, and
incubation was at 37 °C for 30 min. P
was determined by
the method of Fiske and SubbaRow(24) . For measurement of
vanadate-sensitive ATPase activity (plasma membrane), Hepes and ATP
were buffered to pH 6.7 with BTP; 50 mM KCl, 0.1
µM bafilomycin A, and 5 mM sodium
azide (NaN) were included in all assays. Vanadate-sensitive
activity was calculated as the difference in activity in the absence
and presence of 0.1 mM sodium vanadate
(NaVO). For measurement of bafilomycin
A-sensitive ATPase activity (vacuolar membrane), Hepes and
ATP were buffered to pH 7.5 with BTP; 50 mM BTP-Cl, 0.1 mM vanadate, and 5 mM azide were included in all assays.
Bafilomycin A at a concentration of 0.1 µM was
used to determine bafilomycin A-sensitive activity. , 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
)4HO)
in 2 N HSO plus 1% SDS at room
temperature.[
Membranes were incubated at 20 °C in a solution
containing 20 mM Hepes-NaOH (pH 7.5), 0.1 mM phenylmethylsulfonyl fluoride, 5 mM KCl, 1 mM
CaClH]Azidopine Binding
Assays, and 10 nM [H]azidopine
at a final protein concentration of 0.066 mg/ml. Nonspecific binding,
measured by preincubation of membranes with 100 µM of the
DHP antagonist, nifedipine, on ice for 60 min, was <10% of total
binding at a 10 nM concentration of ligand. Nonspecific
binding was subtracted from total binding to yield specific binding.
Variations of these conditions for individual experiments are indicated
in the appropriate figures and tables. Experiments were initiated by
addition of membrane vesicles to reactions; 60 min incubations were
used in standard equilibrium binding experiments. Glass fiber filters
(Whatman GF/C) used for filtration were presoaked for at least 1 h in
0.3% polyethylenimine. Under these conditions,
[H]azidopine bound to filters in the absence of
membranes and could be displaced by nonradioactive ligand. Control
experiments were performed without vesicles for each experiment and the
binding data was corrected for nonspecific filter interactions.
Incubations were stopped by rapid filtration of reaction aliquots
(usually 450 µl) under reduced pressure. The filters were
immediately washed twice with 5 ml of an ice cold solution containing
100 mM Tris-HCl (pH 7.5) and 0.1% bovine serum albumin.
Duplicate experiments were systematically carried out using at least
three different membrane preparations. Ten-µl samples of the
incubation mixture were taken for measurement of the total amount of
[H]azidopine present; radioactivity was measured
by scintillation counting. All experiments were carried out under dim
light due to the light sensitivity of the dihydropyridines. Solvent
controls were included in all assays where necessary. In the
experiments to determine sensitivity of
[H]azidopine binding to sulfhydryl reagents,
membranes were preincubated with the reagents for 30 min at 37 °C
before addition of [H]azidopine. Inhibition of
[H]azidopine binding by different drugs was
measured under equilibrium conditions as described above in the
presence of 10 nM [H]azidopine.),
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.
Materials
[H]Azidopine
(70-90 Ci/mmol) was purchased from DuPont NEN. Unlabeled
nifedipine, bepridil, diltiazem, and Bay K8644 were purchased from
Calbiochem. Verapamil was purchased from Sigma. Bafilomycin A was obtained from Dr. Karl Heinz Altendorf, University of
Osnabrück, Germany. All other chemicals were of the
highest quality available from commercial sources.
Specific Binding of
[
To understand the interaction of the moss channel
with the dihydropyridine ligand, equilibrium binding of
[H]Azidopine to Sites in Moss
MembranesH]azidopine to membranes was carried out in the
presence and absence of 100 µM of the DHP antagonist,
nifedipine (Fig. 1A). The specific binding, defined as
the difference between total and nonspecific binding, was a saturable
function of [H]azidopine concentration. Specific
binding of [H]azidopine varied linearly with
increasing concentrations of moss membranes in the range of
10-130 µg of membrane protein/ml (data not shown); all
binding studies were carried out in the linear range. In typical
experiments using 0.066 mg of protein/ml, total binding in the presence
of 10 nM [H]azidopine was approximately
6500 cpm, while nonspecific binding in the presence of 100 µM nifedipine was approximately 700 cpm (data not shown). The
linearity of the Scatchard plot shown in Fig. 1B indicates that the results are consistent with specific binding to
a single class of sites and gives a value of 35.6 pmol/mg of protein
for the maximum number of [H]azidopine sites (B) and 5.2 nM for the equilibrium
dissociation constant (K
) of the
azidopine-receptor complex.
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.
Localization of
[
To determine the cellular location of the
[H]Azidopine Binding
SitesH]azidopine binding sites, a crude microsomal
suspension was separated with a three-step dextran gradient. The
0-4% interface was enriched in tonoplast vesicles as shown by the
presence of bafilomycin A-sensitive (vanadate- and
azide-insensitive) ATPase activity (Table 1). UDPase and
antimycin A-insensitive NADH cytochrome c reductase, markers
for the Golgi and endoplasmic reticulum, respectively, were found
predominantly in the 4-6% interface. Vanadate-sensitive
(bafilomycin A- and azide-insensitive) ATPase activity, a
marker for the plasma membrane, was highly enriched in the 6-12%
interface. Some nifedipine-sensitive [H]azidopine
binding was found in all fractions as indicated by the B values shown in Table 1. However, levels
were significantly higher in the membranes recovered from the
6-12% interface, suggesting that
[H]azidopine binding is primarily localized in
plasma membranes. The K values calculated for the
binding reactions from the 0-4 and 4-6% interfaces (5.5 and
6.2 nM, respectively) were very similar to the value
calculated for binding in the 6-12% fraction (5.2 nM).
Temperature Dependence and Reversibility of
[
The
temperature dependence of the interaction of the dihydropyridine with
its receptor in moss plasma membranes was determined by direct binding
experiments at 4 and 20 °C. [H]Azidopine BindingH]Azidopine
binding was temperature-dependent; 60-min incubation periods indicated
that specific [H]azidopine binding was maximum at
20 °C, with approximately half as much binding at 4 °C (data
not shown). Kinetic studies indicated a single phase for the
association and dissociation of [H]azidopine. The
association reaction of [H]azidopine at pH 7.5
and 20 °C rapidly reached a steady state within 20 min when 10
nM ligand and 0.066 mg/ml membrane protein were used (Fig. 2A). Kinetic data for association were plotted
semilogarithmically (Fig. 2B) and show a linear
relationship as would be expected for a pseudo-first order reaction.
Using the first-order rate equation, this representation gives a k of 9.6 10M
s
(n = 3).
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
10M
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
.
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).pH Dependence of [
Specific binding of
[H]Azidopine
BindingH]azidopine to moss plasma membrane receptors
increased with increasing pH between pH 5 and 7.5 and remained constant
above pH 7.5 (Fig. 3). The relationship between specific binding
of [H]azidopine and pH resembled a simple
titration curve, which indicated that there is one ionizable group with
a pK
value near 7.1 which is important for the
association of [H]azidopine with its receptor.
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.
Interaction of Calcium Channel Antagonists, Sulfhydryl
Reagents, and Reducing Agents with the Moss Azidopine
Receptor
In addition to DHP analogs, L-type calcium channels in
animal cells are modulated by distinct chemical classes of calcium
antagonists including phenylalkylamines and benzothiazepines. These
drugs exert their effect by binding to distinct allosterically coupled
drug receptors residing on the 
subunit of the calcium
channel and inhibiting binding of DHPs(29) . To determine
whether these agents inhibit azidopine binding to receptors in moss
plasma membranes, a number of agents including unlabeled
dihydropyridines, verapamil, diltiazem, and bepridil were examined ( Fig. 4and Fig. 5). These titration curves were performed
in the presence of 10 nM [H]azidopine.
Increasing concentrations of most of the molecules (in the range of
picomolar to micromolar) gradually inhibited
[H]azidopine binding to moss plasma membranes.
Mean I values (concentration of inhibitors producing
half-maximal inhibition of [
H]azidopine binding)
and the maximal inhibition (percent) observed at the highest inhibitor
concentrations are given in the legends to the figures. Bound
[H]azidopine was displaced with the following
rank order of compounds: Bay K8644 > verapamil > nifedipine >
bepridil > diltiazem. Due to limited solubilities, total
displacement of [H]azidopine binding by
verapamil, diltiazem, or bepridil did not occur, so molar affinities of
these drugs for the binding sites were not estimated.
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.
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) .
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.
Effects of Divalent Cations on
[
To determine
if DHPs bind sites linked to calcium channels, the effect of calcium on
[H]Azidopine BindingH]azidopine binding to moss plasma membranes was
evaluated. While substantial amounts of calcium may be bound to
membranes, moss plasma membranes were isolated in the presence of 3
mM EGTA which should remove most bound calcium. Binding was
dependent on the presence of calcium ions (Fig. 7A); as
little as 10 nM calcium increased the binding and maximum
stimulation of binding occurred at 1 mM calcium.
[H]Azidopine binding was stimulated by 302
± 30% (n = 4) at 1 mM calcium (Table 2), and half-maximal stimulation occurred at a
concentration of 16 µM (n = 3) free
calcium (Fig. 7B).
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.
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.Effects of Hormones on
[
Cytokinins,
which provide the primary stimulus for bud formation in
mosses(33) , were tested for their ability to stimulate or
inhibit [H]Azidopine BindingH]azidopine binding to receptors in moss
plasma membranes. Kinetin stimulated [H]azidopine
binding in a concentration-dependent manner (Fig. 8A)
with half-maximal stimulation at 0.13 nM (Fig. 8B). Other cytokinins also stimulated
[H]azidopine binding with the rank order of
kinetin > trans-zeatin > cis-zeatin (Table 3). The hormonally inactive but chemically related adenine
and the hormones indoleacetic acid and gibberellic acid did not
stimulate [H]azidopine binding.
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 Kfor kinetin of 0.13
nM.
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.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) .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. 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.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.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.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.
)
We are grateful to Dr. H. Sze (University of
Maryland), Dr. M. Dietrich, Dr. R. Leonard, Dr. J. Verbeke, and W.
Hable (University of Arizona) for comments on the manuscript and to Dr.
S. Smith (University of Arizona) for comments on the manuscript and
help with the figures.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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