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From the
The activation of a monovalent cation current was studied in rat
megakaryocytes using patch clamp techniques combined with photometric
measurements of intracellular concentrations of Ca
Megakaryocytes are large cells located in the bone marrow that
are responsible for producing blood platelets, yet little is known of
the cellular mechanisms underlying their function. Uneyama and
co-workers (1, 2) have shown that rat megakaryocytes possess a novel
purinergic receptor, which recognizes the ionized forms of ATP and ADP.
Stimulation of this receptor leads to sustained oscillations of
intracellular Ca
Under conditions where SBFI was introduced into the cell through the
recording pipette, background-corrected 340/380 ratios were used to
provide an indication of [Na
The above whole cell patch clamp
experiments represent conditions that are far from physiological and
dialyze important cytoplasmic factors. We therefore turned to the
nystatin-perforated patch technique to further assess the magnitude of
the ADP-evoked [Na
The present study demonstrates that both extracellularly
applied ADP and internally perfused 1,4,5-IP
In many nonexcitable cells, including the megakaryocyte, IP
The
physiological function of the ADP-evoked monovalent cation channels in
the rat megakaryocyte remains speculative. We could not detect any
significant Ca
Platelets have little or no capacity to manufacture proteins, thus
its progenitor, the megakaryocyte, must eventually express most, if not
all, platelet ion channels. Therefore, the IP
In conclusion, we have
demonstrated the existence of a plasma membrane conductance activated
by 1,4,5-IP
We thank Andres Floto for helpful discussion, in
particular on noise analysis methods, and thank Dr. Stewart Sage for
comments on the manuscript.
Volume 270,
Number 28,
Issue of July 14, pp. 16638-16644, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
([Ca]
) and
Na
. ADP evoked a release of
[Ca] and transiently
activated a monovalent cation-selective channel, which, at negative
potentials and under physiological conditions, would be expected to
carry an inward Na current. The single channel
conductance, estimated by noise analysis from whole cell currents at
-50 to -60 mV was 9 picosiemens. Thapsigargin-induced
[Ca] increases failed
to stimulate the monovalent cation current, suggesting that neither
[Ca] nor the depletion
of internal Ca stores were activators of this
conductance. However, buffering of
[Ca] changes with
1,2-bis-(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid showed that
both activation and inactivation of the current were accelerated by a
rise in [Ca]. The
monovalent cation conductance was activated by internal perfusion with
inositol 1,4,5-trisphosphate, both in the presence and in the absence
of a rise in [Ca].
Internal perfusion with inositol 2,4,5-trisphosphate, the poorly
metabolizable isomer of inositol trisphosphate, similarly activated the
monovalent cation current, whereas 1,3,4,5-tetrakisphosphate neither
activated a current nor modified the ADP-induced monovalent current.
Heparin, added to the pipette, blocked activation of the channel by
ADP. The intracellular concentration of Na, monitored
by sodium-binding benzofuran isopthalate, increased by 10-20
mM in response to ADP under pseudophysiological conditions. We
conclude the existence of a novel nonselective cation channel in the
plasma membrane of rat megakaryocytes, which is activated by IP
and can lead to increases in cytosolic Na after
stimulation by ADP.
concentration
([Ca])
()and activation of Ca-dependent
K channels. We have recently found that ATP also
activates Na and Ca-permeable
channels in rat megakaryocytes via two distinct classes of purinergic
receptor(3) . One receptor is activated by ATP but not
noticeably by ADP and causes rapid, transient opening of a nonselective
cation channel. A second purinoceptor is stimulated by ATP
and ADP
and activates both a monovalent
cation-selective channel and a channel highly selective for
Ca. Stimulation via this second receptor also causes
a release of Ca from intracellular stores and is most
likely the same receptor as that responsible for the generation of
Ca oscillations in the experiments of Uneyama et
al.(1) . The Ca-selective conductance is
activated by depletion of internal Ca stores via an
as yet undetermined messenger and is indistinguishable from the
store-regulated Ca current found in other
nonexcitable cell types(4, 5, 6) . The
monovalent cation-selective current is also activated at the time of
internal Ca release via IP or another
inositol lipid metabolite(3) . This channel is particularly
interesting because, at resting membrane potentials, the current will
be mostly carried by Na, and changes in
[Na] have been proposed
to play a role in the spreading reaction in megakaryocytes(7) .
The spreading reaction may represent the physiological mechanism
whereby megakaryocytes invade the bone sinusoids to reach the blood
vessels and release platelets(8) . In the present study we have
used a combination of patch clamp and fluorescent indicators of
[Ca] and [Na] to
study the monovalent cation-selective current activated by ADP and
inositol phosphates in the plasma membrane of rat megakaryocytes.
Preparation
Adult male Wistar rats weighing
200-300 g were killed by cervical dislocation. Bone marrow from
the femoral and tibial bones was removed by gentle lavage using a
standard external solution containing 20 mg ml
apyrase and 0.1% bovine serum albumin. After filtration through a
fine cotton mesh, the suspension was spun and washed twice before
storage in the same standard solution. Megakaryocytes were
distinguished from other bone marrow cells by their distinctive size
(30-60 µm) and multilobular nucleus. Recordings were made at
room temperature (20-23 °C) within 3-24 h of isolation.
Solutions
The standard external solution contained
140 mM NaCl, 5 mM KCl, 1 mM MgCl
, 2 mM CaCl, 10 mM
glucose, 10 mM HEPES (pH 7.4 adjusted with Tris).
K-, Na-, and
Ca-free external media were obtained by replacing
these ions with Cs, NMDG, and
Mg, respectively. For low Cl
external solutions, all Cl, except for that
added with the divalent cation salts, was replaced by gluconate. In
conventional whole cell patch recordings, Cs replaced
K in order to block K currents and
contained 140 mM cesium gluconate, 5 mM NaCl, 2
mM MgCl, 10 mM HEPES, 0.2 mM NaGTP, 0.05 mM K
fura-2, (pH
7.4 adjusted with Tris). 0.1 mM
(NH
)SBFI replaced the
Kfura-2 in experiments where
[Na] and membrane
current were measured simultaneously. For internal dialysis of inositol
phosphates, the pipette tip was dipped in inositol phosphate-free
pipette solution and then backfilled with pipette solution containing
10 µM inositol 1,4,5-trisphosphate (1,4,5-IP),
50 µM 2,4,5-IP, or 20 µM inositol
1,3,4,5-tetrakisphosphate (IP). Highly calcium-buffered
pipette solution was obtained by replacing 40 mM cesium
gluconate with 10 mM CsBAPTA. In
nystatin-perforated patch recordings, the pipette contained 100 mM KCl, 40 mM KSO, 1 mM MgCl, 10 mM HEPES (pH 7.4 adjusted with
Tris), 150 µg ml nystatin. In these perforated
patch experiments, cells were loaded with SBFI by incubation with 10
µM SBFI-acetoxymethyl ester with 0.04% pluronic acid for
60 min at room temperature. Fura-2, SBFI, CsBAPTA and
pluronic F-127 were from Molecular Probes, Inc. (Eugene, OR).
1,4,5-IP, 2,4,5-IP, and IP were a
gift of Dr. R. F. Irvine (Biotechnology and Biological Sciences
Research Council, Babraham Institute, Cambridge, UK). All other
reagents were from Sigma. A pressure injector was used to administer
agonists from a patch pipette placed 150 µm from the cell. The time
delay for arrival of agonists at the cell was measured and subtracted.
Electrophysiology
Patch clamp experiments were
performed in conventional whole cell (9) or nystatin-perforated
patch (10) configurations by means of an Axopatch 200A patch
clamp amplifier (Axon Instruments, Inc., Foster City, CA). Pipettes
were pulled from borosilicate glass tubing (Clark Electromedical
Instruments) and had filled resistances of 2-3 megaohms. Series
resistances were in the range of 7-30 megaohms, and 40-60%
series resistance compensation was employed. Membrane currents during
voltage ramps (0.6 mV/ms) were filtered at 2 kHz and sampled at 100
µs using Axon Instruments hardware (Digidata 1200) and software
(pClamp). Where stated, voltage ramps prior to agonist application were
used to subtract linear leak currents from ADP-evoked currents. A
holding potential of -40 mV was used in many experiments because
this was close to the membrane potential measured in perforated patch
experiments under pseudophysiological conditions (see Fig. 6). A
less negative potential permitted longer recordings, thus -20 mV
was used in experiments where large currents were detected; however,
holding potentials in the range of -70 to 40 mV were used in some
experiments, depending upon the conditions, to check for measurable
currents. For noise analysis, currents were filtered at 0.5 kHz and
sampled at 2.5 kHz. Currents were also acquired at a rate of 60 Hz
(filtered at approximately 30 Hz) by the Cairn spectrophotometer (see
below) for simultaneous display alongside the fura-2 and SBFI
fluoresence and signal-indicating agonist injection. Liquid junction
potentials were measured by reference to a 3 M KCl agar
bridge, and membrane potentials were adjusted accordingly in
conventional but not nystatin whole cell recordings.
Figure 6:
IP- and ADP-evoked
[Na]
changes. A, whole
cell current (middle trace) and
[Na] (bottom trace) during internal
perfusion of 10 µM Ins-1,4,5-P. The top
trace shows the membrane potential.
[Na] is indicated by the SBFI 340/380 nm
excitation ratio on a linear scale. External solution was nominally
Ca-free with 3 mM 4-aminopyridine saline. B, membrane potential (lower trace) and
[Na] (upper trace) during a
nystatin whole cell recording under current-clamp mode. SBFI 340/380 nm
excitation ratio has been calibrated for
[Na] as described under ``Materials and
Methods.'' Membrane potential and [Na]
axes are both linear over the range shown.
Fluorescence Recordings
Fura-2 and SBFI
fluoresence measurements were made by single cell photometry using a
Cairn spectrophotometer system (Cairn Research Ltd., Kent, UK) coupled
to a Nikon Diaphot inverted microscope. Excitation light passed through
a spinning filter wheel assembly containing four 340-nm and two 380-nm
bandpass excitation filters. Emitted light (400-600 nm) was
selected by two dichroic filters and further filtered by a 485-nm long
pass filter. The combined output from all 340 and 380 nm excitation
filters provided a 340/380 nm ratio for each revolution of the filter
wheel. The signal was then averaged to obtain a ratio value every 67
ms. Background and cell autofluoresence was measured in the
cell-attached recording mode and subtracted to give fura-2 or SBFI
fluoresence. [Ca] was
calculated according to Grynkiewicz et al.(11) using a
dissociation constant for fura-2 of 250 nM(12) .
] changes. In experiments where SBFI was loaded from its
acetoxymethyl ester, [Na] was clamped at different levels by perfusing solutions of
known extracellular Na concentrations in the presence
of a mixture of sodium ionophores (5 µM each of
gramicidin, nigericin, and monensin; Ref. 13). The extracellular
solutions were made from appropriate mixtures of high Na and K solutions. The former consisted of 110
mM sodium gluconate, 30 mM NaCl, 2 mM
CaCl, 1 mM MgCl, 10 mM Na-HEPES (pH 7.4). The high K solution was
identical except for substitution of all Na for
K. A plot of SBFI 340/380 nm intensity ratio versus [Na] was linear in the range
of 0-40 mM. The experimental 340/380 ratio values fell
within this linear range, and therefore
[Na]values were
directly obtained from the calibration curve.
ADP Evokes Calcium Release and Activates a Nonselective
Current
At negative potentials and under conditions that blocked
K currents (see ``Materials and Methods''),
5 µM ADP activated a transient inward current and a
concurrent large increase in [Ca] (Fig. 1A). The initial
[Ca] increase reached
a peak of 0.5-1.5 µM within 1-2 s and then
returned to basal levels (approximately 50-100 nM) in
the continued presence of ADP or was followed by further smaller
increases in [Ca] that
sometimes fused to give a plateau, as shown in the cell of Fig. 1. In the absence of external calcium, an inward current and
[Ca]increase were
still activated by ADP(3) . This suggests that the rise in
[Ca] is at least
partly due to the release of internal stores and that the current is
not selective for Ca. However, variability between
cells did not allow us to quantify the extent to which Ca influx contributed to the response.
Figure 1:
ADP-evoked currents and
[Ca] changes. A,
effect of 5 µM ADP on whole cell current at -40 mV (upper trace) and [Ca] (lower
trace; linear axes) in the presence of 2 mM external Ca. The bath contained 140 mM NaCl, 5 mM CsCl saline, and the pipette contained 140
mM cesium gluconate saline with 0.05 mM fura-2. B, whole cell ramp current, plotted as a function of membrane
potential before the addition of ADP (a) and during the
ADP-evoked inward current (b). C, ramp current after
digital subtraction of background current to display the I-V
relationship of the ADP-dependent current (b-a).
To further examine the
conductance changes in reponse to ADP, membrane currents were recorded
during 0.6 mV/ms voltage ramps within the range of potentials
-100 to 90 mV. A ramp applied prior to agonist application (Fig. 1B, trace a) was used to subtract
background currents from ramp currents obtained during the ADP-evoked
transient (Fig. 1B, trace b). Fig. 1C shows the difference current (b-a) representing the
I-V relationship for the ADP-evoked conductance. In 140 mM Na external solution and 140 mM cesium
gluconate internal solution, the I-V relationship reversed at about
-5 mV, was reasonably linear over the voltage range -90 to
40 mV, and, in most cells, displayed a distinct increase in slope at
more positive potentials. The I-V relationship obtained by ramps at
different times during the ADP-evoked current differed only in
amplitude and not in reversal potential, suggesting that the response
was due to activation of a single ionic conductance.
Ionic Selectivity
The ionic selectivity of the
ADP-evoked conductance was investigated by substitution of internal and
external ions. In Fig. 2, ADP-evoked I-V relationships are shown
for each of four ionic conditions, with the membrane currents and
Ca responses at a single negative holding potential
in the insets. These results were obtained from the first
ADP-evoked response in four different cells and were confirmed in at
least five cells for each condition. Replacement of the majority of the
internal and external Cl had little effect on the
current and Ca response at -40 mV or on the
difference I-V relationship (Fig. 2A) compared with
corresponding Cl-containing salines (see for example Fig. 1C). Therefore, the ADP-evoked conductance does not
appear to be significantly permeant to anions. On the other hand,
replacement of all internal and external monovalent cations with the
impermeant cation NMDG abolished all current at -40 mV, in the
presence of an ADP-evoked Ca response (Fig. 2B, inset). No significant current
developed within the voltage range -80 to 80 mV, as shown by the
overlapping ramp currents before and during the Ca response in Fig. 2B, indicating that the
ADP-evoked current is carried by monovalent cations. 2 mM Ca and 1 mM Mg were
present in the external media throughout, which indicates little or no
permeability to divalent cations at these physiological concentrations.
Increasing external Ca decreases the level of
ADP
(1) and abolishes the ADP response(3) ;
therefore we were unable to increase the external Ca concentration to test for any underlying permeability to
Ca. The absence of membrane current in the experiment
of Fig. 2B was not due to direct block by
NMDG or the requirement of permeant ion on both sides
of the membrane, because current was activated by ADP unidirectionally
when either the external or internal NMDG was replaced by Cs (Fig. 2, C and D). In the presence of
symmetrical 140 mM Cs, the ADP-evoked I-V
relationship was similar to that observed in
Na/Cs salines and reversed at about
-5 mV (not shown), indicating similar Na and
Cs permeabilities.
Figure 2:
Ionic selectivity of the ADP-evoked
current. The I-V relationships of the ADP-evoked current were obtained
by subtraction of background currents as described in Fig. 1. The major
external and internal ions were, respectively: A, sodium
gluconate and cesium gluconate; B, NMDG chloride and NMDG
chloride; C, cesium gluconate and NMDG chloride; D,
NMDG chloride and cesium gluconate. The external solution also
contained 2 mM CaCl, 1 mM MgCl, 10 mM HEPES, 10 mM glucose,
and the internal solution contained 2 mM MgCl, 0.2
mM GTP, 0.05 mM fura-2. The insets show 10-s
ADP-evoked whole cell voltage clamp currents (upper trace) and
[Ca] (lower trace); the vertical bars represent 50 pA of current (upper
trace) and 0-0.5 mM
[Ca] (lower trace), respectively,
and the horizontal bar represents 10 s (upper trace).
The holding potentials were -40 mV in A, B, and C and 40 mV in D.
Single Channel Activity
Clear single channel
events could not be clearly resolved during the off-phase of the
ADP-evoked whole cell current. This was due to the noise generated by
the high capacitance of the megakaryocyte (20-100 picofarads),
although it also indicates that the ADP-evoked events are of relatively
short duration(14) . We therefore turned to noise analysis to
obtain an estimate of single channel conductance. When the number of
channels opening is small, the variance (
) of the
current is linearly related to the mean current with a slope equal to
the single channel current(15, 16) . For this analysis
we used cells that displayed a small ADP-evoked current, as shown in Fig. 3A. This reduced response was most likely the
result of receptor desensitization (e.g. by ADP and ATP from
damaged cells during the cell preparation) rather than a low number of
total channels. As expected, the variance of the mean of the whole cell
current increased in response to ADP (Fig. 3A, lower
record). A plot of variance against the mean current during the
ADP response could be well fitted by a linear relationship with a slope
of 0.49 pA. The holding potential was -60 mV, and the reversal
potential under these conditions was -5 mV, giving a single
channel conductance of approximately 9 picosiemens. Within the range of
holding potentials -50 to -60 mV, the average conductance
was 8.6 ± 0.4 picosiemens (n = 3). This analysis
assumes the existence of a uniform single channel conductance and that
all channels open independently. The estimate must be considered a
lower estimate for the single channel conductance, and, in practice,
direct measurements of channel conductance are higher(17) .
Figure 3:
Noise analysis of the ADP-evoked
Na current. A, whole cell current (lines) and variance of the mean current (
) recorded at
-60 mV in response to a brief application of 2 µM ADP. Current was low-pass filtered at 100Hz for display purposes
only. B, plot of variance of the mean current during the
ADP-evoked current. Variance and mean current were calculated for three
204.8-ms periods every 2 s (current low-pass filtered at 2.5 kHz and
sampled at 0.5kHz). The solid line is the result of a linear
regression fit and has a slope of 0.49 pA.
Mechanism of Activation
The close association of
the ADP-evoked current with the rise in
[Ca]suggested that
this current may be modulated by Ca or a
Ca-dependent process. To test this, the current was
activated by ADP when internal Ca levels were
strongly buffered by 10 mM BAPTA in the pipette saline. In
order to eliminate the store-dependent inward current that is amplified
under such conditions(3) , Ca was omitted from
the external saline. Fig. 4A compares the
[Ca] changes and
membrane currents activated by a 30-s application of 5 µM ADP in normal (Fig. 4Ai) and enhanced (Fig. 4Aii) calcium buffering in nominally
Ca-free salines. With low buffering, the current was
activated 0.6 ± 0.3 s (n = 10) after ADP
application, peaked within 1-3 s and inactivated to 10% of peak
current after 3.5 ± 2 s (n = 10). In most cells
tested the current was inactivated well before
[Ca] returned to basal
levels (Fig. 4Ai). In the absence of any increase in
[Ca], ADP could still
activate an inward current, although its kinetics were very different (Fig. 4Aii). The current was activated more slowly,
reaching a peak after 5-10 s, and was inactivated more slowly
(time to 10% of peak current was 18.5 ± 6 s; n =
6) compared with the currents activated in the unbuffered cells. This
implies that Ca or a Ca-dependent
process, although not required for activation of the ADP-evoked
current, accelerates the rate of both activation and inactivation. In
order to test if this current could be activated by a rise in
[Ca] alone,
[Ca] was continuously
elevated to micromolar levels using the endoplasmic Ca-ATPase inhibitor
thapsigargin (Fig. 4B). This agent results in a
permanent loss of Ca from IP-sensitive
stores and store-dependent (capacitative) calcium
entry(18, 19) . The absence of any current in the
megakaryocyte after thapsigargin treatment in Ca-free
saline suggests that the ADP-evoked current cannot be triggered by an
increase in [Ca] alone; neither is this current activated as a result of
depletion of internal Ca stores.
Figure 4:
Kinetics and Ca
-dependence of the ADP-evoked current. Simultaneous recordings of
membrane current (top traces) and
[Ca] (lower traces) during
exposure to 5 µM ADP (Ai, Aii, and C) or 1 µM thapsigargin (B).
[Ca] axes are linear. Application of ADP
was identical for Ai and Aii. External saline was 140
mM NaCl and was nominally Ca-free. Internal
salines were 140 mM cesium gluconate saline with 0.05 mM fura-2 (Ai, B, and C) or 80 mM cesium gluconate saline with 10 mM CsBAPTA, 0.05 mM fura-2 (Aii)
(see ``Materials and Methods'' for full details of saline
composition). Holding potential was -40 mV in A and C and -70 mV in B.
Repeated
exposures to ADP could reactivate the monovalent cation current,
provided there was an interval of 1-2 min between successive
applications. Under conditions of high internal Ca buffering, thus removing Ca-dependent
inhibition of the current, both activation and inactivation of the
current became progressively slower with repeated ADP additions, as
shown in the experiment of Fig. 4C. This suggested that
dialysis of the cytoplasm removes factors responsible for activation
and inactivation of the current. These factors may, for example,
generate and metabolize the second messenger involved in channel
activation.
Inositol Phosphate-activated Currents
Release of
internal Ca in nonexcitable cells appears to
ubiquitously involve an increase in cytoplasmic IP levels
and IP-dependent Ca stores(20) .
IP is therefore a candidate for the second messenger
involved in the activation of the monovalent cation channel in the rat
megakaryocyte. A previous study provided preliminary evidence for a
role for IP, because dialysis with 1,4,5-IP activated a monovalent cation current with similar
characteristics to that activated by ADP(3) . However, it was
not shown whether IP was acting alone or in synergism with
other second messengers. Fig. 5A compares the currents
activated by dialysis of 1,4,5-IP with (5Ai) and
without (5Aii) an increase in intracellular Ca levels. For these dialysis experiments, 3 mM 4-aminopyridine was added to the external medium to accelerate the
blockade of K currents. As shown by the
current-voltage relationships acquired at three timepoints during each
experiment, a current similar to that observed with ADP was activated
by 1,4,5-IP and did not require an increase in
[Ca].
Figure 5:
Effect of inositol phosphates and heparin
on IP and ADP-evoked currents. A continuous recording of
membrane current (upper traces) and
[Ca] (lower traces) is shown
during internal perfusion of 10 µM Ins-1,4,5-IP (A), 50 µM Ins-2,4,5-IP (B), 20 µM IP (C), and
10 mg/ml heparin (D). 5 µM ADP was applied
externally in B, C, and D at the times
indicated. In A, Bi, C, and D, I-V
relationships obtained by 0.6-mV/ms voltage ramps are displayed at up
to three time points (a, b, and c) indicated
on the continuous current recording. In A and B, the
continuous records of membrane current and
[Ca] start immediately after the block of
voltage-gated K currents, whereas in C and D, the first 90 s and 5 min of the whole cell recording,
respectively, are not shown. Holding potential was -20 mV in A and Bi and -40 mV in Bii, C, and D; in Bi, the cell was depolarized to
0 mV for approximately 17 s between ramps at B and C.
Current scale bars are 100 pA in A, B, and C and 50 pA in D. [Ca] axes are
linear. The pipette saline in Aii and Bii contained
10 mM CsBAPTA to increase the cytosolic
Ca buffering power. The external saline was 140
mM NaCl, 5 mM CsCl saline throughout which was
nominally Ca-free in A and B but
contained 2 mM CaCl in C and D.
3 mM external 4-aminopyridine was also present in A and B.
1,4,5-IP is rapidly metabolized to other inositol lipid
products, including IP(20) . Its isomer,
2,4,5-IP is also active at IP receptors on the
Ca stores, although at higher
concentrations(21) , and is experimentally useful because it is
a poor substrate for the 1,4,5-IP-kinase. Therefore
2,4,5-IP can be used to stimulate IP-dependent
processes, whereas cytoplasmic levels of IP remain low. As
shown in Fig. 5B (i, left panel),
dialysis with 2,4,5-IP released internal Ca and activated the monovalent cation current in a manner
indistinguishable from that seen with 1,4,5-IP. In the
presence of high Ca buffering power, 2,4,5-IP evoked the inward current as expected and application of ADP
failed to activate further current (Fig. 5Bii). These
results suggest that IP on its own is sufficient to
activate the monovalent cation current without a need for other
inositol lipid products such as IP. Furthermore, following
internal dialysis with 20 µM IP, no inward
current was observed and subsequent exposure to ADP produced a normal
response (Fig. 5C).
Block of the ADP and
IP
1,4,5-IP-activated
Channel-activated channels located on
the membrane of internal Ca stores and those on the
plasma membrane of olfactory receptor neurons are both blocked by
heparin(22, 23) . Fig. 5D shows that
internal perfusion of megakaryocytes with 10 mg ml heparin for 5 min virtually abolished both the
[Ca] and membrane
current response to ADP. Cd has also been shown to
block the olfactory neuron IP-dependent plasma membrane
channel(24) ; however, neither Cd nor
Zn, added to the bath at 1 mM, affected the
monovalent cation currents activated by ADP, 1,4,5-IP, or
2,4,5-IP (not shown). Tetrodotoxin, a blocker of
voltage-dependent Na channels, was also ineffective at
concentrations up to 5 µM added to the bath saline (data
not shown).
ADP- and 1,4,5-IP
In physiological salines,
given a normal negative resting potential, the IP-activated
Na
Entry and
ADP-evoked current would be inward and carried mainly by
Na. To detect whether this conductance can result in
significant changes in
[Na], the
Na-sensitive indicator, SBFI(25) , was added to
the pipette saline in place of fura-2 and 1,4,5-IP dialyzed
from the pipette (Fig. 6A). The inward current that
developed at the holding potential of -40 mV, which we have shown
above to be induced by 1,4,5-IP, was associated with a
gradual increase in
[Na]. Depolarization to
0 or 20 mV prevented the [Na] increase, an effect that was fully reversible, although more
negative potentials were required to produce similar rates of
Na increase once a substantial increase in the 340/380
ratio signal had occurred.
] increase. In these experiments, represented by that in Fig. 6B, a K-based pipette saline and
current-clamp conditions were also used in an attempt to further mimic
physiological conditions. SBFI was loaded prior to patch clamp by
incubation with the acetoxymethyl ester (see ``Materials and
Methods''). Application of 5 µM ADP produced a
regular oscillation in membrane potential from the resting level of
-40 mV to -75 mV (approximately 6 times/min), known to
result from oscillations of [Ca] and activation of Ca-dependent
K channels(1, 2) . In this cell, which
is typical of 5 other experiments,
[Na] increased
gradually by 3-4 mM during the 3 min ADP application and
then continued to increase after agonist removal. Further
[Na] increases were
observed in response to a second exposure to ADP. In 5 cells, after a
3-min application of 5 µM ADP,
[Na] increased from a
resting level of 15 ± 6 mM to 28 ± 13
mM, measured 2 min after removal of the agonist.
evoke a
monovalent cation-selective current in rat megakaryocytes. The
similarity between the I-V relationships, reversal potential,
stimulation of Na influx, and failure of ADP to evoke
a current on top of the IP-induced response, suggests
strongly that these two agents activate the same channel. Although
there is no direct evidence for ADP-induced 1,4,5-IP production in megakaryocytes, ADP is known to stimulate
1,4,5-IP production in platelets (26), and both ADP and
IP induce a similar
[Ca] oscillation in
the rat megakaryocyte (27). In addition, the lack of effect of ADP on
[Ca] after internal
perfusion of heparin, a known blocker of IP receptors,
suggests that this agonist acts via phospholipase C to increase
intracellular IP and internal Ca levels,
as in many other nonexcitable cells (20, 28). The activation of the
monovalent cation current by 2,4,5-IP, a poor substrate for
1,4,5-IP-kinase(21) , and not by IP is
strong evidence for direct stimulation by 1,4,5-IP rather
than by any of its metabolites. The rate of inactivation of the current
was reduced in the absence of a
[Ca] increase, which
may be explained in part by slower hydrolysis of 1,4,5-IP because 1,4,5-IP-kinase is
calcium-dependent(29) . Direct inactivation of the current by
calcium cannot be not ruled out, although this is unlikely because, in
experiments where IP was introduced directly into the
cells, fluctuations in [Ca] had little or no effect on the IP-dependent
current(3) . The reduced activation rate of the ADP-evoked
current in the presence of BAPTA can be explained by the calcium
dependence of phospholipase C activity because IP production is accelerated by an increase in
[Ca](28, 30) .
The lack of effect of thapsigargin-induced rise in
[Ca], which does not
involve an increase in IP(18, 19) , suggests
that the monovalent cation current cannot be activated by
[Ca] alone. The
observation that ADP-evoked currents inactivated more slowly and
incompletely the longer a whole cell recording was made suggests loss
by dialysis of a factor responsible for current inactivation. One
likely candidate for this labile factor is 1,4,5-IP kinase.
stimulates a plasma membrane current indirectly by releasing
Ca from internal stores(3, 4) . This
pathway cannot account for the whole cell currents gated by internal
perfusion of IP in this study, with low internal
Ca buffering, because thapsigargin, which releases
internal Ca without generation of
IP(18) , failed to elicit a significant current.
Furthermore, the store-regulated Ca currrent is
highly selective for divalent cations, is blocked by Zn and Cd, and is amplified by buffering of
internal Ca with BAPTA or
EGTA(4, 31) , none of which applied to the
IP-dependent response under our experimental conditions.
1,4,5-IP receptors have been found in the plasma membrane
of T lymphocytes(32) , platelets (33), and olfactory
cilia(34) , and channels activated by 1,4,5-IP have
been identified in patch clamp recordings from Jurkat T
cells(35) , A431 cells(36) , and olfactory
neurons(23, 24, 37, 38) . In the T cell,
the A431 cell, and insect or channel catfish olfactory neurons, only
divalent cation currents were reported, implying a different
selectivity from the IP-gated channel in the megakaryocyte,
which conducts little, if any Ca. Nonselective cation
currents were activated by IP in rat olfactory neurons,
although, unlike in the megakaryocyte, these were
Cd-sensitive(24) . IP also gates
ion channels in the membranes of internal organelles, including the
sarcoplasmic reticulum (39) and nucleus(40) , which
possess a higher permeability to calcium than monovalent cations. Thus,
the IP-dependent channel in the megakaryocyte may represent
a new class of ion channel gated by this second messenger.
permeability, thus the conductance is
unlikely to play a role in agonist-evoked Ca signaling. Furthermore, the rat megakaryocyte has a
store-dependent influx pathway that is highly selective for
Ca(3, 4) , and this is likely to
account for most if not all of the Ca influx that
occurs during IP-dependent Ca release(3) . Application of ADP caused a 10-20
mM increase in
[Na], which,
considering the large volume of the megakaryocyte, amounts to a
considerable Na influx. The continued, slow increase
in [Na], after removal
of ADP, may be explained if IP levels remain elevated for
some time. This certainly does appear to be the case because repetitive
hyperpolarizations in the membrane potential, known to arise from
IP-induced Ca release and activation of
Ca-dependent K channels(27) ,
continued for several minutes after ADP application. Agonist-evoked
Na influx may outlast the Ca responses if IP levels are higher at the plasma
membrane, where this messenger is produced, than deeper in the
cytoplasm near the Ca stores or if the threshold for
activation of the plasma membrane channel by IP is lower
than for that of store Ca channel. An alternative
explanation is that the gradual increase in
[Na] is due to slow
equilibration of Na within the cytoplasm following
IP-evoked Na entry. This would imply a
much greater increase in [Na] next to the plasma membrane and may be detectable by
confocal ratiometric measurements of SBFI fluorescence. The monovalent
cationic conductance that we report here is of particular interest
since a previous study by Leven et al.(7) concluded
that the ADP and thrombin-evoked spreading reaction in the rat
megakaryocyte, which may be a functional response leading to platelet
formation, depended upon an increased Na conductance.
Further work, however, is needed to assess whether the
IP-activated Na influx is indeed involved
in the cell spreading reaction because the present study did not assess
the possible contribution of ADP-dependent stimulation of
Na/Ca exchange or inhibition of
Na/K exchange to the observed
[Na] increase.
-dependent
cation current, in addition to playing a role in the megakaryocyte, may
be important for platelet signaling. With a much larger surface area to
volume ratio in the platelet, this current may result in large
alterations of [Na]. In
fact, in human platelets, thrombin induces a relatively greater
production of IP than ADP (26) and a greater increase in
[Na](41, 42) .
The ADP-induced rise in [Na] has been shown to be mostly via ADP-activated
receptor-operated channels(421) ; however, the mechanism of the
thrombin-induced Na influx is not known and may
involve the channel we report here in the rat megakaryocyte if this is
also expressed in human megakaryocytes.
that carries Na into the cell
at resting membrane potentials and may have a functional role in
megakaryocyte signaling or be expressed for later use in platelet
responses.
], cytosolic Ca concentration; SBFI, sodium-binding benzofuran isopthalate;
IP, myo-inositol trisphosphate (with positional
determinants of the phosphate groups as specified); IP, myo-inositol 1,3,4,5-tetrakisphosphate; BAPTA,
1,2-bis-(2-aminophenoxy)ethane-N,N,N`,N`-tetraacetic acid; NMDG, n-methyl-D-glucamine;
[Na], cytosolic Na concentration; I-V, current-voltage.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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