Research Service, Houston Veterans Affairs Medical Center, and Departments of Medicine and of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030
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ABSTRACT |
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Dami human leukemia cells express G protein-coupled thrombin
receptors that operate through the phospholipase C pathway. When these
receptors are activated by -thrombin or by thrombin
receptor-activating peptide, an elevation in cytosolic
Ca2+ concentration develops that
is accompanied by hyperpolarization of the plasma membrane. This
transitory phase of hyperpolarization is primarily mediated by inwardly
rectifying, Ca2+-activated
K+ channels that have an inward
conductance of ~24 pS. In cell-attached patches the channels open
within seconds after superfusion of the cell with thrombin
receptor-activating peptide. In inside-out patches, perfusion of
submicromolar Ca2+ onto the
cytosolic surface of the membrane is sufficient to activate the
channels. In outside-out patches, channel opening can be blocked by
nanomolar concentrations of charybdotoxin. The function of these
intermediate-sized inwardly rectifying,
Ca2+-activated
K+ channels has not been
established; however, by analogy with other cell systems, they may
serve to regulate cell volume during cellular activation or to increase
the electromotive drive that sustains Na+ and/or
Ca2+ influx through ligand-gated
cation channels.
platelet; megakaryocyte; hematopoietic cell; blood cell
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INTRODUCTION |
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IT WAS FOUND IN 1978 by Horne and Simons (13) that hyperpolarizing and depolarizing membrane potential changes accompanied platelet activation after stimulation with thrombin. Their study, which was done using fluorescent membrane potential dyes, predicted that more than one type of ion channel participated in the platelet's "release reaction."
Through use of the patch-clamp technique, which came into wide use in
the 1980s, it is now clear that platelets contain an impressive array
of plasma membrane ion channels. These include Cl channels (18),
nonselective cation channels that conduct
Ca2+ as well as monovalent cations
(2), and Ca2+-activated (19) and
voltage-gated (20) K+ channels.
Despite the fact that these channels have been identified under static
conditions in platelets, evaluating their function during the intricate
process of cellular activation has proven to be a much more nettlesome
task. Owing to their minuscule size, the technical impediments to
studying ion channel operation in patch-clamped platelets after agonist
stimulation are significantly greater than in many other types of blood
cells. For that reason, with very few exceptions (21, 22), information
pertaining to the function of platelet membrane ion channels in the
activated platelet has remained limited.
Because of its central importance to the platelet, we wished to study the ion channels activated through the G protein-coupled thrombin receptor pathway. For this purpose, we chose the Dami human leukemia cell line (9). As a model for this receptor pathway, Dami cells offer several advantages. First, the heptahelical thrombin receptors they express exist on human platelets (4). Second, the receptor activates the phosphoinositide-specific phospholipase C pathway and stimulates a rise in cytosolic Ca2+ concentration ([Ca2+]i) (8) in several types of cells that is similar to that seen in platelets during the release reaction. Finally, the cells are large and lend themselves well to experiments using a patch-clamp electrode (29).
By directly measuring membrane potential under current-clamp conditions
in single Dami cells, we found that activation of thrombin receptors by
perfusion with thrombin receptor-activating peptide (TRAP) resulted in
a complex, triphasic potential change consisting of marked, immediate
hyperpolarization, gradual depolarization, and then repolarization
toward the resting potential (29). In whole cell experiments under
voltage-clamp conditions, we determined that the initial phase of
hyperpolarization was mediated largely by a
Ba2+-sensitive,
Ca2+-dependent, inwardly
rectifying K+ current (29). The
hyperpolarization induced by that current developed within seconds
after perfusion of Dami cells with TRAP or with -thrombin and
coincided with the peak in
[Ca2+]i.
In this report we have focused on the electrophysiological properties
of the channels responsible for this
K+ conductance.
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MATERIALS AND METHODS |
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Cells. The Dami cell line was obtained from the American Type Culture Collection (Rockville, MD). The cells were grown in RPMI 1640 medium containing 10% FCS and 1% penicillin and streptomycin, as previously described (29). Dami cells from stock cultures were replated in 35-mm plastic culture dishes before use. Immediately before each experiment, culture plates were washed and resuspended in bath solution.
Agonists.
The amino acid sequence of the synthetic TRAP used throughout these
experiments was NH+3-Ser-Phe-Leu-
Leu-Arg-Asn-Pro-Asn-Asp-Lys-Tyr-Glu-Pro-Phe. In a prior
study, we established that 1) the
whole cell current that developed after perfusion of a single Dami cell
with TRAP was similar to that after perfusion with -thrombin and
2) perfusion with a peptide similar
to TRAP that did not contain the activating hexapeptide sequence had no
effect on cell current (29). The synthesis and preparation of these
peptides were described in that report (29).
Solutions and reagents. Analytic-grade salts were dissolved in distilled, deionized water. All electrolyte solutions were buffered with HEPES (pH ~7.4), and their osmolarity was adjusted with glucose (~300 mosM). Solutions were sterilized by Millipore filtration and stored at 4°C. Nystatin and EGTA were obtained from Sigma Chemical (St. Louis, MO). Charybdotoxin was purchased from Calbiochem (La Jolla, CA).
Ca2+
concentrations.
Solutions containing
108-10
3
M Ca2+ were prepared to determine
the threshold of activation of the inwardly rectifying,
Ca2+-activated
K+
[K+ir(Ca)] channel in
inside-out patches. Aliquots of one solution that contained (in mM) 150 sodium aspartate, 2 MgCl2, 10 HEPES, and 2.2 EGTA were mixed with aliquots of another containing (in
mM) 150 sodium aspartate, 2 MgCl2,
10 HEPES, and 2 CaCl2. From known
ratios of EGTA and CaCl2, free
Ca2+ concentrations were
derived using a computer program that applied stability constants of
binding reported by Fabiato and Fabiato (5).
Patch-clamp procedure. All data were obtained using an Axopatch-1D patch-clamp amplifier (Axon Instruments, Foster City, CA). Electrodes were pulled from Corning 7052 glass capillary tubes (Garner Glass, Claremont, CA). All experiments were done at room temperature.
Whole cell experiments were carried out under voltage-clamp conditions. Patches were broken by suction or perforated using the antibiotic nystatin, as previously described (29). Cells were clamped at an appropriate holding potential and then switched at 1-s intervals through a series of voltage steps. Pipette tip resistance varied from 1 to 12 M
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Statistics. Combined data are expressed as means ± SE.
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RESULTS |
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Dami cells express barely detectable transmembrane ion currents in the
resting state. However, in a previous study we found that a strongly
hyperpolarizing current develops within seconds after activation of G
protein-coupled thrombin receptors with TRAP or -thrombin (29). This
hyperpolarizing current coincides with transient elevation
of
[Ca2+]i
that results from activation of this receptor pathway (29). In Fig.
1, a representative experiment is shown
that illustrates that a significant fraction, but not all, of the
hyperpolarizing current is inhibited by charybdotoxin. Under the
conditions of this experiment, whole cell current was monitored in Dami
cells under voltage-clamp conditions using the perforated-patch
technique to permit
[Ca2+]i
to rise undisturbed after receptor activation. TRAP alone induced a
strongly hyperpolarizing current that shifted the reversal potential (Erev) from
33 ± 2 to
71 ± 2 mV
(n = 5), whereas in the presence of
charybdotoxin, the increment was reduced from
36 ± 2 to
42 ± 1 mV (n = 4). In prior
experiments we established that current induced by TRAP is primarily
comprised of two components: a
Ca2+-independent, outwardly
rectifying Cl
current and a
Ca2+-dependent,
Ba2+-sensitive, inwardly
rectifying K+ current (29). Our
findings suggest that most of the
K+ current is charybdotoxin
sensitive.
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Although the mechanism through which the
Cl current becomes
activated is unclear, we found that an elevation in
[Ca2+]i
was sufficient to induce the K+ir(Ca)
conductance. As shown in Fig. 2, the
K+ current could be activated by
dialyzing the cell through a broken patch with pipette solution that
contained Ca2+. In Fig.
2A, each cell was suspended in 150 mM
potassium aspartate solution containing EGTA and dialyzed through a
broken patch with the same solution. Under these conditions in which
intracellular Ca2+ was chelated
with EGTA, very little transmembrane current was detected under
voltage-clamp conditions. In Fig. 2B,
each cell was suspended in a solution containing 145 mM potassium
aspartate, 5 mM sodium aspartate, and 2 mM
Ca2+ and dialyzed in a similar
fashion with the same solution through a patch pipette. Under these
conditions in which 2 mM Ca2+ was
introduced into the cell's interior, a strong current developed that
exhibited inward rectification. As would be predicted for a
K+ conductance, the current
reversed at 0 mV when
[K+]o
and
[K+]i
were identical. To determine whether the channels that
carried this current were selective for
K+, the
Na+ and
K+ concentrations in the bath were
reversed. Under these conditions, [K+]o
was 5 mM and
[K+]i
was 145 mM. The results of this manipulation indicated that the current
was carried primarily by K+, since
its Erev was
strongly affected by the external concentration of that cation.
Erev determined
from these experiments (
74.9 ± 1.0 mV,
n = 3) was slightly more positive than
EK predicted by
the Nernst equation (
84.8 mV). Although the current rectified inwardly, some current was carried in the outward direction.
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We then sought to isolate the channels responsible for the Ca2+-activated K+ current. To identify the channel in the intact cell, we first used the cell-attached patch configuration. Under these conditions, in which the cell remains relatively undisturbed, brief perfusion of the cell with TRAP activated an inwardly rectifying "flickery" channel that opened ~5-10 s after perfusion with the agonist and remained active for 30-60 s (Fig. 3). Thereafter, the channel activity slowly dissipated. The period of time during which the channel was active coincided approximately with the transient elevation of [Ca2+]i, which we previously observed in fura 2-loaded Dami cells after activation with TRAP (29). In some cell-attached patches the channel could be reopened by perfusion of the cell a second time with TRAP.
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We then wished to determine whether the channel we observed in
cell-attached patches conducted K+
current and was activated by increases in
[Ca2+]i.
Toward that end, we isolated a channel in cell-free, inside-out patches
that exhibited very similar properties of rectification and
conductance. In these preparations the channel was studied using
Ca2+-free 150 mM potassium
aspartate solution in the pipette, which faced the extracellular
surface of the patch. The bath solution, to which the cytosolic surface
was exposed, contained the same solution as the pipette or a solution
in which Na+ replaced
K+. In
Ca2+-free solutions, most patches
were electrically "silent." However, occasional patches conducted
current under these conditions, presumably through
Ca2+-independent channels, such as
nonselective cation channels or Cl channels. In
electrically silent patches,
Ca2+-dependent channels could be
identified by perfusion of
Ca2+-enriched bath solution onto
the cytosolic surface of the membrane. Ca2+-dependent
K+ channels were identified by the
appearance of current under these conditions where
Erev depended on
the concentration of K+ in the
bath solution. By using this approach, we found that most patches
contained two to four
Ca2+-dependent
K+ channels through which the
current reversed at 0 mV and rectified inwardly when the concentration
of K+ was identical on both sides
of the patch (Fig.
4B).
When the interior surface of the patch was bathed with 150 mM
Na+ (Fig.
4A) instead of
K+ (Fig.
4B), most of the measurable current
was inward, and the current-voltage relationship was practically
asymptotic. The marked shift of the
Erev under these
conditions indicated that the permeability of
K+ through the channel was
substantially greater than that of
Na+. The conductance of the
channel in the inward direction (calculated between 0 and
80 to
140 mV) was 24 ± 1.4 pS (n = 6) in inside-out patches, 23 ± 2.2 pS
(n = 3) in outside-out patches, and 24 ± 3.2 pS (n = 4) in cell-attached
patches. These channels, which exhibited flickery kinetics
of opening, dependence on Ca2+ on
the cytosolic surface of the patch, and conductance at negative potentials of ~24 pS, appeared to be very abundant in the membrane of
the Dami cell.
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The sensitivity of the K+ channel
to
[Ca2+]i
was determined by isolating inside-out patches and perfusing
the cytosolic surface of the patch with bath solutions containing
various concentrations of Ca2+
(Fig. 5). In these preparations the
channels typically began to open when the
Ca2+ concentration rose above
107 M. The dependence of the
channel on a critical internal concentration of
Ca2+ can be seen in Fig.
6, in which the cytosolic surface of the patch was alternately perfused with 8.1 × 10
8 M Ca2+,
at which the channel was closed, and 9.7 × 10
7
M Ca2+, at which three
K+ir(Ca) channels operated.
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As shown in Fig. 7, the channel was inhibited when 50 nM charybdotoxin was perfused onto the outer surface of isolated outside-out patches. Inhibition typically began within a few seconds after perfusion was initiated with the toxin. The inhibitory effect of charybdotoxin was difficult to reverse by washing the patch with bath solution, but usually after 30-60 s, channel openings would gradually resume.
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DISCUSSION |
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Our data indicate that activation of the G protein-coupled thrombin receptor pathway strongly hyperpolarizes Dami cells through the opening of K+ir(Ca) channels. Their conductance of ~24 pS and sensitivity to charybdotoxin categorize them as "intermediate-sized" K+ir(Ca) channels. Similar channels have now been reported in lymphocytes (11), red blood cells (12), macrophages (7), HL-60 cells (31), and platelets (19), supporting the idea that this class of K+ir(Ca) channel may be highly conserved in hematopoietic cells. Because of their rectification properties and the range of Ca2+ concentrations to which they respond, the channels appear to be ideally designed to operate at the negative membrane potentials at which most of these cells have been shown to function (6).
We found the Dami K+ir(Ca) channel to be highly sensitive to [Ca2+]i. In inside-out patches the channels were activated by submicromolar Ca2+ concentrations perfused onto the inner surface. At higher concentrations, the channel showed no evidence of Ca2+-induced block. These findings are in agreement with those of Varnai et al. (31), who described a similar K+ir(Ca) channel in HL-60 cells induced toward granulocyte differentiation. In their study, K+ir(Ca) channels became active when formyl peptide receptors, which operate through a signal transduction network similar to that of thrombin receptors on Dami cells, were stimulated.
The thrombin receptor pathway that we have studied is prototypical of
agonist-activated receptor pathways that operate in many types of blood
cells. For example, with the exception of the erythrocyte, all other
mature blood cells derived from the myeloid pathway remain functionally
dormant until they are stimulated by cell-specific, surface-acting
ligands. The platelet provides an excellent example of this process.
Agonist-induced activation of these cells, which usually involves rapid
cytoskeletal reorganization and release of stored cytoplasmic granules,
is accompanied by a prominent elevation in
[Ca2+]i,
which results from the release of
Ca2+ from intracellular storage
sites and from Ca2+ influx across
the plasma membrane through ligand-gated, voltage-independent Ca2+ channels and nonselective
cation channels (24). In the resting state, the
[Ca2+]i
in these cells is between
108 and
10
7 M (24). The data that
we now report combined with those of Vernai et al. (31) predict that
whenever
[Ca2+]i
rises much above 10
7 M in
cells that contain K+ir(Ca) channels, the channels open and remain active until the
[Ca2+]i
falls to the resting level.
This concept is supported by the fact that distinct phases of
hyperpolarization after agonist stimulation have been observed in
neutrophils (17), platelets (13), mast cells (16), and macrophages (7).
Hyperpolarizing currents have been isolated from the quantitatively
greater depolarizing conductances in some of these cells by altering
the concentration of agonist (13, 17) or by examining the cells during
different phases of ontogenetic development (30). In addition to
hyperpolarization due to K+ir(Ca) currents, Ca2+-dependent and
-independent Cl channels
have been described in a number of hematopoietic cells (15, 18, 25).
Although the precise purpose of hyperpolarizing currents induced by
K+ir(Ca) and/or
Cl
channels in blood cells
is still not entirely resolved, three potential functions, for each one
of which experimental support exists, seem logical.
First, maintenance of a negative membrane potential may be of critical
importance during the process of cellular activation in hematopoietic
cells. After agonist stimulation, most of these cells sustain
significant influxes of Na+ (3,
32) or Ca2+ (32) through a variety
of cation channels and transporters. Yet, despite their small sizes and
consequently large input resistances, depolarization during activation
is usually modest (6, 14) and only rarely gives rise to membrane
potentials more positive than 15 mV. It is likely that the
influx of these cations through electrogenic pathways would be rapidly
impeded by depolarization were it not counteracted by the simultaneous
operation of hyperpolarizing Cl
or
K+ currents.
Second, hyperpolarization may specifically serve to augment
transmembrane Ca2+ and/or
Na+ influx. In 1988, Penner et al.
(23) provided strong evidence that hyperpolarizing currents that
developed in hematopoietic cells during activation increase the
electromotive drive that supports
Ca2+ influx through
voltage-independent Ca2+ channels.
They studied this phenomenon in rat peritoneal mast cells, which bear
substance P receptors that activate the phospholipase C pathway (23).
In that particular cell system,
Ca2+ influx was driven by
hyperpolarization sustained by cAMP-activated Cl channels that opened
during cellular activation (23). However, subsequent reports have made
it clear that Ca2+ influx through
ligand-gated channels is enhanced by hyperpolarization irrespective of
its cause (20, 26). In general, less is known about the function of
Na+ influx during cellular
activation, and, owing to the relative difficulty in quantitating the
intracellular Na+ concentration,
similar studies concerning the effect of membrane potential on
Na+ influx have not been
forthcoming. Nonetheless, in view of the conspicuous elevation of
intracellular Na+ concentration
that occurs during agonist-induced activation of hematopoietic cells
(3), it is reasonable to infer that hyperpolarizing K+ and/or
Cl
currents ought to
provide electromotive support for
Na+ influx through pathways such
as nonselective cation channels and electrogenic
Na+ transporters.
Finally, hyperpolarizing currents may participate in regulation of cell
volume. In many types of blood cells, cell swelling induced by a
hyposmolar environment results in simultaneous increases in
K+ and
Cl conductances across the
plasma membrane (10). At the usual resting potential of these cells,
efflux of both of these ions is favored. The efflux of
K+ and
Cl
under these conditions
is accompanied by osmotically obligated water molecules. The net result
of these events is a reduction in cell volume (10). In addition to
transmembrane shifts in water that result from changes in the osmotic
strength of the cells' environment, platelets undergo complex volume
changes during cellular activation. Within 5 s after stimulation with
thrombin, cytoskeletal reorganization is initiated, heralded by
swelling and rounding of the platelet and culminating in the release of stored granular contents (27). During this process the platelet is
transformed from its original discoid shape initially into a sphere and
finally into a flattened configuration, which facilitates its adherence
to other surfaces. It is plausible that
K+ir(Ca) and/or
Cl
channels may participate
in the cytoplasmic volume shifts that take place during this process.
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ACKNOWLEDGEMENTS |
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We thank Elkin Romero and Marjorie Withers for excellent technical assistance, Dr. Robert Handin for permission to use Dami cells, and Drs. Michael Kroll, Lucie Parent, and Mary Hamra for critical review of the experiments reported.
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FOOTNOTES |
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This work was supported by Veterans Administration Medical Research funds (merit review) and by National Heart, Lung, and Blood Institute Grant HL-45880.
Data in this manuscript have been presented in abstract form (28).
Present address of D. L. Kunze: Rammelkamp Center for Education and Research, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109.
Address for reprint requests: R. Sullivan, Hematology-Oncology, Mail Stop 111-H, Houston VA Medical Center, 2002 Holcombe, Houston, TX 77030.
Received 18 February 1997; accepted in final form 30 July 1998.
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