Journal
of
Physiology
(1990),
431,
pp.
187-206
187
With
8
figures
Printed
in
Great
Britain
VOLTAGE-GATED
CALCIUM
AND
POTASSIUM
CURRENTS
IN
MEGAKARYOCYTES
DISSOCIATED
FROM
GUINEA-PIG
BONE
MARROW
BY
KAZUYOSHI
KAWA
From
the
Department
of
Pharmacology,
Gunma
University
School
of
Medicine,
Maebashi
371,
Japan
(Received
19
March
1990)
SUMMARY
1.
The
electrophysiological
properties
of
the
cell
membrane
of
guinea-pig
megakaryocytes
were
studied
using
the
whole-cell
patch-clamp
technique.
The
megakaryocytes
(diameter,
17-42
,um)
were
dissociated
mechanically
from
the
bone
marrow
of
adult
guinea-pigs.
2.
In
a
proportion
of
cells,
spike-like
action
potentials
were
generated
in
response
to
depolarization
when
the
cells
were
immersed
in
standard
saline
containing
10
mM-
Ca2
.
Under
voltage
clamping,
a
transient
inward
current
followed
by
a
slowly
developing
outward
current
was
produced
when
the
membrane
potential
was
made
more
positive
than
-55
mV.
3.
The
inward
currents
were
identified
as
Ca2+-carried
current,
since
the
amplitude
depended
distinctly
on
external
Ca2+
concentration
and
since
replacement
of
external
Ca2+
with
Mn2+
reversibly
diminished
the
current.
The
Ca2+
channels
involved
are
most
probably
of
the
transient
type
(T-type).
4.
The
reversal
potential
of the
outward
current
changed
from
-87
to
-46
and
-7
mV
when
the
external
K+
concentration
was
raised
from
5
to
25
and
125
mm.
5.
The
outward
current
was
insensitive
to
chelation
of
internal
Ca2+
but
was
blocked
by
external
application
of
quinine,
4-aminopyridine
and
tetraethyl-
ammonium,
and
was
thus
very
probably
a
membrane
potential-dependent
K+
current.
The
dependence
of
the
current
activation
and
inactivation
on
the
membrane
potential
was
consistent
with
that
of
a
delayed
K+
rectifier.
6.
The
amplitudes
of
the
Ca2+
currents
and
K+
currents
showed
considerable
intercell
variation.
However,
the
density
of
the
Ca2+
current
showed
a
tendency
to
increase
with
megakaryocyte
size,
presumably
accompanying
maturation.
The
roles
of
these
currents
in
cellular
function
remain
to
be
elucidated.
INTRODUCTION
Recent
studies
on
the
membrane
properties
of
blood
cells
using
patch
electrodes
have
revealed
the
presence
of
various
types
of
voltage-dependent
ion
channels
(Chandy,
DeCoursey,
Cahalan
&
Gupta,
1985;
Gallin,
1986;
Lewis
&
Cahalan,
1988).
Some
of
these
channels
seem
to
play
a
crucial
role
in
the
regulation
of
blood
cell
functions
(Lewis
&
Cahalan,
1988).
MS
8355
In
mammals,
platelets
are
the
smallest
cells
or
cell
fragments
in
the
body
and
perform
multiple
functions
in
haemostasis,
phagocytosis
and
serotonin
metabolism
(Zucker,
1980).
Maruyama
(1987)
has
recently
investigated
the
electrical
properties
of
mammalian
platelets
using
patch-clamp
techniques.
Although
neither
voltage-
dependent
inward
currents
nor
chemically
induced
currents
were
detected
in
the
platelet
membrane,
one
type
of
K+
channel
was
identified,
which
resembled
the
delayed
rectifier
K+
channel
found
in
other
tissues.
As
mammalian
platelets
are
small
and
fragile,
some
ion
channels
or
chemical
receptors
might
be
lost
or
undergo
functional
modification
during
platelet
isolation
or
immediately
after
establishment
of
the
whole-cell
configuration.
For
example,
in
thrombocytes
of
the
newt,
which
are
analogous
to
mammalian
platelets,
the
gating
properties
of
one
type
of
K+
channel
in
the
membrane
are
markedly
modifiable;
after
chemical
or
mechanical
irritation,
the
inactivation
property
of
the
transient
K+
channel
is
almost
eliminated
(Kawa,
1987
a).
In
order
to
avoid
such
probable
drawbacks
associated
with
mammalian
platelets
and
to
further
characterize
their
membrane
properties,
we
have
employed
the
progenitors
of
mammalian
platelets,
i.e.
megakaryocytes,
freshly
dissociated
from
the
bone
marrow
of
the
guinea-pig.
The
megakaryocytes
release
fragments
of
cells
into
the
circulating
blood
while
maturing
in
the
bone
marrow
(Fedorko,
1978).
Upon
application
of
depolarizing
current
pulses,
megakaryocytes
are
able
to
generate
action
potential-like
responses
(Miller,
Sheridan
&
White,
1978).
Most
of
the
functions
of
platelets,
such
as
phagocytotic
activity
(Fedorko,
1977
b),
responsiveness
to
bioactive
substances
(Leven
&
Nachmias,
1982;
Miller,
1983)
and
uptake
of
serotonin
(Fedorko,
1977
a),
are
reportedly
present
in
megakaryocytes.
Isolated
guinea-pig
megakaryocytes
are
spherical
in
shape
with
a
diameter
of
17-42
gm
and
permit
stable
whole-cell
recordings
with
a
patch
electrode.
In
this
study
we
have
identified
voltage-dependent
Ca2+
and
K+
currents
in
the
megakaryocyte
membrane.
Their
amplitudes,
however,
showed
remarkable
variation
among
the
cells
studied.
Characterization
of
these
two
voltage-dependent
ion
channels
and
comparison
with
previous
observations
obtained
in
guinea-pig
megakaryocytes
using
conventional
microelectrodes
(Miller
et
al.
1978)
are
described
here.
METHODS
Preparation
Adult
male
guinea-pigs
of
the
Hartley
strain
weighing
350-630
g
were
anaesthetized
by
intraperitoneal
injection
of
urethane
(1000
mg/kg;
Tokyo
Kasei,
Japan)
and
then
killed
by
an
overdose
of
vapourized
ether.
Tibial
or
femoral
bones
were
isolated
from
each
animal.
Under
a
dissecting
microscope
(
x
8-40),
the
bone
marrow
was
isolated
by
gently
cutting
the
osterous
tissues.
The
bone
marrow
was
roughly
dissociated
with
a
pair
of
foreceps
in
a
plastic
dish
containing
0
mM-Ca2+
saline
(Table
1)
and
then
stored
at
4
°C
until
use.
The
recording
chamber
(volume,
0-2
ml)
employed
had
a
base
made
of
a
cover-slip
0-15
mm
thick.
The
base
was
coated
beforehand
with
adhesive
polypeptide
(Cell-Tak,
Collaborative
Research
Inc.,
MA,
USA;
Waite
&
Tanzer,
1981)
by
adding
200
,u
of
diluted
solution
(final
concentration
of
polypeptide,
0
005
%)
to
the
chamber.
A
few
pieces
of
bone
marrow
were
then
transferred
to
the
recording
chamber
containing
the
external
standard
saline,
and
the
cells
were
further
dissociated
gently
with
a
pair
of
fine
forceps.
To
facilitate
stable
settlement
of
cells
on
the
base
of
the
chamber,
the
chamber
containing
dissociated
cells
was
placed
in
the
basket
of
a
rotor
and
centrifuged
at
about
20
g
for
188
K.
KA
WA
Ca2+
AND
K+
CURRENTS
IV
MEGAKAR
YOCYTES
5
min,
after
the
upper
surface
of
the
chamber
had
been
sealed
with
a
plastic
plate.
Then,
the
chamber
was
mounted
on
the
movable
stage
of
a
phase-contrast
inverted
microscope
(final
magnification
x
600,
Diaphot-TMD,
Nikon)
and
was
perfused
with
experimental
saline
at
a
rate
of
1-2
ml/min.
The
temperature
of
the
chamber was
monitored
with
a
thermistor
probe
and
maintained
at
25
+
1
'C.
TABLE
1.
Composition
of
solutions
(mM)
External
solutions*
NaCl
KCl
CaCl2
MgCl2
Standard
saline
120
5
10
1
0
mM-Ca2+
saline
135
5
1
3
mM-Ca2+
saline
130
5
3
1
30
mM-Ca2+
saline
92
5
30
1
25
mM-K+
saline
100
25
10
1
125
mM-K'
saline
125
10
1
EGTA
(potassium
Internal
solutionst
KCl
CsCl
CaCl2
salt)
MgCl2
Simple
KCl
saline
150
1
Ca/EG-01
KCl
saline
150
005
01
1
Ca/EG-0-5
KCl
saline
150
0-25
05
1
Ca/EG-5
KCl
saline
150
2-5
5
1
EG-5
KCl
saline
150
5
1
Ca/EG-01
CsCl
saline
150
005
01
1
EG-5
CsCl
saline
150
5
1
*
In
external
media,
each
saline
also
contained
10
mM-D-glucose
and
10
mM-HEPES
(sodium
salt)
to
give
a
final
pH
of
7-3+0-1
at
25+1
°C.
t
In
internal
media,
each
saline
also
contained
10
mM-HEPES
(potassium
salt)
to
give
a
final
pH
of
7-2+0-1.
Electrical
recordings
The
whole-cell
variation
of
the
patch-electrode
voltage-clamp
technique
was
employed
(Marty
&
Neher,
1983).
The
details
of
this
technique
were
similar
to
those
used
previously
(Kawa,
1987
a,
b).
In
brief,
the
patch-clamp
amplifier
used
was
an
EPC-7
(List,
FRG)
with
a
05
GQ
feedback
resistor.
The
resistance
of
the
patch
electrodes
ranged
between
2
and
10
MQ
when
filled
with
an
internal
solution.
The
configuration
of
whole-cell
recording
was
usually
established
in
the
standard
saline.
After
a
gigaohm
seal
with
a
cell
had
been
obtained,
negative
pressure
of
up
to
120
cmH2O
was
applied
to
rupture
the
membrane
at
the
tip
of
the
patch
electrode.
When
the
megakaryocyte
being
recorded
was
to
be
exposed
to
a
different
external
saline,
a
glass
micropipette
(orifice
diameter,
4-8
,um)
containing
the
different
external
saline
was
positioned
within
a
distance
of
100
,um
from
the
cell.
Then,
the
cell
was
locally
perfused
with
the
saline
by
applying
pneumatic
pressure
of
about
3
kPa
to
the
micropipette.
No
compensation
for
leakage
at
the
tip
of
the
patch
electrode
was
made,
unless
otherwise
stated.
Errors
caused
by
liquid
juention
potentials
were
corrected
as
described
previously
(Kawa,
1987
a,
b).
Membrane
potentials
and
current
signals
were
displayed
on
a
storage
oscilloscope
(5111,
Tektronix)
and
photographed.
Staining
for
acetylcholinesterase
Dissociated
bone
marrow
cells
in
the
chamber
were
stained
for
acetycholinesterase
using
a
modification
of
the
method
of
Karnovsky
&
Roots
(1964).
In
some
mammals
including
guinea-pigs,
the
cellular
activity
of
acetylcholinesterase
has
been
used
to
confirm
the
identification
of
megakaryocytes
(Zajicek,
1957;
Jackson,
1973;
Stenberg
&
Levin,
1987).
The
procedures
of
cell
isolation
and
centrifugation
were
the
same
as
those
for
electrical
recordings.
The
reaction
substrate
medium
consisted
of
10
mg
acetylthiocholine
iodide
dissolved
in
15
ml
of
0
1
M-sodium
phosphate
buffer
(pH
60).
With
constant
stirring,
1
ml
of
01
M-sodium
citrate,
2
ml
of
30
mM-copper
sulphate
and
2
ml
of
5
mM-potassium
ferricyanide
were
added
in
order.
After
the
substrate
medium
had
been
applied
to
the
cells
in
the
chamber,
the
cells
were
incubated
at
37
'C
for
120-145
min
189
under
100%
humidity.
Preliminary
experiments
on
the
incubation
time
had
suggested
that
for
megakaryocytes
isolated
from
guinea-pigs,
the
above
incubation
time
was
most
adequate,
whereas
for
megakaryocytes
from
mice
(adult
male,
ddy
strain),
60
min
was
sufficient
for
the
reaction.
After
the
incubation,
the
cells
were
fixed
for
10
min
in
10%
formalin
and
rinsed
twice
with
distilled
water.
The
preparation
was
then
covered with
glass
and examined
using
a
transmission
microscope
(
x
600,
Optiphot,
Nikon,
Japan).
Solutions
The
compositions
of
the
external
and
internal
(inside
the
patch
electrode)
solutions
are
listed
in
Table
1.
The
osmolalities
of
external
and
internal
salines
were
measured
with
an
automatic
osmometer
using
freezing-point
depression
(OM-6020,
Kyoto
Daiichi
Kagaku,
Kyoto,
Japan)
and
their
values
were
280
+
5
and
290
+
5
mosmol/kg
H20,
respectively.
This
combination
of
osmolalities
was
found
to
be
most
suitable
for
obtaining
a
high
seal
resistance
between
the
cell
and
the
tip
of
the
patch
electrode.
The
free
Ca2+
concentrations
in
the
internal
media
were
estimated
by
calculation
with
a
personal
computer
as
described
previously
(Kawa,
1987
b),
and
were
61,
61,
59
nm
and
less
than
1
nm
for
Ca/EG-0
1,
Ca/EG-0-5,
Ca/EG-5
and
EG-5
KCl
salines,
respectively.
For
Ca/EG-0
1
and
Ca/EG-5
CsCl
salines,
the
calculated
values
were
61
nm
and
less
than
1
nm,
respectively.
Chemicals
for
the
staining
solution,
ethyleneglycol-bis-(,8-amino-ethylether)N,N'-
tetraacetic
acid
(EGTA),
N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic
acid
(HEPES)
and
4-
aminopyridine
(4-AP),
were
obtained
from
Sigma
Chemical
Co.
(St
Louis,
MO,
USA).
Quinine
sulphate
and
tetraethylammonium
chloride
(TEA)
were
obtained
from
Nakarai
Chemical
Co.
(Kyoto,
Japan).
RESULTS
Cell
identification
and
passive
membrane
properties
Morphological
characteristics
of
megakaryocytes
Under
phase-contrast
microscopy,
megakaryocytes
isolated
from
adult
guinea-
pigs
appeared
almost
spherical
with
a
diameter
of
17-42
,um.
The
cells
which
settled
on
the
base
of
the
chamber
were
solitary
and
exhibited
a
bright
cell
surface.
Since
other
contaminant
cells
had
a
small
diameter
of
less
than
12
,um,
it
was
possible
to
discriminate
megakaryocytes
from
others
without
difficulty.
To
further
confirm
their
identification,
specific
staining
for
acetylcholinesterase
was
performed
(Fig.
1).
After
incubation
in
the
staining
solution,
almost
all
the
cells
showing
the
characteristic
morphological
features
described
above
were
stained
dark
brown.
Other
contaminant
cells
remained
almost
unstained
or
only
faintly
stained,
presumably
due
to
non-
specific
esterase
activity.
These
observations
were
consistent
with
those
reported
previously
for
guinea-pig
megakaryocytes
(Zajicek,
1957;
Fedorko,
1978;
Leven
&
Nachmias,
1982).
It
is
possible,
however,
that
a
fraction
of
megakaryocytes,
especially
those
at
the
undifferentiated
stage,
might
have
a
smaller
diameter
and
only
slight
acetylcholinesterase
activity,
thus
making
them
unsuitable
for
the
above
identification
method
(see
Stenberg
&
Levin,
1987).
In
this
study,
no
efforts
were
made
to
identify
such
small
megakaryocytes,
if
present,
and
only
megakaryocytes
with
definite
features
recognizable
by
phase-contrast
microscopy
were
used
(i.e.
a
diameter
of
17
,cm
or
more
and
a
bright
cell
surface).
Resting
membrane
potentials
In
megakaryocytes
immersed
in
standard
external
saline,
the
zero-current
potential
was
measured
immediately
after
the
establishment
of
whole-cell
recordings.
The
value
ranged
between
-67
and
-19
mV,
with
a
mean
of
-48
+
15
mV
(mean
+
S.D.,
n
=
21).
The
mean
diameter
of
recorded
megakaryocytes
was
23
+
6
,um
190
K.
KA
WA
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
191
(mean
+S.D.,
n
=
21),
which
was
estimated
before
patch
electrode
application.
In
twelve
megakaryocytes
with
diameters
of
23
+
2
gm
(mean
+S.D.),
the
input
resistance
of
the
cell
was
measured
with
a
hyperpolarizing
current
pulse
(20
pA,
300
ms).
The
input
resistance
was
1-5+1-2
G
(mean+s.D.,
n
12).
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patch
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negligible.
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the
same
consideration,
the
real
resting
potential
in
the
absence
of
leakage
may
be
more
negative
than
the
mean
zero-current
potential
obtained
above.
Generation
of
action
potentials
All-or-none
action
potentials
in
megakaryocytes
In
some
megakaryocytes
immersed
in
the
external
standard
saline,
it
was
possible
to
generate
spike-like
action
potentials
under
current-clamp
conditions
when
the
cell
was
held
hyperpolarized
at
around
-80
mV
and
stimulated
with
depolarizing
current
pulses
(Fig.
2A).
The
threshold
for
the
action
potentials
was
about
-55
mV.
The
duration
of
each
action
potential
was
50-80
is,
and
was
usually
followed
by
an
K.
KA
WA
.,
_ _
--.--4
0
100
ms
-3
mV
1--
-13
II
-23
I500
pA
Potential
(mV)
-1000
CL
c
*0
-500
C-)
Fig.
2.
A,
action
potential
in
a
guinea-pig
megakaryocyte.
Two
traces
of
membrane
potentials
recorded
under
current-clamp
conditions
are
shown.
With
a
DC
current
of
60
pA,
the
cell
was
held
at
hyperpolarization
around
-80
mV.
Open
bar
under
traces
indicates
the
duration
of
overlapping
current
pulses
used
for
depolarization.
With
a
pulse
of
20
pA,
the
cell
showed
an
almost
passive
response
(lower
trace)
but
with
a
pulse
of
doubled
intensity,
the
cell
generated
a
spike-like
action
potential
(upper
trace).
Dashed
line
indicates
the
0
mV
level.
External
medium,
standard
saline.
Cell
diameter,
35
,um.
Patch
pipette
contained
Ca/EG-5
KCI
saline.
B,
whole-cell
membrane
currents
of
the
same
megakaryocyte
under
voltage
clamping.
Insets
show
specimen
records
of
membrane
currents.
Two
and
three
traces
are
superimposed
in
upper
and
lower
records,
respectively.
Numbers
indicate
membrane
potential
(mV)
during
the
pulse.
Holding
potential,
-83
mV.
Time
scale
applies
to
all
traces.
Lower
right,
current-voltage
relations
at
the
peak
inward
current
(@)
and
at
the
end
of
the
pulse
(0).
Vh
represents
the
holding
potential.
after-hyperpolarization
lasting
several
tens
of
milliseconds.
The
action
potential
could
not
be
generated
repetitively
even
with
prolonged
depolarizing
current
pulses
(500
ms).
It
was
noted
that
the
occurrence
of
action
potentials
showed
considerable
intercell
variation
among
the
megakaryocytes
investigated;
some
megakaryocytes
could
generate
only
abortive
hump-like
potentials
(duration,
70-100
ms),
while
other
192
A
-40
.5°
c
0
0
B
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
cells
showed
no
regenerative
potentials
even
though
the
cells
were
hyperpolarized
below
-80
mV
to
remove
the inactivation
and
were
stimulated
with
depolarizing
pulses
(10-50
pA,
200-500
ms).
Membrane
currents
under
voltage
clamping
To
further
characterize
the
electrical
properties
of
megakaryocytes,
membrane
currents
were
recorded
using
whole-cell
patch
clamping.
Figure
2B
shows
one
example
of
the
current
recordings
and
I-V
relations
obtained
from
a
cell.
Transient
inward
currents
were
evoked
when
the
membrane
potential
was
made
more
positive
than
-55
mV
from
a
holding
potential
of
-83
mV.
The
amplitude
of
the
inward
current
increased
as
the
membrane
potential
was
made
more
positive,
and
reached
a
maximum
peak
amplitude
at
about
-20
to
-30
mV
(0
in
Fig.
2B,
right).
When
the
potential
exceeded
-40
mV,
an
outward
current
followed
the
inward
current.
Its
amplitude,
observed
at
the
end
of the
140
ms
voltage
pulse,
increased
as
the
membrane
potential
was
made
more
positive
(O
in
Fig.
2B).
Negative
voltage
pulses
down
to
-
153
mV
did
not
produce
appreciable
voltage-dependent
currents;
there
was
no
sign
of
inward
rectification.
The
amplitudes
of
inward
currents
as
well
as
those
of
the
outward
currents
showed
remarkable
intercell
variation;
in
about
half
of
the
megakaryocytes
examined,
no
measurable
inward
currents
(>
5
pA)
were
evoked.
The
amplitude
of
outward
currents
measured
at
-3
mV
differed
by
more
than
10-fold
among
the
megakaryocytes
studied
(see
below,
Fig.
8).
Identification
of
voltage-dependent
Ca2+
channels
Effects
of
changes
in
external
Cia2+
concentration
In
the
following
experiments,
we
used
patch
electrodes
filled
with
EG-5
CsCl
saline,
since
internal
Cs+
was
able
to
block
the
outward
currents
(see
Fig.
7B)
and
facilitated
the
isolation
of
inward
currents.
The
amplitude
of
inward
currents
was
remarkably
increased
as
the
concentration
of
external
Ca2+
([Ca2+]O)
was
raised.
In
the
cell
shown
in
Fig.
3A
(diameter,
27
,um)
the
maximum
peak
amplitude
of
the
inward
currents
increased
from
250
pA
in
3
mM-Ca2+
saline
(upper
inset
and
EC
in
Fig.
3A)
to
440
pA
in
the
standard
saline
(containing
10
mM-Ca2
,
lower
inset
and
0).
The
I-V
relationship
shifted
along
the
voltage
axis
in
a
positive
direction
when
[Ca2+].was
increased,
which
would
be
expected
for
a
change
in
the
surface
potential
(Ohmori
&
Yoshii,
1977).
In
another
megakaryocyte
(diameter,
27
,cm)
for
which
[Ca2+]0
was
raised
from
10
to
30
mm,
the
maximum
peak
amplitude
of
the
inward
current
increased
from
320
pA
(depolarization
at
-18
mV)
to
380
pA
(depolarization
at
-8
mV).
These
results
suggest
a
definite
dependence
of
inward
currents
on
[Ca2+]0.
In
contrast,
the
contribution
of
external
Na+
to
the
inward
currents
in
megakaryocytes
seemed
negligible;
when
the
external
NaCl
in
the
standard
saline
was
replaced
with
equimolar
choline
chloride,
the
I-V
relations
for
both
inward
and
outward
currents
remained
almost
unaltered.
Although
non-specific
leakage
currents
seemed
to
increase
reversibly,
the
maximum
peak
inward
currents
measured
after
the
leakage
subtraction
changed
by
less
than
10%.
These
observations
strongly
indicate
that
the
inward
currents
evoked
in
megakaryocytes
are
carried
pre-
dominantly
by
Ca2
.
In
Fig.
3B,
effects
of
Mn2
,
a
specific
Ca2+
channel
blocker
7
PH
Y
431
193
(Hagiwara
&
Byerly,
1981),
were
studied.
External
Mn2+
at
10
mm
selectively
eliminated
the
inward
currents
of
a
megakaryocyte.
The
elimination
was
completely
reversible
after
washing
in
the
standard
saline.
In
the
presence
of
Mn2+,
the
threshold
of
the
outward
currents
appeared
to
shift
in
a
positive
direction
(inset
traces
in
Fig.
3B),
presumably
due
to
stronger
effects
of
Mn2+
than
those
of
Ca2+
on
the
external
surface
charge
of the
membrane
(Ohmori
&
Yoshii,
1977;
Hille,
1984).
A
B
Potential
(mV)
Potential
(mV)
100
Vh
-50
Vh
-0
-0
ms0
0
-28Im\
-100
m
\
0
0aline-200
(1
3
-50
-18
--
d28
an
-400
m
e
-100
pA
V
mv~~~~~~~~p
lOOms
~~~~~~~~-23
20p
-33
I-
Fig.
3.
Characterization
of
inward
currents.
A,
dependence
on
external
Cal2
concentration.
I-V
relations
at
the
peak
inward
current
obtained
from
one
cell
immersed
in
3
mm-Cat2
saline
(El)
and
in
standard
saline
(containing
10
mm-Call,
0).
Vh
represents
the
holding
potential
of
-83
mV.
Patch
electrode
contained
EG-5
CsCl
saline.
Cell
diameter,
27
/tm.
Insets
are
specimen
membrane
current
records
for
the
cell
obtained
in
3
mm-Cab2
saline
(upper)
or
in
10
mm-Ca2e
saline
(lower),
which
showed
near-maximum
inward
currents
at
depolarization
to
-28
and
-18
mV,
respectively.
B,
disappearance
of
inward
current
after
replacement
of
10
mm-Can2
with
isomolar
Mn
2
in
external
saline.
I-V
relations
before
(0)
and
after
(0)
the
replacement
of
external
Cat2
with
Mnt2.
Recovery
of
the
inward
current
was
almost
complete
after
washing
again
in
the
external
standard
saline
(A).
Vth
represents
the
holding
potential
of
-83
mV.
Patch
electrode
contained
EG-5
KCI
saline.
Cell
diameter,
24
/rm.
Insets
are
superimposed
current
traces
obtained
before
(upper)
and
after
(lower)
the
replacement
of
Ca
2+
with
Mn
2+.
Numbers
indicate
membrane
potential
(mV)
during
the
pulse.
Scales
apply
to
both
sets
of
traces.
Voltage-dependent
inactivation
One
of
the
characteristics
of
the
inward
currents
is
that
they
decayed
during
maintained
voltage
pulses
(Fig.
2B).
This
was
also
the
case
when
the
overlapping
outward
currents
were
reduced
using
internal
saline
containing
Cs+
(Fig.
3A).
To
further
characterize
the
inactivation
property,
the
holding
potential
was
altered
and
the
amplitudes
of
the
inward
currents
were
examined
(Fig.
4A).
The
plots
in
Fig.
4A
show
that
steady-state
inactivation
started
around
a
membrane
potential
of
-70
mV
and
became
almost
complete
at
a
membrane
potential
more
positive
than
-40
mV.
Comparison
of
Figs
3A
and
4A
obtained
in
the
standard
saline
indicates
K.
KA
WA
194
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
A
a;
b
-83
mV
-53
_,
Iwave
--
JLWO
*4q%
U
I
4
.
-100
Potential
(mV)
-
.
.
-50
V
vt
B
-33
mV
-13
mi
100
Ms
O.cc
-100
-50
c
-43
100
I100
m
A
100
ms
-1
0
0
Q
.oE
0.5
E2
oc
0
on
-100
E
0
E
0
_
-
50
-O
co
I
XXJOOO-
0
Potential
(mV)
Fig.
4.
Inactivation
properties
of
inward
currents.
A,
steady-state
inactivation.
Upper
traces,
specimen
records
showing
changes
in
amplitude
of
inward
currents
at
various
holding
potentials
from
-83
mV
(a)
to
-53
mV
(b)
to
-43
mV
(c).
Test
voltage
pulses
used
for
evoking
currents
were
unchanged
(at
-23
mV,
270
ms).
Cell
diameter,
24
rtm.
External
medium,
standard
saline.
Internal
medium,
Ca/EG-0
1
CsCl
saline.
Lower
plot,
inactivation
curve
of
inward
currents.
Amplitudes
of
peak
inward
currents
were
normalized
to
the
value
obtained
at
-83
mV
and
their
values
were
plotted
along
the
ordinate
against
the
holding
potentials
along
the
abscissa.
Two
sets
of
data
from
different
cells
are
shown
by
@
and
*,
respectively.
The
latter
includes
the
values
illustrated
on
the
upper
traces.
V,
represents
the
levels
of
depolarization
of
test
pulses
used
for
obtaining
these
data.
The
curve
was
drawn
by
eye.
B,
half-decay
time
of
the
inward
currents
plotted
against
the
voltage
of
depolarization.
The
curve
was
drawn
by
eye.
External
medium,
30
mM-Ca2+
saline.
Internal
medium,
EG-5
CsCl
saline.
Holding
potential,
-73
mV.
Cell
diameter,
27
,um.
Inset
shows
specimen
records
of
the
plotted
currents.
Three
traces
are
superimposed,
which
were
evoked
by
depolarization
to
-33,
-23
and
-13
mV.
In
both
A
and
B,
linear
leakage
currents
have
been
subtracted
electrically.
7-2
195
196
K.
KAWA
-
1000
A
I
+17
mV
0
~~~~~~~~I
1
400
*
pA
500
C-)
-
-43
50
ms
Vh
/
-100
-50
0
50
B
Potential
(mV)
0
/
E
/
+4
mV
.x
/00
a
4,7_
eI
aopA
o
-6
-16
.
/
50
ms
I..
/
>
/a/
Ub
-76mV>
-100
-86_
;I
50
pA
-96/
5
25
125
External
K'
concentration
(mM)
Fig.
5.
Outward
currents
in
megakaryocytes.
A:
left,
specimen
records
showing
seven
current
traces
superimposed.
Figures
(mV)
on
each
trace
indicate
the
membrane
potential
during
the
pulse
(160
ms).
Holding
potential,
-83
mV.
Cell
was
immersed
in
the
standard
saline,
and
the
patch
electrode
contained
Ca/EG-01
KCl
saline.
Linear
leakage
has
been
subtracted.
Note
that
in
this
cell
no
obvious
inward
currents
were
recorded.
Cell
diameter,
23
,um.
Right,
I-V
relations
at
the
peaks
of
evoked
current
in
the
cell.
B,
reversal
potential
of
outward
current
and
its
dependence
on
K+
concentration.
Right,
specimen
records
used
for
measuring
reversal
potentials.
a,
three
superimposed
current
traces
obtained
from
a
cell
immersed
in
125
mM-K+
saline.
The
evoked
current
was
inward
at
depolarization
to
-16
mV
but
was
outward
at
depolarization
to
+4
mV.
It
was
almost
flat
at
-6
mV,
indicating
the
reversal
potential.
Cell
diameter,
19
,um.
Holding
potential,
-66
mV.
b,
three
superimposed
current
traces
obtained
from
the
same
cell
immersed
in
standard
saline
(containing
5
mM-K+).
Tail
current
method
was
used
to
determine
the
reversal
potentials.
Namely,
from
a
holding
potential
of
-66
mV,
conditioning
depolarization
to
-6
mV
(duration,
140
ms)
was
applied
to
activate
outward
currents
(upper
parts
truncated
at
dashed
line);
the
depolarization
was
followed
by
test
pulses
to
obtain
tail
currents.
The
tail
currents
were
outward
and
inward
at
test
pulses
to
-76
and
-96
mV,
respectively.
The
tail
current
was
almost
flat
at
-86
mV,
indicating
the
reversal
potential.
The
reversal
potential
for
25
mM-K+
was
similarly
determined
by
tail
currents.
Relationship
between
reversal
potential
and
external
K+
concentration
is
shown
at
the
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
that
the
Ca2+
current
was
mostly
inactivated
at
membrane
potentials
more
negative
than
the
potential
range
at
which
the
current
was
activated.
Therefore,
the
decay
of
inward
Ca2+
currents
is
not
probably
due
to
Ca2+
influx,
and
thus
the
decay
is
most
probably
caused
by
voltage-dependent
inactivation
(Hagiwara
&
Byerly,
1981).
The
decay
of
the
inward
currents
evoked
by
maintained
voltage
pulses
approximated
a
single
exponential
in
most
of
the
cases
examined
(Fig.
4B).
We
measured
the
half-decay
times
of the
inward
currents
as
a
quantitative
measure
of
the
inactivation
rate.
The
values,
plotted
against
the
pulse
potential,
indicated
that
inactivation
became
faster
as
the
membrane
potential
became
more
positive,
attaining
20
ms
at
0
mV.
The
value
shown
in
Fig.
4B
was
obtained
in
30
mM-Ca2+
saline,
since
a
high
concentration
of
external
Ca2+
induced
larger
inward
currents,
and
a
more
stable
seal
resistance
was
secured
than
in
the
standard
saline.
These
two
features
facilitated
more
accurate
measurements
of
the
half-decay
time.
In
the
standard
saline,
the
relationship
between
the
half-decay
time
and
the
membrane
potential
might
have
been
shifted
by
10
mV
in
a
negative
direction
due
to
changes
in
surface
charge
effects,
as
judged
from
comparison
of
the
I-V
curve
in
standard
saline
with
that
in
30
mM-Ca2+
saline
(not
shown).
Identification
of
voltage-dependent
K+
channels
I-V
relationship
under
voltage
clamping
In
the
following
experiments,
analyses
of
outward
currents
were
performed
in
megakaryocytes
having
no
obvious
inward
current
component.
This
was
done
in
order
to
reduce
contamination
by
Ca2+-carried
currents
in
the
analyses.
With
regard
to
outward
currents,
we
had
noticed
no
qualitative
differences
among
cells
with
or
without
measurable
inward
currents.
When
the
membrane
potential
was
shifted
to
a
level
more
positive
than
-40
mV
from
a
holding
potential
of
-83
mV,
obvious
outward
currents
were
evoked
(Fig.
5A).
The
outward
currents
developed
slowly
and
their
amplitude
reached
a
steady
level
within
30-80
ms.
The
amplitude
of
the
outward
current
measured
at
the
end
of
a
140
or
160
ms
voltage
pulse
increased
steadily
as
the
membrane
potential
was
made
more
positive
(Fig.
5A,
right).
At
stronger
depolarizations
exceeding
0
mV,
the
amplitude
of
evoked
currents
showed
slow
decay
during
the
voltage
pulse,
possibly
suggesting
the
appearance
of
inactivation
at
intense
depolarizations.
Negative
voltage
pulses
down
to
-
153
mV
did
not
produce
appreciable
voltage-dependent
membrane
currents.
Dependence
on
external
K+
concentration
Figure
5B
shows
the
dependence
of
outward
currents
on
the
external
K+
concentration
([K+]0).
The
reversal
potentials
shown
on
the
left
were
determined
from
the
reversal
of
tail
currents.
For
the
standard
saline
(containing
5
mM-K+)
as
well
as
for
25
mM-K+
saline,
tail
currents
were
induced
by
stepping
back
the
left.
Open
circles
and
bars
indicate
the
average
and
the
range
of
observed
values,
respectively.
Number
of
measurements
are
three,
two
and
three
for
5,
25
and
125
mM-K+,
respectively.
The
dashed
line
represents
a
slope
of
59
mV
for
a
10-fold
change
in
external
K+
concentration.
The
megakaryocytes
used
in
these
figures
showed
no
obvious
Ca2+-
carried
currents.
197
membrane
potential
from
the
depolarization
(-10
mV
or
more
positive
potential)
to
various
levels,
as
illustrated
in
Fig.
5Bb.
For
125
mM-K+
saline,
the
reversal
potential
was
determined
directly
by
observing
changes
in
the
direction
of
evoked
currents
(Fig.
5Ba).
Mean
values
of
the
reversal
potentials
thus
obtained
were
-87,
-46
and
-7
mV
for
external
K+
concentrations
of
5,
25
and
125
mm,
respectively;
the
slope
of
changes
in
the
potential
fitted
fairly
well
the
value
expected
from
the
Nernst
equation
for
the
K+
equilibrium
potential
(59
mV/decade
change
in
[K+]0;
dashed
line
in
Fig.
5B).
Thus,
it
seems
safe
to
conclude
that
the
outward
currents
are
carried
predominantly
by
K+.
Properties
of
activation
and
inactivation
To
further
confirm
and
characterize
the
K+
currents,
their
kinetic
properties
were
studied
under
voltage
clamping.
The
voltage
dependence
of
activation
of
the
K+
currents
was
determined
by
recording
evoked
membrane
currents
in
125
mM-K+
saline
and
calculating
the
chord
conductances
from
their
amplitudes.
This
strategy
had
two
merits:
first,
depolarization
up
to
+
40
mV
could
be
applied
to
the
cell
without
saturating
the
current-recording
system.
Second,
with
the
similar
intra-
and
extracellular
K+
concentrations,
the
K+
current
vs.
membrane
potential
relation
predicted
by
the
constant-field
equation
was
approximately
linear;
the
chord
conductance
due
to
K+
currents
becomes
almost
directly
proportional
to
K+
permeability
as
defined
by
the
constant-field
equation
(Hodgkin
&
Katz,
1949;
Hille,
1984).
Figure
6A
shows
that
the
threshold
of
the
conductance
was
about
-40
mV,
and
that
the
conductance
increased
gradually
as
the
membrane
potential
became
more
positive.
The
conductance
reached
a
maximum
value
at
around
0
mV.
Similar
results
were
obtained
from
two
other
cells.
In
Fig.
6B,
the
steady-state
inactivation
and
its
dependence
on
the
membrane
potential
is
illustrated.
The
currents
began
to
be
inactivated
at
around
-50
mV
and
were
almost
completely
inactivated
at
potentials
of
0
mV
or
more
positive.
When
depolatization
steps
lasting
several
seconds
were
applied
to
the
mega-
karyocyte,
the
outward
currents
showed
a
slow
decline
in
their
amplitudes
(Fig.
6
C).
The
outward
currents
decayed
almost
completely
at
depolarization
more
positive
than
0
mV,
if
the
leakage
currents
were
subtracted
from
the
recordings.
The
time
course
of
current
decay
consisted
of
two
components
of
exponential
decay
in
most
cases.
For
example,
the
current
trace
at
+
17
mV
in
Fig.
6C
decayed
with
fast
and
slow
time
constants
of
0-6
and
1'3
s,
respectively.
As
the
time
constants
showed
some
intercell
variations,
no
further
qualification
was
attempted.
These
features
of
voltage-dependent
activation
and
inactivation
of
the
K+
currents
were
consistent
with
those
of
K+
currents
in
other
tissues
supposedly
passing
through
voltage-dependent
delayed
rectifier
K+
channels
(Hille,
1984;
Fukushima,
Hagiwara
&
Henkart,
1984;
Maruyama,
1987;
Lewis
&
Cahalan,
1988).
Sensitivities
to
K+
channel
blockers
The
outward
currents
were
reversibly
suppressed
by
external
application
of
quinine,
4-AP
and
TEA
(Fig.
7A).
Among
these
blockers,
quinine
was
the
most
potent.
The
dose
giving
50
%
inhibition
was
2-3
/LM
for
quinine,
50
JM
for
4-AP
and
4
mm
for
TEA.
The
effectiveness
of
quinine
on
the
K+
current
of
megakaryocytes
is
comparable
to
that
on
mammalian
T-lymphocytes
(Fukushima
et
al.
1984)
and
198
K.
KA
WA
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
199
A
B
400
+31mV
pA
2
1500
-100
Vh-5
NJ
400
-83
mV
-28
500
pA
Potential50
ms
(D
50-ms
200
50
-0.5
E2
Potential
(mV)
-100
6
50
A
t
0
50
z
Vh
-1004lJ
Potential
(mV)
i
-100
-50
0
50~~~~c
-0
actvaedconucane.
ppr
lot
crrnt-olag
reltio
o
meaayctsi
current
traces.
Seven
traces
were
superimposed,
which
were
evoked
by
voltage
pulses
to
+
32,
+22,
+
12,
+2,
-8,
-18
and
-28
mV
(from
upper
to
lower
in
the
inset).
Patch
electrode
contained
EG-5
KCl
saline.
Cell
diameter,
19
,um.
Lower
plot,
the
relationship
between
the
chord
conductance
at
the
peak
of the
evoked
current
(determined
using
a
reversal
potential
of
-6
mV)
and
the
membrane
potential
for
the
same
cell.
Linear
portion
of
leakage
currents
have
been
subtracted.
B,
steady-state
inactivation.
In
the
upper
part
are
specimen
current
traces
obtained
by
applying
a
long
(15
s)
pre-pulse
to
-83
mV
(left)
and
-28
mV
(right),
respectively,
followed
by
a
140
ms
test
pulse
to
+
7
mV.
Linear
portion
of
leakage
currents
has
been
subtracted.
In
the
lower
part
is
the
inactivation
curve
thus
obtained.
The
amplitudes
of
evoked
currents,
which
were
normalized
to
the
maximum
value
of
1280
pA
(evoked
with
a
pre-pulse
to-63
mV),
were
plotted
against
the
pre-pulse
potential.
U
represent
records
illustrated
above.
Cell
diameter,
17
,um.
C,
time
course
of
inactivation.
Typical
example
of
outward
currents,
which
decayed
slowly
during
prolonged
depolarization
pulses
(5
s).
Six
current
traces
were
superimposed.
These
were
obtained
with
depolarization
to
+
17,
+
7,
-3,
-13,
-23
andP-43
mV.
Holding
potential,
-83
mV.
Cell
diameter,
17
,um.
The
cells
shown
in
A,
B
and
C
had
no
obvious
Ca2i-carried
currents
in
the
standard
saline.
mammalian
platelets
(Maruyama,
1987).
The
sensitivities
to
TEA
were
rather
weak
and
the
slope
of
the
dose-effect
curve
was
shallow
for
unknown
reasons.
Even
in
the
presence
of
external
100
mM-TEA,
outward
currents
could
still
be
observed.
Little
effect
of
reduced
[Ca2n]
of
megakaryocyte
on
K+
currents
Since
the
outward
currents
were
highly
sensitive
to
quinine,
which
is
a
K+
channel
inhibitor
reportedly
potent
for
Ca2u-dependent
K+
channels
in
certain
tissues
including
erythrocytes
and
hepatocytes
(Armando-Hardy,
Ellory,
Ferreira,
Flem-
K.
KA
WA
A
.
1.
.)_
Q
a)
0
0-
B
1000
-
500
-3
mV
-13
10-4
M
4-AP
10-3
M
00
pA
_
5n
ms
-
I3
-if
-0---fj
0
0
~~0
0~~
0
\O~~
Cotrl0,-~
9.-
.-..-
Control
-6
-5
~~~~~~-4
-3
-2-1
log
concentration
(M)
C/
\(,OR,
a
-3
mV
a
0
0-0-
J
-43
I_-
b
u
-
I
I
0
2
4
bI
-
500
pA
,
50
ms
6
8
Time
(min)
Fig.
7.
Characterization
of
the
outward
currents.
A,
suppression
of
outward
currents
by
K+
channel
blockers.
Upper
part,
specimen
records
obtained
from
a
megakaryocyte
immersed
in
the
control
standard
saline
(left-hand
traces),
in
the
presence
of
10-4
M
and
10-3
M-4-AP
(middle
and
right-hand
traces,
respectively).
In
each
set
of
traces,
superimposed
are
three
current
traces
evoked
at
depolarizations
to
-3,
-13
and
-23
mV
from
the
holding
potential
of
-83
mV.
Cell
diameter,
24
,tm.
Lower
part,
dose-effect
relation
for
quinine
(E1),
4-AP
(M)
and
TEA
(0).
Amplitudes
of
evoked
outward
currents
at
-3
mV
were
normalized
to
those
in
the
control
saline
and
were
plotted
against
the
dose
of
blockers.
For
quinine
and
4-AP,
each
point
represents
an
average
of
two
different
cells;
the
curves
through
the
data
were
drawn
assuming
one-to-
one
binding
with
dissociation
constants
of
2-3
and
50
/LM
for
quinine
and
4-AP,
respectively.
For
TEA,
data
were
obtained
from
two
megakaryocytes
at
different
doses;
the
curve
was
drawn
by
eye.
B,
little
effect
of
decrease
in
internal
Ca2+
concentration
on
outward
currents.
Patch
electrode
was
filled
with
EG-5
KCl
saline
(Table
1)
containing
5
mM-EGTA
and
applied
to
a
megakaryocyte.
After
establishment
of
the
whole-cell
configuration
(i.e.
zero
time
on
the
abscissa),
the
amplitudes
of
outward
currents
evoked
by
voltage
pulses
to
-3
mV
were
successively
measured
and
plotted
along
the
ordinate
against
time
of
measurement
along
the
abscissa.
Holding
potential,
-63
mV.
Cell
diameter,
18
,um.
External
medium,
standard
saline.
Tip
resistance
of
patch
electrode,
2-5
MQ.
On
the
right,
specimen
records
obtained
at
zero
time
(a)
and
at
5
min
58
s
(b)
are
shown.
In
each
set
of
traces,
superimposed
are
two
current
traces
evoked
at
200
0.
Q
41)
a)
L..)
Ilia.
nI
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
inger
&
Lew,
1975;
Burgess,
Claret
&
Jenkinson,
1981),
the
possibility
remained
that
the
K+
currents
in
megakaryocytes
might
be
Ca2"-dependent
K+
currents.
To
examine
this
possibility,
patch
electrodes
filled
with
internal
saline
containing
5
mM-EGTA
and
no
Ca2+
(EG-5
KCl
saline
in
Table
1)
were
used
for
current
recordings.
The
concentration
of
free
Ca2+
in
the
saline
was
estimated
to
be
less
than
1
nm.
The
tip
resistance
of
the
patch
electrode
was
about
2-5
MQ2.
After
establishment
of
the
whole-cell
configuration,
the
internal
saline
might
diffuse
into
the
cell
interior
from
the
patch
electrode
and
dialyse
the
cell.
During
the
observation
time
of
8
min,
the
amplitude
of
the
outward
current
of
the
dialysed
cell
showed
only
small
changes
(Fig.
7B).
This
small
decline
in
the
amplitude
was
probably
due
to
non-specific
deterioration
of
ion
channels,
which
was
also
described
in
delayed
K+
channels
in
T-
lymphocytes
(Fukushima
et
al.
1984).
To
evaluate
the
efficacy
of
internal
perfusion,
a
patch
electrode
with
similar
tip
diameter
was
filled
with
EG-5
CsCl
saline
and
applied
to
another
megakaryocyte.
Since
Cs+
is
non-permeant
through
various
types
of
K+
channel,
internal
Cs+
might
block
the
K+
currents
(Hagiwara,
1983;
Hille,
1984;
Kawa,
1987
a).
As
shown
by
0
in
Fig.
7B,
the
outward
currents
decreased
with
time
and
after
4
min
the
amplitudes
became
less
than
10
%
of
the
initial
value.
These
results
indicate
that
the
outward
currents
in
megakaryocytes
were
unaffected
by
lowering
[Ca2+]i,
and
that
the
currents
were
most
likely
to
permeate
through
the
voltage-dependent
K+
channels
of
the
delayed
rectifier
type.
Variations
of
Ca2`
and
K+
current
amplitudes
in
megakaryocytes
The
amplitudes
of
the
peak
inward
currents
and
those
of
the
outward
currents
showed
remarkable
intercell
variation.
In
Fig.
8A,
peak
amplitudes
of
inward
currents
measured
at
-23
mV
are
plotted
against
cell
diameters.
Measurement
at
-23
mV
was
chosen
since
the
peak
inward
currents
attained
their
maximum
at
around
this
potential
(Figs
2B
and
3A),
while
the
activation
of
outward
currents
was
slow
and
the
overlapping
on
the
peak
inward
current
seemed
minor
(Figs
2B
and
5A).
Half
of
the
measurements
in
Fig.
8A
(0)
were
obtained
using
Ca/EG-0
1
or
EG-
5
CsCl
saline
(Table
1)
in
order
to
reduce
the
contamination
of
the
K+
current
more
completely.
As
an
index
of
the
magnitude
of
the
outward
currents,
their
amplitude
measured
at
the
end
of
a
140
ms
voltage
pulse
to
-3
mV
was
used.
At
this
potential
and
time,
contamination
of
outward
currents
with
Ca2+
currents
seemed
minor,
since
the
Ca2+
currents
appeared
to
have
declined
during
140
ms
to
almost
zero
(Figs
3A
and
4).
Although
variation
of
amplitude
among
cells
with
similar
diameter
was
substantial,
the
plots
for
both
the
inward
and
outward
currents
in
Fig.
8
seem
to
allow
speculation
that
the
current
amplitude
has
a
tendency
to
increase
with
the
diameter
of
the
cell.
In
order
to
evaluate
the
current
densities
of
the
cell,
the
recorded
megakaryocytes
depolarizations
to
-3
and
-43
mV.
Note
that
leakage
currents
evaluated
at
-43
mV
were
small
and
unaltered
during
the
whole
experiment.
In
another
cell,
changes
in
outward
currents
were
measured
with
a
patch
electrode
containing
EG-5
CsCl
saline;
their
amplitudes
(@)
are
plotted
against
the
time
of
measurement.
Cell
diameter,
24
4Um.
Test
depolarization,
-3
mV.
Holding
potential,
-83
mV.
Tip
resistance
of
patch
electrode,
4-2
MQ.
201
202
K.
KA
WA
were
classified
into
three
groups
depending
on
their
size:
megakaryocytes
with
a
diameter
of
less
than
20
/tm
(group
I),
those
with
a
diameter
of
20
or
26
,tm
(group
II),
and
those
with
a
diameter
of
more
than
26
,tm
(group
III).
The
area
attached
by
the
patch
electrode
could
be
considered
as
negligible
since
the
tip
diameter
of
the
A
B
35001
600
-..
0
400-
0
2000-
o
0o
3
200
0
O
1000
C
l
@]0
0
0
0
w
0
_ _ _ _ _ _ _ _ _
0
I
8
I
*
20
30
40
Cell
diameter
(,m)
20
30
40
Cell
diameter
(gm)
Fig.
8.
Variation
in
magnitude
of
inward
and
outward
currents
in
megakaryocytes.
A,
the
amplitudes
of
the
peak
inward
currents
are
plotted
along
the
ordinate
against
the
diameter
of
cells
along
the
abscissa.
Each
circle
represents
one
cell;
the
internal
saline
filling
the
patch
electrode
was
Ca/EG-01
KCI
saline
for
*
and
Ca/EG-01
or
EG-5
CsCl
saline
for
0.
Cells
producing
no
measurable
inward
currents
are
plotted
in
the
lower
part
between
the
two
horizontal
lines.
Holding
potential,
-83
mV.
External
medium,
standard
saline.
B,
the
amplitudes
of
outward
currents
measured
at
-3
mV
are
plotted
as
in
A.
Patch
electrode
contained
Ca/EG-O
1
KCI
saline.
Holding
potential,
-83
mV.
External
medium,
standard
saline.
electrode
was
less
than
3
gum,
and
thus
any
error
induced
in
the
megakaryocytes
(diameter
range,
17-42
,um)
would
be
less
than
2%.
The
maximum
peak
inward
current
per
unit
area
of
the
cell
was
undetectable,
namely
00
+
00
(S.D.,
n
=
14),
2-4+541
(n
=
26)
and
5-6+6-2
,uA/cm2
(n
=
15)
for
megakaryocytes
in
groups
I,
II
and
III,
respectively.
The
density
of
the
outward
current
measured
at
-3
mV
was
47-5+2216
(S.D.,
n
=
11),
44-3+25-0
(n
=
10)
and
383+238
uA/cm2
(n
=
7)
for
megakaryocytes
in
groups
I,
II
and
III,
respectively.
The
Ca2`
current
density
was
largest
in
group
III
followed
by
those
in
group
II,
while
the
K+
current
density
showed
no
significant
differences
among
these three
groups
(Student's
t
test,
P>
005).
As
megakaryocytes
may
have
fine
indentations
and
processes
in
their
plasma
membrane
(Fedorko,
1978;
Stenberg
&
Levin,
1987),
the
actual
surface
area
will
be
Ca2+
AND
K+
CURRENTS
IN
MEGAKAR
YOCYTES
somewhat
larger
than
those
calculated
above.
The
calculation
of
current
density
should
therefore
be
considered
a
tentative
rough
approximation.
With
these
reservations,
it
can
be
speculated
that
as
megakaryocytes
become
larger,
their
Ca2+
current
densities
tend
to
increase,
whereas
those
of
the
K+
current
remain
almost
unchanged.
DISCUSSION
In
guinea-pig
megakaryocytes
dissociated
from
bone
marrow,
voltage-dependent
Ca2+
and
K+
channels
are
present
in
the
membrane.
Generation
of
Ca2+-dependent
action
potentials
with
an
all-or-none
property
was
observed
in
a
fraction
of
megakaryocytes.
Using
conventional
microelectrodes,
Miller
et
al.
(1978)
observed
that
action
potential-like
responses
could
be
generated
in
guinea-pig
megakaryocytes
with
depolarizing
current
pulses.
In
their
study,
however,
the
electrical
excitability
of
the
megakaryocytes
seemed
to
deteriorate,
presumably
due
to
leakage
at
the
site
of
electrode
penetration.
This
effect
might
have
caused
the
action
potential
to
be
graded
rather
than
all-or-none,
as
described
in
the
present
study.
Recent
studies
have
revealed
various
types
of
Ca2+
channels
in
both
excitable
and
non-excitable
tissues
(Hagiwara
&
Byerly,
1981;
Gallin,
1986;
Tsien,
Lipscombe,
Madison,
Bley
&
Fox,
1988).
The
Ca2+
channels
in
the
megakaryocytes
are
most
likely
to
be
of
the
transient
type
(T-type;
Tsien
et
al.
1988)
judging
from
their
relatively
low
threshold
of
activation
(Figs
2
and
3)
as
well
as
the
rapid
decay
of
the
evoked
current
during
depolarization
(Figs
2
and
4B)
and
steep
voltage
dependence
of
steady-state
inactivation
(Fig.
4A).
The
classification
of
voltage-dependent
Ca2+
channels
may,
however,
be
phenomenological
at
present
and
will
require
a
more
definite
molecular
basis
(Hagiwara,
1983;
Hille,
1984;
Tsien
et
al.
1988).
T-type
Ca2+
channels
have
been
found
in
many
neural
and
muscular
cells
(Tsien
et
al.
1988)
and
also
in
haematocytes
(Fukushima
&
Hagiwara,
1983),
endocrine
cells
(Kawa,
1987
b;
Hiriart
&
Matteson,
1988)
and
other
non-excitable
tissues
(Hagiwara
&
Kawa,
1984;
Hirano
&
Takahashi,
1987;
Chen,
Corbley,
Roberts
&
Hess,
1988).
The
K+
current
of
megakaryocytes
was
characterized
by
its
delayed
activation
(Figs
2
and
5),
and
slow
voltage-
and
time-dependent
inactivation
(Fig.
6)
and
no
definite
dependence
on
[Ca2+]i
(Fig.
7B).
This
type
of
K+
current,
presumably
one
subtype
of
the
delayed
rectifier
K+
current,
has
been
reported
in
other
haematocytes
such
as
human
and
mouse
T-lymphocytes
(Fukushima
et
al.
1984;
Lewis
&
Cahalan,
1988),
mouse
cultured
macrophages
(Ypey
&
Clapham,
1984)
and
mammalian
platelets
(Maruyama,
1987).
The
sensitivity
of
the
K+
current
in
megakaryocytes
to
quinine
was
comparable
to
that
of
mammalian
platelets
and
mammalian
T-
lymphocytes.
It
is
of
interest
that
in
the
newt,
nucleated
thrombocytes
have
voltage-
dependent
K+
channels
of
a
transient
type
in
the
membrane,
and
that
during
activation
of
the
cell,
the
inactivation
property
of
the
K+
channels
is
selectively
eliminated
(Kawa,
1987a).
In
the
present
preparation,
however,
the
presence
of
transient
K+
current
was
never
recognized,
and
the
K+
currents
observed
were
stable
and
seemed
to
consist
of
only
one
type.
Our
present
observation
is
almost
consistent
with
those
in
mammalian
platelets
(Maruyama,
1987).
1203
The
magnitudes
of the
Caa2+-carried
currents
and
K+-carried
currents
in
the
investigated
cells
seemed
to
vary
considerably
(Fig.
8).
We
have
no
clear
explanation
for
this
phenomenon.
As
the
megakaryocytes
increase
in
size
during
maturation
in
the
bone
marrow
(Fedorko,
1978;
Levine,
Hazzard
&
Lamberg,
1982;
Schick,
Schick
&
Williams-Gartner,
1989),
the
tendency
for
a
larger
density
of
Ca2+
current
to
occur
in
larger
megakaryocytes
prompts
speculation
that
the
number
of
functional
Ca2+
channels
per
unit
area
might
increase
during
megakaryocyte
maturation.
Changes
in
current
density
during
cellular
maturation
have
been
reported
in
other
tissues
(Hagiwara
&
Kawa,
1984;
Ypey
&
Clapham,
1984;
Hirano
&
Takahashi,
1987;
Chen
et
al.
1988).
Although
the
biological
significance
of
Ca2+
and
K+
currents
has
yet
to
be
clarified,
one
possibility
is
that
these
channels
may
regulate
the
resting
membrane
potential
and/or
intracellular
Ca2+
and
K+
concentrations,
thus
regulating
the
metabolic
activity
of
the
cell
(Rasmussen
&
Goodman,
1977;
Lewis
&
Cahalan,
1988;
Hoffmann
&
Simonsen,
1989).
An
increase
in
current
density
might
have
some
relevance
to
changes
in
biochemical
activities
at
different
stages
of
development
(e.g.
Schick
&
Filmyer,
1985;
Schick,
Walsh
&
Jenkins-West,
1988).
In
mammalian
platelets,
influx
of
extracellular
Ca2+
into
the
cytoplasm
during
activation
has
been
suggested
(Hallam
&
Rink,
1985).
The
pathway
of
Ca2+
influx
through
the
membrane
might
be
voltage-dependent
Ca2+
channels
and/or
receptor-
operated
Ca2+
channels
(Zschauer,
van
Breemen,
Biihler
&
Nelson,
1988).
A
previous
patch-clamp
study
using
mammalian
platelets
detected
no
voltage-dependent
Ca2+
channels
in
the
membrane
(Maruyama,
1987;
see
also
Doyle
&
Riiegg,
1985).
Although
the
possibility
cannot
be
excluded
that
during
application
of
the
patch
electrode
wash-out
or
deterioration
of
voltage-dependent
Ca2+
channels
might
have
occurred,
rendering
the
Ca2+
channels
undetectable
(Hagiwara
&
Byerly,
1983;
Marty
&
Neher,
1983),
the
roles
of
receptor-operated
Ca2+
channels
would
presumably
be
predominant
for
passing
external
Ca2+
into
the
cytoplasm.
If
this
speculation
is
indeed
the
case,
then
how
and
when
the
voltage-dependent
Ca2+
channels
disappear
and
the
receptor-operated
Ca2+
channels
become
functional
in
the
megakaryo-
cyte/platelet
membranes
would
be
one
of
the
next
key
concerns
in
these
preparations.
I
thank
Ms
Y.
Aoki
and
Ms
Y.
Roppongi
for
their
secretarial
assistance.
This
work
was
supported
by
Grant-in-Aid
for
Scientific
Research
from
the
Ministry
of
Education,
Science
and
Culture
of
Japan.
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