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Hypotonic solution increases the slowly activating potassium current IsK expressed in Xenopus oocytes
Vol.
184,
No.
2, 1992
April
30,
1992
BIOCHEMICAL
Andreas
RESEARCH
COMMUNICATIONS
INCREASES THE SLOWLY ACTIVATING POTASSIUM EXPRESSED IN XENOPUS OOCYTES
Busch,
E.
Michael
Institute,
Received
BIOPHYSICAL
Pages
HYPOTONIC SOLUTION
Vellum
AND
Varnum,
Oregon
February
27,
Health
John
P. Adelman,
Sciences
CURRENT I,
and R. Alan
University,
804-810
Portland,
North OR 97201
1992
SUMMARY. A slowly activating potassium current was expressed in Xenopus oocytes by injection of RNA transcribed from a rat kidney cDNA clone. Hypotonic solutions (160 mOsmol/l; control was 220 mOsmol/l) increased the current by increasing the rate of activation and by decreasing the depolarization needed to activate the current. This effect of hypotonicity was not observed in calcium-free solution, but was unaffected by staurosporine or the calmodulin antagonist W7. Cytochalasin D reduced the current and prevented the increase by hypotonic solution. The results suggest that the increase in this potassium current by hypotonic solution might result from calcium entry and changes in the actin network. ri 1992 Acadrmlc Press, Inc.
Many cell pressure
osmotic
the
types
regulate
their
by activating
ion
intracellular
decrease the
ion
when
pathways
of
(2-7).
to
potassium
Changes
and
in
processes
apical I,,;
this
induces
the
Xenopus
oocytes
as
whether
I,,
underlying
The purpose
affected
by
in
activating of
the
changes
in
mechanism.
0006-291X/92 Copyright All rights
of a slowly
0
$1.50
1992 hi Academic Press, of reproduction in any form
Inc. reserved.
channel
of the rat
804
thereby
which
vitro c
present osmotic
from current
volume
different
is
(10,ll)
and
in activating in
has become kideny
(I)
when
strength
transport
expressed
rat
experiments
with
([Ca"],),
key roles that
kidney
changing
coincides
concentration to play
potassium
of abnormal
a regulatory
through
calcium
tubule
undergo
an effect efflux
RNA transcribed
expression
was
chloride
One kidney
because
(10).
cells
solution,
intracellular
of the proximal is
Renal
to solutions
the membrane,
have been suggested
(2,8,9).
membrane
when exposed across
(1).
a hypotonic
in cytoskeletalorganization, these
fluxes
concentration
exposed
activation
volume
known
cDNA clone injected
was to and
the
to
into
determine seek
the
Vol.
184,
No.
MATERIALS
BIOCHEMICAL
2, 1992
and
expression
voltage-dependent described
of
potassium
described
voltage
three
clamp
solution
contained
(pH 7.4,
220
were
to six
currents
voltages
was 50 s and 20 s for
Therefore, fitting
evoked D,
-90
the
a single
staurosporine
between
of
were
by
current
does
not the
-90
to
on the
than
of the mean. same day,
a
oocyte
from
more
negative
estimated
Numerical
comparisons groups
of
were
cytochalasin
Incorporated).
paired
such
currents
A23187,
a
(12).
were
RBKl
Statistical
from
48 mM
steady-state
current
were
Biochemicals
only
0 mV. Even with
current.
used
been
1 mM and HEPES 5 mM
mV and
maximum
to 0 mV. Drugs
W7 (Research
-20
reach
the
have
The external
the
to
the
in vitro
ng RNA (14).
depolarizing
positive
of
by two electrode
contained
more
function
recorded
mOsmol/l)
steps
and
and
(RBKl)
1.8 mM, MgCl,
(160
of
channel
RNA transcribed
of 0.5
potentials
as mean f s.e. obtained
donor
currents
evoked
activation
from
(Sigma),
results
capped
Duration
exponential
are reported
single
COMMUNICATIONS
family
with
solution
the
rate
by 250 ms steps
values
mV.
pulses
both
Shaker
injection
were
potential
depolarizing
the
injected
Hypotonic
of
of
days after
holding
by
RESEARCH
potassium
96 mM, KC1 2 mM, CaClz
mOsmol/l).
I SK potassium
kidney
Membrane
(14).
NaCl
the
channel
Oocytes
(12,14).
as previously
long
BIOPHYSICAL
AND METHODS
Cloning
NaCl.
AND
were made
oocytes
from
a
frog.
RESULTS An
outward
depolarized
that
cDNA;
the
(I& the
(1 - 2.5
quickly
constant
conductance
40 mV, respectively
(Fig.
10 min,
within
with
and reversed a
solution
voltage-dependence
of
1).
seen
several
the
activation
seconds
in mock-injected
of activation
was 24.1+
maximal
current
was increased
by about reached
tonicity. to
The a more 805
hypotonic negative
the
rat
were kidney
at -20 mV and 6.0 considerably
the
current
among activated
50 and 20 % for
a steady
when the
oocytes
As previously
3.4
varied
solution,
effect
from oocytes.
In a hypotonic
This
when
RNA transcribed
30 min completely
normal of
with
and 40 mV; the
pA at 40 mV).
and the
over
injected was not
time
s (n - 4) at -20
oocytes more
(12),
developed
had been
current
described +_ 0.9
current
oocyte
level
and
within
about
was perfused
again
solution potential
-20
shifted (Fig.
1B);
the the
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A Hypotomc .A
ji!ziij- fr;500 nA -20 mV
40 mV
10s
B 35
Potential (mV)
Fig.
1. Reduction
mV in control Holding
solution
potential
hypotonic
at which
changed
from
-17.0
substitution)
unchanged application Currents
by
mV to -25.8
+ 1.9
s at -20
did calcium-free
in oocytes
+ 1.3
I,,
expressing
+ 0.5
of
for
normal
not
control
(see
methods)
was
constants
for
at 40 mV (n - 5). no change
not
affected
in
of
osmotic did
and
each point.
The properties
solution
KBKl were
in
The time
48 mM with
(n - 4).
hypotonic
oocytes
at -20 and 40 (160 mOsmol/l).
I,,
mV (n - 5).
to
solution
of a calcium-free
underlying
mV and 3.6
affect
solution
was half-maximal
concentration
not
A, I,, recorded
-90 mV. Five
conductance
3.2
sodium
I,,.
and in hypotonic
potential
underlying
f
of
increases
mV. B, conductance
Holding
13.0
Reduction (Tris
was -90
the
were
strength
(220 mOsmol/l)
solutions.
voltage
activation
in ionic
tonicity
I,,
were
strength; increase
also
however, I,,
by hypotonic
(Fig.
2A).
solution
(n
- 5). Hypotonic
shock
cytoskeletal
organization
cytochalasin
D,
Pretreatment
with
of
control
Cytochalasin Currents
which
level, D did in oocytes
and
changes (15,16),
binds
cytochalasin and prevented not
in
change
expressing
[Ca'+],
and
to
the
the the
these
barbed
D (1 #i
have
for
increase input
IU3Kl were 806
also
changes ends
of
been can actin
resistance unaffected
of
be
to
I,,
solution the
oocyte
by cytochalasin
alter
inhibited
filaments
2 - 4 h) decreased by hypotonic
shown
to
by (17).
about (Fig.
40% 2B).
membrane. D (1 FM, 2
Vol.
BIOCHEMICAL
184, No. 2, 1992
AND BIOPHYSICAL
A
RESEARCH COMMUNICATIONS
Hypotonic Control Hypotonic Control i
- -20 mV
/ ‘40mV
B
n, Control 500 nA - -20 mv
Fig.
2.
The
increase
calcium-free potential
W7 (10 pM) did
I,,
(A) was
The kinase
by about
of
solutions
Holding
- 4 h).
10s
-90
by
(160
or
after
treatment
mV;
steps
were
inhibitor
not
hypotonicity
with to
-20
staurosporine
prevent
the
30% as previously
was
cytochalasin mV or
40
of I,,
(n = 4);
not
seen
D (1
mV as
(3 PM) and the
increase
described
mOsmol/l)
PM,
2 h).
indicated.
calmodulin
both
in
antagonist
agents
inhibited
I,,
(12).
DISCUSSION The
experiments
expressed of
the
several of the
in oocytes
minutes rate
of
assuming osmotic
solution
The maximum a potassium
conductance a physiological
is
potassium
equilibrium
on g,, is
modulation
I,,
reach
was not
maximal
current.
The time
time
constant
of
was to
increase
by about
conductance
larger
at of
by about
channel 807
osmolarity
was used
-20 the
to estimate
two-fold
The effect at
of
I,,,
by
which
consistent to
of
low
40 mV. This at
is
rate
from
potential
close
The main the
mV than
10 mV. It
at potentials
estimates
(r,,,).
mV (10).
after
of activation
was calculated
of -100
(1=x)
even
course
activation
(g,)
a shift
by the
to make precise
and this
in
the
possible
current
steady-state
function
activated, of
did not
potential
reflected
potassium
is affected
was relatively
half-maximally
activating
cDNA clone
exponential and the
hypotonic
voltage-dependence
it
and the
(I,,)
slowly
Because
of depolarization,
current
strength
the kidney
solution.
by a single
the
activation.
that a rat
of activation
maximum
effect
from
extracellular
was well-fitted the
show
rest.
the with
Vol.
184,
No.
2,
1992
BIOCHEMICAL
The mechanism it
is
selective,
because
currents
through
a Shaker-like
of
was also
increase because
I,,
a calcium-free
finding
implies
that that
entry
observation How might
(13);
an increase
protein
after
treatment
phorbol
of in
role
volume
the
in
regulate
the
filaments
of
potassium
is
is
Cytochalasin
inhibited
and prevented
D had
no
effect
KBKl.
epithelial
and protein
residue than
remained W7.
This
kinase
C by
as a result
suggested
the
on
a Ser rather
D prevents
I,,
(12).
solutions
been
our
can be inhibited
increased
as has
through with
stimulate
of
I,,.
hypotonicity
[Ca'+],
of
to
I,,
change
consistent
The current
The
strength,
perhaps
in
activation
possible
to play
increase
input
Cytochalasin
D has
also
Na+ channels
via
its
of a
reorganization
the
conductances
species
have
been
reported
increase
of
[Ca2+li
(2,5),
potassium SK’
effect,
not
caused
by
resistance
or
been
to
shown
effects
on
actin
(21). Different
I
of
staurosporine
the
(16,19,20).
expressing
effect
of hypotonic
organization,
both
not
expected
effect
blocks
cytochalasin
activity
be
inhibitors
this
oocytes
would
It
did
by phosphorylation
kinase
(12).
strength
is
I,,?
did RBKl).
ionic
by an increase
the
regulation
But
currents
[Ca2+Ji
strength
in
the
However,
(KvV1.1;
the
in
increase
known.
a change
interpretation
increased
cytoskeletal
filaments;
hypotonicity.
prevented
the
oocytes
in
of actin
solution
this
is
of
step
is not
channel
a result
first
I,,
COMMUNICATIONS
Low osmotic
same ionic
the
staurosporine
some change in cell
brain
C. Moreover,
with
esters
rat
in [Cazfli
in
kinase
concentration
of
and oleoylacylglycerol,
an increase
inhibit
I,,
solutions
the
(18);
that
esters
but
is
channels
by phorbol
of
RESEARCH
increases
not
a calcium-free
stretch-activated previous
probably solution
calcium
BIOPHYSICAL
hypotonicity
somewhat
affect
The
by which
AND
The
functional
conductances present role
as found
results to
to AEB)
be activated
in proximal
this
ACKNOWLBDCNBNTS. This (Bu 704/l-2
to
might unusual
work
in
for
and by U.S.
However, cells be
potassium
current.
helpful
808
of Health
from
solutions are
have in
by the Deutsche
Department
cells
there
that
therefore
was supported
tubule
by hypotonic
I,,.
tubule
proximal
through
no reports
kinetics the
various an of
similar
assignment
to of
Forschungsgemeinschaft
and Human Services
grants
a
Vol.
184,
No.
DA03160 Mike
2,
1992
BIOCHEMICAL
(RAN) andNS28504
Kavanaugh
(JPA).
AND
BIOPHYSICAL
We thankYan-Na
and Jim Maylie
for
helpful
RESEARCH
COMMUNICATIONS
technical
assistance
Wu for
and
discussion.
REFERENCES 1.
Chamberlin,
2.
Ubl,
M.E.,
J.,
Murer,
and Strange,
K.
and Kolb,
H.,
(1989)
H.-A.
Am. J.
(1988)
Physiol.
Pfluegers
257,
C159-C173.
Archiv.
412,
551-553. 3.
Lopez,
A.G.,
4.
Ritter,
M.,
and Guggino, Paulmichl,
W.B. M.,
(1987)
J.
and Lang,
Membr.
F.
(1991)
Biol.
97,
Pfluegers
117-125. Archiv.
418,
35-39. 5.
Kirk,
DiBona,
K.L.,
D.R.,
and Schafer
J.A.
(1987)
Am. J.
Physiol.
252,
F933-F942. 6.
Kawahara,
K.,
Hunter,
M.,
and Giebisch,
G. (1987).
Am. J.
Physiol.
253,
F488-F494. 7.
Welling,
P.A.,
249,
M.A.,
and Sullivan,
L.P
(1985).
Am. J.
Physiol.
F20-F27.
8.
Christensen,
9.
Linshaw,
0.
(1987)
M.A.,
and Okada, 10.
Linshaw,
Takumi,
MacAlister,
Y.
T.,
Nature,
(1990)
330,
T.J.,
Welling,
Biochem.
Ohkubo,
H.,
66-68. L.W.,
Biophys.
Res.
and Nakanishi,
S.
Bauman,
Commun. (1988)
C.A.,
167,
Hazama,
A,,
287-293.
Science
242,
1042-1045 11.
Sugimoto, H .,
12.
Busch,
14.
A.E., (1992)
Busch, (in
Tanabe,
and Nakanishi,
R.A. 13.
T.,
Shigemoto,
S. (1990)
Kavanaugh, J.
A.E.,
Y.,
M.P.,
Physiol.
Varnum,
J.
(in M.D.,
R.,
Membr.
Iwai, Biol.
Varnum,
M.D.,
M., 113,
Takumi,
T.,
Ohkubo,
39-47.
Adelman,
J.P.,
and North,
press). North,
R.A.,
and Adelman,
J.P.
(1992)
Science
press).
Christie, Science
M.J., 244,
15.
Forscher,
P.
16.
Foskett,
J.K.,
Adelman,
J.P.,
Douglass,
J.,
and North,
R.A.
(1989)
221-224. (1989)
Trends
and Spring,
Neurosci. K.R.
(1985)
809
12,
468-474.
Am. J.
Physiol.
248,
C27-C36.
Vol.
184,
No.
2,
1992
17.
Cooper,
18.
Young,
19.
Pierce,
S.K,
Calcium
9,
Pewitt,
E.B.,
20.
Chem. 21.
J.A.
Cantiello, J.
(1987)
X.C.,
265,
Physiol.
BIOCHEMICAL
AND
BIOPHYSICAL
J.
Cell
Biol.
and Sachs,
F.,
(1989)
Science
Smith,
L.H.,
Politis,
A.D.,
105,
RESEARCH
COMMUNICATIONS
1473-1478 243,
1068-1071.
and Rowland,
L.M.
(1988)
Cell
129-140. Hedge,
R.S.,
Haas,
M.,
and Palfrey,
H.C.
(1990)
J.
Biol.
20747-20756. H.F., vol,
Stow,
J.L.,
Prat,
C882-888.
810
A.G.,
and Ausiello,
D.A.
(1991)
Am.
184,
No.
2, 1992
April
30,
1992
BIOCHEMICAL
Andreas
RESEARCH
COMMUNICATIONS
INCREASES THE SLOWLY ACTIVATING POTASSIUM EXPRESSED IN XENOPUS OOCYTES
Busch,
E.
Michael
Institute,
Received
BIOPHYSICAL
Pages
HYPOTONIC SOLUTION
Vellum
AND
Varnum,
Oregon
February
27,
Health
John
P. Adelman,
Sciences
CURRENT I,
and R. Alan
University,
804-810
Portland,
North OR 97201
1992
SUMMARY. A slowly activating potassium current was expressed in Xenopus oocytes by injection of RNA transcribed from a rat kidney cDNA clone. Hypotonic solutions (160 mOsmol/l; control was 220 mOsmol/l) increased the current by increasing the rate of activation and by decreasing the depolarization needed to activate the current. This effect of hypotonicity was not observed in calcium-free solution, but was unaffected by staurosporine or the calmodulin antagonist W7. Cytochalasin D reduced the current and prevented the increase by hypotonic solution. The results suggest that the increase in this potassium current by hypotonic solution might result from calcium entry and changes in the actin network. ri 1992 Acadrmlc Press, Inc.
Many cell pressure
osmotic
the
types
regulate
their
by activating
ion
intracellular
decrease the
ion
when
pathways
of
(2-7).
to
potassium
Changes
and
in
processes
apical I,,;
this
induces
the
Xenopus
oocytes
as
whether
I,,
underlying
The purpose
affected
by
in
activating of
the
changes
in
mechanism.
0006-291X/92 Copyright All rights
of a slowly
0
$1.50
1992 hi Academic Press, of reproduction in any form
Inc. reserved.
channel
of the rat
804
thereby
which
vitro c
present osmotic
from current
volume
different
is
(10,ll)
and
in activating in
has become kideny
(I)
when
strength
transport
expressed
rat
experiments
with
([Ca"],),
key roles that
kidney
changing
coincides
concentration to play
potassium
of abnormal
a regulatory
through
calcium
tubule
undergo
an effect efflux
RNA transcribed
expression
was
chloride
One kidney
because
(10).
cells
solution,
intracellular
of the proximal is
Renal
to solutions
the membrane,
have been suggested
(2,8,9).
membrane
when exposed across
(1).
a hypotonic
in cytoskeletalorganization, these
fluxes
concentration
exposed
activation
volume
known
cDNA clone injected
was to and
the
to
into
determine seek
the
Vol.
184,
No.
MATERIALS
BIOCHEMICAL
2, 1992
and
expression
voltage-dependent described
of
potassium
described
voltage
three
clamp
solution
contained
(pH 7.4,
220
were
to six
currents
voltages
was 50 s and 20 s for
Therefore, fitting
evoked D,
-90
the
a single
staurosporine
between
of
were
by
current
does
not the
-90
to
on the
than
of the mean. same day,
a
oocyte
from
more
negative
estimated
Numerical
comparisons groups
of
were
cytochalasin
Incorporated).
paired
such
currents
A23187,
a
(12).
were
RBKl
Statistical
from
48 mM
steady-state
current
were
Biochemicals
only
0 mV. Even with
current.
used
been
1 mM and HEPES 5 mM
mV and
maximum
to 0 mV. Drugs
W7 (Research
-20
reach
the
have
The external
the
to
the
in vitro
ng RNA (14).
depolarizing
positive
of
by two electrode
contained
more
function
recorded
mOsmol/l)
steps
and
and
(RBKl)
1.8 mM, MgCl,
(160
of
channel
RNA transcribed
of 0.5
potentials
as mean f s.e. obtained
donor
currents
evoked
activation
from
(Sigma),
results
capped
Duration
exponential
are reported
single
COMMUNICATIONS
family
with
solution
the
rate
by 250 ms steps
values
mV.
pulses
both
Shaker
injection
were
potential
depolarizing
the
injected
Hypotonic
of
of
days after
holding
by
RESEARCH
potassium
96 mM, KC1 2 mM, CaClz
mOsmol/l).
I SK potassium
kidney
Membrane
(14).
NaCl
the
channel
Oocytes
(12,14).
as previously
long
BIOPHYSICAL
AND METHODS
Cloning
NaCl.
AND
were made
oocytes
from
a
frog.
RESULTS An
outward
depolarized
that
cDNA;
the
(I& the
(1 - 2.5
quickly
constant
conductance
40 mV, respectively
(Fig.
10 min,
within
with
and reversed a
solution
voltage-dependence
of
1).
seen
several
the
activation
seconds
in mock-injected
of activation
was 24.1+
maximal
current
was increased
by about reached
tonicity. to
The a more 805
hypotonic negative
the
rat
were kidney
at -20 mV and 6.0 considerably
the
current
among activated
50 and 20 % for
a steady
when the
oocytes
As previously
3.4
varied
solution,
effect
from oocytes.
In a hypotonic
This
when
RNA transcribed
30 min completely
normal of
with
and 40 mV; the
pA at 40 mV).
and the
over
injected was not
time
s (n - 4) at -20
oocytes more
(12),
developed
had been
current
described +_ 0.9
current
oocyte
level
and
within
about
was perfused
again
solution potential
-20
shifted (Fig.
1B);
the the
Vol.
184,
No.
2, 1992
BIOCHEMICAL
AND
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
A Hypotomc .A
ji!ziij- fr;500 nA -20 mV
40 mV
10s
B 35
Potential (mV)
Fig.
1. Reduction
mV in control Holding
solution
potential
hypotonic
at which
changed
from
-17.0
substitution)
unchanged application Currents
by
mV to -25.8
+ 1.9
s at -20
did calcium-free
in oocytes
+ 1.3
I,,
expressing
+ 0.5
of
for
normal
not
control
(see
methods)
was
constants
for
at 40 mV (n - 5). no change
not
affected
in
of
osmotic did
and
each point.
The properties
solution
KBKl were
in
The time
48 mM with
(n - 4).
hypotonic
oocytes
at -20 and 40 (160 mOsmol/l).
I,,
mV (n - 5).
to
solution
of a calcium-free
underlying
mV and 3.6
affect
solution
was half-maximal
concentration
not
A, I,, recorded
-90 mV. Five
conductance
3.2
sodium
I,,.
and in hypotonic
potential
underlying
f
of
increases
mV. B, conductance
Holding
13.0
Reduction (Tris
was -90
the
were
strength
(220 mOsmol/l)
solutions.
voltage
activation
in ionic
tonicity
I,,
were
strength; increase
also
however, I,,
by hypotonic
(Fig.
2A).
solution
(n
- 5). Hypotonic
shock
cytoskeletal
organization
cytochalasin
D,
Pretreatment
with
of
control
Cytochalasin Currents
which
level, D did in oocytes
and
changes (15,16),
binds
cytochalasin and prevented not
in
change
expressing
[Ca'+],
and
to
the
the the
these
barbed
D (1 #i
have
for
increase input
IU3Kl were 806
also
changes ends
of
been can actin
resistance unaffected
of
be
to
I,,
solution the
oocyte
by cytochalasin
alter
inhibited
filaments
2 - 4 h) decreased by hypotonic
shown
to
by (17).
about (Fig.
40% 2B).
membrane. D (1 FM, 2
Vol.
BIOCHEMICAL
184, No. 2, 1992
AND BIOPHYSICAL
A
RESEARCH COMMUNICATIONS
Hypotonic Control Hypotonic Control i
- -20 mV
/ ‘40mV
B
n, Control 500 nA - -20 mv
Fig.
2.
The
increase
calcium-free potential
W7 (10 pM) did
I,,
(A) was
The kinase
by about
of
solutions
Holding
- 4 h).
10s
-90
by
(160
or
after
treatment
mV;
steps
were
inhibitor
not
hypotonicity
with to
-20
staurosporine
prevent
the
30% as previously
was
cytochalasin mV or
40
of I,,
(n = 4);
not
seen
D (1
mV as
(3 PM) and the
increase
described
mOsmol/l)
PM,
2 h).
indicated.
calmodulin
both
in
antagonist
agents
inhibited
I,,
(12).
DISCUSSION The
experiments
expressed of
the
several of the
in oocytes
minutes rate
of
assuming osmotic
solution
The maximum a potassium
conductance a physiological
is
potassium
equilibrium
on g,, is
modulation
I,,
reach
was not
maximal
current.
The time
time
constant
of
was to
increase
by about
conductance
larger
at of
by about
channel 807
osmolarity
was used
-20 the
to estimate
two-fold
The effect at
of
I,,,
by
which
consistent to
of
low
40 mV. This at
is
rate
from
potential
close
The main the
mV than
10 mV. It
at potentials
estimates
(r,,,).
mV (10).
after
of activation
was calculated
of -100
(1=x)
even
course
activation
(g,)
a shift
by the
to make precise
and this
in
the
possible
current
steady-state
function
activated, of
did not
potential
reflected
potassium
is affected
was relatively
half-maximally
activating
cDNA clone
exponential and the
hypotonic
voltage-dependence
it
and the
(I,,)
slowly
Because
of depolarization,
current
strength
the kidney
solution.
by a single
the
activation.
that a rat
of activation
maximum
effect
from
extracellular
was well-fitted the
show
rest.
the with
Vol.
184,
No.
2,
1992
BIOCHEMICAL
The mechanism it
is
selective,
because
currents
through
a Shaker-like
of
was also
increase because
I,,
a calcium-free
finding
implies
that that
entry
observation How might
(13);
an increase
protein
after
treatment
phorbol
of in
role
volume
the
in
regulate
the
filaments
of
potassium
is
is
Cytochalasin
inhibited
and prevented
D had
no
effect
KBKl.
epithelial
and protein
residue than
remained W7.
This
kinase
C by
as a result
suggested
the
on
a Ser rather
D prevents
I,,
(12).
solutions
been
our
can be inhibited
increased
as has
through with
stimulate
of
I,,.
hypotonicity
[Ca'+],
of
to
I,,
change
consistent
The current
The
strength,
perhaps
in
activation
possible
to play
increase
input
Cytochalasin
D has
also
Na+ channels
via
its
of a
reorganization
the
conductances
species
have
been
reported
increase
of
[Ca2+li
(2,5),
potassium SK’
effect,
not
caused
by
resistance
or
been
to
shown
effects
on
actin
(21). Different
I
of
staurosporine
the
(16,19,20).
expressing
effect
of hypotonic
organization,
both
not
expected
effect
blocks
cytochalasin
activity
be
inhibitors
this
oocytes
would
It
did
by phosphorylation
kinase
(12).
strength
is
I,,?
did RBKl).
ionic
by an increase
the
regulation
But
currents
[Ca2+Ji
strength
in
the
However,
(KvV1.1;
the
in
increase
known.
a change
interpretation
increased
cytoskeletal
filaments;
hypotonicity.
prevented
the
oocytes
in
of actin
solution
this
is
of
step
is not
channel
a result
first
I,,
COMMUNICATIONS
Low osmotic
same ionic
the
staurosporine
some change in cell
brain
C. Moreover,
with
esters
rat
in [Cazfli
in
kinase
concentration
of
and oleoylacylglycerol,
an increase
inhibit
I,,
solutions
the
(18);
that
esters
but
is
channels
by phorbol
of
RESEARCH
increases
not
a calcium-free
stretch-activated previous
probably solution
calcium
BIOPHYSICAL
hypotonicity
somewhat
affect
The
by which
AND
The
functional
conductances present role
as found
results to
to AEB)
be activated
in proximal
this
ACKNOWLBDCNBNTS. This (Bu 704/l-2
to
might unusual
work
in
for
and by U.S.
However, cells be
potassium
current.
helpful
808
of Health
from
solutions are
have in
by the Deutsche
Department
cells
there
that
therefore
was supported
tubule
by hypotonic
I,,.
tubule
proximal
through
no reports
kinetics the
various an of
similar
assignment
to of
Forschungsgemeinschaft
and Human Services
grants
a
Vol.
184,
No.
DA03160 Mike
2,
1992
BIOCHEMICAL
(RAN) andNS28504
Kavanaugh
(JPA).
AND
BIOPHYSICAL
We thankYan-Na
and Jim Maylie
for
helpful
RESEARCH
COMMUNICATIONS
technical
assistance
Wu for
and
discussion.
REFERENCES 1.
Chamberlin,
2.
Ubl,
M.E.,
J.,
Murer,
and Strange,
K.
and Kolb,
H.,
(1989)
H.-A.
Am. J.
(1988)
Physiol.
Pfluegers
257,
C159-C173.
Archiv.
412,
551-553. 3.
Lopez,
A.G.,
4.
Ritter,
M.,
and Guggino, Paulmichl,
W.B. M.,
(1987)
J.
and Lang,
Membr.
F.
(1991)
Biol.
97,
Pfluegers
117-125. Archiv.
418,
35-39. 5.
Kirk,
DiBona,
K.L.,
D.R.,
and Schafer
J.A.
(1987)
Am. J.
Physiol.
252,
F933-F942. 6.
Kawahara,
K.,
Hunter,
M.,
and Giebisch,
G. (1987).
Am. J.
Physiol.
253,
F488-F494. 7.
Welling,
P.A.,
249,
M.A.,
and Sullivan,
L.P
(1985).
Am. J.
Physiol.
F20-F27.
8.
Christensen,
9.
Linshaw,
0.
(1987)
M.A.,
and Okada, 10.
Linshaw,
Takumi,
MacAlister,
Y.
T.,
Nature,
(1990)
330,
T.J.,
Welling,
Biochem.
Ohkubo,
H.,
66-68. L.W.,
Biophys.
Res.
and Nakanishi,
S.
Bauman,
Commun. (1988)
C.A.,
167,
Hazama,
A,,
287-293.
Science
242,
1042-1045 11.
Sugimoto, H .,
12.
Busch,
14.
A.E., (1992)
Busch, (in
Tanabe,
and Nakanishi,
R.A. 13.
T.,
Shigemoto,
S. (1990)
Kavanaugh, J.
A.E.,
Y.,
M.P.,
Physiol.
Varnum,
J.
(in M.D.,
R.,
Membr.
Iwai, Biol.
Varnum,
M.D.,
M., 113,
Takumi,
T.,
Ohkubo,
39-47.
Adelman,
J.P.,
and North,
press). North,
R.A.,
and Adelman,
J.P.
(1992)
Science
press).
Christie, Science
M.J., 244,
15.
Forscher,
P.
16.
Foskett,
J.K.,
Adelman,
J.P.,
Douglass,
J.,
and North,
R.A.
(1989)
221-224. (1989)
Trends
and Spring,
Neurosci. K.R.
(1985)
809
12,
468-474.
Am. J.
Physiol.
248,
C27-C36.
Vol.
184,
No.
2,
1992
17.
Cooper,
18.
Young,
19.
Pierce,
S.K,
Calcium
9,
Pewitt,
E.B.,
20.
Chem. 21.
J.A.
Cantiello, J.
(1987)
X.C.,
265,
Physiol.
BIOCHEMICAL
AND
BIOPHYSICAL
J.
Cell
Biol.
and Sachs,
F.,
(1989)
Science
Smith,
L.H.,
Politis,
A.D.,
105,
RESEARCH
COMMUNICATIONS
1473-1478 243,
1068-1071.
and Rowland,
L.M.
(1988)
Cell
129-140. Hedge,
R.S.,
Haas,
M.,
and Palfrey,
H.C.
(1990)
J.
Biol.
20747-20756. H.F., vol,
Stow,
J.L.,
Prat,
C882-888.
810
A.G.,
and Ausiello,
D.A.
(1991)
Am.