Vol. 179, No. 3, 1991 September 30, 1991
BIOCHEMICAL
CURRENT INACTIVATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1384-1390
INVOLVES A HISTIDINE
RESIDUE IN
THE PORE OF THE RAT LYMPHOCYTE POTASSIUM CHANNEL RGK5 A.E.
Busch,
Vellum
Received
R.S.
Institute,
August
5,
Hurst,
R.A.
North,*
Oregon
Health
J.P.
Sciences
Adelman
and M.P.
University,
Kavanaugh
Portland,
OR 97201
1991
RGK5 is a rat genomic DNA clone that encodes the n-type potassium channel found Current through this channel declines in T-lymphocytes and other cells. (inactivates) over a period of hundreds of milliseconds during a maintained depolarizing pulse, whether in lymphocytes or when expressed in Xenopus oocytes. Here we demonstrate that an amino acid residue near the outer pore of the channel, histidine401, is involved in the inactivation process. Replacement of this residue by tyrosine, the amino acid found in the equivalent position of the homologous but non-inactivating channel RBKl, reduced inactivation of RGK5 over a 5 s depolarizing pulse from 84.3 t 1.9% to 18.3 f 1.1%. Conversely, replacement of this tyrosine in RBKl (Ty?" ) by histidine increased its inactivation from 21.6 k 1.1% to 42.3 k 1.5%. These results suggest a mechanism of channel inactivation distinct from that previously described for the A-type potassium 0 1991 Academic Press, Inc. channel.
Voltage-dependent proteins
potassium
(Tempel
conducting
et
pore
membrane
is
is
or inactivates
among
channel
Abbreviations: a rat
0006-291X/91 Copyright All rights
several
that should
The
transient
channels
time
(or
in Xenopus
and
it
1991). opens
open
in others
cloned studies
mechanism
for
the
channel
inactivate
from
the
using the rapid
the
during
little
currents
The
when
of inactivation
inactivate A-)
membrane
MacKinnon,
course
oocytes;
a molecular
tetrameric
remain
whereas
subunits
varies during within locus
Shaker
site-directed inactivation
be addressed. RGK5, a rat
cDNA clone,
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
ions,
currents
TEA, tetraethylammonium; channel
potassium
seconds,
channel
of
1989;
rectifier
whereas
a family al.,
the
1984).
Delayed
indicated
potassium
et
for
Hille,
Potassium
*To whom correspondence RBKl,
(see
can be expressed have
Stuhmer
selective
for
depolarizations
of Drosophila
are
In some cases,
types.
of milliseconds.
mutagenesis
highly
maintained
closes
tens
1987;
depolarized.
depolarization
maintained
al.,
channels
1384
genomic
DNA potassium
channel;
Vol.
179,
No.
of this
3, 1991
channel appears
that
occlude
BIOCHEMICAL
type
involves
to undergo
the
internal
slows
intracellular
blocking
The n type the
molecular
potassium
channel
is expressed 1990;
has been the
from
cations
was to
channels
this
has been TEA,
shown is
known
histidine
(HGK5;
et al.,
1989b).
lymphocyte which
channel
competing
kinetically
but
for
the
this
inactivation but
al.,
et
when
1989).
n type channel
the
In both
cases,
cations
Grissmer
or has a much lower
site
for
affinity
purpose
of
it
present
& Cahalan,
is a binding
A general
the
a
RCK3 and MK3)(Douglass
by divalent
there
suggests of
also
(RGKS,
from
of milliseconds
milliseconds;
1985;
that
different
hundreds
lymphocyte
al.,
lies
in
1989a; divalent
in channels
these
experiments
Therefore,
external
These
studies
1990), However, it
experiments
sensitivity
to the
channel have
position rat
only
slows
by TEA cannot were
mouth
designed 1385
S5 and S6. This channel
there
TEA
(Tyr3"
of
the
lymphocyte et al., the
inactivation, inactivate to
& Yellen,
in
RBKl);
1990), current
(Grissmer
this
channels
suggesting
specifically
blocker
is one residue
to
reduces
channels
(MacKinnon
shown that
(RGK5: Douglass
TEA not
markedly
open and blocked
segments
sensitivity
equivalent
et al., 1991).
the
potassium
transmembrane
influence
determining the
of voltage-dependent
between
that
1991).
channels, is
chain
to occlude
in
Grissmer
channel
pore
in
mouse (MK3:
type
of
channel
is absent
polypeptide
et al.,
involved
Cai
the et
this
by
is
oocytes
inactivation.
by mutations
Kavanaugh
is
of
intracellular
such a site. of the
critically residue
of
of
over
Stuhmer
suggests
that
the external
which
1990;
form
identify
in the
(DeCoursey
on these
tens
1990;
of
so as to
1991).
cDNA in Xenopus
This
current
characteristic
only
segment
activation
Application
inactivates
The
to blockade
1990).
forms
it
peptide
following
apparently
than
et al.,
et al.,
The region that
cloned
solution
showing
not
that
rather
Grissmer
external
Douglass
in
seen not
attributed
peak
et al.
COMMUNICATIONS
intracellular
of T lymphocytes
mechanism. is
the
(Choi
channel
1985)
al.,
different
et al.,
site
RESEARCH
1990).
al.,
inactivation,
channels
et
terminal change
et
reduces
its
potassium
Shaker-like
(Cahalan
(Hoshi
(TEA)
additionally
an amino
BIOPHYSICAL
a conformational pore
tetraethylammonium
AND
of
and human through
n
that
a
& Cahalan, examine
the
Vol.
179, No. 3, 1991
possibility
that
BIOCHEMICAL
the sites
involved
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in current
inactivation
and TEA binding
were
related.
MATERIALS The
AND METHODS
methods
described
1985).
et al.,
DNAs were with
et al.,
1990).
Hepes
out
with
two
5 mM (pH 7.2). a holding was 5 s.
function
except
fit.
observations
chain
currents
of
-80
Inactivation
were
evoked
0 and
of the current
are
reported
Oocytes
described
(Christie
in
et
most
as mean
al.,
+ s.e.
currents The
1989).
mM, MgCl,
1.0 mM and the
experiments
oocyte
the
by a single
provided
pulse
exponential
a significantly mean
were
(Christie
by depolarizing
was fitted
the sum of two exponentials
values
Kunkel,
of 0.5 ng RNA, membrane
clamp
mV to
1991;
method.
as previously
previously
Site-directed
et al.,
termination
injection
been
1990).
(Kavanaugh
in vitro
voltage
Potassium
where
Numerical
by dideoxy
et al.,
NaCl 96 mM, KC1 2 mM, CaC12 1.8
potential
duration
before
electrode
and RGK5 have
Douglass
as reported
days after
contained
RBKl
1990;
RNA transcribed
One to four
solution
subcloning
1989;
sequenced
capped
recorded
external
and
was carried
injected
from
cloning
(Christie
mutagenesis
were
of
for
the
better number
of
in parentheses.
RESULTS The properties reported with
of currents
(Douglass
et al.,
a dissociation
1990).
constant
Christie
et
Cahalan,
1989b),
TEA also
expressed
in
oocyte
potassium
current
the
and this
slowed
directly
with
decreases Lowering decay
1990).
al.,
during
wild-type
(Ko)
of
markedly (Figure with
RGK5 channels
TEA inhibited
As seen with
decayed
about
the current
1).
potassium
currents
time
inactivation
In
oocytes
constant
TEA;
specific
of the channel
expressing
947 ? 134
a 5 s depolarizing
pH reduced pulse
to block also
(Figure 1386
RGK5 channels et
al.,
1990;
(Grissmer
ms
current
in control
In this
interacts
extracellular et al.
a decrease series
the
conditions
In RGK5, His401
in
and
RGK5 channels,
by TEA (Kavanaugh
resulted 2).
as previously
of the remaining
of His 401 by lowering
protonation
extracellular
through Douglass
lymphocyte slowed
were
10 mM (see
to 2050 ? 121 ms in 10 mM TEA (n - 3).
the sensitivity the
through
in
pH 1991). current
of experiments,
Vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
h kL PH 7.2
0
1
-I
FIGURE 1. TEA slows
IS
inactivation.
evoked by a depolarizing and in the presence FIGURE 2. Effect a depolarizing
similar
reduction
of extracellular
declined
In
order
directed
RBKl,
that
channels 72
test
shows
(n - 6).
showed
little
constant
(holding
is observed
-60 mV, 5
are superimposed
reduced
for
decay during
potential
indicated
is also
+ 1.1% at pH 7.6
directly
for
in
little
the
pH
as
is
on a
lowered;
the non-inactivating
and
decline
the
to replace
this
equivalent
the
105;
a
channel
was reduced
to 42.7
current
of
current
declined showed
tyrosine
a homologous
channel,
current
with
time
peak
value
mutant pulses
18.3
+ 1.1%
in
constant at
of
end
channel
current
unaltered
site-
tyrosine;
experiments,
the
by
with of
its
through
depolarizing
channel
these
+ 1.9%
RGK5 inactivation,
residue
exponential
during
mutant
in
position In
by 84.3
In contrast,
The
of His401
to a single
declined
*
a role
inactivation.
according
2065
3).
Current
1991).
inactivation
was
s)(Figure
et al.
found
at 0 mV; it
pulse
Peak current
was employed
acid
decayed
ms
(Control)
(n = 4).
to
amino
at pH values
of peak current
mutagenesis
the
is
as pH is reduced.
traces
by 80.0
k 1.5% at pH 6.4
are membrane currents
pH on RGK5 inactivation.
decreases
at pH 7.6.
RBKl (see Kavanaugh
current
traces
command from -80 mV to 0 mV, in the absence
s step tp 0 mV.) Current trace
Two superimposed
3 uA 5s
of TEA (10 mu).
pulse
control
02
100 nA
PH 6.6
876 ?
of a 5 s
RGK5(H401Y)
lasting of
RGK5
its
5 s (time peak
in
voltage-dependence
5 of
activation. Currents 1989;
Christie
by 21.6 step.
through
+ 1.1%
Currents
et al.
RBKl show only 1990).
(n = 6; in oocytes
In this
time
constant
expressing
slight series 1574
inactivation of experiments, f
the mutant
1387
in 5 s (Christie
67 ms)
during
RBKl(Y379H)
the current
et al. declined
a 5 s depolarizing inactivated
by 42.3
vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RGKS
RGK5
RESEARCH COMMUNICATIONS
H40lY
RBKl
RBKl
Y37W-l
L - 7500
nA
2500
FIGURE 3. Effects
of amino
depolarizing
from -80 mV to 0 mV. (A) Typical
pulse
inactivation.
(B) Current
acid
of similar
RGKs(H401Y) shows much less Wild-type
RBKl current
+ 1.5%
(n - 6; time
no apparent
channel
current
amplitude comparable
little
during
in RGK5 shows marked in the mutant
to that
channel
seen in RBKl.
(D) Increased
inactivation.
a 5 s
(C)
inactivation
RBKl(Y379H).
constants
difference
on inactivation
initial
inactivation,
showing
is seen in the mutant
substitutions
ms
5080
between
+ 310 and 497 + 30 ms)(Figure
RBKl
and RBKl(Y379H)
in
3).
There
was
voltage-dependence
of
activation.
DISCUSSION The histidine determinant
residue
of inactivation
TEA (Kavanaugh slows
the
et al.,
currents
in Xenopus
low
inactivation
pH,
by is
that
specific also
& Cahalan
blocked
similarly;
inactivation.
inhibited
experiments
as a critical
shown
to interact
with
(1989b)
found
channels
not of
by TEA cannot in addition
RGK5(H401Y)]
His401;
by decreasing
1388
that
the pH.
TEA markedly
found
that
channels
addition,
their
inactivate,
to reducing
present In
external
and explained
We previously
RGK5 [though protonation
these
potassium
a channel behave
TEA slows through
Grissmer
T lymphocyte
oocytes
amplitude,
by TEA of current at
1991). of
by postulating
in
has been previously
inactivation
results
current
in RGK5 identified
RGK5 the peak the
block
was decreased
results when
show His401
that was
Vol.
replaced
by
These
tyrosine,
results
Both depend the
in
suggest
lymphocytes
al.,
His401 causes
necessary
(DeCoursey
takes
part
decreased.
in both
to test
this
type
channels
involving
an extracellular that
TEA binding
The functional
volume
will
or hypotonic
stimuli;
conservation
of
cells
located
of
within
the the
have
roles
(Grinstein tend
et
to limit
where
histidine
this
channel
that
extracellular
potassium
efflux
under
advantage
might
expressed.
of
Health
ion
will
be
inactivation blocking of mechanism further
of the
channel.
in lymphocytes
(DeCoursey The
by
as occurs
and provide
currents
1982).
is
that
mouth
division
position
a blocking
type
pore
low
Douglass
studies
another
when in
"tethered"
for
to
mechanism
mouth,
showed
al.,
in this
the
Further
channel
in cell
slower 1989a;
the channel
(1990)
basis
is
directly is
shown
more rapid
al.,
an intracellular,
such a functional
the
et
of voltage-gatedpotassium
regulation
undergo
and
1990).
et al.
from
blockade
they
address
Chakrabati,
a structural
significance
discussed;
becomes
Cahalan
within
Hoshi
resulted
is
has been
increased,
1985;
residues (see
suggest
His401
is
do not
hypothesis.
Our results
inactivation
inactivation
et al.
by histidine
particle.
other
residue
dramatically
of RGK5; one possibility
of metalloproteins
Shaker
channels
RESEARCH COMMUNICATIONS
was
oocytes,
studies
inactivation
bound
in a number
in
this
of calcium
The present
is coordinately
and
inactivation
cations;
concentration
1990).
has been
that
divalent
calcium
evidence
of
and in Xenopus
upon extracellular
extracellular
in
amount
AND BIOPHYSICAL
inactivation.
extracellular
which
the
strongly
and channel
et
BIOCHEMICAL
179, No. 3, 1991
et al.,
inactivation
1984) that
maintained
have
contributed
and may play
a similar
the
mitogenic to the role
in
ACKNOWLEDGMENTS Supported DA03161, thank
by
U.S.
HD24562
Yan-Na
Department and NS28504
Wu for
injecting
and
and by the RNA into
Human
Deutsche
Services
grants
Forschungsgemeinschaft.
oocytes.
REFERENCES Cahalan, Physiol.
M.D., 358,
Chandy,
K.G.,
DeCoursey,
T. & Gupta,
197-237.
1389
S.
(1985)
J.
Gen.
DAO3160, We
Vol.
179, No. 3, 1991
Chakrabati,
P. (1990)
Choi,
Aldrich,
K.L.,
BIOCHEMICAL Protein
Engin.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4, 57-63.
R.W. 6 Yellen,
G. (1991)
Proc.
Natl.
Acad.
Sci.
U.S.A.
88,
5092-5095. Christie,
M.J.,
Adelman,
M.J.,
North,
J.P.,
J.
Douglass,
6 North,
R.A.
(1989)
Science
244,
221-224. Christie, Neuron
Osborne,
P.B.,
Douglass,
J.
& Adelman,
M.D.
(1984)
M.,
Christie,
Gen.
Physiol.
J.P.
(1990)
2, 405-411.
DeCoursey, 307,
T.E.,
Chandy,
K.G.,
Gupta,
S. & Cahalan,
Nature
465-468.
Douglass,
J.,
Adelman,
Osborne,
J.P.
Grinstein, 79,
R.A.,
P.B.,
(1990)
S.,
J.
Dupre,
Cai,
Y.-C.,
Wilkinson,
Immunol.
144,
4841-4850.
A. & Rothstein,
A. (1982)
J.
M.J.
&
849-868.
Grissmer,
S. 6 Cahalan,
M.D.
(1989a)
J.
Grissmer,
S. & Cahalan,
M.D.
(1989b)
Biophys.
Grissmer,
S.,
B.,
Wasmuth,
Cahalan,
Dethlefs,
M.D.
& Chandy,
K.G.
Gen. Physiol.
J.J.,
(1990)
J.
93,
55,
203-206.
Goldin,
A.L.,
Natl.
Acad.
Proc.
609-630.
Gutman, Sci.
G.A.,
U.S.A.
87,
9411
-9415. Hille,
B. (1984)
Ionic
Massachusetts: Hoshi,
T.,
M.P.,
Adelman, Kunkel,
J.P. T.A. R.
MacKinnon, Stiihmer, Giese,
W.N. & Aldrich, Varnum,
& North,
(1985)
MacKinnon,
in Excitable
M.D., R.A.
Proc.
(1991)
Osborne, (1991)
Natl.
Nature
R.W.
J.
Acad.
232-235.
R. 6 Yellen,
G. (1990)
Science
W., Ruppersberg,
J.P.,
Perschke,
A.,
(1990)
Sunderland,
Christie,
Biol.
Chem. 266,
Schroter, Baumann,
Science
P.B.,
Sci.
350,
K.P.,
Membranes.
Sinauer.
Zagotta,
Kavanaugh,
Channels
U.S.A.
250,
82,
250,
533-538.
M.J.,
Busch,
A.E.,
7583-7587.
488-492.
276-279.
K.H., A. 6 Pongs,
Sakmann, 0.
B.,
(1989)
Stocker, EMBO J.
M., 8, 3235
-3244. Tempel, Science
B.L., 237,
Papazian,
D.M.,
Schwarz,
T.T.,
770-775.
1390
Jan,
Y.N.
6 Jan L.Y.
(1987)
BIOCHEMICAL
CURRENT INACTIVATION
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1384-1390
INVOLVES A HISTIDINE
RESIDUE IN
THE PORE OF THE RAT LYMPHOCYTE POTASSIUM CHANNEL RGK5 A.E.
Busch,
Vellum
Received
R.S.
Institute,
August
5,
Hurst,
R.A.
North,*
Oregon
Health
J.P.
Sciences
Adelman
and M.P.
University,
Kavanaugh
Portland,
OR 97201
1991
RGK5 is a rat genomic DNA clone that encodes the n-type potassium channel found Current through this channel declines in T-lymphocytes and other cells. (inactivates) over a period of hundreds of milliseconds during a maintained depolarizing pulse, whether in lymphocytes or when expressed in Xenopus oocytes. Here we demonstrate that an amino acid residue near the outer pore of the channel, histidine401, is involved in the inactivation process. Replacement of this residue by tyrosine, the amino acid found in the equivalent position of the homologous but non-inactivating channel RBKl, reduced inactivation of RGK5 over a 5 s depolarizing pulse from 84.3 t 1.9% to 18.3 f 1.1%. Conversely, replacement of this tyrosine in RBKl (Ty?" ) by histidine increased its inactivation from 21.6 k 1.1% to 42.3 k 1.5%. These results suggest a mechanism of channel inactivation distinct from that previously described for the A-type potassium 0 1991 Academic Press, Inc. channel.
Voltage-dependent proteins
potassium
(Tempel
conducting
et
pore
membrane
is
is
or inactivates
among
channel
Abbreviations: a rat
0006-291X/91 Copyright All rights
several
that should
The
transient
channels
time
(or
in Xenopus
and
it
1991). opens
open
in others
cloned studies
mechanism
for
the
channel
inactivate
from
the
using the rapid
the
during
little
currents
The
when
of inactivation
inactivate A-)
membrane
MacKinnon,
course
oocytes;
a molecular
tetrameric
remain
whereas
subunits
varies during within locus
Shaker
site-directed inactivation
be addressed. RGK5, a rat
cDNA clone,
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
ions,
currents
TEA, tetraethylammonium; channel
potassium
seconds,
channel
of
1989;
rectifier
whereas
a family al.,
the
1984).
Delayed
indicated
potassium
et
for
Hille,
Potassium
*To whom correspondence RBKl,
(see
can be expressed have
Stuhmer
selective
for
depolarizations
of Drosophila
are
In some cases,
types.
of milliseconds.
mutagenesis
highly
maintained
closes
tens
1987;
depolarized.
depolarization
maintained
al.,
channels
1384
genomic
DNA potassium
channel;
Vol.
179,
No.
of this
3, 1991
channel appears
that
occlude
BIOCHEMICAL
type
involves
to undergo
the
internal
slows
intracellular
blocking
The n type the
molecular
potassium
channel
is expressed 1990;
has been the
from
cations
was to
channels
this
has been TEA,
shown is
known
histidine
(HGK5;
et al.,
1989b).
lymphocyte which
channel
competing
kinetically
but
for
the
this
inactivation but
al.,
et
when
1989).
n type channel
the
In both
cases,
cations
Grissmer
or has a much lower
site
for
affinity
purpose
of
it
present
& Cahalan,
is a binding
A general
the
a
RCK3 and MK3)(Douglass
by divalent
there
suggests of
also
(RGKS,
from
of milliseconds
milliseconds;
1985;
that
different
hundreds
lymphocyte
al.,
lies
in
1989a; divalent
in channels
these
experiments
Therefore,
external
These
studies
1990), However, it
experiments
sensitivity
to the
channel have
position rat
only
slows
by TEA cannot were
mouth
designed 1385
S5 and S6. This channel
there
TEA
(Tyr3"
of
the
lymphocyte et al., the
inactivation, inactivate to
& Yellen,
in
RBKl);
1990), current
(Grissmer
this
channels
suggesting
specifically
blocker
is one residue
to
reduces
channels
(MacKinnon
shown that
(RGK5: Douglass
TEA not
markedly
open and blocked
segments
sensitivity
equivalent
et al., 1991).
the
potassium
transmembrane
influence
determining the
of voltage-dependent
between
that
1991).
channels, is
chain
to occlude
in
Grissmer
channel
pore
in
mouse (MK3:
type
of
channel
is absent
polypeptide
et al.,
involved
Cai
the et
this
by
is
oocytes
inactivation.
by mutations
Kavanaugh
is
of
intracellular
such a site. of the
critically residue
of
of
over
Stuhmer
suggests
that
the external
which
1990;
form
identify
in the
(DeCoursey
on these
tens
1990;
of
so as to
1991).
cDNA in Xenopus
This
current
characteristic
only
segment
activation
Application
inactivates
The
to blockade
1990).
forms
it
peptide
following
apparently
than
et al.,
et al.,
The region that
cloned
solution
showing
not
that
rather
Grissmer
external
Douglass
in
seen not
attributed
peak
et al.
COMMUNICATIONS
intracellular
of T lymphocytes
mechanism. is
the
(Choi
channel
1985)
al.,
different
et al.,
site
RESEARCH
1990).
al.,
inactivation,
channels
et
terminal change
et
reduces
its
potassium
Shaker-like
(Cahalan
(Hoshi
(TEA)
additionally
an amino
BIOPHYSICAL
a conformational pore
tetraethylammonium
AND
of
and human through
n
that
a
& Cahalan, examine
the
Vol.
179, No. 3, 1991
possibility
that
BIOCHEMICAL
the sites
involved
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
in current
inactivation
and TEA binding
were
related.
MATERIALS The
AND METHODS
methods
described
1985).
et al.,
DNAs were with
et al.,
1990).
Hepes
out
with
two
5 mM (pH 7.2). a holding was 5 s.
function
except
fit.
observations
chain
currents
of
-80
Inactivation
were
evoked
0 and
of the current
are
reported
Oocytes
described
(Christie
in
et
most
as mean
al.,
+ s.e.
currents The
1989).
mM, MgCl,
1.0 mM and the
experiments
oocyte
the
by a single
provided
pulse
exponential
a significantly mean
were
(Christie
by depolarizing
was fitted
the sum of two exponentials
values
Kunkel,
of 0.5 ng RNA, membrane
clamp
mV to
1991;
method.
as previously
previously
Site-directed
et al.,
termination
injection
been
1990).
(Kavanaugh
in vitro
voltage
Potassium
where
Numerical
by dideoxy
et al.,
NaCl 96 mM, KC1 2 mM, CaC12 1.8
potential
duration
before
electrode
and RGK5 have
Douglass
as reported
days after
contained
RBKl
1990;
RNA transcribed
One to four
solution
subcloning
1989;
sequenced
capped
recorded
external
and
was carried
injected
from
cloning
(Christie
mutagenesis
were
of
for
the
better number
of
in parentheses.
RESULTS The properties reported with
of currents
(Douglass
et al.,
a dissociation
1990).
constant
Christie
et
Cahalan,
1989b),
TEA also
expressed
in
oocyte
potassium
current
the
and this
slowed
directly
with
decreases Lowering decay
1990).
al.,
during
wild-type
(Ko)
of
markedly (Figure with
RGK5 channels
TEA inhibited
As seen with
decayed
about
the current
1).
potassium
currents
time
inactivation
In
oocytes
constant
TEA;
specific
of the channel
expressing
947 ? 134
a 5 s depolarizing
pH reduced pulse
to block also
(Figure 1386
RGK5 channels et
al.,
1990;
(Grissmer
ms
current
in control
In this
interacts
extracellular et al.
a decrease series
the
conditions
In RGK5, His401
in
and
RGK5 channels,
by TEA (Kavanaugh
resulted 2).
as previously
of the remaining
of His 401 by lowering
protonation
extracellular
through Douglass
lymphocyte slowed
were
10 mM (see
to 2050 ? 121 ms in 10 mM TEA (n - 3).
the sensitivity the
through
in
pH 1991). current
of experiments,
Vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
h kL PH 7.2
0
1
-I
FIGURE 1. TEA slows
IS
inactivation.
evoked by a depolarizing and in the presence FIGURE 2. Effect a depolarizing
similar
reduction
of extracellular
declined
In
order
directed
RBKl,
that
channels 72
test
shows
(n - 6).
showed
little
constant
(holding
is observed
-60 mV, 5
are superimposed
reduced
for
decay during
potential
indicated
is also
+ 1.1% at pH 7.6
directly
for
in
little
the
pH
as
is
on a
lowered;
the non-inactivating
and
decline
the
to replace
this
equivalent
the
105;
a
channel
was reduced
to 42.7
current
of
current
declined showed
tyrosine
a homologous
channel,
current
with
time
peak
value
mutant pulses
18.3
+ 1.1%
in
constant at
of
end
channel
current
unaltered
site-
tyrosine;
experiments,
the
by
with of
its
through
depolarizing
channel
these
+ 1.9%
RGK5 inactivation,
residue
exponential
during
mutant
in
position In
by 84.3
In contrast,
The
of His401
to a single
declined
*
a role
inactivation.
according
2065
3).
Current
1991).
inactivation
was
s)(Figure
et al.
found
at 0 mV; it
pulse
Peak current
was employed
acid
decayed
ms
(Control)
(n = 4).
to
amino
at pH values
of peak current
mutagenesis
the
is
as pH is reduced.
traces
by 80.0
k 1.5% at pH 6.4
are membrane currents
pH on RGK5 inactivation.
decreases
at pH 7.6.
RBKl (see Kavanaugh
current
traces
command from -80 mV to 0 mV, in the absence
s step tp 0 mV.) Current trace
Two superimposed
3 uA 5s
of TEA (10 mu).
pulse
control
02
100 nA
PH 6.6
876 ?
of a 5 s
RGK5(H401Y)
lasting of
RGK5
its
5 s (time peak
in
voltage-dependence
5 of
activation. Currents 1989;
Christie
by 21.6 step.
through
+ 1.1%
Currents
et al.
RBKl show only 1990).
(n = 6; in oocytes
In this
time
constant
expressing
slight series 1574
inactivation of experiments, f
the mutant
1387
in 5 s (Christie
67 ms)
during
RBKl(Y379H)
the current
et al. declined
a 5 s depolarizing inactivated
by 42.3
vol.
179, No. 3, 1991
BIOCHEMICAL
AND BIOPHYSICAL
RGKS
RGK5
RESEARCH COMMUNICATIONS
H40lY
RBKl
RBKl
Y37W-l
L - 7500
nA
2500
FIGURE 3. Effects
of amino
depolarizing
from -80 mV to 0 mV. (A) Typical
pulse
inactivation.
(B) Current
acid
of similar
RGKs(H401Y) shows much less Wild-type
RBKl current
+ 1.5%
(n - 6; time
no apparent
channel
current
amplitude comparable
little
during
in RGK5 shows marked in the mutant
to that
channel
seen in RBKl.
(D) Increased
inactivation.
a 5 s
(C)
inactivation
RBKl(Y379H).
constants
difference
on inactivation
initial
inactivation,
showing
is seen in the mutant
substitutions
ms
5080
between
+ 310 and 497 + 30 ms)(Figure
RBKl
and RBKl(Y379H)
in
3).
There
was
voltage-dependence
of
activation.
DISCUSSION The histidine determinant
residue
of inactivation
TEA (Kavanaugh slows
the
et al.,
currents
in Xenopus
low
inactivation
pH,
by is
that
specific also
& Cahalan
blocked
similarly;
inactivation.
inhibited
experiments
as a critical
shown
to interact
with
(1989b)
found
channels
not of
by TEA cannot in addition
RGK5(H401Y)]
His401;
by decreasing
1388
that
the pH.
TEA markedly
found
that
channels
addition,
their
inactivate,
to reducing
present In
external
and explained
We previously
RGK5 [though protonation
these
potassium
a channel behave
TEA slows through
Grissmer
T lymphocyte
oocytes
amplitude,
by TEA of current at
1991). of
by postulating
in
has been previously
inactivation
results
current
in RGK5 identified
RGK5 the peak the
block
was decreased
results when
show His401
that was
Vol.
replaced
by
These
tyrosine,
results
Both depend the
in
suggest
lymphocytes
al.,
His401 causes
necessary
(DeCoursey
takes
part
decreased.
in both
to test
this
type
channels
involving
an extracellular that
TEA binding
The functional
volume
will
or hypotonic
stimuli;
conservation
of
cells
located
of
within
the the
have
roles
(Grinstein tend
et
to limit
where
histidine
this
channel
that
extracellular
potassium
efflux
under
advantage
might
expressed.
of
Health
ion
will
be
inactivation blocking of mechanism further
of the
channel.
in lymphocytes
(DeCoursey The
by
as occurs
and provide
currents
1982).
is
that
mouth
division
position
a blocking
type
pore
low
Douglass
studies
another
when in
"tethered"
for
to
mechanism
mouth,
showed
al.,
in this
the
Further
channel
in cell
slower 1989a;
the channel
(1990)
basis
is
directly is
shown
more rapid
al.,
an intracellular,
such a functional
the
et
of voltage-gatedpotassium
regulation
undergo
and
1990).
et al.
from
blockade
they
address
Chakrabati,
a structural
significance
discussed;
becomes
Cahalan
within
Hoshi
resulted
is
has been
increased,
1985;
residues (see
suggest
His401
is
do not
hypothesis.
Our results
inactivation
inactivation
et al.
by histidine
particle.
other
residue
dramatically
of RGK5; one possibility
of metalloproteins
Shaker
channels
RESEARCH COMMUNICATIONS
was
oocytes,
studies
inactivation
bound
in a number
in
this
of calcium
The present
is coordinately
and
inactivation
cations;
concentration
1990).
has been
that
divalent
calcium
evidence
of
and in Xenopus
upon extracellular
extracellular
in
amount
AND BIOPHYSICAL
inactivation.
extracellular
which
the
strongly
and channel
et
BIOCHEMICAL
179, No. 3, 1991
et al.,
inactivation
1984) that
maintained
have
contributed
and may play
a similar
the
mitogenic to the role
in
ACKNOWLEDGMENTS Supported DA03161, thank
by
U.S.
HD24562
Yan-Na
Department and NS28504
Wu for
injecting
and
and by the RNA into
Human
Deutsche
Services
grants
Forschungsgemeinschaft.
oocytes.
REFERENCES Cahalan, Physiol.
M.D., 358,
Chandy,
K.G.,
DeCoursey,
T. & Gupta,
197-237.
1389
S.
(1985)
J.
Gen.
DAO3160, We
Vol.
179, No. 3, 1991
Chakrabati,
P. (1990)
Choi,
Aldrich,
K.L.,
BIOCHEMICAL Protein
Engin.
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
4, 57-63.
R.W. 6 Yellen,
G. (1991)
Proc.
Natl.
Acad.
Sci.
U.S.A.
88,
5092-5095. Christie,
M.J.,
Adelman,
M.J.,
North,
J.P.,
J.
Douglass,
6 North,
R.A.
(1989)
Science
244,
221-224. Christie, Neuron
Osborne,
P.B.,
Douglass,
J.
& Adelman,
M.D.
(1984)
M.,
Christie,
Gen.
Physiol.
J.P.
(1990)
2, 405-411.
DeCoursey, 307,
T.E.,
Chandy,
K.G.,
Gupta,
S. & Cahalan,
Nature
465-468.
Douglass,
J.,
Adelman,
Osborne,
J.P.
Grinstein, 79,
R.A.,
P.B.,
(1990)
S.,
J.
Dupre,
Cai,
Y.-C.,
Wilkinson,
Immunol.
144,
4841-4850.
A. & Rothstein,
A. (1982)
J.
M.J.
&
849-868.
Grissmer,
S. 6 Cahalan,
M.D.
(1989a)
J.
Grissmer,
S. & Cahalan,
M.D.
(1989b)
Biophys.
Grissmer,
S.,
B.,
Wasmuth,
Cahalan,
Dethlefs,
M.D.
& Chandy,
K.G.
Gen. Physiol.
J.J.,
(1990)
J.
93,
55,
203-206.
Goldin,
A.L.,
Natl.
Acad.
Proc.
609-630.
Gutman, Sci.
G.A.,
U.S.A.
87,
9411
-9415. Hille,
B. (1984)
Ionic
Massachusetts: Hoshi,
T.,
M.P.,
Adelman, Kunkel,
J.P. T.A. R.
MacKinnon, Stiihmer, Giese,
W.N. & Aldrich, Varnum,
& North,
(1985)
MacKinnon,
in Excitable
M.D., R.A.
Proc.
(1991)
Osborne, (1991)
Natl.
Nature
R.W.
J.
Acad.
232-235.
R. 6 Yellen,
G. (1990)
Science
W., Ruppersberg,
J.P.,
Perschke,
A.,
(1990)
Sunderland,
Christie,
Biol.
Chem. 266,
Schroter, Baumann,
Science
P.B.,
Sci.
350,
K.P.,
Membranes.
Sinauer.
Zagotta,
Kavanaugh,
Channels
U.S.A.
250,
82,
250,
533-538.
M.J.,
Busch,
A.E.,
7583-7587.
488-492.
276-279.
K.H., A. 6 Pongs,
Sakmann, 0.
B.,
(1989)
Stocker, EMBO J.
M., 8, 3235
-3244. Tempel, Science
B.L., 237,
Papazian,
D.M.,
Schwarz,
T.T.,
770-775.
1390
Jan,
Y.N.
6 Jan L.Y.
(1987)
