Funnel-web spider venom and a toxin fraction block calcium current expressed from rat brain mRNA in Xenopus oocytes

Abstract

Injection of rat brain mRNA into Xenopus oocytes has been shown to induce a calcium current (ICa) that is insensitive to dihydropyridine and omega-conotoxin. We examined the effect of funnel-web spider venom on two aspects of this expressed ICa: (i) the calcium-activated chloride current [ICl(Ca)] and (ii) the currents carried by barium ions through calcium channels (IBa). In the presence of 1.8 mM extracellular calcium, ICl(Ca) tail current became detectable between -30 and -40 mV from a holding potential of -80 mV and reached a maximal amplitude between 0 and +10 mV. Total spider venom partially (83%) and reversibly blocked the calcium-activated chloride current without changing its voltage sensitivity. A chromatographic toxin fraction from the venom also blocked this current (64%). The venom had a minimal effect on INa and IK. Direct investigation of inward current mediated by calcium channels was carried out in high-barium solution. IBa had a higher threshold of activation (-30 to -20 mV) and reached its maximal amplitude at about +20 mV. Total venom or a partly purified chromatographic toxic fraction blocked IBa partially and reversibly without changing its current-voltage characteristics. Furthermore, the extent of the total venom block depended on the concentration of extracellular barium. Only 35% of the IBa was blocked in 60 mM Ba2+, whereas the block increased to 65% and 71%, respectively, for 40 and 20 mM Ba2+. On the basis of these results, we propose that the calcium channels expressed from rat brain mRNA in Xenopus oocytes is similar to the recently discovered P-type channels.

Full text

Proc.
Nadl.
Acad.
Sci.
USA
Vol.
87,
pp.
4538-4542,
June
1990
Neurobiology
Funnel-web
spider
venom
and
a
toxin
fraction
block
calcium
current
expressed
from
rat
brain
mRNA
in
Xenopus
oocytes
J.-W.
LIN,
B.
RUDY,
AND
R.
LLINAS
Department
of
Physiology
and
Biophysics,
New
York
University
Medical
Center,
550
First
Avenue,
New
York,
NY
10016
Contributed
by
R.
Llinds,
March
12,
1990
ABSTRACT
Iijection
of
rat
brain
mRNA
into
Xenopus
oocytes
has
been
shown
to
induce
a
calcium
current
('Ca)
that
is
insensitive
to
dihydropyridine
and
c-conotoxin.
We
exam-
ined
the
effect
of
funnel-web
spider
venom
on
two
aspects
of
this
expressed
ICa:
(i)
the
calcium-activated
chloride
current
[Ic'(ca)]
and
(it)
the
currents
carried
by
barium
ions
through
calcium
channels
(IB,).
In
the
presence
of
1.8
mM
extracellular
calcium,
'CI(Ca)
tail
current
became
detectable
between
-30
and
-40
mV
from
a
holding
potential
of
-80
mV
and
reached
a
maximal
amplitude
between
0
and
+10
mV.
Total
spider
venom
partially
(83%)
and
reversibly
blocked
the
calcium-activated
chloride
current
without
changing
its
voltage
sensitivity.
A
chromato-
graphic
toxin
fraction
from
the
venom
also
blocked
this
current
(64%).
The
venom
had
a
minimal
effect
on
IN.
and
IK.
Direct
investigation
of
inward
current
mediated
by
calcium
channels
was
carried
out
in
high-barium
solution.
IBa
had
a
higher
threshold
of
activation
(-30
to
-20
mV)
and
reached
its
maximal
amplitude
at
about
+20
mV.
Total
venom
or
a
partly
purified
chromatographic
toxic
fraction
blocked
1B.
partially
and
reversibly
without
changing
its
current-voltage
character-
istics.
Furthermore,
the
extent
of
the
total
venom
block
de-
pended
on
the
concentration
of
extracellular
barium.
Only
35%
of
the
1B.
was
blocked
in
60
mM
Ba2+,
whereas
the
block
increased
to
65%
and
71%,
respectively,
for
40
and
20
mM
Ba2+.
On
the
basis
of
these
results,
we
propose
that
the
calcium
channels
expressed
from
rat
brain
mRNA
in
Xenopus
oocytes
is
similar
to
the
recently
discovered
P-type
channels.
The
role
of
calcium
channels
in
central
nervous
system
neurons
is
important
and
diverse.
Somadendritic
calcium
currents
control
firing
patterns
by
way
of
rebound
activation
of
low-threshold
currents
(1,
2)
or
by
the
activation
of
calcium-dependent
potassium
currents
(3).
Dendritic
calcium
channels
provide
local
active
responses
and
expand
the
complexity
of
neuronal
integration
(4).
Calcium
currents
located
in
the
presynaptic
terminals
are
essential
for
trigger-
ing
transmitter
release
(5,
6).
Finally,
calcium
influx
through
voltage-sensitive
calcium
channels
can
play
the
role
of
a
second
messenger,
by
triggering
protein
phosphorylation
(7)
or
the
inositol
trisphosphate
pathway
(8),
and
produce
long-
lasting
effects
on
neuronal
behavior.
These
functions
are
probably
mediated
by
various
types
of
Ca
channels
and
require
their
strategic
distribution
on
the
plasma
membrane.
Present
classification
of
mammalian
central
nervous
sys-
tem
neuronal
calcium
channels,
which
includes
L,
N,
and
T
types,
is
based
on
physiological
and
pharmacological
criteria
established
from
studies
of
dorsal
root
ganglion
neurons
(2).
Following
this
classification,
the
L-type,
dihydropyridine
(DHP)-sensitive
channels
have
been
demonstrated
in
disso-
ciated
hippocampal
neurons
(9),
whereas
low-threshold
cal-
cium
current
recorded
from
inferior
olivary
and
thalamic
neurons
corresponds
to
T-type
channels
(10).
However,
the
classification
appears
to
be
too
restricted
because
many
studies
have
revealed
Ca
channels
that
do
not
conform
to
the
existing
three
types.
Studies
of
calcium
flux
in
brain
synap-
tosomes
revealed
a
combination
of
kinetic
and
pharmaco-
logical
properties
that
did
not
fit
L-,
N-,
or
T-type
behavior
(11,
12).
Dendritic
calcium
spikes
of
cerebellar
Purkinje
cells
(13)
and
Ca
currents
expressed
in
Xenopus
oocytes
from
rat
brain
mRNA
are
totally
insensitive
to
DHP
or
w-conotoxin
(14),
whereas
both
currents
have
N-type
kinetics-i.e.,
un-
detectable
until
-40
mV
and
showed
little
inactivation.
Thus,
a
broader
classification
scheme
is
needed
to
incorporate
these
channel
types,
and
the
identification
of
new
pharma-
cological
tools
to
differentiate
Ca
channels
has
become
an
urgent
task.
A
fraction
of
funnel-web
spider
venom
has
been
charac-
terized
recently
(15)
and
shown
to
block
the
calcium
current
in
the
squid
giant
synapse
and
calcium
spikes
in
cerebellar
Purkinje
cells
(13,
16).
Because
the
calcium
channels
in
both
preparations
are
insensitive
to
DHP
and
w-conotoxin
and
have
a
threshold
higher
than
T-type
channels,
these
venom-
sensitive
channels
have
been
assumed
to
represent
another
class
and
have
been
named
P
(for
Purkinje
cells)
channels
(13).
Calcium
channels
of
similar
characteristics
have
also
been
observed
in
neurohypophysis
and
cerebellar
granular
cells
(17,
18).
In
this
report,
we
characterize
the
effect
of
the
funnel-web
spider
venom
and
its
toxin
fraction
(FTX)
on
the
Ca
current
expressed
from
rat
brain
mRNA
in
Xenopus
oocytes.
Our
results
show
that
they
can
block
the
expressed
Ca
current,
and
the
level
of
the
block
depends
upon
the
concentration
of
divalent
cations
extracellularly.
METHODS
Adult
Xenopus
laevis
were
maintained
in
fresh
water
(200C)
and
fed
weekly.
Surgical
removal
of
oocytes
was
performed
under
anesthesia
(0.17%
MS222).
After
isolation,
oocytes
were
treated
with
collagenase
(2
mg/ml,
Sigma
type
1A)
dissolved
in
OR(2)
(82.5
mM
NaCl/2.0
mM
KCl/1.0
mM
MgCl2/5.0
mM
Hepes,
titrated
to
pH
7.4).
Collagenase
was
washed
out
after
45-60
min.
The
oocytes
were
then
selected
[stage
V
and
VI
(19)]
and
transferred
to
PS
solution
(96
mM
NaCl/2.0
mM
KCl/1.8
mM
CaCl2/1.0
mM
MgCl2/5.0
mM
Hepes/2.5
mM
pyruvate,
pH
7.4,
containing
penicillin
at
100
units/ml
and
streptomycin
at
100
gg/ml).
Injection
of
RNA
[50
nl
of
RNA
(1
,ug/,ul)
per
oocyte]
was
carried
out
24
hr
later,
and
oocytes
were
maintained
in
PS
solution
thereafter.
Electrophysiological
study
of
the
oocytes
was
performed
48-96
hr
after
the
injection.
RNA
Preparation.
Whole
brain
RNA
was
isolated
from
16-day-old
rats
after
the
procedure
of
Dierks
et
al.
(20).
Poly(A)+
RNA
was
purified
from
total
RNA
on
oligo(dT)-
Abbreviations:
FTX,
funnel-web
spider
toxin
fraction;
DHP,
dihy-
dropyridine;
I,
current;
V,
voltage;
ICa,
'Ba'
INa,
IK,
currents
of
calcium,
barium,
sodium,
and
potassium,
respectively;
ICI(ca)'
cal-
cium-activated
chloride
current.
4538
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
4539
cellulose
type
III
(Collaborative
Research)
according
to
established
protocols
(21).
Electrophysiology.
Two-electrode
voltage
clamp
was
used
to
characterize
the
currents
mediated
by
ion
channels
ex-
pressed
from
injected
mRNA.
Both
voltage
recording
and
current
electrodes
were
filled
with
3
M
KCl
when
lcl(ca)
was
studied.
In
those
experiments
where
barium
currents
were
isolated,
current
electrodes
were
filled
with
a
1:1
mixture
of
3
M
tetraethylammonium
chloride/3
M
cesium
chloride.
The
current
electrode
holder
was
attached
to
a
pressure
source
to
inject
K
channel
blockers.
Electrode
resistance
ranged
from
0.5-2
Mfl.
Typically,
the
oocytes
were
held
at
-80
mV
and
stepped
up
to
+50
mV
in
10-mV
increments;
the
pulse
duration
was
400
msec.
For
analysis
of
the
calcium-activated
chloride
current,
the
recordings
were
obtained
in
ND96
(ND96
is
identical
to
PS
solution
except
that
penicillin,
streptomycin,
and
pyruvate
were
omitted).
To
study
calcium
current
in
isolation,
the
extracellular
solution
was
replaced
by
high-Ba
and
Cl-free
solution
(BaMS:
60
mM
Ba(OH)2/20
mM
NaOH/
2
mM
KOH/5
mM
Hepes,
titrated
to
pH
7.4
with
methane-
sulfonic
acid).
Furthermore,
1
AM
tetrodotoxin
was
used
to
block
INa,
and
10
mM
tetraethylammonium
chloride
was
added
to
the
bath
to
block
IK
and
to
stabilize
the
bath
potential.
Pressure
injection
of
the
tetraethylammonium/
cesium
mixture
from
the
current
electrodes
also
facilitated
IK
blockade.
In
some
experiments,
the
Ba2+
concentration
was
varied
systematically;
the
detailed
ionic
compositions
of
these
solutions
are
specified
in
the
figure
legends.
Data
acquisition
and
analysis
were
performed
with
the
pClamp
system
(Axon
Instruments,
Burlingame,
CA).
When
IBa
was
studied,
four
to
eight
traces
were
routinely
averaged
to
improve
the
signal-to-noise
ratio.
All
experiments
were
per-
formed
at
a
bath
temperature
of
18-20°C.
Crude
spider
venom
(Agelenopsis
aperta
or
Hololena
curta)
or
toxin
partially
purified
by
chromatography
(FTX,
from
A.
aperta)
(13)
was
applied
to
the
oocytes
by
perfusion.
The
volume
of
the
chamber
was
300
to
400
,l,
and
the
effect
of
the
toxin
was
recorded
after
5-8
ml
of
the
solution
containing
venom
was
washed
in.
Unless
otherwise
indi-
cated,
experiments
illustrated
in
the
Results
section
were
obtained
with
the
crude
venom.
RESULTS
Effect
of
the
Spider
Venom
on
ICI(ca).
The
effect
of
the
spider
venom
on
the
'CICa,
was
studied
in
ND96
solution.
The
Ici(Ca)
is
an
intrinsic
current
of
Xenopus
oocytes
(22,
23)
and
can
be
A
activated
by
calcium
channels
intrinsic
to
the
oocytes
and/or
the
calcium
channels
synthesized
from
the
injected
RNA.
The
contribution
of
the
native
calcium
current
to
ICI(Ca)
is
small
because
the
amplitude
of
this
current
in
noninjected
oocytes
was
10
to
100
times
smaller
than
that
recorded
in
injected
oocytes.
The
oocytes
were
typically
held
at
-80
mV
and
depolarized
in
10-mV
increments
by
400-msec
pulses.
Depolarizations
above
-30
to
-40
mV
started
to
activate
a
tail
current
after
termination
of
the
voltage
steps
(Fig.
LA).
Because
the
holding
potential
was
near
EK,
the
tail
current
mostly
reflected
the
ICI(Ca)
activated
by
the
depolarizing
pulses.
Furthermore,
previous
studies
have
demonstrated
that
ICI(Ca)
was
not
voltage
dependent
in
the
potential
range
investigated
here
(24).
Thus,
a
plot
of
the
tail-current
ampli-
tude
against
the
voltage
steps
activating
it
provides
an
indication
on
the
voltage
sensitivity
of
the
underlying
Ca
channels.
In
the
I-V
plot
shown
in
Fig.
1B,
this
Cl
tail
current
became
visible
between
-30
to
-40
mV
and
reached
peak
amplitude
at
0
mV.
Further
depolarization
led
to
a
reduction
of
this
current.
The
tail
current
and
the
outward
current
during
the
pulse
(Fig.
LA)
were
partially
and
reversibly
blocked
when
the
crude
venom
was
washed
into
the
record-
ing
chamber.
Most,
if
not
all,
of
the
remaining
outward
current
during
the
pulse
was
probably
due
to
expressed
K
channels
that
were
insensitive
to
the
venom.
The
block
was
a
simple
reduction
of
the
current
amplitudes,
whereas
the
shape
of
the
I-V
curve
remained
unchanged
(Fig.
1B).
On
average
83%
(n
=
13)
of
the
'CI(ca)
was
blocked
by
a
1:1000
dilution
of
the
crude
venom,
and
higher
concentrations
did
not
produce
additional
block. Similar
results
were
obtained
when
FTX
was
used
(64%
block,
n
=
5).
The
effect
of
the
spider
venom
on
INa
and
IK
seemed
minimal
because
the
amplitude
and
waveform
of
both
currents
were
changed
little
after
the
toxin
(25).
The
reduction
of
ICI(Ca)
is
not
due
to
the
effect
of
the
venom
on
the
Cl
channels
directly
because
pressure
injections
of
Ca
could
evoke
this
current
in
com-
parable
amplitudes
before
and
after
the
toxin
(25).
Therefore,
the
venom
blocked
ICi(ca)
reversibly
by
interfering
with
the
underlying
Ca
channels.
The
spider
venom
blocks
ICI(Ca)
in
a
dose-dependent
man-
ner.
The
I-V
plot
in
Fig.
2A
shows
that
ICI(Ca)
was
blocked
to
different
levels
in
the
presence
of
two
toxin
concentrations.
In
this
case,
dose
1
was
equivalent
to
a
1
,ul/ml
dilution
of
FTX
purified
chromatographically,
and
higher
concentra-
tions
generally
did
not
produce
further
block.
A
dose-
response
relationship
obtained
from
a
similar
experiment
is
shown
in
Fig.
2B.
The
fitting
curve
was
drawn
by
hand
and
does
not
imply
any
binding
characteristics.
With
a
10-fold
V
(mV)
-50
a
control
&
FTX
*
wash
B
-100
IC
Ho
100
msec
(nA)
L
500
FIG.
1.
Spider
venom
blocks
ICI(ca).
(A)
Voltage-activated
currents
recorded
before
and
after
the
venom.
These
currents
were
activated
by
voltage
pulses
stepped
to
0
mV
from
a
holding
potential
of
-80
mV.
After
application
of
total
spider
venom
(1:1000
dilution),
the
chloride
component
of
the
outward
current
is
blocked,
leaving
the
IK
component.
The
blocking
effect
is
clearer
for
the
tail
current,
where
there
is little
contamination
of
potassium
current.
Washing
out
the
venom
for
1
hr
resulted
in
partial
recovery.
(B)
I-V
plot
Of
ICl(Ca).
Tail
current
amplitudes
were
plotted
against
voltage
steps.
Maximal
Icl(ca)
tail
was
activated
by
pulses
stepped
to
0
mV.
The
venom
reduced
the
current
amplitude
without
changing
the
voltage
sensitivity.
The
plot
was
obtained
from
the
same
oocytes
used
in
A.
Neurobiology:
Lin
et
al.
<

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
0~~~
0~~~
I
.1
Dose
FIG.
2.
Dose-dependent
block
of
ICI(ca)
by
FTX.
(A)
I-V
plot
Of
ICI(Ca)
under
control
conditions
and
after
application
of
two
concentrations
of
partially
purified
toxin.
The
maximal
quantity
of
toxin
added
to
the
2-ml
bath
was
2
jl;
the
other
dose
was
2/5ths
of
this
maximal
concentration.
Note
that
the
I-V
characteristics
were
independent
of
the
level
of
the
block.
All
data
were
collected
from
the
same
oocyte.
We
started
with
the
lowest
concentration
and
increased
the
concentration
by
adding
venom
without
washing.
Recordings
were
collected
at
least
15
min
after
each
dose
was
added.
(B)
Dose-dependent
block
of
maximal
ICI(ca)
measured
at
0
mV.
The
x
axis
corresponds
to
concentrations
of
FTX
in
;J/ml.
Percentage
of
remaining
current
was
normalized
by
the
current
measured
before
addition
of
any
toxin.
increase
in
the
FTX
concentration
(right
half
of
the
graph),
the
remaining
current
decreased
from
85
to
10%.
Effect
of
the
Spider
Venom
on
IBa*
The
direct
effect
of
the
spider
venom
on
ICa
was
examined
by
isolating
inward
currents
in
a
high-barium
solution
where
Cl-
was
replaced
by
methanesulfonate
and
tetrodotoxin
and
tetraethylammonium
were
added
to
block
Na
and
K
channels.
Under
these
conditions,
the
only
inward
current
will
be
carried
by
Ba
ions
moving
through
Ca
channels.
Depolarizing
pulses
activated
an
inward
current
showing
little
inactivation
(Fig.
3A).
This
current
started
to
appear
at
-20
mV
and
reached
peak
amplitude
at
about
+20
mV
(Fig.
3
A
and
C).
The
rightward
shift
of
the
voltage
sensitivity
in
comparison
with
the
I-V
A
control
FTX
curve
of
Ici(ca)
shown
in
Fig.
1B
is
due
to
high
concentration
of
divalent
cations
(26)
because
a
similar
shift
was
seen
for
ICI(Ca)
when
extracellular
calcium
concentration
was
raised
from
1.8
to
15.6
mM
(data
not
shown,
see
also
ref.
23).
The
application
of
FTX,
at
1:1000
dilution,
reversibly
reduced
the
amplitude
of
IBa
(Fig.
3A),
whereas
the
waveform
and
voltage
sensitivity
of
the
remaining
currents
were
not
affected
(Fig.
3).
Because
the
'Ba
block
was
noted
to
be
less
extensive
than
that
seen
for
ICI(Ca)
(Fig.
1B,
for
example),
we
examined
the
possible
effect
of
Ba2+
concentration
on
this
block.
Signifi-
cant
changes
in
'Ba
amplitude
were
seen
when
the
extracel-
lular
Ba2+
concentration
was
varied
(Fig.
4A).
The
peak
inward
current
in
20
mM
Ba2+
was
-40%
of
that
recorded
in
wash
100
mec
I
°C
C
B
10
mV
-100
-50
V
(mV)
0
50
FTX
k
wash
control
0
200n
A
50
msec
=
control
n
FTX
*
wash
I-N
&I
-400
(nA)
C
-800
\0
FIG.
3.
Barium
current
is
partially
and
reversibly
blocked
by
FTX.
(A)
Examples
of
an
IBa
series
recorded
before
and
after
FTX
and
after
washout.
Inward
current
started
to
appear
between
-20
to
-30
mV
and
generally
showed
little
inactivation.
(The
slight
decline
of
the
larger
currents
during
the
pulses
was
probably
from
incomplete
block
of
IK.)
Applications
of
the
toxin
reduced
the
current
amplitudes
without
changing
current
waveform.
One-hour
wash
partially
recovered
the
toxin-blocked
currents.
(B)
Superimposed
traces
to
provide
direct
comparison
of
control,
blocked,
and
recovered
currents.
These
traces
were
activated
by
voltage
pulses
stepped
to
+10
mV.
(The
residual
current
and
leakage
were
corrected
by
subtracting
residual
currents
measured
in
100
,um
Cd.)
(C)
I-V
plot
of
IBa
obtained
from
the
same
oocyte
as
in
A
and
B.
The
toxin
reduced
the
current
without
changing
the
voltage
sensitivity
of
the
Ca
channels.
A
B
mV
-50
a
:Control
*
:2/5
x
:1
50
.0
0
-
0
c
-I.
C
0
0
1.0-
0.8-
0.6-
0.4
0.2-
0.04
0.0
.01
1
4540
Neurobiology:
Lin
et
al.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
4541
A
20
mM
-10mV
+10mV
40
mM
60
mM
100
msec
B
100
N
co
80
0
z
.
60
cts
40
m
°3-
00
0
/
5
20
40
60
[Ba]o
(mM)
FIG.
4.
IBa
dependence
on
extracellular
Ba2+
concentration
([Bal]).
(A)
IBa
recorded
from
the
same
oocyte
in
three
different
Ba2+
concentrations.
Each
pair
of
currents
was
activated
by
voltage
step
to
-10
and
+
10
mV.
Larger
IBa
was
recorded
as
[Ba]0
was
increased.
There
is
a
more
apparent
decline
in IBa in
low
[Ba]0,
(compare
the
+
10
mV
traces
of
20
mM
and
60
mM
Ba2+
results);
this
is
probably
from
less
complete
block
of
IK
in
20
mM
Ba2+
rather
than
from
inactivation
of
Ca
channels.
(B)
[Ba]0
dependence
of
barium
current
amplitude.
Data
points
were
pooled
from
four
oocytes,
and
each
oocyte
was
exposed
to
several
Ba2+
concentrations.
Currents
were
normalized
by
the
maximal
current
recorded
in
60
mM
Ba2+
for
individual
oocytes
before
the
data
were
pooled
for
this
graph.
The
maximal
current
occurred
at
different
voltages
as
[Ba]0
was
changed;
normally
there
is
a
shift
of
20-30
mV
to
the
right
as
[Ba]0
was
increased
from
10
to
60
mM.
The
ionic
composition
of
20
and
40
mM
Ba2+
solutions
are
as
follows:
20
mM
Ba
solution-20
mM
Ba(OH)2/50
mM
NaOH/2
mM
KOH/5
mM
Hepes/30
mM
tetraethylammonium
hydroxide,
titrated
to
pH
7.4
with
methanesulfonic
acid;
40
mM
solution-40
mM
Ba(OH)2/20
mM
NaOH/2
mM
KOH/5
mM
Hepes/30
mM
tetraethylammonium
hydroxide,
titrated
to
pH
7.4
with
methanesulfonic
acid.
60
mM
Ba2+
(Fig.
4B).
A
rightward
shift
of
the
voltage
steps
that
activated
maximal
current
was
seen
as
Ba2+
concentra-
tion
was
increased
(data
not
shown).
The
same
concentration
of
the
crude
venom
was
less
effective
in
blocking
IBa
in
60
mM
Ba2+
than
in
lower
Ba2+
concentrations.
The
examples
illustrated
in
Fig.
5
A-C
show
that
the
total
venom
blocked
most
'Ba
in
20
mM
Ba2+
(A),
whereas
only
30%
of
the
current
was
blocked
in
60
mM
Ba2+
(C).
Results
obtained
from
22
oocytes
are
pooled
in
Fig.
SD
where,
on
average,
the
spider
venom
blocked
71%
of
IBa
in
A
20
mM
c
60
mM
20
mM
Ba2+,
and
65%
and
35%
of
the
current
were
blocked
in
40
and
60
mM
Ba2+,
respectively.
DISCUSSION
The
characterization
of
mammalian
central
nervous
system
Ca
channels
is
clearly
just
beginning.
The
study
of
induced
channels
in
Xenopus
oocyte
circumvents
major
technical
difficulties
associated
with
the
accessibility
of
ion
channels
in
dendrites
and
synaptic
terminals.
However,
the
application
B
40
mM
I1<
c
To
D
80
60
0
4o
I0-
40
50
msec
20
in-5
l
T
n-5
\4n-12
40
[Ba]o
(mM)
60
FIG.
5.
[Ba]0
dependence
of
the
venom
blocks.
(A-C)
Barium
current
blocked
by
the
venom
under
different
[Ba]0
levels.
Each
pair
of
traces
shows
'Ba
before
and
after
blockade.
Recordings
were
obtained
from
three
different
oocytes,
and
the
traces
shown
here
were
the
maximal
current
of
each
voltage
clamp
series:
0
mV
for
20
mM,
and
20
mV
for
40
mM
and
for
60
mM
Ba2+.
Most
of
the
inward
current
was
blocked
in
20
mM
Ba2+
(A),
whereas
only
a
30%o
reduction
occurred
in
60
mM
Ba2+
(C).
(D)
Effect
of
[Ba]0
on
venom
potency
at
1
,ul/ml
concentration.
About
71%
Of
'Ba
was
blocked
by
the
venom
in
20
mM
Ba2+,
whereas
only
65%
and
35%
of
the
current
was
blocked
in
40
and
60
mM
Ba2+,
respectively.
Sample
size
and
SD
are
indicated.
Data
were
collected
from
22
different
oocytes,
and
percentages
of blocks
were
calculated
from
the
voltage
steps
that
activated
maximal
current.
Neurobiology:
Lin
et
al.

Proc.
Natl.
Acad.
Sci.
USA
87
(1990)
of
this
approach
to
Ca
channels
is
still
in
an
early
stage.
Studies
of
Ca
channels
expressed
from
human
temporal
cortex
mRNA
demonstrated
a
single
ICa
component
that
is
w-conotoxin
sensitive
(27).
In
contrast,
Leonard
et
al.
(14)
reported
that
rat
brain
mRNA-induced
'Ca
was
insensitive
to
DHP
or
w-conotoxin
(however,
see
ref.
28).
Our
results
demonstrate
that
a
toxin
present
in
Agelenopsis
spider
venom
blocks
the
calcium
currents
expressed
in
Xenopus
oocytes
after
injection
of
rat
brain
mRNA.
Because
the
native
venom
and
the
toxin
fraction
FTX
have
similar
blocking
properties,
we
will
assume
for
the
purpose
of
this
discussion
that
FTX
is
the
calcium
channel-blocking
agent.
On
the
basis
of
its
abundance
of
expression
and
its
similar
slow
inactivation
kinetics
to
that
measured
by
Ca
flux
studies
of
brain
synaptosomes
(29),
Leonard
et
al.
(14)
suggested
that
the
calcium
channels
expressed
in
the
oocytes
injected
with
rat
brain
mRNA
could
mediate
neuronal
transmitter
release.
However,
review
of
published
studies
on
the
spider
toxin
indicated
that
the
toxin-sensitive
calcium
channels
play
func-
tional
roles
broader
than
simply
triggering
transmitter
re-
lease.
Calcium
spikes
in
the
dendrites
of
cerebellar
Purkinje
cells
exhibit
identical
pharmacological
and
electrophysiolog-
ical
properties-i.e.,
insensitive
to
DHP
or
c-conotoxin,
block
by
FTX,
and
slow
inactivation
kinetics
(13).
Additional
examples
of
the
spider
venom-sensitive
calcium
current
include
the
following:
(i)
the
presynaptic
calcium
current
in
the
squid
giant
synapse
(13);
(it)
presynaptic
calcium
spikes,
measured
optically,
of
the
frog
neurohypophysis
(17);
(iii)
soma-dendritic
calcium
current
in
cerebellar
granular
cells
in
tissue
culture
(18);
and
(iv)
calcium
currents
measured
in
Drosophila
neurons
(30).
Among
the
examples
listed,
when
ICa
was
measured,
the
spider
venom-sensitive
calcium
cur-
rents
all
exhibited
a
similar
noninactivating
kinetics
(howev-
er,
see
ref.
31).
Therefore,
although
the
spider
venom-
sensitive
channels
studied
thus
far
may
all
belong
to
the
P-channel
family,
their
distribution
and
functional
roles
may
be
diverse.
We
found
that
the
spider
toxin
block
of
the
calcium
current
expressed
from
rat
brain
RNA
depends
upon
the
concentra-
tion
of
Ba
ions.
The
toxin
may
block
Ca
channels
by
plugging
the
permeation
pore.
In
this
case,
the
toxin
and
Ba2+
would
compete
for
a
site
at
the
channel
mouth.
Alternatively,
Ba2+
may
screen
negative
charges
at
or
close
to
the
venom-binding
sites
and
compete
with
its
binding,
such
as
in
the
competition
between
charybdotoxin
and
monovalent
cations
for
calcium-
activated
potassium
channels
(30).
These
sites
need
not
be
the
permeation
pore
itself,
and
the
toxin
may
block
either
by
a
plugging
mechanism
or
by
modulating
the
gating
of
the
Ca
channels.
The
divalent
cation
dependence
may
also
explain
why
the
native
venom
or
FTX
block
was
never
complete,
either
in
ND96
or
in
high
Ba2+.
This
may
simply
be
due
to
a
baseline
competition
between
the
toxin
and
existing
divalent
cations
in
the
solution.
A
related
observation
is
that
changing
the
divalent
cation
concentration
had
a
quantitatively
differ-
ent
effect
on
the
spider
venom
block
and
the
IBa.
Specifically,
the
level
of
block
was
reduced
to
half
when
Ba2+
was
changed
from
40
to
60
mM
(Fig.
5).
In
contrast,
there
was
only
a
20%
change
in
the
'Ba
amplitude
for
the
same
concentration
range
(Fig.
4).
This
behavior
suggests
that,
regardless
of
where
the
binding
site
is
located,
the
spider
toxin
has
a
shallower
binding
curve
for
calcium
channels
than
Ba
ions
have
for
the
ion
permeation
pore.
This
work
was
supported
by
Grant
NS13742
from
the
National
Institute
of
Neurological
and
Communicative
Disorders
and
Strokes,
by
the
Fidia
Research
Foundation
for
funding
and
venom,
and
by
Grant
26976
from
the
National
Institute
of
General
Medical
Sciences.
1.
Llinas,
R.
&
Yarom,
Y.
(1981)
J.
Physiol.
(London)
315,
569-584.
2.
Nowycky,
M.
C.,
Fox,
A.
P.
&
Tsien,
R.
W.
(1985)
Nature
(London)
316,
440-443.
3.
Lancaster,
B.
&
Nicoll,
R.
A.
(1987)
J.
Physiol.
(London)
389,
187-203.
4.
Llinds,
R.
&
Sugimori,
M.
(1980)
J.
Physiol.
(London)
305,
171-195.
5.
Katz,
B.
(1969)
The
Release
of
Neural
Transmitter
Substances
(Thomas,
Springfield,
IL).
6.
Llin.s,
R.,
Steinberg,
I.
Z.
&
Walton,
K.
(1981)
Biophys.
J.
33,
323-352.
7.
Nestler,
E.
J.
&
Greengard,
P.
(1984)
Protein
Phosphorylation
in
the
Nervous
System
(Wiley,
New
York).
8.
Eberhard,
D.
A.
&
Holz,
R.
W.
(1988)
Trends
NeuroSci.
11,
517-520.
9.
Doerner,
D.,
Pitler,
T.
A.
&
Alger,
B. E.
(1988)
J.
Neurosci.
8,
4069-4078.
10.
Llinas,
R.
(1988)
Science
242,
1654-1664.
11.
Woodward,
J.
J.,
Rezazadeh,
S.
M.
&
Leslie,
S.
W.
(1988)
Brain
Res.
475,
141-145.
12.
Suszkiw,
J.
B.,
Murawsky,
M.
M.
&
Shi,
M.
(1989)
J.
Neuro-
chem.
52,
1260-1269.
13.
Llinas,
R.,
Sugimori,
M.,
Lin,
J.-W.
&
Cherksey,
B.
(1989)
Proc.
Natl.
Acad.
Sci.
USA
86,
1689-1693.
14.
Leonard,
J.
P.,
Nargeot,
J.,
Snutch,
T.
P.,
Davidson,
N.
&
Lester,
H.
A.
(1987)
J.
Neurosci.
7,
875-881.
15.
Cherksey,
B.,
Llinds,
R.,
Sugimori,
M.
&
Lin,
J.-W.
(1990)
Biol.
Bull.
177,
321.
16.
Sugimori,
M.
&
Llinas,
R.
(1987)
Soc.
Neurosci.
Abstr.
13,
228.
17.
Salzberg,
B.
M.,
Obaid,
A.
L.,
Stanley,
K.,
Lin,
J.-W.,
Sugi-
mori,
M.,
Cherksey,
B.
&
Llinis,
R.
(1990)
Biophys.
J.
Abstr.
57,
305a.
18.
Bertolino,
M.,
Vicini,
S.,
Costa,
E.
&
Llinas,
R.
(1990)
FASEB
J.
Abstr.
4,
A1202.
19.
Dumont,
J.
N.
(1972)
J.
Morphol.
136,
153-180.
20.
Dierks,
P.,
van
Ooyen,
A.,
Mantei,
N.
&
Weissmann,
C.
(1981)
Proc.
Natl.
Acad.
Sci.
USA
78,
1411-1415.
21.
Maniatis,
T.,
Fritsch,
E.
F.
&
Sambrook,
J.
(1982)
Molecular
Cloning:
A
Laboratory
Manual
(Cold
Spring
Harbor
Lab.,
Cold
Spring
Harbor,
NY).
22.
Miledi,
R.
(1982)
Proc.
R.
Soc.
London
Ser.
B
215,
491-497.
23.
Barish,
M.
E.
(1983)
J.
Physiol.
(London)
342,
309-325.
24.
Miledi,
R.
&
Parker,
I.
(1984)
J.
Physiol.
(London)
357,
173-183.
25.
Lin,
J.-W.,
Rudy,
B.,
Cherksey,
B.,
Sugimori,
M.
&
Llinas,
R.
(1989)
Soc.
Neurosci.
Abstr.
15,
652.
26.
Hille,
B.,
Woodhull,
A.
M.
&
Shapiro,
B.
I.
(1975)
Philos.
Trans.
R.
Soc.
London
Ser.
B
270,
301-318.
27.
Gundersen,
C.
A.,
Umbach,
J.
A.
&
Swartz,
B. E.
(1988)
J.
Pharmacol.
Exp.
Ther.
247,
824-829.
28.
Kaneko,
S.
&
Nomura,
Y.
(1987)
Neurosci.
Lett.
83,
123-127.
29.
Suszkiw,
J.
B.,
O'Leary,
M.
E.,
Murawsky,
M.
M.
&
Wang,
T.
(1986)
J.
Neurosci.
6,
1349-1357.
30.
Anderson,
C.
S.,
MacKinnon,
R.,
Smith,
C.
&
Miller,
C.
(1988)
J.
Gen.
Physiol.
91,
317-333.
31.
Leung,
J.-T.,
Branton,
W.
D.,
Phillips,
H.
S.,
Jan,
L.
&
Byerly,
L.
(1989)
Neuron
3,
767-772.
4542
Neurobiology:
Lin
et
al.