BRIEF
COMMUNICATION
CURRENTS
RECORDED
THROUGH
SMALL
AREAS
OF
SQUID
AXON
MEMBRANE
WITH
AN
INTERNAL
VIRTUAL
GROUND
VOLTAGE
CLAMP
Jos
LOPEZ-BARNEO,
DONALD
R.
MAT ESON,
AND
CLAY
M.
ARMSTRONG,
Depa men
o
Physiology,
G4,
Uni e si y
o
Pennsyl ania,
School o
Medicine,
Philadelphia,
Pennsyl ania
19104,
and
Ma ine
Biological
Labo a o y,
Woods
Hole,
Massachuse s
02543
ABSTRACT
A
new
ol age-clamp
appa a us
o
he
squid
axon
has
been
implemen ed
o
enable
eco ding
o
cu en s
h ough
small
a eas
o
axon
memb ane.
The
pe o mance
o
his
clamp
was
es ed
by
eco ding
o al
sodium
cu en s
om
pe used
axons
(IQ,)
and
sodium
cu en s
om
small
memb ane
pa ches
(Ip Ch),
which
we e
eco ded
om
inside
he
axon
wi h
an
L-shaped
pipe e.
The
Ip,,,h
eco ds,
al hough
ou
o de s
o
magni ude
smalle
han
1 o ,
we e
s able
and
showed
no mal
kine ics
and
ol age
dependence,
and
appea ed
o
e lec
he
ac i a ion
o
a
small
popula ion
o
no mal
sodium
channels.
The
size
o
he
cu en
eco ded
om
he
pa ch
was
mainly
a
unc ion
o he
ip
diame e
o
he
L-shaped
pipe e
and
o
he
shun
esis ance
be ween
inside
he
pipe e
and
he
axoplasm.
In
s udying
cu en
luc ua ions
ela ed
o
ionic
channels
in
exci able
memb anes,
i
is
ad an ageous
o
isola e
and
eco d
om
a
e y
small
memb ane
pa ch
(Fishman,
1975;
Nehe
e
al.,
1978).
Reducing
he
eco ding
a ea
enhances
channel- ela ed
luc ua ions
ela i e
o
noise
om
o he
sou ces.
Se e al
echniques
ha e
ecen ly
been
de eloped
o
elec ical
isola ion
o
small
pa ches.
Success ul
applica ion
o
hese
echniques
in
squid
gian
axons
will
make
i
possible
o
apply
he
ensemble
me hod
o
measu ing
cu en
luc ua ions
(Sigwo h,
1977),
and
o
eco d
cu en
om
single
memb ane
channels
(Con i
and
Nehe ,
1980;
Sigwo h
and
Nehe ,
1980).
We
epo
he e
he
gene al
ea u es
as
well
as
he
pe o mance
o
a
ol age-clamp
echnique
o
he
squid
gian
axon
which
can
be
used
o
eco d
cu en
h ough
small
a eas
o
memb ane.
30-mm-long
segmen s
o
axon
we e
placed
in
a
chambe
and
in e nally
pe used.
The
main
ea u es
o
he
ol age-clamp
ci cui
a e
illus a ed
in
Fig.
1.
In e nal
ol age
(VJi)
was
eco ded
h ough
a
0.6
M
KCl- illed
pipe e
(1).
Vin
was
ed
back
o
an
in e nal
con ol
ampli ie
(ICA)
which
held
Vi.
a
g ound
po en ial
by
passing
he
app op ia e
cu en
h ough
a
pla inized-pla inum
wi e
(3)
placed
inside
he
axon.
Ex e nal
ol age
(Vou )
was
eco ded
h ough
a
sil e -sil e
chlo ide
elec ode
(2).
V
-Vin
was
ed
o
an
ampli ie
(ECA)
which
con olled
memb ane
ol age
by
a ying
he
ba h
po en ial
ela i e
o
in e nal
g ound
h ough
wo
la ge
pla inum
elec odes
(4)
placed
longi udinally
along
he
ou side
o
he
axon.
Vol age-clamp
command
pulses
we e
gene a ed
om
a
PDP-8
compu e
(Digi al
Equipmen
BIOPHYS.
J.
e
Biophysical
Socie y
*
0006-3495/81/12/811/05
$1.00
811
Volume
36
Decembe
1981
811-816
Via
FIGURE
1
Vol age
clamp
and
cu en
eco ding
ci cui .
The
numbe s
in
he
diag am
e e
o
he
ollowing
elec odes:
1,
in e nal
ol age
pipe e;
2,
ex e nal
ol age
elec ode;
3,
in e nal
pla inized-
pla inum
axial
cu en
passing
wi e;
4,
ex e nal
pla inized-pla inum
elec odes;
and
5,
L-shaped
pa ch
pipe e.
The
la ge
ex e nal
elec odes
(4)
we e
15
mm
long
and
ex ended
he
en i e
leng h
o
he
axon
be ween
he
ai
gaps,
so
I,.w
was
eco ded
om
his
leng h
o
axon.
Co p.,
Mayna d,
Mass.)
h ough
a
D/A
con e e
and
ed
o
he
summing
junc ion
o
ECA.
The
cu en
gene a ed
by
he
whole axon
(I o)
was
moni o ed
by
eco ding
he
ol age
d op
ac oss
a
100
Q
esis o
connec ed
in
se ies
wi h
he
ou pu
o
ECA.
Cu en s
h ough
small
pa ches
o
memb ane
we e
eco ded
simul aneously
wi h
I w
using
a
me hod
simila
o
he
one
desc ibed
by
Con i
and
Nehe
(1980).
The
me hod
in ol es
placing
an
L-shaped
pipe e
inside
he
axon
and
maneu e ing
he
ip
o
he
inside
su ace
o
he
memb ane.
The
ben
pa
o
he
pipe e
was
100-200
,um
in
leng h
and
had an
ou side
diame e
o
70-100
,um.
As
he
pipe e
ip
was
i e
polished
he
inside
diame e
dec eased
o
a
inal
diame e
o
10-25
,m.
This
pa ch
pipe e
(5)
was
connec ed
o
an
ope a ional
ampli ie
wi ed
as
a
cu en - o- ol age
con e e
o
measu e
cu en
h ough
he
pipe e.
The
pa icula
pa ch
pipe e
used
in
he
expe imen s
epo ed
he e
had
an
inside
ip
diame e
o
20-25
Mm
and
a
DC
esis ance
o
-0.5
MO.
A
loa ing
25-,um
Diam
pla inum
wi e
was
placed
inside
he
pipe e
o
dec ease
impedance
and
he eby
minimize
se ies
esis ance
e o s
caused
by
pa ch
elec ode
esis ance.
The
pa ch
pipe es
we e
illed
wi h
a
solu ion
con aining
150
mM
e ame hylammonium
(TMA)
glu ama e,
50
mM
TMA
luo ide,
20
mM
sodium
glu ama e,
10
mM
T is
and
450
mM
suc ose.
All
eco ds
we e
ob ained
a
80C.
Linea
leakage
and
capaci a i e
cu en s
we e
sub ac ed
ou
elec onically
using
he
P/4
echnique
(A ms ong
and
Bezanilla,
1974).
Fo
ou
pu poses
he
majo
ad an age
ha
his
pa icula
ol age-clamp
design
o e s
o e
he
mo e
commonly
used
design
(Cole
and
Moo e,
1960;
Hodgkin
e
al.,
1952)
is
he
main enance
o
he
in e io
o
he
axon
a
i ual
g ound
po en ial,
which
elimina es
capaci a i e
cu en s
ac oss
he
wall
o
he
pa ch
pipe e
he eby
imp o ing
he
equency
esponse.
Clamping
he
axoplasm
o
i ual
g ound
was
also
done
in
ol age-clamp
echniques
de eloped
o
he
node
o
Ran ie
(Dodge
and
F ankenhaeuse ,
1958;
Nonne ,
1969)
and
sugges ed
o
he
squid
gian
axon
by
Le is
(1979).
The
pe o mance
o
his
ol age-clamp
was
assessed
by
compa ing
cu en s
eco ded
om
BIOPHYSICAL
JOURNAL
VOLUME
36
1981
812
he
whole
memb ane
(I o al)
wi h
cu en
gene a ed
by
he
pa ch
(Ip, ch).
Al hough
he
gene al
cha ac e is ics
o
l
a e
simila
o
sodium
cu en s
eco ded
wi h
he
mo e
usual
ol age-
clamp
con igu a ion,
ou
chambe
lacked
gua d
elec odes;
o al
memb ane
cu en s
eco ded
wi h
his
clamp
a e
he e o e
no highly
accu a e,
since
he
ends
o
he
p epa a ion
a e
no
well
con olled.
To
eco d
accu a e
o al
cu en s,
he
measu ing
egion
could
be
es ic ed
o
a
cen al
egion
and
gua d
elec odes
in
he
pe iphe y
could
be
d i en
by
a
sepa a e
con ol
ampli ie .
Fig.
2
A
shows
supe imposed
sodium
cu en s,
eco ded
as
' o al
om
15
mm
o
axon,
gene a ed
by
ol age-clamp
s eps
om
-80
mV
o
he
indica ed
ol ages.
The
cu en - ol age
ela ionship
om
hese
eco ds
is
shown
by
he
illed
symbols
in
Fig.
2
C.
Pa ch
eco dings
o
sodium
cu en s
om
he
same
axon
a e
shown
in
Fig.
2
B,
and
hei
cu en - ol age
ela ionship
is
plo ed
as
he
open
symbols
in
Fig.
2
C.
The
impo an
poin
o
no e
is
ha
he
kine ics
and
ol age
dependence
o
hese
cu en s
indica e
ha
hey
we e
gene a ed
by
a
no mal
popula ion
o
sodium
channels,
and
he
ampli ude
o
he
cu en s
sugges s
ha
he
numbe
o
channels
was
small.
P e ious
a emp s
o
eco d
pa ch
cu en s
om
he
inside
o
he
axon
upon
s eps
in
memb ane
ol age
ha e
ailed
(Con i
and
Nehe ,
1980).
Con i
and
Nehe
e e
o
he
appea ance
o
la ge
and
slow
a i ac s
ha
con amina e
hei
cu en
eco ds.
Al hough
he
gene al
shape
and
posi ion
o
he
I-V
cu es
a e
simila
he e
a e
se e al
appa en
di e ences
be ween
he
se s
o
cu en s
shown
in
Fig.
2.
Fi s ,
he
peak- o-
s eady-s a e
sodium
cu en
a io
is
no iceably
smalle
o
I o al
han
o
Ip ch.
Second,
he
e e sal
po en ial
o
he
I o al
cu en
is
-
12
mV
less
posi i e
han
o
Ip' ch*
We
eel
ha
he
pa ch
eco ds
a e
a
mo e
accu a e
ep esen a ion
o
he
memb ane
sodium
cu en s
o
he
4
! ' '
|^
i;
.
:
d.
FIGURE
2
A,
Na
cu en s
(Iw)
gene a ed
by
I
0-ms
ol age-clamp
pulses
o
he
indica ed
ol ages
om
a
holding
po en ial
o
-
80
mV.
This
axon
was
-440
um
in
diame e
so
ha
he
o al
a ea
eco ded
om
was
app oxima ely
0.21
cm2.
B,
Na
cu en s
h ough
a
25
Am
(inside
ip
diame e )
pa ch
elec ode
gene a ed
by
10-ms
ol age
clamp
pulses
o
he
indica ed
ol ages
om
a
holding
po en ial
o
-80
mV.
Solu ions
o
A
and
B
we e
he
same.
Ex e nal
solu ion:
225
mM
NaCl,
50
mM
CaCl2
and
240
mM
T is
7.0.
In e nal
solu ion:
150
mM
TMA
glu ama e,
50
mM
TMA
luo ide,
20
mM
Na
glu ama e,
10
mM
T is
7.0,
and
540
mM
suc ose.
C,
Cu en - ol age
ela ionships
o
peak
cu en s
om
he
eco ds
shown
in
A
(-)
and
B
(O).
LOPEZ-BARNEO
ET
AL.
Cu en s
Reco ded
Th ough
Squid
Axon
Memb ane
813
ollowing
easons.
Unco ec ed
I,.,w
eco ds
we e
ound
o
ha e
a
much
highe
leakage
cu en
han
he
pa ch
eco ds
and
his
leak
p esumably
lowed
ou
he
cu
ends
o
he
axon.
I
his
cu en
inc eases
nonlinea ly
wi h
ol age,
due
in
some
unknown
way
o
he
damaged
ends
o
he
axon,
i
could
p oduce
a
la ge
s eady-s a e
cu en
and
an
appa en ly
lowe
e e sal
po en ial.
The
e e sal
po en ial
p edic ed
by
he
Ne s
equa ion
is
58.8
mV,
e y
close
o
he
e e sal
po en ial
o
he
pa ch
eco ds.
The
pa ch
cu en s
shown
in
Fig.
2
B
we e
eco ded
wi h
a
25-,um
ip-Diam
pipe e
ha
was
ouching
ligh ly
agains
he
memb ane.
Wi h
his
la ge
diame e
pipe e
i
is
impossible
o
ob ain
he
high
shun
esis ances
(RSh)
which
can
be
ob ained
wi h
smalle
diame e
pipe es
(c .
Nehe
e
al.,
1978).
The e o e,
he
Ip, ch
eco ds
a e
no
an
accu a e
e lec ion
o
he
o al
cu en
gene a ed
by
he
pa ch,
since
much
o
his
cu en
appa en ly
lows
h ough
Rh.
The
Ip,, ch
cu en
gene a ed
a
0
mV
ep esen s
a
cu en
densi y
o
-3.7
mA/cm2
assuming
he
a ea
eco ded
om
was
490
,um2
(i.e.
a
ci cle
o
25
,um
Diam).
Since
he
Ih.,w
cu en
a
0
mV
was
-0.42
mA/cm2,
he
Ipa ch
eco ds
mus
ha e
been
eco ded
om
a
much
la ge
a ea
han
he
25-,um
Diam
ci cle
we
assumed.
An
imp o emen
in
he
isola ion
o
he
memb ane
pa ch
can
be
ob ained
unde
he
app op ia e
condi ions
and
Fig.
3
illus a es
he
echnique
we
ha e
used.
The
ou
cu en s
illus a ed
a e
pa ch
eco dings
o
ol age
s eps
o
0
mV
om
a
holding
po en ial
o
-80
mV.
I
was
ob ained
wi h
he
pipe e
placed
in
he
cen e
o
he
axon
and
he
ip
-100
Am
away
om
he
memb ane.
The
pipe e
was
hen
mo ed
un il
i
jus
ouched
he
memb ane
and
he
cu en
was
ound
o
inc ease
app eciably
(2).
An
e en
highe
cu en
ampli ude
could
be
ob ained
by
p essing
he
pipe e
ip
mo e
i mly
agains
he
memb ane
(3),
which
p esumably
inc eases
R,h.
The
in e nal
pe usion
o
he
axon
was
hen
swi ched
o
a
low
ionic
s eng h
solu ion
(wi hou
changing
he
solu ion
in
he
cu en
eco ding
pipe e)
and
he e
was
a
d ama ic
inc ease
in
cu en
ampli ude
(4),
al hough
R,h
was
only
inc eased
o
-1
Mg.
S ill
highe
alues
o
R,h
could
mos
likely
be
ob ained
by
using
smalle
diame e
pa ch
pipe es
and
by
cleaning
he
inside
su ace
o
he
memb ane
wi h
p onase
as
was
done
by
Con i
and
Nehe
(1980).
Since
we
do
no
ha e
pe ec
isola ion
o
he
memb ane
pa ch,
some
o
he
low
ionic
s eng h
suc ose
solu ion
may
each
he
memb ane
a ea
unde
he
pa ch
elec ode.
This
could
explain
he
ela i ely
la ge
s eady-s a e
cu en
seen
in
suc ose
(4
in
Fig.
3)
since
i
has
p e iously
been
shown
ha
pe usion
wi h
low
ionic
s eng h
solu ion
shi s
he
s eady-s a e
FIGURE
3
Na
cu en s
h ough
a
25-,um
Diam
pa ch
elec ode
as
he
shun
esis ance
om
pipe e
in e io
o
axon
in e io
inc eased.
Numbe s
e e
o
successi e
10-ms
ol age
s eps
o
0
mV
om
a
holding
po en ial
o
-80
mV.
Reco ds
1-3
we e
eco ded
wi h
he
same
solu ions
as
in
Fig.
2
and
he
pipe e
in
he
ollowing
posi ions:
1,
cen e
o
he
axon;
2,
jus
ouching
he
axon
memb ane;
and
3,
p essing
agains
he
memb ane.
4
was
eco ded
a e
swi ching
o
an
in e nal
solu ion
o
he
ollowing
composi ion:
850
mM
suc ose,
50
mM
TMA
luo ide,
and
10
mM
T is
7.0.
BIOPHYSICAL
JOURNAL
VOLUME
36
1981
814
inac i a ion
cu e
o
mo e
posi i e
ol ages
(Chandle
e
al.,
1965;
Moo e
e
al.,
1964).
The e o e,
in
he
suc ose
solu ion
inac i a ion
may
be
less
comple e
a
0
mV,
esul ing
in
mo e
s eady-s a e
sodium
cu en .
A
second
possibili y
is
ha
he e
is
a
change
in
he
junc ion
po en ial
a
he
ip
o
he
in e nal
ol age
elec ode
when
he
in e nal
pe usion
is
swi ched
o
low
ionic
s eng h
solu ion.
The e o e,
he
ue
memb ane
po en ial
in
his
solu ion
may
be
a
a
ol age
whe e
inac i a ion
is
less
comple e.
The
majo
poin
o
his
communica ion
is
ha
he
sodium
cu en
eco ds
p esen ed
illus a e
he
abili y
o
ou
i ual
g ound
ol age-clamp
con igu a ion
o
p oduce
s able
sodium
cu en s
whose
ampli ude
is
limi ed
mainly
by
he
size
o
he
pipe e
ip
and
by
he
alue
o
he
shun
esis ance.
Since
he
in e nal
i ual
g ound
ci cui
e ec i ely
elimina es
he
s ay
capaci ance
ac oss
he
wall
o
he
pa ch
pipe e,
he
equency
esponse
o
he
eco ding
sys em
is
imp o ed.
This
may
be
why
we
ha e
no
seen
he
long
las ing
a i ac s
obse ed
by
Con i
and
Nehe
(1980)
upon
s ep
changes
in
memb ane
ol age.
By
using
smalle
diame e
pa ch
pipe es,
we
should
be
able
o
achie e
accep able
alues
o
RSh
so
ha
his
ol age-clamp
con igu a ion
will
be
use ul
o
eco ding
memb ane
cu en
luc ua ions
and
es ima ing
single
channel
conduc ance
(Sigwo h,
1977).
In
addi ion,
i
should
be
possible
o
eco d
he
cu en
h ough
single
memb ane
channels.
Recei ed o
publica ion
19
Decembe
1980
and
in
e ised o m
22
Augus
1981.
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a
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A ch.
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815