Sodium and Calcium Cu en s in
Dispe sed Mammalian Sep al Neu ons
ANTONIO CASTELLANO and
Jos~
L6PEZ-BARNEO
F om he Depa amen o de Fisiolog a y Bio lsica, Facul ad de Medicina, 41009 Se illa, Spain
A B S T R A C T
Vol age-ga ed Na ÷ and Ca 2÷ conduc ances o eshly dissocia ed sep al
neu ons we e s udied in he whole-cell con igu a ion o he pa ch-clamp echnique.
All cells exhibi ed a la ge Na + cu en wi h cha ac e is ic as ac i a ion and
inac i a ion ime cou ses. Hal - ime o peak cu en a -20 mV was 0.44 _ 0.18 ms
and maximal ac i a ion o Na + conduc ance occu ed a 0 mV o mo e posi i e
memb ane po en ials. The a e age alue was 91 -+ 32 nS (~ 11 mS cm-2). A all
memb ane ol ages inac i a ion was well i ed by a single exponen ial ha had a
ime cons an o 0.44 _+ 0.09 ms a 0 inV. Reco e y om inac i a ion was comple e
in ~900 ms a -80 mV bu in only 50 ms a -120 mV. The decay o Na ÷ ail
cu en s had a single ime cons an ha a -80 mV was as e han 100 p.s.
Depola iza ion o sep al neu ons also elici ed a Ca ~+ cu en ha peaked in ~ 6-8
ms. Maximal peak Ca ~÷ cu en was ob ained a 20 mV, and wi h 10 mM ex e nal
Ca ~+ he ampli ude was 0.35 - 0.22 hA. Du ing a main ained depola iza ion his
cu en pa ially inac i a ed in he cou se o 200-300 ms. The Ca ~+ cu en was due
o he ac i i y o wo ypes o conduc ances wi h di e en deac i a ion kine ics. A
-80 mV he closing ime cons an s o slow (SD) and as (FD) deac i a ing channels
we e, espec i ely, 1.99 -+ 0.2 and 0.11 +_ 0.03 ms (25°C). The wo kinds o channels
also di e ed in hei ac i a ion ol age, inac i a ion ime cou se, slope o he
conduc ance- ol age cu e, and esis ance o in acellula dialysis. The p opo ion
o SD and FD channels a ied om cell o cell, which may explain he di e en ial
elec ophysiological esponses o in acellula ly eco ded sep al neu ons.
INTRODUCTION
Mammalian cen al neu ons possess in insic elec ical p ope ies ha make hem
able o gene a e complex i ing pa e ns. This implies he exis ence o a a ie y o
memb ane ionic conduc ances which, wi h a ew excep ions, a e poo ly cha ac e ized.
Some p ope ies o Na + channels ha e ecen ly been s udied in dissocia ed hippo-
campal (Sah e al., 1988) and neoco ical (Huguena d e al., 1988) neu ons, as well as
in Pu kinje cells o o gano ypic ce ebella cul u es (G~ihwile and Llano, 1989), bu
addi ional wo k is s ill needed o ully in es iga e he biophysical p ope ies o
ce eb al Na ÷ channels and o asce ain whe he hei cha ac e is ics a e homoge-
neous in di e en b ain egions. Ca ~+ channels, on he o he hand, ha e been
Add ess ep in eques s o D . J. L6pez-Bameo, Depa amen o de Fisiologia M dica y Bio sica,
Facuhad de Medicina, A da. S~inchez Pizju in, 4, 41009 Se illa, Spain.
J. GEN. PHYSIOL. © The Rocke elle Uni e si y P ess • 0022-1295/91/02/0303/18 $2.00
Volume 97 Feb ua y 1991 303-320
303
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THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME
97 . 1991
in ensi ely s udied in neu ons o he pe iphe al ne ous sys em (Fedulo a e al.,
1985; Ca bone and Lux, 1987; Fox e al., 1987; Swandulla and A ms ong, 1988),
bu wi hin he b ain hey ha e been only pa ially cha ac e ized (He u~indez-C uz
and Pape, 1989; Meye s and Ba ke , 1989). We ha e p e iously shown ha in acel-
lula ly eco ded sep al neu ons in in i o b ain slices display di e en ial elec ical
esponses due o he a iabili y in he dis ibu ion o memb ane ionic conduc ances
(L6pez-Ba neo e al., 1985; Al a ez de Toledo and L6pez-Bameo, 1988). This a icle
con inues ou p eceding wo k and ocuses on he cha ac e iza ion o he ionic
cu en s o eshly dissocia ed sep al neu ons om adul guinea pigs. Due o hei
size and egula geome y hese cells p o ide an excellen p epa a ion o s udying
he ol age-ac i a ed ionic channels in a well-clamped neu onal memb ane using he
whole-ceU a ian o he pa ch-clamp echnique (Hamill e al., 1981). This esea ch
no only has biophysical in e es , i is also o impo ance because an accu a e analysis
TABLE I
Composi ien o Solu ions
NaCl KC! CaClz MgCi~ BaCI~ CoCI 2 T izma* HEPES
Ex~ nal
130 Na 130 2.7 2.5 1 -- -- -- 10
80 Na 80 2.7 2.5 1 -- -- 70 --
I0 Ca 70 2.7 10 -- -- -- 60 --
10 Ba 70 2.7 -- -- I0
-- 60 --
Mg/Co 70 2.7 1 7.5 -- 1.5 60 --
NaCI KCI K-glu ama~ KF MgCI, CsCI CsF HEPES EGTA
In~ nal
130 K -- 30 80 20 2 -- -- 10 5
130 Cs .... 2 110 20 10 5
40 Na 40 -- -- -- 2 70 20 10 5
All alues a e gi en in miUimola .
pH was 7.4 in ex e nal and 7.3 o 7.35 in in e nal solu ions
*T is (hyd oxyme hyl)aminome hane (pH = 7.4).
o he ionic conduc ances o cen al neu ons is equi ed be o e unde aking he
in es iga ion o hei modula ion by neu omodula o s and d ugs. This a icle
concen a es on he desc ip ion o he p ope ies o Na ÷ and Ca ~÷ cu en s; he K ÷
cu en is only b ie ly p esen ed and he e o e no s udied in de ail.
METHODS
Cells we e ob ained om co onal slices, 300-400 p~m hick, o he adul guinea pig sep al
egion. The p ocedu e ollowed o slicing was he same as p e iously desc ibed (Al a ez de
Toledo and L6pez-Bameo, 1988). Fo cell dissocia ion, six slices con aining only he sep al
nucleus we e placed in a ial wi h 3 ml o Ea le solu ion (Flow Labo a o ies, Inc., McLean, VA)
con aining 0.2 mg/ml o ypsin ype IX and 0.05 mg/ml o DNase ype II (Sigma Chemical Co.,
S . Louis, MO). This solu ion was con inuously bubbled wi h a mix u e o 95% O~ and 5% CO2
and he issue was incuba ed o 20 min a 37°C. A e his ime pe iod he issue was washed
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CASTELLANO AND I.XSPEZ-BARNEO Na + and Ca 2+ Channels o Sep al Neu ons 305
wice and he enzymes we e emo ed. Cells we e mechanically dispe sed using i e-polished
Pas eu pipe es, cen i uged, and esuspended in MEM cul u e medium supplemen ed wi h
glu amine, an ibio ics, and bo ine e al se um. Cells we e pla ed on agmen s o glass
co e slips ea ed wi h poly-L-lysine and main ained in an incuba o un il use (be ween 2 and 6
h a e pla ing). The gene al p ocedu es and he se up used o eco ding o whole-cell
cu en s we e he same as p e iously desc ibed (U e ia e al., 1989a). The composi ion o he
eco ding solu ions is gi en in Table I. Juncdon po en ials we e co ec ed once he elec ode
was in he ba h and immedia ely be o e es ablishing a seal. In he ex and in he igu e legends
solu ions a e indica ed as ex e nal//in e nal. Expe imen s we e pe o med a oom empe a u e
(20-25°C).
Fo eco ding we used a pa ch-clamp ampli ie buil in ou labo a o y ollowing he design o
Sigwo h (1983). A as and uni o m con ol o he memb ane po en ial was a o ed by he use
o low esis ance elec odes (be ween 0.8 and 2 M ~), a eedback esis ance o only 100 Ml'l in
he cu en - o- ol age con e e , and elec onic compensa ion o se ies esis ance. Wi h his
eco ding con igu a ion capaci y ansien s ypically had ime cons an alues be ween 40 and
50 ~s. Clamp speed was u he enhanced by ballis ic cha ging o memb ane capaci ance
(Ma eson and A ms ong, 1984). Compensa ion o se ies esis ance was done empi ically; in
mos expe imen s we compensa ed o 1.5 imes elec ode esis ance. An IBM-PC/AT compu e
in e aced o he analog elec onics was used o pulse gene a ion and o acquisi ion, display,
s o age, and analysis o he da a. The in e ace and he associa ed so wa e we e de eloped and
ab ica ed in ou labo a o y (U e ia e al., 1989b) and allowed us sub ac ion o he linea ionic
and capaci y cu en s. Sampling in e al was ei he 10 o 20 Ws. Tail cu en s we e i ed wi h
one o he sum o wo exponen ials using a leas -squa es p ocedu e.
RESULTS
Cells subjec ed o ol age-clamp had an almos sphe ical shape and no app eciable
p ocesses unde he ligh mic oscope. The diame e o he dispe sed neu ons a ied
be ween 12 and 25 Ixm. Memb ane capaci ance, calcula ed om he ime in eg al o
he ansien capaci y cu en s, was 8 - 3 pF (mean _ SD,
n = 87).
Inpu esis ance
was 1.5 -+
0.6 G /.
Whole-Cdl Cu en s
Fig. 1 A is a ep esen a i e example o he majo cu en componen s eco ded in
sep al neu ons du ing a s ep depola iza ion o 20 mV. A he beginning o he pulse
he cell gene a es a as inwa d cu en ha peaks in less han 0.5 ms. In he cou se o
he nex 2 ms he cu en changes om inwa d o ou wa d and ises slowly un il he
end o he pulse. On e u n o he holding po en ial he e is an almos ins an aneous
jump in he cu en (which in his eco ding las s less han 80 V,s) and a la ge ail
cu en is obse ed. Fig. 1 B shows he cu en eco ded a e blockade o K +
channels by in e nal Cs +. The ou wa d cu en is supp essed, and a e he ini ial as
inwa d componen a second, mo e s eady componen is disclosed, which, when he
pulse is u ned o , is ollowed by he ail cu en . Ex e nal addi ion o e odo oxin
(TTX, 0.5-1 ~M) abolishes he as inwa d cu en , while he s eady componen and
he ail emain unal e ed (Fig. 1 C). I will be shown la e ha bo h he slow inwa d
cu en and he ail disappea a e emo al o ex e nal Ca 2+. These indings indica e
ha he di e en componen s o he whole-cell cu en a e due o he ac i i y o Na ÷,
Ca 2÷, and K ÷ channels. The h ee cu en componen s we e eco ded in e e y neu on
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306
: /
"-: /
./
u
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME
97
• 1991
B
#.
2 ms
C
,.-..
FIGURE 1. Majo cu en compo-
nen s in sep al neu ons. The h ee
cu en sweeps we e eco ded du ing
6-ms ol age s eps o 20 mV om a
holding po en ial o -80 mV. Solu-
ions: 130 Na//130 K (A), 10 Ca//130
Cs (B and C). In C, 1 IxM TIXwas
added o he ex e nal solu ion.
s udied (n > 150), al hough he ampli ude o each a ied om cell o cell. In he
ollowing sec ions we analyze he p ope ies o Na + and Ca *+ cu en s.
Na + Channels
Ac i a ion and conduc ance- ol age ela ions. Na + cu en was s udied in isola ion in cells
dialyzed wi h he 130 mM Cs +'solu ion and ba hed in an ex e nal solu ion wi h 130
mM Na +, whe e 90% o he Ca ~+ was eplaced by Mg ~+ and Co s+ (see Table I). In
hese solu ions depola iza ion elici ed la ge Na + cu en s ha a 0 mV had a peak
ampli ude o 4.6 + 1.6 nA (n = 21). Assuming no memb ane in olding, cu en
densi y was 0.6 mAcm -~. Mos expe imen s we e, howe e , pe o med wi h he 80
Na ex e nal solu ion (Table I) o a o an accu a e con ol o he memb ane
po en ial. Fig. 2 illus a es a cu en ace ob ained in low ex e nal Na +. The
eco ding is e y ep esen a i e o a well-con olled Na + cu en wi h as ac i a ion
80 Na. TTX
:
' l
• -
-80 Na
•
:" 1ms
• i
FIGURE 2. Na + cu en eco ded on
isola ion du ing a ol age pulse om
-80 o 0 mV. The a ows indica e he
onse and he e mina ion o he
pulse. Solu ions we e 80 Nail130 Cs.
The cu en was comple ely sup-
p essed by 1 p,M TFX.
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CASTELLANO AND LOPEZ-BARNEO
Na + and
Ca 2*
Channels o Sep al Neu ons
307
and comple e inac i a ion in 5-6 ms. The igu e also shows ha addi ion o TTX
p oduced a comple e blockade o he cu en . Wi h 80 mM ex e nal Na ÷, peak
cu en ampli ude a 0 mVwas 2.8 - 1.3 nA (n = 49). Good con ol o he memb ane
po en ial was also indica ed by a conduc ance- ol age cu e wi h a sigmoid ising
phase (no shown), and because cu en s o di e en ampli ude eco ded a he same
memb ane po en ial (as, o ins ance, hose in Fig. 6A) had, a e scaling, almos
iden ical ime cou ses.
Fig. 3 A is a amily o Na ÷ cu en s gene a ed by depola iza ion o he indica ed
memb ane po en ials. In his expe imen we added 40 mM Na + o he in e nal
solu ion o ob ain a well-de ined e e sal po en ial (EN=). Cu en is inwa d a
/.~
• ~ -50 o +90
:"J /
"" l
." /
:), 2 nA
~j
2 ms
B I M.nA
-40 -20 20 /40
_. I I I ._[ I
-0.7
-1.4
-1.4 /
-0.7
6O
I VM,mV
FIGURE 3. Na +
I-V
ela ions.
(A) Family o cu en sweeps
elici ed by depola iza ions o
-50, -30, -10, 10, 30, 50, 70,
and 90 mV om a holding po-
en ial o -80 inV. (B) Peak
cu en ampli ude (o dina e) a
a ious memb ane po en ials
(abscissa). In e sec ion o he
con inuous line wi h he ab-
scissa gi es he alue o he e-
e sal po en ial (31 mV). Solu-
ions: 130 Na//40 Na.
memb ane po en ials mo e nega i e han EN, and ou wa d a mo e posi i e po en-
ials. The cu en u ns on apidly and inac i a es comple ely a all ol ages.
Ac i a ion kine ics speed up wi h memb ane depola iza ion and hal - ime o peak
cu en is 0.44 + 0.18 ms a -20 mV bu only 0.21 --. 0.06 ms a +20 mV (n = 10).
Fig. 3 B is he
I-V cu e
ob ained by measu ing he peak cu en a a ious
memb ane po en ials. This igu e shows ha a measu able cu en can be eco ded a
-50 mV and ha he maximal peak cu en is ob ained be ween -20 and -10 mV.
The e e sal po en ial o he cu en occu s a +31 mV, which is simila o he Na ÷
equilib ium po en ial calcula ed by he Ne ns equa ion. This sugges s ha , as in
o he p epa a ions (Fenwick e al., 1982; Ma eson and A ms ong, 1984; U e ia e
al., 1989a), he aqueous componen s o he cy osol equilib a e comple ely wi h he
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308 THE JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 97 • 1991
pipe e solu ion. Na + conduc ance (gN:), calcula ed by he o mula gN: ----
IN:~
(I'M- ENd) (Hodgkin and Huxley, 1952), was maximal a 0 mV o mo e posi i e
ol ages. The a e age alue om ou sep al neu ons was 91 - 32 nS (~ 11 mS
cm-2), and hal o he o al conduc ance was ac i a ed a -35 mV.
Inac i a ion.
Du ing a main ained depola iza ion he inac i a ion o Na + cu -
en s could be well i ed by a single exponen ial unc ion. This is illus a ed by he
eco ding o Fig. 4 A. In he ange be ween -20 and +60 mV inac i a ion ime
cons an diminishes wi h memb ane depola iza ion (Fig. 4 B) and a 0 mV i has an
a e age alue o 0.44 --+ 0.09 ms (n = 5).
The ol age dependence o s eady-s a e inac i a ion is illus a ed in Fig. 5. In A i is
B
0.6-
E
~0,4-
.~ 0,2-
A
1ms
4._
i i i i i
-40 -20 0 210 40 60
V M,m
FIGURE 4. Time cou se o
Na + cu en inac i a ion. (A)
Single sweep o a Na ÷ cu en
eco ded du ing a depola iza-
ion o 0 inV. A single exponen-
ial ex apola ed o he onse o
he pulse was i ed o inac i a-
ion ime cou se. To acili a e
compa ison, he exponen ial is
d awn supe imposing he cu -
en ace. (B) Dependence o
inac i a ion ime cons an on
he pulse memb ane po en ial.
Ci cles a e he mean and he
ba s he s anda d de ia ion o
alues om i e cells. Solu ions:
Mg/Co//130 Cs.
shown ha he ampli ude o he cu en eco ded by a es depola iza ion o 0 mV
depends on he memb ane po en ial du ing a condi ioning p epulse las ing 50 ms.
Fig. 5 B shows he ela ion be ween he noninac i a ed ac ion o channels (o dina e)
and he p epulse memb ane po en ial (abscissa) wi h mean alues ob ained om six
cells. A -80 mV only a small (5-10%) ac ion o he channels a e inac i a ed, bu all
o hem a e inac i a ed a ol ages mo e posi i e han -20 inV. The a e age hal
s eady-s a e inac i a ion occu s a a memb ane po en ial o abou -50 mV; his
pa ame e a ied be ween -45 and -70 mV in he ceils s udied.
Reco e y om inac i a ion was s udied by applying a 20-ms condi ioning pulse o 0
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CASTELLANO AND L6PEZ-BARNEO
Na + and Ca 2+ Channels o Sep al Neu ons
309
mV o inac i a e he Na ÷ cu en and, a e a a iable ime in e al a he holding
po en ial, applying a es pulse o open he channels al eady deinac i a ed. The
aces o Fig. 6 A a e Na ÷ cu en s elici ed by depola iza ion o 0 mV in he absence
o p epulse (con ol) and 10, 25, and 900 ms a e he p epulse; inac i a ion is almos
comple ely emo ed in he cou se o 900 ms. In Fig. 6 B a e shown he ime cou ses
o eco e y om inac i a ion when he memb ane po en ial du ing he in e al is
held a -80
(ci cles), -100 (squa es),
o -120 mV
( iangles).
Reco e y is abou six
imes as e a -120 mV han a -80 inV. In e es ingly, a -80 mV he eco e y o
he las 15-20% o he cu en equi ed as much as 900 ms. This indica es he
p esence o a as inac i a ing componen ha eco e s ex emely slowly.
Closing kine ics.
Closing kine ics o mammalian neu onal Na + channels ha e no
been p e iously s udied. We o en ob ained eco dings whe e he as Na ÷ ail
A -20
/
•
'" 1hA
1 ms
B
1,0-
<E0.5-
-100 -60 -20
M , mV
FIGURE 5.
S eady-s a e inac i a ion
o Na ÷ cu en s. (A) Cu en sweeps
du ing depola iza ions o 0 mV p e-
ceded by a 50-ms p epulse o he
indica ed memb ane po en ials.
Holding po en ial, -80 InV. No mal-
ized ac ion o peak cu en eco ded
du ing he pulse (o dina e) as a unc-
ion o he p epulse memb ane po-
en ial. Ci cles and ba s a e he mean
and s anda d de ia ion alues om
i e cells. The inse illus a es he
pulse p o ocol. Solu ions: Mg/Co//130
Cs.
cu en s could be esol ed. The p o ocol consis ed o he applica ion o a b ie
depola iza ion (0.3-0.5 ms) o open Na ÷ channels ha ended a he ime when he
cu en was maximal (Fig. 7 A ). A he ins an o epola iza ion he d i ing o ce o
Na ÷ suddenly inc eases and hus a ail cu en is eco ded. The decay o he ail
e lec s he p og essi e deac i a ion o he channels open du ing he pulse. In ou
expe imen s Na ÷ ail cu en s we e always well i ed by a single exponen ial unc ion
(Fig. 7 B), which a -80 mV had a ime cons an as e han 100 Izs (n = 5).
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THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME
97
.
1991
A /10 ms
~~ lnA
con ol ~ ms
>.
0.5 i i I -//
I i
25 50 75 100 250 9()0
ime. ms
FIGURE 6. Reco e y om in-
ac i a ion o Na ÷ channels. (el)
Na + cu en s elici ed by ol age
s eps o 0 mV a he indica ed
ime in e als a e a p epulse
o 0 mV las ing 20 ms. The
con ol ace was ob ained in
he absence o p epulse. Hold-
ing po en ial, -80 mV. (B)
No malized pulse cu en am-
pli ude (o dina e) as a unc ion
o in e al du a ion (abscissa).
Memb ane po en ials du ing
he p epulse we e -80 mV
(ci -
cles),
-100 mV
(squa es),
and
-120 mV
( iangles).
Lines a e
i ed by eye. Solu ions: Mg/Co//
130 Cs.
Ca 2+ Channels
Iden i ica ion and I-V ela ions. In he p esence o TTX depola iza ion elici ed in all
neu ons a slowly inac i a ing inwa d cu en ha was eadily iden i ied as a Ca ~+
cu en . Fig. 8 A is a ypical eco ding o he slow inwa d cu en . This ace shows
ha he cu en u ns on slowly and almos eaches a maximum by he end o he
6-ms pulse. On epola iza ion he e is a ail cu en due o he inc ease in he
elec ochemical g adien o Ca 2÷ ions. As in mos p epa a ions s udied, he cu en
was la ge when Ba 2+ ions ins ead o Ca 2÷ we e used as cha ge ca ie (Fig. 8 B).
Cu en du ing he pulse and a he pulse end ( ail) was supp essed a e emo al o
A
• ,. /
loA
:: 0,5 ms
gle exponen ial wi h a ime cons an o 60 p~s. The exponen ial
epola iza ion. Solu ions: Mg/Co//130 Cs.
FIGURE 7. Closing kine ics o
ce eb al Na ÷ channels. (A) Na +
cu en eco ded du ing a
0.5-ms ol age pulse (indica ed
be ween he a ows) o 0 mV
om a holding po en ial o
-80 inV. When he pulse is
u ned o a as ail cu en is
eco ded. (B) Fi ing o he de-
ac i a ion ime cou se by a sin-
is ex apola ed o he ins an o
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CASTEL .~O AND L6PEz-B~Eo Na + and Ca z+ Channels o Sep al Neu ons 311
ex e nal Ca ~+ o a mix u e o Mg 2÷ and Co ~+, ca ions p ac ically impe mean
h ough Ca ~+ channels (Fig. 8 C).
Fig. 9 shows a amily o Ca ~÷ cu en s eco ded on depola iza ion o he indica ed
memb ane ol ages. Cu en du ing he pulse, which is indica ed be ween he a ows,
inc eases in ampli ude and ac i a es mo e apidly a mo e depola ized le els. A e a
maximum he pulse cu en diminishes wi h posi i e po en ials due o he dec ease in
he elec ochemical g adien o Ca 2+. Ca ~+ cu en measu ed a he end o he 6-ms
s eps a a ious memb ane po en ials is illus a ed in Fig. 10. This/-V cu e shows
ha he cu en becomes de ec able a -40 o -50 mV, eaches a maximum a abou
+20 mV, and a e wa ds declines as he d i ing o ce o Ca ~+ p og essi ely
dec eases. In 10 mM ex e nal Ca 2+ he ampli ude o he cu en measu ed a he end
o 6-ms pulses o 20 mVwas 0.35 -+ 0.22 nA (n = 12).
Sepa a ion o wo Ca 2÷ channel ypes. I is clea ly seen in Fig. 9 ha a e small
depola iza ions he decay o he ails ollows a simple, slow ime cou se, bu a e
depola iza ions o po en ials mo e posi i e han 0 mV a la ge as componen also
A 10 Ca
";~"~.,,4.~_~. " ..... 1,-,;-.~-~""- FIGURE 8. Iden i ica ion o Ca 2+ cu -
-~'''-~ .¢ en s. (A) Cu en p oduced by depo-
' la iza ion o 10 mV du ing 6 ms wi h
Ca ~÷ as cha ge ca ie . (B) Cu en
B eco ded in he same cell and wi h he
10 Ba .-, same pulse, using Ba ~+ ins ead o
. ,,~ Ca 2÷ in he ex e nal solu ion. (C)
: Blockade o he cu en a e eplace-
: men o Ca 2+ by Mg ~+ and Co ~÷.
: 1 nA Holding po en ial, -80 mV. The di -
: 2 ms e en ex e nal solu ions a e indica ed
C in he igu e; he in e nal solu ion was
Mg. Co 130 Cs.
~"*"~'~"~'~"~'~'--'~.2" "
appea s. Bo h he slow and as componen s o he ails g ow la ge as he memb ane
po en ial is made mo e posi i e. The sudden appea ance o he as componen o
he ails a ol ages be ween 0 and 20 mV pa allels he sha p ansi ion seen in he
I-V cu e a he same ol ages (Fig. 10). These obse a ions sugges he exis ence in
sep al neu ons o a leas wo di e en Ca 2÷ conduc ances wi h di e en ac i a ion
ol ages and deac i a ion kine ics. These conduc ances a e p obably due o he
ac i i y o wo kinds o Ca ~÷ channels.
Sepa a ion o he wo componen s o he Ca ~+ cu en was done ollowing he
me hod illus a ed in Fig. 11 (Co a, 1986; Ma eson and A ms ong, 1986; Taba es e
al., 1989). T ace A is a ail cu en eco ded on epola iza ion o -80 mV a e a
pulse o 40 mV las ing 6 ms. An exponen ial is i ed o he slow componen and
ex apola ed back o he ins an o epola iza ion. Sub ac ion o he slow exponen-
ial om he o al cu en yields a as componen on isola ion which could be i ed
by ano he exponen ial wi h a as e ime cons an . Thus, each exponen ial unc ion
ep esen s he closing ime cou se o slow (SD) and as (FD) deac i a ing Ca 2÷
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318 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 - 1991
ansien ac i a ion o SD channels and hei low ac i a ion ol age make some sep al
neu ons capable o i ing ei he onically o in a bu s mode, depending on he
memb ane po en ial o he cells (Al a ez de Toledo and L6pez-Ba neo, 1988). I is
wo h men ioning ha ou p e ious da a al eady sugges ed ha in some sep al
neu ons a signi ican p opo ion o FD channels migh be loca ed nea he soma. The
cha ac e is ic slow, high h eshold Ca 2+ spikes o hese cells appea ed du ing small
cu en injec ions, which is no a common inding in o he CNS neu ons, and i was
ela i ely usual o ob ain eco dings whe e an ini ial Na + spike o small ampli ude was
ollowed by la ge and b oade Ca ~+ ac ion po en ials. These, conside ed as p esump-
i e in adend i ic eco dings (L6pez-Ba neo e al., 1985), we e p obably impale-
men s placed in he egion be ween he soma and he p ima y dend i es.
The au ho s wish o hank J. Toledo o his collabo a ion in some expe imen s.
This wo k was suppo ed by he Di ecci6n Gene al de In es igaci6n Cien i ica y T6cnica (g an
PB-86-0250). D . An onio Cas eUano is a ellow o FISss.
O iginal e sion ecei ed 20 Feb ua y 1990 and accep ed e sion ecei ed 22 Augus 1990.
REFERENCES
Al a ez de Toledo, G., and J. L6pez-Ba neo. 1988. Ionic basis o he di e en ial neu onal ac i i y o
guinea-pig sep al nucleus s udied
in i o. Jou nal o Physiology.
396:399-415.
Ba es, B. A., L. L. Chun, and D. P. Co ey. 1989. Glial and neu onal o ms o he ol age-dependen
sodium channel: cha ac e is ics and cell- ype dis ibu ion.
Neu on.
2:1375-1388.
Bezanilla, F., and C. M. A ms ong. 1977. Inac i a ion o he sodium channel. I. Sodium cu en
expe imen s.
Jou nal o Gene al Physiology.
70:549-566.
Ca bone, E., and H. D. Lux. 1987. Kine ics and selec i i y o a low- ol age-ac i a ed calcium cu en
in chick and a senso y
neu ons.Jou nal o Physiology.
386:547-570.
Chiu, S. Y. 1977. Inac i a ion o sodium channels: second o de kine ics in myelina ed
ne e.Jou nal
o Physiology.
273:573-596.
Co a, G. 1986. Calcium channel cu en s in pa s in e media cells o he a pi ui a y
gland.Jou nal o
Gene al Physiology.
88:83-105.
Dubinsky, J. M., and G. S. Ox o d. 1984. Ionic cu en s in wo s ains o a an e io pi ui a y umo
cells.Jou nal o Gene al Physiology.
83:309-339.
Fedulo a, S. A., P. G. Kos yuk, and N. S. Veselo sky. 1985. Two ypes o calcium channels in he
soma ic memb ane o new bo n a do sal oo ganglion neu ons.
Jou nal o Physiology.
359:431-
446.
Fenwick, E. M., A. Ma y, and E. Nehe . 1982. Sodium and calcium channels in bo ine ch oma in
cells.Jou nal o Physiology.
331:599-635.
Fe n~indez, J. M., A. P. Fox, and D. K asne. 1984. Memb ane pa ches and whole-cell memb anes: a
compa ison o elec ical p ope ies in a clonal pi ui a y (GH~) cells.
Jou nal o Physiology.
356:565-585.
Fo sche , P., and G. S. Ox o d. 1985. Modula ion o calcium channels by no epineph ine in
in e nally dialyzed a ian senso y neu ons.
Jou nal o Gene al Physiology.
85:743-763.
Fox, A. P., M. C. Nowycky, and R. W. Tsien. 1987. Kine ic and pha macological p ope ies
dis inguishing h ee ypes o calcium cu en s in chick senso y neu ons.
Jou nal o Physiology.
394:149-172.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Feb ua y 1, 1991

CASTELLANO AND L PEz-BARNEO
Na + and Ca 2+ Channels o Sep al Neu ons
319
G~ihwile , B. H., and I. Llano. 1989. Sodium and po assium conduc ances in soma ic memb anes o
a Pu kinje cells om o gano ypic ce ebella
cul u es.Jou nal o Physiology.
417:105-122.
Gilly, W. F., and C. M. A ms ong. 1984. Th eshold channels: a no el ype o sodium channel in
squid gian axon.
Na u e.
309:448-450.
Hagiwa a, S., and H. Ohmo i. 1982. S udies o calcium channels in a clonal pi ui a y cells wi h
pa ch elec ode ol age-clamp.
Jou nal o Physiology.
331:231-252.
Hamill, O. P., A. Ma y, E. Nehe , B. Sakmann, and F. Sigwo h. 1981. Imp o ed pa ch-clamp
echniques o high- esolu ion cu en eco ding om cells and cell- ee memb ane pa ches.
P l ige s A chi .
391:85-100.
He n indez-C uz, A., and H. C. Pape. 1989. Iden i ica ion o wo calcium cu en s in acu ely
dissocia ed neu ons om he a la e al genicula e nucleus.
Jou nal o Neu ophysiology.
61:1270-
1283.
Hodgkin, A. L., and A. F. Huxley. 1952. A quan i a i e desc ip ion o memb ane cu en and i s
applica ion o conduc ion and exci a ion in ne e.
Jou nal o Physiology.
117:500-544.
Huguena d, J. R., O. P. HamiU, and D. A. P ince. 1988. De elopmen al changes in Na + conduc ances
in a neoco ical neu ons: appea ance o a slowly inac i a ing componen .
Jou nal o Neu ophysiol-
0gy. 59:778-795.
Llin is, R. 1988. The in insic elec ophysiological p ope ies o mammalian neu ons: insigh s in o
cen al ne ous sys em unc ion.
Science.
242:1654-1664.
Liinhs, R., and M. Sugimo i. 1980. Elec ophysiological p ope ies o
in i o
Pu kinje cell soma a in
mammalian ce ebella slices.
Jou nal o Physiology.
305:171-195.
L pez-Ba neo, J., G. Al a ez de Toledo, and Y. Ya om. 1985. Elec ophysiological p ope ies o
guinea-pig sep al neu ons.
B ain Resea ch.
347:358-362.
Ma eson, D. R., and C. M. A ms ong. 1984. Na and Ca channels in a ans o med line o an e io
pi ui a y cells.
Jou nal o Gene al Physiology.
83:371-394.
Ma eson, D. R., and C. M. A ms ong. 1986. P ope ies o wo ypes o calcium channels in clonal
pi ui a y cells.
Jou nal o Gene al Physiology.
81:161-182.
Meye s, D. E., and J. L. Ba ke . 1989. Whole-cell pa ch-clamp analysis o ol age-dependen calcium
conduc ances in cul u ed emb yonic a hippocampal neu ons.
Jou nal o Neu ophysiology.
61:467-
477.
Noda, M., T. Ikeda, T. Kayano, H. Suzuki, H. Takeshima, M. Ku asaki, H. Takahashi, and S. Numa.
1986. Exis ence o dis inc sodium channel messenge RNA's in a b ain.
Na u e.
320:188-192.
Sah, P., A. J. Gibb, and P. Gage. 1988. The sodium cu en unde lying ac ion po en ials in guinea-pig
hippocampal CAI
neu ons.Jou nal o Gene al Physiology.
91:373-398.
Sigwo h, F.J. 1983. Elec onic design o he pa ch-clamp
In
Single Channel Reco ding. B. Sakmann
and E. Nehe , edi o s. Plenum Publishing Co p., New Yo k. 3-35.
S a s om, C. E., P. C. Schwind , M. C. Chubb, and W. E. C ill. 1985. P ope ies o pe sis en sodium
conduc ance and calcium conduc ance o laye V neu ons om ca senso imo o co ex
in i o.
Jou nal o Neu ophysiology.
53:153-170.
S iihme , W., C. Me h essel, B. Sakmann, and S. Numa. 1987. Pa ch clamp cha ac e iza ion o
sodium channels exp essed om a b ain cDNA.
Eu opean Biophysics Jou nal.
14:131-138.
SwanduUa, D., and C. M. A ms ong. 1988. Fas -deac i a ing calcium channels in chick senso y
neu ons.Jou nal o Gene al Physiology.
92:197-218.
Taba es, L, J. U e ia, and J. L6pez-Ba neo. 1989. P ope ies o calcium and po assium cu en s o
clonal ad enoco ical cells.
Jou nal o Gene al Physiology.
93:495-519.
U e ia, J., J. R. L6pez-L6pez, C. Gonz~ilez, and J. L6pez-Ba neo. 1989a. Ionic cu en s in dispe sed
chemo ecep o cells o he mammalian ca o id
body.Jou nal o Gene al Physiology.
93:979-999.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Feb ua y 1, 1991
320 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 97 - 1991
U e ia, J., J. c. Ma eos, and J. L6pez-Ba neo. 1989b. Low-cos sys em o au oma ed acquisi ion,
display and analysis o ansmemb ane ionic cu en s.
Medical & Biological Enginee ing & Compu -
ing.
27:94-98.
Vandenbe g, C. A., and R. Ho n. 1984. Inac i a ion iewed h ough single sodium
channels.Jou nal
o Gene al Physiology.
84:535-564.
Yaa i, Y., B. Hamon, and H. D. Lux. 1987. De elopmen o wo ypes o calcium channels in cul u ed
mammalian hippocampai neu ons.
Science.
235:680-682.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Feb ua y 1, 1991