Po assium Channel Types in A e ial
Chemo ecep o Cells and Thei Selec i e
Modula ion by Oxygen
MAR A DOLORES GANFORNINA and
Josg LOPEZ-BARNEO
F om he Depa amen o de Fisiolog a y Bio lsica, Facuhad de Medicina, Uni e sidad de
Se illa, 41009 Se illa, Spain
ABSTRACT
Single K + channel cu en s we e eco ded in excised memb ane
pa ches om dispe sed chemo ecep o cells o he abbi ca o id body unde
condi ions ha abolish cu en low h ough Na + and Ca 2+ channels. We ha e ound
h ee classes o ol age-ga ed K + channels ha di e in hei single-channel
conduc ance (~/), dependence on in e nal Ca 2+ (Cai2+), and sensi i i y o changes in
02 ension (Po2). Ca2+-ac i a ed K + channels (Kca channels) wi h ~/ ~ 210 pS in
symme ical K + solu ions we e obse ed when [Ca2+]i was > 0.1 IzM. Small conduc-
ance channels wi h ~/= 16 pS we e no a ec ed by [Ca2+]i and hey exhibi ed slow
ac i a ion and inac i a ion ime cou ses. In hese wo channel ypes open p obabili y
(Pop,n) was una ec ed when exposed o no moxic (Po2 = 140 mmHg) o hypoxic
(P02 - 5-10 mmHg) ex e nal solu ions. A hi d channel ype ( e e ed o as Ko 2
channel), ha ing an in e media e ,/( ~ 40 pS), was he mos equen ly eco ded. Ko~
channels a e s eeply ol age dependen and no a ec ed by [Ca2+]i, hey inac i a e
almos comple ely in < 500 ms, and hei Pope, e e sibly dec eases upon exposu e
o low P02. The e ec o low Po2 is ol age dependen , being mo e p onounced a
mode a ely depola ized ol ages. A 0 mV, o example,
Popen
diminishes o ~ 40%
o he con ol alue. The ime cou se o ensemble cu en a e ages o Ko 2 channels
is ema kably simila o ha o he O2-sensi i e K + cu en . In addi ion, ensemble
a e age and mac oscopic K + cu en s a e a ec ed simila ly by low P02. These
obse a ions s ongly sugges ha Ko 2 channels a e he main con ibu o s o he
mac oscopic K + cu en o glomus cells. The e e sible inhibi ion o Ko 2 channel
ac i i y by low P02 does no desensi ize and is no ela ed o he p esence o F-,
ATP, and GTP--¢-S a he in e nal ace o he memb ane. These esul s indica e ha
Ko 2 channels con e upon glomus cells hei unique chemo ecep o p ope ies and
ha he O2-K + channel in e ac ion occu s ei he di ec ly o h ough an O2 senso
in insic o he plasma memb ane closely associa ed wi h he channel molecule.
INTRODUCTION
Al hough i has been known o decades ha he mammalian ca o id bodies
pa icipa e in he egula ion o b ea hing by adjus ing he en ila o ), a e o he le el
Add ess ep in eques s o D . J. L6pez-Ba neo, Depa amen o de Fisiolog a y Bio isica, Facul ad de
Medicina, A enida S~ nchez Pizjm n, 4, 41009 Se illa, Spain.
j. GEN. PHYSIOL. C) The Rocke elle Uni e si y P ess • 0022-1295/92/09/0401/26 $2.00
Volume 100 Sep embe 1992 401-426 401
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
402 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992
o oxygen ension (Po2) in a e ial blood (De Cas o, 1926; Heymans, Bouckae , and
Dau ebande, 1930; Fidone and Gonz ilez, 1986; Fi zge ald and Lahi i, 1986) he
mechanisms in ol ed in he p ocess o 02 sensing ha e emained unknown. The e
has been a gene al consensus ha ype I (o glomus) cells, he mos nume ous in he
ca o id body, a e he elemen s esponsible o chemo ansduc ion since hey make
synapses wi h a e en ne e endings, ha e cy osolic g anules con aining ca echola-
mines, and sec e e dopamine in esponse o hypoxia and high ex e nal K ÷ (Fidone,
Gonz ilez, and Yoshizald, 1982; Fishman, G eene, and Pla ika, 1985; Alma az,
Gonz~ilez, and Obeso, 1986; Rigual, Gonz ilez, Gonz ilez, and Fidone, 1986; Obeso,
Fidone, and Gonz ilez, 1987). Di ec p oo o he chemo ecep i e p ope ies o ype
I ceils has come, howe e , om ecen elec ophysiological expe imen s. I has been
shown ha ype I cells can i e ac ion po en ials epe i i ely and, as in o he
elec ically exci able cells, hey gene a e ol age-dependen Na +, Ca z+, and K +
cu en s (Duchen, Caddy, Ki by, Pa e son, Pon e, and Biscoe, 1988; L6pez-Ba neo,
L6pez-L6pez, U e ia, and Gonz ilez, 1988; U e ia, L6pez-L6pez, Gonz ilez, and
L6pez-Ba neo, 1989a). Fu he mo e, i has also been ound ha he ol age-ga ed
K ÷ cu en o ype I cells is e e sibly a enua ed by lowe ing en i onmen al Po2,
whe eas Na s and Ca e+ cu en s emain unal e ed (L6pez-Ba neo e al., 1988). These
indings, con i med by he pa allel wo k o o he in es iga o s on se e al mammalian
species (Delpiano and Heschele , 1989; Heschele , Delpiano, Acke , and Pie uschka,
1989; Pee s, 1990; S ea and Nu se, 1991), ha e p o ided a amewo k o unde -
s anding he basic mechanisms unde lying senso y ansduc ion in he ca o id body.
Inhibi ion o he O2-sensi i e K ÷ cu en unde hypoxic condi ions p oduces an
inc ease in he i ing equency o glomus cells (L6pez-L6pez, Gonz ilez, U e ia, and
L6pez-Ba neo, 1989), which could lead o Ca 2+ in lux, enhanced ansmi e elease,
and ac i a ion o he a e en ibe s o he sinus ne e. This basic scheme is also
suppo ed by wo k on ype I cells loaded wi h luo escen Ca 2s indica o s showing an
inc ease in cy osolic Ca 2s in esponse o low Po2 (Biscoe and Duchen, 1990a; Beno ,
A., J. U e ia, and J. L6pez-Ba neo, unpublished obse a ions).
The mac oscopic K ÷ cu en eco ded in ype I cells has a small Ca2S-dependen
componen ha disappea s a e wash-ou o Ca 2+ channels and when in e nal
solu ions wi h high Ca2+-bu e ing capaci y a e used. In addi ion, he Ca2S-indepen -
den componen o he cu en inac i a es almos en i ely in 200 ms bu he deg ee o
inac i a ion a ies among di e en cells (U e ia e al., 1989a). The e o e, i can be
expec ed ha , as in o he exci able cells (Ma y and Nehe , 1985; Hoshi and Ald ich,
1988), glomus cells possess se e al classes o K s channels wi h speci ic biophysical
p ope ies ( o e iew, see Rudy, 1988). The p esen esea ch was unde aken o
es ablish a i s classi ica ion o K + channels in ype I cells and o asce ain whe he a
speci ic K s channel class is esponsible o he O2 sensi i i y o he cells' elec ical
p ope ies. These ques ions a e o c i ical impo ance o elucida ing he molecula
mechanisms unde lying 02 sensing. The iden i ica ion o he p ima y si e in ol ed in
02 de ec ion is also o in e es because i has been a gued ha he a enua ion o he
mac oscopic K + cu en on lowe ing Po2 could be a seconda y phenomenon a he
han an ini ial s ep in he p ocess o chemo ansduc ion (Biscoe and Duchen, 1989,
1990a, b).
In his pape we p esen a sys ema ic analysis o he single K s channel ypes ound
in glomus cells. We show ha in excised memb ane pa ches he e a e h ee di e en
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND L PEZ-BARNEO
Single K + Channel Modula ed by 02 in Glomus Cells
403
K + channel ypes and ha only one o hem ( e e ed o as "Ko~ channel") is
selec i ely and e e sibly modula ed by hypoxia. The cha ac e is ics o hese channels
ully accoun o he p ope ies o he mac oscopic O2-sensi i e K + cu en . Ou
expe imen al esul s indica e ha Ko 2 channels a e di ec ly egula ed by 02 and
s ongly sugges ha he 02 senso o chemo ecep o cells is in, o closely associa ed
wi h, he plasma memb ane. In he ollowing a icle we ocus on a mo e de ailed
desc ip ion o he kine ic p ope ies o he Ko 2 channel and p opose a minimal
model ha .explains he e ec s o hypoxia on channel ga ing.
A b ie epo o pa o he wo k p esen ed in his pape has been published
(Gan o nina and L6pez-Ba neo, 1991).
METHODS
Cell P epa a ion
Expe imen s we e pe o med on ype I cells isola ed om abbi ca o id bodies. The
p ocedu es ollowed o enzyma ic cell dispe sion and cul u e we e he same as desc ibed
TABLE I
Composi ion o Solu ions
Ex e nal
NaCl KCI CaCle MgCI2 HEPES Glucose
S anda d Na 140 2.7 2.5-5 2 10 5
140 K -- 140 2.5-5 2 10 5
80 K 60 80 2.5-5 2 10 5
In e nal
KCI K-glu ama e KF HEPES EGTA MgCI~
S anda d K 30 80 20 10 10 2.58
130 K 130 -- -- 10 10 2.58
All alues a e gi en in millimola . Te odo oxin a a concen a ion o 0.6 IxM was
added o he s anda d Na and 80 K solu ions. Solu ions wi h a iable [Ca 2+] we e
made by mixing CaCI2, MgClz, and EGTA. The inal ee [Ca 2÷] was calcula ed wi h a
compu e p og am ha akes in o accoun he a ini y cons an s o EGTA o Ca 2+ and
Mg 2+ a di e en pH (Taba es, U e ia, and L6pez-Ba neo, 1989). In hose solu ions
wi h 10 mM EGTA and no Ca 2+ added he es ima ed [Ca ~+] was < 10 -9 M.
p e iously (L6pez-Ba neo e al., 1988; U e ia e al., 1989a). Cells we e pla ed on sli e s o glass
co e slips ea ed wi h poly-l-lysine and used o eco ding be ween 12 h and 2 d a e
dissocia ion. Du ing he expe imen s a co e slip was ans e ed o a small chambe o ~ 0.2 ml
olume wi h con inuous low o solu ions ha could be eplaced in 10-15 s.
Solu ions
The composi ion o solu ions used in he expe imen s is shown in Table I. Solu ions we e
adjus ed o a pH o 7.3 (in e nal) o be ween 7.35 and 7.4 (ex e nal) and had an osmola i y o
290-300 mosmol/kg. In he ex and in he igu e legends solu ions a e gi en as ex e nal//
in e nal wi h speci ica ion o he inal [Ca z÷] o [EGTA] used. Expe imen s we e pe o med a
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
404 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992
oom empe a u e (22-25°C). Du ing he expe imen s he ex e nal solu ions we e equilib a ed
wi h ei he ai , N2, o a mix u e o bo h, in o de o ob ain he desi ed Po2 in he eco ding
chambe (see below).
Reco ding Techniques
The da a p esen ed in his a icle a e mainly based on single K + channel cu en s eco ded
om memb ane pa ches o glomus cells using he pa ch clamp echnique (Hamill, Ma y,
Nehe , Sakmann, and Sigwo h, 1981). In some expe imen s whole-cell K + cu en s we e
s udied be o e es ablishing he ou side-ou pa ch con igu a ion ollowing he me hodology
p e iously epo ed (U e ia e al., 1989a). Fo single-channel eco ding we used i e-polished
glass pipe es ab ica ed om bo osilica e glass (Kimax 51) ha once illed wi h solu ion had a
esis ance o 4-10 MI'~. The eco ding bandwid h o he ampli ie was 10 kHz, howe e i s
ou pu signal was low-pass il e ed by an 8-pole Bessel il e (model 902; F equency De ices
Inc., Ha e hill, MA) wi h cu o equencies be ween 1 and 2 kHz, gi ing an e ec i e cu o
equency o 0.95-1.29 kHz (Colquhoun and Sigwo h, 1983). The ime esolu ion o ou
eco ding sys em and i s in luence on he measu emen o he ampli ude and du a ion o
single-channel e en s is u he explained in he accompanying pape .
Da a Acquisi ion and Analysis
An IBM-PC-AT compu e in e aced o he analog elec onics was used o da a acquisi ion,
display, and analysis. In mos expe imen s we eco ded ionic cu en s in esponse o ol age
s eps. In hese cases he cu en signal was digi ized on-line by an inpu -ou pu in e ace buil
in ou labo a o y (U e ia, Ma eos, and L pez-Ba neo, 1989b). A sweep was de ined by ei he
500 o 1,000 digi al poin s. Single-channel cu en s gene a ed in esponse o long-las ing
(> 220 ms) o s a iona y depola iza ions we e ini ially s o ed on ideo ape. The segmen s o
he signal equi ed o igu es o analysis we e eplayed on a cha eco de o con e ed in o
digi al o m using a GPIB-PC ca d plugged in o he compu e expansion slo s. The sample
equency a ied acco ding o he expe imen al p o ocol and is gi en in he igu e legends.
Leakage and uncompensa ed capaci y cu en s we e digi ally sub ac ed using scaled empla es
cons uc ed by i ing smoo h unc ions o ei he eco ds wi h no openings o o he a e age o
20 consecu i e cu en sweeps gene a ed by 20-mV hype pola iza ions om a holding po en ial
o -80 mV. Ensemble a e ages we e ob ained om o iginal aces a e capaci y and leakage
sub ac ion.
Single-channel cu en ampli ude was measu ed by a e aging alues ob ained om 20-40
well- esol ed single e en s. We used a 50% ampli ude c i e ion o de ec opening and closing
ansi ions (Colquhoun and Sigwo h, 1983). Unless o he wise no ed, no co ec ion o
un esol ed e en s was pe o med. The numbe o ac i e channels in a pa ch (N) was
de e mined by obse ing o long ime pe iods he maximum numbe o simul aneous cu en
s eps ha appea ed a s ongly depola ized ol ages. In pa ches whe e one o wo simul aneous
openings we e obse ed, he p obabili y ha his numbe could be smalle han he ac ual alue
o N was s a is ically es ed by he binomial dis ibu ion me hod as indica ed by Pa lak and
Ho n (1982). In hose eco dings whe e he es ima ed alue o N was one, a e age channel
open p obabili y (Pop,n) was calcula ed by di iding he ime spen in he open s a e by he o al
du a ion o he eco ding. In pa ches wi h mo e han one channel, Pop~n was es ima ed by he
o mula:
Popen =
(N'i'T)- ' :l( ) "d
(1)
whe e i is he single-channel cu en ampli de, T is he du a ion o he pulse o o he
obse a ion pe iod in s a iona y condi ions, and
I( )
is he ne cu en du ing he eco ding
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND L6PEZ-BARNEO
Single K + Channel Modula ed by 02 in Glomus Cells
405
pe iod. In channels ac i a ed du ing depola izing pulses, we ob ained an ensemble cu en
a e age
(l(a))
gi en by:
I (a) = N'i'Po( )
(2)
whe e
Po( )
is he open p obabili y as a unc ion o ime. Signi icance o di e ences be ween
mean alues ob ained (e.g., o di e en Po2) was de e mined wi h a S uden 's es o pai ed
samples. Unless o he wise indica ed, he le el o signi icance (cx) o he es was se a 0.05.
A
>
0
>
2.0
1.5
1.0
0.5
0
B
1.0
'~ I 0.8
i i >
0.6
o
0.4
I I >
I I
I 0.2
0
~-
0 0.3 0.6 0.9 1.2 0
V
bo e y (V)
I I I
30 60 90
%
02
i
120
C D
N2 N2 N 2 PO2(mmHg)
lOO
50
o
Ai Ai Ai
1 min
FIGURE 1. Pe o mance and calib a ion o he O2-sensing elec ode. (A) Ou pu ol age o he
cu en - o- ol age con e e as a unc ion o he nega i e pola izing po en ial and 02 ension.
150 mmHg
( illed ci cles),
87 mmHg
(open ci cles),
and 0--5 mmHg
( illed squa es).
A a
pola izing ol age be ween -0.6 and -0.8 V he ou pu ol age is linea ly ela ed o he Po 2 in
he solu ion ( ange be ween dashed lines). (B) Ou pu ol age s. %02 ela i e o ai a a
pola izing ol age o -0.7 V. (C and D) Responses o he 02 elec ode in he chambe (C) and
du ing ins an aneous imme sions in solu ions equilib a ed wi h N2 and ai (D).
Measu emen o Oxygen Tension
Because some expe imen al p o ocols equi ed epe i i e exposu e o a memb ane pa ch o
ex e nal solu ions wi h a ep oducible Po2 alue, we buil an O2-measu ing elec ode o
es ima e Po2 alues in he icini y o he cu en - eco ding pipe e. We used a nega i ely
pola ized 100- .m- hick pla inum wi e insula ed by an O2-impennean enamel excep a he
end sec ion (Tsacopoulos and Lehmenkiihle , 1977; Tsacopoulos, Poi y, and Bo sellino,
1981). Cu en gene a ed in he wi e in esponse o a iable PO 2 alues was eco ded by an
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992

406
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 "
1992
ope a ional ampli ie wi ed as an
1/V
con e e . The nega i e pole o a d.c. ba e y was
connec ed o he nonin e ing inpu o he
I/V
con e e o main ain he pola izing ol age a
a cons an alue. The majo cha ac e is ics as well as he pe o mance o he O2-measu ing
elec ode a e illus a ed in Fig. 1. Plo A shows he changes in he ou pu ol age o he
1/V
con e e (Vou0 as a unc ion o he nega i e po en ial applied o he pla inum elec ode (Vba e~y)
o h ee di e en Po~ alues. In he Vba e~y ange be ween -0.6 and -0.8 V, Vou was
p opo ional o he 02 concen a ion; hus we used -0.7 V as he mos app op ia e alue o
pola ize he pla inum elec ode. A his pola izing ol age, which was used in all he
expe imen s, he ou pu ol age o he eco ding elec ode is linea ly ela ed o he O2
concen a ion and he e o e a calib a ion cu e could be done using solu ions equilib a ed wi h
known concen a ions o 02 (Fig. 1 B ). Du ing he expe imen al p o ocol Vou was con inuously
moni o ed and s o ed on ape. The esponse o he elec ode is illus a ed in Fig. 1, C and D.
When imme sed in solu ions equilib a ed wi h N2 o ai , he change in ol age was almos
ins an aneous (D), swi ching o simila solu ions when placed in he expe imen al chambe
p oduced a as change (~ 80% o he maximum) in a ew seconds bu comple e equilib a ion
equi ed >40 s (C). The ol age signal om he Oz-sensing elec ode was una ec ed by
changes in pH o by modi ica ions in he ionic composi ion o he solu ions. Mos o he
expe imen s epo ed he e and in he accompanying epo we e based on epe i i e exposu e
o he K ÷ channels o a ep oducible Poz alue. Gi en ha ou eco ding chambe is in con ac
wi h he ai , we ound ha he mos easily ep oducible Poz le el in he icini y o he cells
(Po2 = ~ 5-10 mmHg) was ob ained by bubbling he es solu ion wi h
N 2.
RESULTS
The e A e Th ee Majo K + Channel Types in Glomus Cells
Single K + channel cu en s eco ded om memb ane pa ches wi h well- esol ed
single-channel e en s allowed he classi ica ion o he K + channels o ype I cells in o
h ee majo classes. To acili a e compa ison, single-channel cu en s ep esen a i e
o he a ious K + channel ypes a e shown in isola ion in Fig. 2. Table II summa izes
he majo p ope ies o each channel popula ion. The aces o Fig. 2 a e om h ee
di e en inside-ou pa ches wi h wo unc ional channels. In all cases he memb ane
was exposed o asymme ical K + solu ions and he cu en was eco ded a a ious
memb ane po en ials (Vm). The h ee se s o eco dings display openings and
closu es o he channels ha appea as cu en s eps o ixed ampli ude. Fas
ansi ions a e pa ially il e ed due o he limi ed eco ding bandwid h. Opening o
each channel ype p oduced cu en s eps o clea ly di e en ampli ude, bu in all
cases channel opening was a o ed by memb ane depola iza ion. I will be shown
below ha besides dis inc single-channel conduc ance alues he h ee channel
popula ions also di e in hei kine ic p ope ies, Ca z+ dependence, and sensi i i y o
changes in Po2 (see Table II). Fo he sake o cla i y abb e ia ions a e used o each
channel ype. Kc, deno es he la ge Ca2+-ac i a ed K + channels, SK a e channels o
small conduc ance, and Ko 2 e e s o he O2-sensi i e K + channels.
Ca2+-dependen K + Channels (Kca Channels)
Kca
channel ac i i y was clea ly obse ed in inside-ou memb ane pa ches (a o al o
24 pa ches) when [Ca 2+] in he solu ion acing he cy osolic side o he memb ane was
>0.1 ~M. These channels we e ac i e du ing main ained depola iza ions and
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND LOPEZ-BARNEO
Kca
V::oV
Single K + Channel Modula ed by 02 in Glomus Cells
SK Ko2
0 2
~
-- 02
01 02 01
__01
C --C C
407
20 ~15p A ....... ~ 2pA
50 ms
FIGURE 2. Rep esen a i e eco dings o he ac i i y o he h ee majo K ÷ channel ypes ound
in ype I cells. The da a we e ob ained om di e en inside-ou excised pa ches, con aining wo
channels each, depola ized a he indica ed memb ane po en ials. Kca = Ca~+-ac i a ed
channels; SK = small conduc ance channels;
K% =
O~-sensi i e channels. In all igu es upwa d
de lec ions om he ze o cu en le el (c) indica e ou wa d cu en . K~ and SK channels we e
eco ded unde s eady depola iza ions and K% channels upon 200-ms s ep depola iza ions
om -80 mV. E ec i e cu o equency = 0.95 kHz and sampling in e al = 500 Ixs.
Solu ions: s anda d Na, TIX//130 K, 10 EGTA. K~ channels we e eco ded wi h an in e nal
solu ion con aining 1 IxM ee Ca 2+.
he e o e hei single-channel cu en - ol age (i-Vm) ela ion and
Popen we e
s udied
a e s eady-s a e changes in he memb ane po en ial. Single Kca channel ac i i y as a
unc ion o he memb ane po en ial is illus a ed in Fig. 3 A. The aces a e om a
pa ch ba hed in symme ical K + solu ions ha con ained a leas h ee ac i e
channels. Single-channel cu en ampli ude a ied in pa allel wi h he elec ochemi-
cal d i ing o ce o K + ions, and he numbe o ac i e channels as well as he ime
TABLE II
Classi ica ion o K ÷ Channels in Glo aus Cells
Channel Conduc ance in 130 K//130 K Conduc ance in 2.7 K//130 K Ca 02
ype Inside-ou Ou side-ou Inside-ou Ou side-ou dependence sensi i i y
K~ 206.7 (10) -- 83.7 (3) -- Yes No
SK 16 (6) -- 6 (2) -- No No
K% 41.5 (1) 41.5 (2) 17.6 (18) 20.1 (14) No Yes
21 (10)* 19.5 (5)*
A e age slope conduc ance alues a e gi en in picosiemens. The numbe o pa ches is in pa en heses. Kc~ =
Ca-ac i a ed channel; SK = small conduc ance channel; K% - oxygen-sensi i e channel. Fo he K%
channel alues a e gi en in con ol and hypoxic ( ) condi ions. Po assium concen a ions a e in millimola
and indica ed as ex e nal//in e nal.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
408
THE JOURNAL OF
GENERAL
PHYSIOLOGY • VOLUME
100
• 1992
spen in he open s a e inc eased wi h depola iza ion. The a e age
i-Vm
ela ions
ob ained om da a pooled om se e al pa ches exposed o symme ical and
asymme ical K ÷ concen a ions ([K÷]) a e plo ed in Fig. :3 B. In symme ical [K ÷]
( illed symbols) he
i-Vm
plo is linea be ween -50 and +60 mV, and he e e sal
po en ial was 0 mV, as expec ed o K+-selec i e channels. A linea eg ession i o
he da a poin s yields a alue o 206.7 pS (n = 10 pa ches) as he a e age single-
A
Vm ~- o2
--01
(mV)
+30
--C
~
--03
--0 2
--01
+20 --C
-10 ........ C
--0 2
-20 -~--C
--01
--0 2
1 O0 ms
B
I
-90
15
9
-30 •
i(pA)
I I
30 90
FIGURE 3. Single-channel cu en - ol age ela ion o he Kca channels. (A) Uni a y e en s
eco ded in an inside-ou excised pa ch wi h a leas h ee simul aneously open channels a
a ious s a iona y memb ane po en ials. E ec i e cu o equency = 0.95 kHz and sampling
in e al = 500 I~s. Solu ions: 130 K, 0.01 ~M Ca2+//130 K, 1 I~M Ca 2+. (B) Plo o
single-channel cu en (i) as a unc ion o he memb ane po en ial (Vm) measu ed in symme i-
cal
( illed symbols,
n = 10 inside-ou pa ches) and asymme ical
(open symbols,
n = 3 inside-ou
pa ches) K + concen a ions. The poin s indica e he mean ± SD alues and he s aigh lines
he linea eg ession i s o he da a. Slope conduc ances a e: 206.7 pS
( illed symbols,
= 0.99)
and 83.7 pS
(open symbols,
= 0.98). Solu ions: 130 K, 0.01 I~M Ca2+//130 K, 1 I~M Ca z+
(symme ical [K+]); s anda d Na, TFX//130 K, 1 I~M Ca 2+ (asymme ical [K+]).
channel conduc ance. In asymme ical [K ÷] (open symbols) he a e age slope
conduc ance, measu ed be ween -30 and +50 mV, is 83.7 pS (n = 3 pa ches).
The possible modula o y e ec o Po2 on he ac i i y o Kca channels was
in es iga ed in inside-ou excised pa ches ha we e ini ially exposed o a iable
in e nal Ca ~+ concen a ions ([Ca2+]i) o es he Ca 2+ dependence o channel
ac i a ion, and he ea e o a ious Po~ le els keeping [Ca2+]i unal e ed. An example
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND L PEZ-BARNEO
Single K + Channel Modula ed by 02 in Glomus Cells
409
o his expe imen al p o ocol is illus a ed in Fig. 4. The memb ane was ba hed in
symme ical high K + solu ions wi h a [Ca2+]i o 1 ~M and held a a po en ial o +20
inV. Unde hese condi ions opening o he wo ac i e channels included in he pa ch
p oduced an ou wa d cu en ha disappea ed comple ely a e swi ching o a
solu ion wi h 0.01 o,M Ca ~+. This e ec was pe ec ly e e sible on ein oduc ion o
1 ~M Ca 2+ in he chambe (Fig. 4 A ). When a a ixed [CaZ+]i o 1 ,M he same pa ch
was exposed o low Po2, no app eciable changes in single-channel ac i i y o uni a y
A
02 --
01 --
C
--
0.01 #.M Ca 2+ 1 /, ,M Ca 2+
i l
B
02--
l
0 --
C
--
10 pA
140
mmHg
N 2 Ai
FIGURE 4. Dependence o Kca channels on in e nal Ca ~+ and lack o e ec o changes in Po2.
(A) Ac i i y a +20 mV o wo Kca channels in an inside-ou pa ch exposed o 1 I~M in e nal
Ca 2+ and e e sible inhibi ion when
[Ca2+]i
is dec eased o 0.01 IxM. (B) A he same
memb ane po en ials and [Ca2+]i = 1 I~M channel ac i i y was una ec ed by lowe ing Po2.
The signal om he O~-measu ing elec ode is shown in he lowe panel. E ec i e cu o e-
quency = 0.95 kHz. Solu ions: 130 K, 0.01 I~M Ca~÷//130 K, 1 IJ.M o 0.01 I~M Ca ~+.
cu en ampli ude we e obse ed (Fig. 4 B). The signal om he Po2-measu ing
elec ode is shown in he lowe panel. Following his same expe imen al p ocedu e,
bu using a ious [Ca2+]i (be ween 0.04 and 1 ~M) and V~ alues (be ween -30 and
+30 mV), exposu e o hypoxia did no al e Kca channel open p obabili y (Popen) in
he 16 pa ches es ed (pai ed es ). Fig. 5 summa izes he e ec s o [Ca2+]i and low
Po 2 on Kca channel ac i i y. In Fig. 5 A, single-channel Po~, as a unc ion o Vm is
plo ed a wo [Ca~+]i. In bo h cases he da a poin s a e he mean +_ SE alues om
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
416
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992
eco dings (panel B) show a peak Pope, a he beginning o he depola iza ion and a
p og essi e dec ease du ing he pulse. Single-channel Pope, in eg a ed h oughou
he pulse du a ion (see Eq. 1) is 0.31 in he con ol solu ion bu only 0.18 du ing
exposu e o low Po2. Thus, hypoxia p oduces a e e sible dec ease in channel open
p obabili y bu , as shown in Fig. 8 B ( iangles), i does no modi y ei he he i-Vm
ela ion o he single-channel conduc ance (see Table II).
A summa y o he e ec o low Po2 on he Popen o Ko 2 channels a di e en
memb ane po en ials is shown in Fig. 11. The a e age Popen alue du ing 200-ms
pulses was measu ed in pa ches wi h one o wo unc ional Ko~ channels. Low Po2
p oduced a dec ease in single-channel Pope. ha was s a is ically signi ican a all
memb ane ol ages (pai ed es , x < 0.01). A +30 mV, o example, Popen is ~0.43
in he con ol solu ion and ~0.31 in low Po2, bu a 0 mV hese alues a e,
espec i ely, ~0.2 and ~0.07. This indica es ha he inhibi ion o K + channel
ac i i y by hypoxia is mo e ma ked a less depola ized memb ane po en ials (see also
GL
0
O-
0.6
0.4
0.2
[~] con ol
low
PO2
0 +10 +20 +30
w (my)
FIGURE 11. Dec ease o Ko 2 channel
Pop, n by hypoxia. A e age open p ob-
abili y du ing 200-ms pulses (o dina e)
was measu ed om ei he inside-ou
o ou side-ou pa ches a a ious
memb ane po en ials. Pop,n alues
measu ed in he con ol and in he
low (<5 mmHg) Po2 solu ions a e
ep esen ed by he mean +-- SE. Num-
be o expe imen s we e: n = 5 (0
mV); n = 3 (+10 mV); n = 23 (+20
mV); and n = 2 (+30 mV). A all
ol ages he di e ences we e s a is i-
cally signi ican (pai ed es ,
c~ < 0.01). Solu ions: s anda d Na +,
TIX//130 KCI, 10 EGTA o s anda d
K, 10 EGTA.
accompanying pape ). Fo simplici y, in his se o expe imen s solu ions we e
bubbled wi h ei he N~ o ai and hus he low Po 2 alues in he icini y o memb ane
pa ches we e ~ 5-10 mmHg (see Me hods). Wi h his p o ocol he dec ease o Pope.
by hypoxia is unde es ima ed since i is known ha on exposu e o ex emely low Po2
he inhibi ion o K + channel ac i i y is ela i ely less p onounced han when he
channels a e exposed o mode a ely low Po2 alues (be ween 60 and 80 mmHg;
L6pez-L6pez e al., 1989; Gan omina and L6pez-Ba neo, 1991).
Di ec modula ion o Ko 2 channels accoun s o he p ope ies o he mac oscopic
O2-sensi i e K + cu en . The kine ic and pha macological p ope ies o he Ko 2
channels indica e ha hey a e he main channels esponsible o he mac oscopic
O~-sensi i e K + cu en o ype I cells. The ensemble a e age cu en s shown ea lie
(see Fig. 10 B) illus a e ha he ime cou se exhibi ed by Ko 2 channels du ing a
depola iza ion closely esembles he kine ics o he mac oscopic K + cu en which
u ns on in a ew milliseconds and inac i a es almos comple ely in 200--300 ms
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992

GANFORNINA AND L6PEZ-BARNEO Single K + Channel Modula ed by 02 m Glomus Cells 417
(L pez-L6pez e al., 1989; see also Fig. 12). In addi ion, TEA + e e sibly blocks he
whole-cell K + cu en (U e ia e al., 1989a) as well as he Koz channels (Gan o nina
and L pez-Ba neo, 1991). The pa allel ime cou ses o Ko~ channel Pope, and he
mac oscopic K + cu en we e clea ly e iden when eco dings in he whole-cell mode
and in ou side-ou mul ichannel pa ches we e ob ained ollowing he same expe i-
men al p o ocol. Fig. 12 illus a es he e e sible inhibi ion o he mac oscopic K +
cu en ( aces in C) and he dec ease in K + channel Popen in an ou side-ou pa ch
wi h a leas i e channels ( aces in A and B ) du ing a ansien exposu e o hypoxia.
The ensemble a e ages o Fig. 12 B, which ep esen he beha io o a ew channels,
ha e a ime cou se compa able o ha o he whole-cell cu en s.
Con ol Hypoxia Reco e y
B
C
50 ms
pA
•
~A
FIGURE 12. Compa ison o he e ec o lowe ing Po2 on single Ko 2 channel and mac oscopic
K + cu en s elici ed by depola iza ion o 0 mV om a holding po en ial o -80 mV. (A and B )
Rep esen a i e single sweeps and ensemble a e ages o cu en eco ded om an ou side-ou
pa ch, con aining a leas i e channels, in he con ol solu ion (Po~ = 150 mmHg; n = 13
consecu i e sweeps; Pope, = 0.29), du ing a 9-min exposu e o hypoxia (Po~ < 5 mmHg;
n = 18 consecu i e pulses; Pop~, = 0.15), and a e eco e y in he no mal Po2 solu ion (n = 18
consecu i e pulses; Po~, = 0.27). E ec i e cu o equency = 0.95 kHz and sampling in e -
al = 500 p.s. (C) Whole-cell cu en s eco ded wi h he same expe imen al p o ocol. E ec i e
cu o equency = 10 kHz and sampling in e al = 500 Ws. Cu en calib a ion ba is 2 pA o
A and B, and 0.6 nA o C. Solu ions: s anda d Na, TI'X//130 K, l0 EGTA.
Wi h a high Ca 2+ bu e capaci y a he in e nal solu ion, mos o he mac oscopic
K + cu en is due o he ac i i y o Ko~ channels since Kca channels canno be
ac i a ed and, in addi ion, he densi y o SK channels is low and hei uni a y
conduc ance is small. The e o e, he numbe o Ko~ channels can be es ima ed by
di iding he peak K ÷ cu en by he alue o he single Ko~ channel cu en
ampli ude a he same ol age and co ec ing o he peak channel Popen (~ 0.8 a
+20 mV). Ou es ima e gi es alues o 720 -+ 80 (mean -+ SD, n = 18) channels pe
cell, which co esponds o wo o ou channels pe squa e mic ome e . This ela i ely
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
418
THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 - 1992
high densi y may explain why we ob ained mul ichannel pa ches in ~ 50% o he
expe imen s (87 o 168) e en hough we used ela i ely high esis ance pipe es (> 8
MI~).
I was shown in a p e ious epo ha he modula ion by 02 o he mac oscopic K +
cu en o ype I cells is independen o in e nal Ca 2÷ o he p esence o exogenous
nucleo ides (L6pez-Ba neo e al., 1988; L6pez-L6pez e al., 1989). Ou esul s a he
single-channel le el con i m and ex end hese obse a ions since e e sible inhibi ion
A
Con ol
B
Hypoxia
_J I
C
~, con ol
~. I1 pA
2 pA
100
ms
FIGURE 13. Lack o desensi iza ion a e main ained exposu e o low Po2. (A and B)
Single-channel cu en s eco ded om an inside-ou pa ch wi h wo Ko~ channels du ing
350-ms depola iza ions o +20 mV om -80 mV. The cell was exposed o 30 min o low Po2
( < 5 mmHg) be o e excising he pa ch om which he sweeps we e ob ained du ing al e na ing
exposu es o con ol and hypoxic solu ion as explained in Fig. 10. (C) Supe imposed ensemble
a e ages o aces eco ded in con ol (n = 25 sweeps, Pop~n = 0.52) and hypoxic (n = 16
sweeps, Po~n = 0.28) condi ions. E ec i e cu o equency = 0.95 kHz and sampling in e -
al = 500 ~s. Solu ions: s anda d Na, TIX//130 K, 10 EGTA.
o Ko~ channel ac i i y was obse ed in excised pa ches wi hou Ca 2+ o nucleo ides
added o he in e nal solu ion. We also es ed whe he 02 could ac h ough he
ac i a ion o a memb ane-bound G p o ein, a amily o p o eins ha a e i e e sibly
ac i a ed by GTP-,/-S (Gilman, 1987) o by A1F 4 o med om luo ide (a no mal
componen o some o ou in e nal solu ions) and aluminum (which could be eleased
om he mic opipe e glass) (S e nweis and Gilman, 1982; Bigay, De e e, P is e ,
and Chab e, 1985). The e e sible modula ion o Ko 2 channels by changes in Poe was
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND LOPEZ-BARNEO
Single K + Channel Modula ed by 02 in Glomus Cells
419
unal e ed in solu ions ee o F- (n = 51) o when up o 200 ,m GTP-',/-S was added
o he in e nal solu ion (n = 22). Thus, he esul s sugges ha soluble cy osolic
media o s o memb ane-bound G p o eins do no pa icipa e in he e ec o 02 on
he Ko 2 channel, and ha Oz may in e ac wi h an in insic senso closely associa ed
wi h he channel p o ein.
Repe i i e exposu e o low Po2 does no p oduce desensi iza ion.
In many examples o
ligand- ecep o in e ac ion, epea ed o pe manen exposu e o he agonis p oduces
an a enua ion o he physiological esponse. This phenomenon, called
"desensi iza ion," has been ypically s udied in some ligand-ac i a ed channels and i
is well known ha , a e wi hd awal o he agonis , eco e y o he es ing condi ions
is slow (see Hille, 1984). Al hough desensi iza ion is a e m applicable o ligand-
ecep o in e ac ions, i is a phenomenon ha could play a pa in he physiological
adap a ion obse ed in some senso y ecep o s (S ebbens, B own, and Pe e son,
1984).
I was desc ibed in ou p e ious wo k ha chemosenso y ansduc ion in he ype I
cell is a nonadap ing, o slowly adap ing, p ocess since e e sible a enua ion o he
mac oscopic K + cu en can be epea edly obse ed in a gi en cell (L6pez-Ba neo e
al., 1988; see also Gan o nina, 1991). Fig. 13 shows single-channel cu en sweeps
eco ded du ing al e na ing exposu e o con ol (A) and low Po2 (B) solu ions in a
pa ch wi h wo Ko 2 channels excised om a cell ha had been p eincuba ed in
ex eme hypoxia (Po2 = 5 mmHg) o 30 min. To acili a e compa ison, ensemble
a e ages in he wo expe imen al condi ions a e shown supe imposed in Fig. 13 C.
Re e sible inhibi ion o Ko 2 channel ac i i y by low Po2 can be obse ed epea edly
a e long-las ing exposu e o ex eme hypoxia, u he sugges ing ha he O2-Ko~
channel in e ac ion does no desensi ize.
DISCUSSION
In his a icle we desc ibe he p ope ies o h ee ypes o K + channels in chemo e-
cep o cells o he ca o id body ha can be dis inguished by hei biophysical
cha ac e is ics. We also demons a e ha in excised memb ane pa ches only he
ac i i y o a speci ic K + channel class, he Ko~ channel, is e e sibly inhibi ed by
lowe ing en i onmen al Po~. Ou indings explain he modula ion by O~ o he
mac oscopic K + cu en o glomus cells and s ongly sugges ha he O2-sensing
mechanism esides in he plasma memb ane.
K + Channel Types in Glomus Cells
Bo h cell-a ached and excised memb ane pa ches o abbi glomus cells con ain
h ee majo K+-selec i e channels (Kca, SK, and Ko~ channels). A Ca~+-independen
and high-conduc ance CI- channel encoun e ed in a ype I cells (S ea and Nu se,
1989) was no s udied. The h ee classes o K ÷ channels di e in hei single-channel
conduc ance and kine ics as well as in hei dependence on in e nal Ca 2+ and 02
sensi i i y. K + channels a e ex ao dina ily di e se (see o e iews Rudy, 1988;
Adams and Nonne , 1989) and i is well known ha di e en subpopula ions coexis
in a gi en memb ane (Dubois, 1983; Con i, Hille, and Nonne , 1984; Ma y and
Nehe , 1985; Hoshi and Ald ich, 1988; Llano, Webb, and Bezanilla, 1988). In his
espec , he single K + channels iden i ied in ype I cells sha e mos o hei p ope ies
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
420 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992
wi h hose classi ied in bo ine ch oma in (Ma y and Nehe , 1985) and mouse
neu oblas oma (Quand , 1988) cells. Equi alen single-channel cu en s a c also
p esen in pheoch omocy oma cells (Hoshi and Ald ich, 1988). In e es ingly, all
hese cell ypes ha e a close emb yological o igin.
The Kca channels ha e, in symme ical high K + solu ions, an a e age conduc ance
o 206.7 pS. These channels a e simila o he maxi-K + Ca2+-dependen channels o
o he p epa a ions (Ma y, 1981; Ba e e al., 1982; Quand , 1988), and, in excised
pa ches, hey a c una ec ed by changes in Po~. These obse a ions con i m ou
p e ious expe imen s showing ha pa o he mac oscopic K + cu en , p esumably a
Ca2+-dcpenden componen , disappea s a e wash-ou o Ca 2+ channels (U e ia e
al., 1989a) and ha unde hese condi ions, and wi h l0 mM EGTA added o he
in e nal solu ion, he K + cu en is s ill e e sibly a enua ed by lowe ing Po2
(L6pez-Ba neo e al., 1988; L6pez-L6pez e al., 1989). I has been epo ed ha
hypoxia speci ically inhibi s he Ca2+-dependen componen o he mac oscopic K +
cu en eco ded in dialyzed ca o id body cells om newbo n a s (Pee s, 1990). The
disc epancy be ween hese da a and ou wholc-ccU and single-channel esul s may
e lec a di e ence be ween animal species; howe e , wc also belie e ha he
conclusion eached by Pee s (1990) may ha e been biased by he expe imen al
p o ocol used in he isola ion o he Ca2+-ac i a ed K + cu en . We know, o
example, ha millimola concen a ions o Cd ~+ and Co 2+, which could p oduce a
dec ease o he Ca2+-ac i a cd K + cu en due o blockade o Ca 2+ channels, can also
p oduce a la ge and e e sible inhibi ion o he Ca2+-independen and O~-sensi i e
componen o he K + cu en (Gan o nina, M.D., and J. L6pez-Ba neo, unpublished
esul s).
We ollowed he e minology o Ma y and Nehe (1985) o deno e a second
popula ion o K+-selec i e channels ha ha e a small conduc ance (SK channels). In
asymme ical K + solu ions he uni a y conduc ance o ca o id body SK channels
(~ 6-7 pS) is simila o he alues epo ed in ch oma in and neu oblas oma cells
(Ma y and Nche , 1985; Quand , 1988). Hoshi and Ald ich (1988) ha e also ound
in pheoch omocy oma cells wo popula ions o K + channels (Ky and Kx) wi h he
same uni a y conduc ance alue. Al hough we did no s udy in de ail he kine ic
p ope ies o SK channels, in acco d wi h p e ious wo k (Ma y and Nchc , 1985;
Quand , 1988), hey beha ed as Ca2+-independen and slowly ac i a ing channels. In
glomus cells inac i a ion o SK channels, i any, mus be also e y slow since channel
ac i i y could be eco ded o minu es a depola ized memb ane po en ials. SK
channels a e only mode a ely ol age dependen (Popen = 0.2 a +20 and 0.05 a -70
mV) and hei Popen a nega i e ol ages sugges s ha hey may con ibu e o he
gcnc a ion o he cs ing po en ial o he cells. In excised pa ches SK channels wc c
unal c cd by changcs in Po2, bu we canno discoun ha in si u hey could be
subjec ed o modula ion. This idea is based on he ac ha in some inside-ou
pa ches he ac i i y o SK channels appea ed ab up ly se e al minu cs a e excision
o he memb ane, which could be explained by he dilu ion o some soluble media o
ha blocks he channels o ha a o s hei closed con o ma ion.
Wc ha e coined he e m Ko 2 o designa e he K+-sclec i c and O2-scnsi i e
channels o ype I cells. These channels we e he mos equen ly obse ed, p obably
because hey a e densely packed in he glomus cell memb ane. Ou es ima e is wo o
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND L PEZ-BARNEO
Single K + Channel Modula ed by 02 in Glomus Cells
421
ou channels pe squa e mic ome e . In asymme ical K + solu ions he uni a y
conduc ance o he Ko 2 channel is ~ 20 pS. This alue is in excellen ag eemen wi h
he conduc ance o as ac i a ing (FK) channels in ch oma in, neu oblas oma, and
pheoch omocy oma cells (Ma y and Nehe , 1985; Hoshi and Ald ich, 1988; Quand ,
1988) as well as o delayed ec i ie and A- ype K + channels desc ibed in a numbe o
p epa a ions (Coope and Sh ie , 1985, 1989; Kasai, Kameyama, Yamaguchi, and
Fukuda, 1986; Llano e al., 1988). K% channels a e no in luenced by changes in
in e nal Ca 2+ bu hey a e s eeply dependen on memb ane ol age; he ac i a ion
h eshold is a ~-50 o -40 mV, and a +20 mV he peak/)open is 0.8. This las
pa ame e is simila o alues epo ed o o he mammalian inac i a ing K ÷
channels (Ma y and Nehe , 1985; Coope and Sh ie , 1989).
Al hough he ac i a ion and inac i a ion kine ics a e s udied in mo e de ail in he
accompanying a icle, he e we show ha K% channels ha e a as ac i a ion. As he
memb ane is mo e depola ized he numbe o ac i e channels inc eases and he
la ency o he i s opening dec eases. Du ing main ained depola iza ions Ko 2
channels inac i a e comple ely in a ew hund ed milliseconds. These p ope ies a e
pe ec ly compa ible wi h he cha ac e is ics o he mac oscopic K + cu en o ype I
cells (U e ia e al., 1989a) and s ongly sugges ha he Ko~ channels a e he main
con ibu o s o his cu en . This is also suppo ed by he close pa allelism exis ing
be ween he ime cou ses o he whole-cell K + cu en and he ensemble a e ages
om pa ches con aining only Ko~ channels.
Modula ion o Ko 2 Channels by 02 Tension
A dis inc p ope y o Ko~ channels is ha hei Popen dec eases on exposu e o low
Po2. The inhibi ion o channel ac i i y by hypoxia is e e sible and concen a ion
dependen (Gan o nina and L6pez-Ba neo, 1991), and is a p ocess ha does no
unde go desensi iza ion. These p ope ies i pe ec ly wi h hose encoun e ed in he
modula ion o he mac oscopic K + cu en by Po2 (L6pez-Ba neo e al., 1988;
L6pez-L6pez e al., 1989). The co espondence be ween he 02 modula ion o
whole-cell and single-channel cu en s is qui e ema kable and can be clea ly seen
when ensemble a e ages om pa ches con aining Ko 2 channels in isola ion a e
compa ed wi h he O2-sensi i e K ÷ cu en (see, o example, Fig. 12). The majo
e ec o lowe ing Po2 is a dec ease in he Pope, o he channels lea ing unal e ed
uni a y conduc ance. In e es ingly, he magni ude o he dec ease o Pope, in hypoxic
condi ions in he ol age ange be ween +10 and +30 mV (~25% o he con ol
alue) is he same as he inhibi ion o he mac oscopic K + cu en by low Po2
(L6pez-Ba neo e al., 1988). The ac ion o low Po2 is mo e p onounced a less
depola ized memb ane ol ages, which is an obse a ion ha , as discussed in he
accompanying pape , could be expec ed i he lack o Oz a o s closed o inac i a ed
con o ma ions o he channels.
Delpiano and Heschele (1989) ha e epo ed in cell-a ached pa ches o glomus
cells om abbi emb yos he exis ence o a K ÷ channel e e sibly inhibi ed by
lowe ing Po2. This channel was eco ded du ing s a iona y memb ane depola iza-
ions and he uni a y conduc ance alue was 137 pS wi h high K ÷ in he pipe e
solu ion. These esul s a e di icul o compa e wi h ou own since in Delpiano and
Heschele 's wo k he O2-sensi i e channel was no cha ac e ized and i does no
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992

422 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992
include da a abou o he possible channel ypes. The kine ics and conduc ance o
Delpiano and Heschele 's channel a e compa able o hose o he Kca channel, which,
as shown be o e, is insensi i e o changes in Po2 in excised pa ches. Al hough we ha e
obse ed in si u he same ypes o K + channels as in excised pa ches, we did no
a emp an ini ial classi ica ion o K + channels in he cell-a ached con igu a ion
because he memb ane po en ial, cy osolic Ca 2+ concen a ion, and o he a iables
a e unknown. In addi ion, we ha e also obse ed ha ype I cells a e elec ically e y
compac and he e o e cu en lowing h ough a cell-a ached pa ch can induce
modi ica ions in he memb ane po en ial o he cell (Gan o nina, 1991). Ne e he-
less, we canno elimina e he possibili y ha he e could be a change in he
O2-sensing mechanisms o ca o id body cells du ing de elopmen (He zbe g, Hell-
s 6m, Lage c an z, and Pequigno , 1990). In his espec i is in e es ing o no e ha
whe eas emb yonic glomus cells seem o lack Na + channels (Heschele e al., 1989),
in he adul issue la ge Na + cu en s can be eco ded (U e ia e al., 1989a).
The ac ha he modula ion o Ko 2 channels by O2 is main ained o long pe iods
o ime in excised pa ches s ongly sugges s ha he O2-Ko 2 channel in e ac ion
occu s h ough an in insic senso o he plasma memb ane which may be pa o he
channels o a molecule closely associa ed wi h hem. This in e ac ion seems o be
di ec wi hou he pa icipa ion o soluble cy osolic media o s. We ha e sough o he
possible in ol emen o memb ane-di usible G p o eins, which a e being ac i a ed
can di ec ly egula e ionic channel ac i i y (Logo he is, Ku achi, Galpe , Nee , and
Claphan, 1987; B own and Bi nbaume , 1988), wi h nega i e esul s. Bo h GTP-~/-S
and F-, agen s ha i e e sibly ac i a e G p o eins (Gilman, 1987), we e ine ec i e in
p e en ing he e e sibili y o he inhibi ion o Ko 2 channels by lowe ing Po2.
None heless, we canno discoun ha he Ko 2 o o he channels o glomus cells migh
be modula ed by cy osolic media o s in si u. Exposu e o hypoxia al e s he con en
o cGMP and cAMP in he ca o id body (Wa y, Cheng, Dinge , and Fidone, 1989;
P6 ez-Ga cia, Alma az, and Gonz~lez, 1990) and hese agen s a e known o egula e
a b oad numbe o ionic channels.
The Ko 2 channel may belong o a amily o 02 senso s b oadly dis ibu ed in
na u e. Apa om he well-known 02 anspo unc ions o heme p o eins, he e
a e, om bac e ia o mammalian cells, examples o heme-linked enzymes, he ac i i y
o which is egula ed by en i onmen al 02 (Goldbe g, Dunning, and Bunn, 1988;
Gilles-Gonz,41ez, Di a, and Helinski, 1991). C oss, Hende son, Jones, Delpiano,
Hen schel, and Acke (1990) ha e ecen ly p oposed ha he ac i i y o a NADPH-
oxidase in glomus cells (con aining a b- ype cy och ome) could be egula ed by 02,
and ha his enzyme could de e mine he edox s a e o hiol g oups o p o eins and
in luence he p ope ies o ionic channels.
Physiological Signi icance o he Ko 2 Channel
The Ko 2 channel ep esen s he i s known example o an ionic channel egula ed by
02. A simila ype o egula ion has been sough , bu no ound, in sep al neu ons
(L6pez-L6pez e al., 1989) and in he small dopamine gic in e neu ons o he
sympa he ic ganglia (S ea and Nu se, 1991), which a e de elopmen ally ela ed o
glomus cells. Ou esul s demons a e he speci ici y o he Ko 2 channels loca ed in an
O2- esponsi e cell and s ongly sugges ha hey ep esen he ini ial s ep in
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANVORNINA AND L6PEZ-BARNEO
Single K + Channel Modula ed by Oe in Glomus Cells
423
chemo ansduc ion and hus con e upon glomus cells hei unique chemo ecep o
p ope ies. Due o ou expe imen al equi emen s (see Me hods), he Poz alues o
he hypoxic solu ions used in his and he accompanying pape a e much lowe han
he ones ha can be a ained unde physiological condi ions; howe e , we ha e
shown be o e ha he
Popen
o Ko~ channels is e e sibly modi ied by changes o Poz
in a physiological ange (Gan o nina and L6pez-Ba neo, 1991). The lack o app e-
ciable desensi iza ion in he O2-Ko 2 channel in e ac ion may ensu e ha changes o
he physico-chemical a iable (02 ension) would be ansla ed in o a main ained
elec ophysiological esponse. This cha ac e is ic could be ela ed o he ac ha
single- ibe chemo ecep o a e en discha ges can be main ained o long pe iods
du ing sus ained hypoxia (Nielsen, Bisga d, and Vid uk, 1988).
The signi icance o Ko 2 channels o espi a o y physiology is ob ious bu hey may
ha e a b oade unc ional, and pe haps pa hophysiological, ele ance. Simila ypes
o channels may exis in lung al eolus and in he ine b anches o he pulmona y
a e y and pa icipa e in he egula ion o egional pulmona y pe usion, o in small
essels o b ain and hea issues whe e hey may con ibu e o he au o egula ion o
blood low.
The au ho s wish o hank D s. R. Ald ich, T. Hoshi, and W. Zago a (S an o d Uni e si y) o
commen s on he manusc ip .
This esea ch was suppo ed by a g an om he Di ecci6n Gene al de In es igaci6n Cien i ica y
T~cnica (PB86-0250).
O iginal e sion ecei ed 18 Decembe 1991 and accep ed e sion ecei ed 16 Ap il 1992.
REFERENCES
Acke , H., and F. Pie uschka. 1977. Meaning o he ype I cell o he chemo ecep i e p ocess: an
elec ophysiological s udy on cul u ed ype I cells o he ca o id body.
In
Chemo ecep ion in he
Ca o id Body. H. Acke , S. Fidone, D. Pallo , C. Eyzagui e, D. W. Liibbe s, and R. W. To ance,
edi o s. Sp inge -Ve lag, Be lin. 92-98.
Adams, D. J., and W. Nonne . 1989. Vol age-dependen po assium channels: ga ing, ion pe mea ion
and block.
In
Po assium Channels: S uc u e, Classi ica ion, Func ion and The apeu ic Po en ial.
D. L. Cook, edi o . Ellis Ho wood Limi ed, New Yo k. 40-69.
Alma az, L., C. Gonz~ilez, and A. Obeso. 1986. E ec s o high po assium on he elease o [3H]
dopamine om he ca ca o id body in i o.
Jou nal o Physiology.
379:293-307.
Ba e , J. N., K. L. Magleby, and B. S. Pallo a. 1982. P ope ies o single calcium-ac i a ed
po assium channels in cul u ed a muscle.
Jou nal o Physiology.
331:211-230.
Bigay, J., P. De e e, C. P is e , and M. Chab e. 1985. Fluo oalumina es ac i a e ansducin-GDP by
mimicking he gamma-phospha e o GTP in i s binding si e.
FEBS Le e s.
191:181-185.
Biscoe, T.J., and M. R. Duchen. 1989. Elec ophysiological esponses o dissocia ed ype I cells o he
abbi ca o id body o
cyanide.Jou nal o Physiology.
413:447-468.
Biscoe, T. J., and M. R. Duchen. 1990a. Responses o ype I cells dissocia ed om he abbi ca o id
body o
hypoxia. Jou nal o Physiology.
428:39--59.
Biscoe, T. J., and M. R. Duchen. 1990b. Cellula basis o ansduc ion in ca o id chemo ecep o s.
Ame ican Jou nal o Physiology.
258:L271-L278.
Bla z, A. L., and K. L. Magleby. 1987. Calcium-ac i a ed po assium channels.
T ends in Neu osciences.
10:463-467.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
424 THE JOURNAL OF GENERAL PHYSIOLOGY • VOLUME 100 • 1992
B own, A. M., and L. Bi nbaume . 1988. Di ec G p o ein ga ing o ion channels.
Ame ican Jou nal o
Physiology.
254:H401-H410.
Colquhoun, D., and F. L. Sigwo h. 1983. Fi ing and s a is ical analysis o single channel eco ds.
In
Single Channel Reco dings. B. Sakmann and E. Nehe , edi o s. Plenum Publishing Co p., New
Yo k. 191-263.
Con i, F., B. Hille, and W. Nonne . 1984. Non-s a iona y luc ua ions o he po assium conduc ance
a he node o Ran ie o he
og.Jou nal o Physiology.
353:199-230.
Coope , E., and A. Sh ie . 1985. Single-channel analysis o as ansien po assium cu en s om a
nodose neu ones.
Jou nal o Physiology.
369:199-208.
Coope , E., and A. Sh ie . 1989. Inac i a ion o A cu en s and A channels on a nodose neu ons in
cul u e.Jou nal o Gene al Physiology.
94:881-910.
C oss, A. R., L. Hende son, O. T. G. Jones, M. A. Delpiano, J. Hen schel, and H. Acke . 1990.
In ol emen o an NAD(P)H oxidase as a Po2 senso p o ein in he a ca o id body.
Biochemical
Jou nal.
272:743-747.
De Cas o, F. 1926. Su la s uc u e e l'inne a ion de la glande in e ca o idienne (glomus
ca o icum) de l'homme e des mami ~ es, e su un nou eau sys ~me d'inne a ion au onome du
ne glossopha yngien.
T abajos del Labo a o io de ln es igaciones Biol6gicas de la Uni e sidad de
Mad /d. 24:365--432.
Delpiano, M. A., andJ. Heschele . 1989. E idence o a Po~-sensi i e K + channel in he yped cell o
he abbi ca o id body.
FEBS Le e s.
249:195-198.
Dubois, J. M. 1983. Po assium cu en s in he og node o Ran ie .
P og ess in Biophysics and
Molecula Biology.
42:1-20.
Duchen, M. R., K. W. T. Caddy, G. C. Ki by, D. L. Pa e son, J. Pon e, and T. J. Biscoe. 1988.
Biophysical s udies o he cellula elemen s o he abbi ca o id body.
Neu oscience.
26:291-311.
Eyzagui e, C., R. S. Fi zge ald, S. Lahi i, and P. Zapa a. 1983. A e ial chemo ecep o s.
In
Handbook o Physiology: The Ca dio ascula Sys em. J. T. Sheppe d, and F. Abboud, edi o s.
Ame ican Physiological Socie y, Be hesda, MD. 557-621.
Fidone, S. J., and C. Gonz~ilez. 1986. Ini ia ion and con ol o chemo ecep o ac i i y in he ca o id
body.
In
Handbook o Physiology: The Respi a o y Sys em II. A. P. Fishman, edi o . Ame ican
Physiological Socie y, Be hesda, MD. 247-312.
Fidone, S.J., C. Gonz~ilez, and K. Yoshizaki. 1982. E ec s o low oxygen on he elease o dopamine
om he abbi ca o id body in i o.
Jou nal o Physiology.
333:93-110.
Fishman, M. C., W. L. G eene, and D. Pla ika. 1985. Oxygen chemo ecep ion by ca o id body cells in
cul u e.
P oceedings o he Na ional Academy o Sciences, USA.
82:1448-1450.
Fi zge ald, R. S., and S. Lahi i. 1986. Re lex esponses o chemo ecep o s imula ion.
In
Handbook
o Physiology: The Respi a o y Sys em II. A. P. Fishman, edi o . Ame ican Physiological Socie y,
Be hesda, MD. 313-362.
Gan o nina, M. D. 1991. Es udio elec o isiol6gico del canal Ko 2, un canal de K ÷ modulado po 02
en las c ilulas quimio ecep o as del cue po ca o ideo. Doc o al hesis. Uni e sidad de Se illa,
Se illa, Spain. 183 pp.
Gan o nina, M. D., and J. L6pez-Ba neo. 1991. Single K + channels in memb ane pa ches o a e ial
chemo ecep o cells a e modula ed by 02 ension.
P oceedings o he Na ional Academy o Sciences,
USA.
88:2927-2930.
Gilles-Gonz~ilez, M. A., G. S. Di a, and D. R. Helinski. 1991. A haemop o ein wi h kinase ac i i y
encoded by he oxygen senso o
Rhizobium melilo i. Na u e.
350:170-172.
Gilman, A. G. 1987. G p o eins: ansduce s o ecep o -gene a ed signals.
Annual Re iew o
Biochemis y.
56:615-649.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992
GANFORNINA AND L6PEZ-BARNEO
Single K + Channel Modula ed by 02 m Glomus Cells
425
Goldbe g, M. A., S. P. Dunning, and H. F. Bunn. 1988. Regula ion o he e y h opoie in gene:
e idence ha he oxygen senso is a heme p o ein.
Science.
242:1412-1415.
HamiU, O. P., A. Ma y, E. Nehe , B. Sakmann, and F. J. 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 zbe g, T., S. Hells 6m, H. Lage c an z, and J. M. Pequiguo . 1990. De elopmen o he a e ial
chemo e lex and u no e o ca o id body ca echolamines in he newbo n a .
Jou nal o Physiology.
425:211-225.
Heschele , J., M. A. Delpiano, H. Acke , and F. Pie nschka. 1989. Ionic cu en s on ype-I cells o
he abbi ca o id body measu ed by ol age-clamp expe imen s and he e ec o hypoxia.
B ain
Resea ch.
486:79--88.
Heymans, C., J. J. Bouckae , and L. Dau ebande. 1930. Sinus ca o idien e 6 lexes espi a oi es. II.
In luences espi a oi es ~ lexes de l'acid6se, de l'alcal6se, de l'anhyd ide ca bonique, de I'ion
hyd og6ne e de I'anox6mie: sinus ca o idiens e 6changes espi a oi es dans les poumons e au
dei~ des poumons.
A chi es In e na ionales de Pha macodynamie e de Thi apie.
39:400--408.
HiUe, B. 1984. Ionic channels o exci able memb anes. Sinaue Associa es, Inc., Sunde land, MA. 426
pP.
Hoshi, T., and R. W. Ald ich. 1988. Vol age-dependen K + cu en s and unde lying single K +
channels in pheoch omocy oma
cells.Jou nal o Gene al Physiology.
91:73-106.
Kasai, H., M. Kameyama, K. Yamaguchi, and J. Fukuda. 1986. Single ansien K channels in
mammalian senso y neu ons.
Biophysical Jou nal.
49:1243-1247.
Llano, I., C. K. Webb, and F. BezaniUa. 1988. Po assium conduc ance o he squid gian
axon.Jou nal
o Gene al Physiology.
92:179-196.
Logo he is, D. E., Y. Ku achi, J. Galpe , E. J. Nee , and D. E. Claphan. 1987. The [3 subuni s o
GTP-binding p o eins ac i a e he musca inic K ÷ channel in hea .
Na u e.
325:321-326.
L6pez-Ba neo, J., J. R. L6pez-L6pez, J. U e ia, and C. Gonz~ilez. 1988. Chemo ansduc ion in he
ca o id body: K ÷ cu en modula ed by Po~ in ype I chemo ecep o cells.
Science.
241:580-582.
L6pez-L6pez, J., C. Gonz~ilez, J. U e ia, and J. L6pez-Ba neo. 1989. Low Po~ selec i ely inhibi s K
channel ac i i y in chemo ecep o cells o he mammalian ca o id body.
Jou nal o Gene al
Physiology.
93:1001-1015.
Ma y, A. 1981. Ca-dependen K channels wi h la ge uni a y conduc ance in ch oma in cell
memb anes.
Na u e.
291:497-500.
Ma y, A., and E. Nehe . 1985. Po assium channels in cul u ed bo ine ad enal ch oma in cells.
Jou nal o Physiology.
367:117-141.
Nielsen, A. M., G. E. Bisga d, and E. H. Vid uk. 1988. Ca o id chemo ecep o ac i i y du ing acu e
and sus ained hypoxia in
goa s.Jou nal o Applied Physiology.
65:1796-1802.
Obeso, A., S. Fidone, and C. Gonz~lez. 1987. Pa hways o calcium en y in o ype I cells: signi icance
o he sec e o y esponse.
In
Chemo ecep o s in Respi a o y Con ol. J. A. Ribei o and D. J.
Pallo , edi o s. C oom Helm, London. 91-97.
Pa lak, J., and R. Ho n. 1982. E ec o N-b omoace amide on single sodium channel cu en s in
excised memb ane
pa ches.Jou nal o Gene al Physiology.
79:333-351.
Pee s, C. 1990. E ec s o D600 on hypoxic supp ession o K + cu en s in isola ed ype I ca o id body
cells o he neona al a .
FEBS Le e s.
271:37-40.
P6 ez-Ga c a, M. T., L. Alma az, and C. Gonz~ lez. 990. E ec s o di e en ypes o s imula ion on
cyclic AMP con en in he abbi ca o id body: unc ional signi icance.
Jou nal o Neu ochemis y.
55:1287-1293.
Quand , F. N. 1988. Th ee kine ically dis inc po assium channels in mouse neu oblas oma cells.
Jou nal o Physiology.
395:401-418.
on Sep embe 22, 2014jgp. up ess.o gDownloaded om
Published Sep embe 1, 1992