Single K+ channels in membrane patches of arterial chemoreceptor cells are modulated by 02 tension

P oc. Na i. Acad. Sci. USA Vol. 88, pp. 2927-2930, Ap il 1991 Neu obiology Single K+ channels in memb ane pa ches o a e ial chemo ecep o cells a e modula ed by 02 ension (02 sensing/glomus cells) MARiA D. GANFORNINA AND Jose L6PEZ-BARNEO Depa amen o de Fisiologia y Bio isica, Facul ad de Medicina, Uni e sidad de Se illa, A da. Sanchez Pizjudn, 4, 41009 Se ille, Spain Communica ed by R. Llinas, Decembe 26, 1990 ABSTRACT Type I cells o he ca o id body a e known o pa icipa e in he de ec ion o 02 ension in a e ial blood bu he p ima y chemo ansduc ion mechanisms a e no well unde - s ood. He e we epo he exis ence in excised memb ane pa ches o ype I cells o a single K+ channel ype modula ed by changes in P02. Open p obabili y o he O2-sensi i e K+ channel e e s- ibly dec eased by a leas 50% on exposu e o hypoxia bu single-channel conduc ance (-20 pS) was unal e ed. In he ange be ween 70 and 150 mmHg (1 mmHg = 133 Pa) he dec ease o single-channel open p obabili y was p opo ional o he Po2 measu ed in he icini y o he memb ane pa ch. The inhibi ion o K+ channel ac i i y by low P02 was independen o he p esence o non-hyd olyzable guanine iphospha e ana- logues a he in e nal ace o he memb ane. The esul s indica e ha he 02 senso o ype I cells is in he plasma memb ane and sugges ha en i onmen al 02 in e ac s di ec ly wi h he K+ channels. Type I, o glomus, cells o he ca o id body ha e been conside ed o decades o be esponsible o he de ec ion o oxygen ension (Po2) in he a e ial blood bu he mechanisms in ol ed in he p ocess o chemo ansduc ion ha e emained obscu e (1, 2). Howe e , i has been ecen ly disco e ed ha ype I cells om adul abbi s can gene a e ac ion po en ials and ha hey ha e a ol age-dependen K+ cu en selec i ely and e e sibly a enua ed by lowe ing Po2 (3-8). Inhibi ion o his K+ cu en unde hypoxic condi ions could p oduce an inc ease in he i ing equency o chemo ecep o cells, leading o Ca2' in lux, enhanced ansmi e elease, and ac i a ion o he a e en ibe s o he sinus ne e (1, 2, 6). The p ima y si e o 02 de ec ion is, howe e , unknown. Exposu e o cyanide o o ex eme hypoxia (<40 mmHg; 1 mmHg = 133 Pa) induces an inc ease o [Ca2+]j in ype I cells, possibly due o Ca2' elease om mi ochond ia, and a subsequen ac i a ion o a Ca2+-dependen K+ cu en (9, 10). The e o e i has been a gued ha he modi ica ion o he K+ cu en by lowe ing Po2 migh be a seconda y phenomenon a he han an ini ial s ep in he p ocess o chemo ansduc ion. Now we epo he iden i ica ion o a single K+ channel ype ha ully accoun s o he p ope ies o he mac oscopic 02-sensi i e K+ cu en . Fu he mo e, we show ha in excised memb ane pa ches he ac i i y o hese K+ channels is e e sibly modula ed by P02. Ou esul s suppo he iew ha he K+ 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 associa ed wi h, he plasma memb ane. This ype o Ke channel egula ion ound in he ca o id body may ha e an e en b oade unc ional in e es because i could also be in ol ed in physiological esponses o hypoxia in o he issues. METHODS Expe imen s we e pe o med on enzyma ically dispe sed ype I cells isola ed om abbi ca o id bodies. The me hods ollowed in cell dissocia ion and cul u e we e he same as p e iously desc ibed (3, 4). Cells we e pla ed on agmen s o glass co e slips ea ed wi h poly(L-lysine). Du ing he expe - imen a co e slip was ans e ed o a small chambe o 0.2 ml wi h con inuous low o solu ion ha could be comple ely eplaced in 10-15 s. 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 02 ension. Po2 in he chambe was di ec ly moni o ed wi h a pola ized 100- ,m- hick pla inum wi e (11) placed in he icin- i y o he pa ch elec ode. We used 5- o 8-M l pa ch pipe es ab ica ed om bo osilica e glass. In mos expe imen s cells we e i s subjec ed o whole-cell ol age clamp and he ea e he ou side-ou excised memb ane pa ch con igu a ion was ob ained by pulling he elec ode away om he cell (12). Howe e , in a ew expe imen s he e ec o hypoxia on K+ channels included in inside-ou excised pa ches was also es ed. Composi ion o solu ions and o he expe imen al a - iables a e gi en in he igu e legends. RESULTS The majo p ope ies o he 02-sensi i e single K+ channels a e summa ized in Fig. 1. A mac oscopic K+ cu en e- co ded in whole-cell mode du ing a ol age s ep o +20 mV is shown in Fig. lA. The cu en ace, shown o compa i- son, illus a es he ypical ime cou se o he 02- egula ed K+ cu en o ype I cells, wi h inac i a ion du ing a main ained depola iza ion (6). A e excision o a memb ane pa ch (Fig. 1B) he same pulse p o ocol p oduced he appea ance o single-channel e en s wi h a uni a y ampli ude o 1.8 ± 0.2 pA (mean ± SD, n = 14). The h ee K+ channels ac i a ed on depola iza ion open p e e en ially a he beginning o he pulse and p og essi ely en e ed an inac i a ed s a e. Channel inac i a ion is clea ly e iden in Fig. 1C, whe e i is shown he a e age o 23 consecu i e single-channel cu en sweeps wi h a ime cou se almos iden ical o he whole-cell cu en . Single-channel ac i i y was blocked by 5 mM e ae hylam- monium (Fig. 1D), which also abolishes he whole-cell cu - en (4). Single K+ channels simila o hose o Fig. 1B we e ound in e e y expe imen and hei es ima ed densi y is =600 pe cell. Uni a y cu en s eco ded om a pa ch wi h a mos one open K+ channel a e shown in Fig. 1E, which also illus a es he inc ease in single-channel cu en ampli ude wi h memb ane depola iza ion, in pa allel wi h he inc ease o he elec ochemical d i ing o ce o K+ mo emen . Single-channel cu en ampli ude (i) e sus memb ane po- en ial (VM) is plo ed in Fig. iF. Mean uni a y cu en alues om 14 expe imen s a e ep esen ed by do s. In hese ionic condi ions (2.7 mM K+ in he ex e nal solu ion and 140 mM K+ in he solu ion a he in e nal ace o he memb ane) he i-VM ela ionship was almos linea in he ange be ween -30 and +50 mV, yielding an es ima e o he single-channel conduc ance o be 20.1 pS. In symme ical 140 mM K+ ( iangles) single-channel cu en e e sed a 0 mV and he uni a y conduc ance was 41.5 pS. These obse a ions indi- ca e ha he channels we e highly selec i e o K+. Ca2+- 2927 The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked "ad e isemen " in acco dance wi h 18 U.S.C. §1734 solely o indica e his ac . 2928 Neu obiology: Gan o nina and L6pez-Ba neo A Cell 1nA _ B 03-----. Pa ch 2 pA c C Ensemble -Jil E VM MV 20 1 i pA 2- 1- 0- -1 - -2- 50 ms I I I I . -40 -20 0 20 40 60 VMmV FIG. 1. P ope ies o he 02-sensi i e K+ channels. (A) Whole-cell K+ cu en elici ed by a 200-ms pulse om -80 o +20 mV. (B) Single-channel e en s eco ded du ing a simila ol age s ep in an ou side-ou excised memb ane pa ch. (C and D) A e age cu en o 23 consecu i e sweeps eco ded om he same pa ch wi h simila pulse p o ocol as in B (C) and blockade o he cu en by he p esence o 5 mM e ae hylammonium (TEA+) in he ex e nal solu ion (D). (E) Single-channel cu en elici ed by ol age s eps om -80 o 0, +20, and +40 mV in an ou side-ou pa ch wi h a mos one open channel. Pulse du a ion was 200 ms. (F) Single-channel cu en ampli ude (i) e sus memb ane po en ial (VM). Do s a e a e age cu en s om 14 pa ches eco ded in asymme ical K+ concen a ions and wi h an ex e nal solu ion equilib a ed wi h he no mal Po2 (150 mmHg). The s aigh line i has a slope o 20.1 pS. Open ci cles a e a e age cu en s om i e pa ches wi h he same solu ions bu wi h low 02 con en (be ween 80 and 20 mmHg). T iangles a e a e age measu emen s om wo pa ches in symme ical K+ concen a ions and no mal Po2. The s aigh line i has a slope o 41.5 pS. Cu en signals we e low-pass- il e ed a 1 kHz (8-pole Bessel) and digi ized wi h a sampling in e al o 500 ,us. In all cu en aces linea ionic and capaci y cu en s we e sub ac ed. Solu ions in A-F (do s and open ci cles) con ained he ollowing (in mM): ex e nal: 140 NaCl, 2.7 KCI, 5 CaC12, 2 MgCl2, 10-3 e odo oxin, 10 Hepes; in e nal: 80 po assium glu ama e, 40 KCI, 20 KF, 2 MgCl2, 10 EGTA, 10 Hepes. In he ex e nal solu ion o D 5 mM NaCl we e eplaced by 5 mM e ae hylammonium chlo ide. In F ( iangles) he ex e nal solu ion con ained he ollowing (in mM): 140 KCI, 5 CaC12, 2 MgCl2, 10 Hepes. In all solu ions pH was be ween 7.3 and 7.4. Holding po en ial was -80 mV and empe a u e was 22-25°C. Cu en calib a ion in B also applies o aces in C-E. dependen maxi-K+ channels wi h uni a y conduc ance o abou 250 pS (in symme ical 140 mM K+ solu ions) we e also p esen in ype I cells. Since he ac i i y o maxi-K+ channels was una ec ed by changes in 02 ension (n = 14), hei ac i a ion was p e en ed in mos expe imen s by main aining [Ca2+] a he in e nal ace o he memb ane below 1 nM. The modula ion o he small K+ channels by Po2 was in es iga ed in excised memb ane pa ches exposed o solu- ions wi h educed 02 con en . Fig. 2 illus a es he e ec o hypoxia on an ou side-ou excised pa ch ha ne e showed mo e han one open channel. Fig. 2A shows h ee se s o cu en aces eco ded du ing ol age pulses o +20 mV in a con ol solu ion equilib a ed wi h ai (Po2 = 150 mmHg), while exposu e o he pa ch o low Po2 (swi ching om 150 o 80 mmHg), and a e e u ning o he solu ion wi h no mal Po2 ( eco e y aces). The a e age o 15-30 consecu i e sweeps eco ded in he h ee di e en expe imen al condi- ions a e shown in Fig. 2B. Open p obabili y o he channel, in eg a ed h oughou he pulse du a ion, in he con ol solu ion (po = 0.61) dec eased ma kedly du ing exposu e o hypoxia (po = 0.28) and e u ned o a high alue (po = 0.74) a e es o a ion o no moxic condi ions. The eco dings clea ly show ha single-channel cu en ampli ude was un- a ec ed by low Po2. The a e age i-VM alues om i e pa ches exposed o hypoxia a e plo ed in Fig. 1F (open ci cles). The da a poin s indica e ha uni a y conduc ance was iden ical o he con ol alue. Simila quali a i e esul s ha e been ob ained in all pa ches (ei he ou side-ou o inside-ou ) whe e he e ec o hypoxia was es ed (n = 36). Al hough a de ailed s udy o he e ec o hypoxia on he kine ics o he K+ channel is necessa y, ou p elimina y da a sugges ha changes in 02 ension speci ically modi y ac i- a ion a e cons an s. Mean open ime (-18 ms a +20 mV) was he same in con ol and in hypoxic condi ions, whe eas mean closed ime (20 ms a he same memb ane po en ial and Po2 = 150 mmHg) inc eased by a leas a ac o o 2.5 on exposu e o hypoxia. Inac i a ion ime cou se, e alua ed om ensemble a e age eco dings in mul ichannel pa ches, seemed o be unchanged by low Po2. The e e sible dec ease o K+ channel open p obabili y occu s oughly wi h he ime cou se o Po2 change in he neighbo hood o he memb ane pa ch. Fig. 3A shows a con inuous elec ical signal p opo ional o he a ia ion o 02 ension in he chambe . Single-channel e en s eco ded om an ou side-ou excised pa ch du ing 1.3-s ol age s eps o +20 mV a e shown in Fig. 3B. The a ows in Fig. 3A indica e he ime a which each pulse was deli e ed. The pa ch seemed o con ain i e channels and he numbe o P oc. Na l. Acad. Sci. USA 88 (1991) P oc. Na l. Acad. Sci. USA 88 (1991) 2929 A Con ol I J ~~"0 h_4 Joj - HI~~~~~~~~~~~~~ -I, B I-ML j2pA 10.7 pA 50 ms FIG. 2. Modula ion o single K+ channels by 02 ension. (A) Rep esen a i e cu en aces elici ed by 200-ms depola iza ions om -80 o +20 mV in an ou side-ou excised memb ane pa ch ha con ained a mos one open channel. The onse and he end o he pulses a e indica ed by he a ows. Reco dings we e ob ained in he con ol ex e nal solu ion (equilib a ed wi h ai ; Po2 = 150 mmHg), in low Po2 (swi ching om 150 o 80 mmHg), and a e e u ning o he solu ion wi h no mal Po2. Pulses we e applied e e y 5 s. Cu en calib a ion was 2 pA. (B) Ensemble a e ages o 15-30 consecu i e sweeps in he di e en expe imen al condi ions. Cu en calib a ion was 0.7 pA. Open channel p obabili y (con ol = 0.61; low Po2 = 0.28; eco e y = 0.74) was calcula ed om he ime spen in he open s a e di ided by he du a ion o he pulses. Solu ions and o he expe imen al a iables we e as in Fig. 1 B-F. simul aneous e en s ma kedly dec eased on exposu e o hypoxia. The a e age open p obabili y (p0), in eg a ed h oughou he pulse du a ion, was 0.34 in no moxic condi- ions (a; Po2 = 150 mmHg) bu only 0.06 (b), 0.09 (c), and 0.12 (d) wi h Po2 alues o 85, 44, and 116 mmHg, espec i ely. Comple e eco e y o single-channel ac i i y (po = 0.25) was ob ained on e u ning o he con ol solu ion (e, Po2 = 145 mmHg). po (o dina e) as a unc ion o 02 ension (abscissa) is plo ed in Fig. 3C. Be ween 70 and 150 mmHg, a ange ha includes he no mal Po2 alues in a e ial blood o he abbi , channel open p obabili y dec eased in pa allel o 02 ension. Lowe ing Po2 below 70 mmHg p oduced, howe e , a ela i e inc ease in p0. These esul s demons a e a concen a ion- dependen e ec o 02 on he K+ channel. DISCUSSION This epo shows ha a speci ic kind o K+ channel o he ype I cell plasma memb ane is e e sibly and selec i ely modu- la ed by changes in Po2. Ca2+-dependen maxi-K+ channels also p esen in he same p epa a ion a e una ec ed by al e - a ions in 02 ension. The 02-sensi i e K+ channels, which explain he modula ion o he mac oscopic K+ cu en by Po2 (3), a e mos likely key elemen s in he ansduc ion o hypoxic s imuli by ype I cells. These K+ channels a e p obably in ol ed in he egula ion o cell i ing and o Ca2' en y h ough ol age-ga ed channels du ing mode a e changes in ca o id a e y Po2 (2-4). A e in oxica ion o ype I cells wi h cyanide o unde ex eme hypoxia (wi h Po2 alues <40 mmHg) sec e ion may be also suppo ed by s ong elease o Ca2+ om mi ochond ia (9, 10); howe e , hese condi ions a e o unlikely physiological occu ence. In ac , we ha e ob- se ed in mos expe imen s a pa ial eco e y o K+ channel ac i i y a e y low Po2 le els ( e . 6 and Fig. 3 in his epo ), which may limi he i ing equency o ype I cells and con ibu e o p e en ing hei ex ensi e deg anula ion. The inhibi ion o K+ channel opening by hypoxia could no be ela ed o he p esence o any dialyzable componen o he memb ane. I was epea edly obse ed in a gi en pa ch ega dless o he ime elapsed a e excision and was inde- penden o he in e nal [Ca2+] o he p esence o MgATP a he in e nal ace o he memb ane. Mo eo e , he e e sible educ ion o K+ channel ac i i y by hypoxia was no al e ed by he addi ion o 20 AM GTP[y-S] (n = 4), which is known o abolish he e e sibili y o G-p o ein-media ed modula ion o ionic channels (13, 14). These obse a ions sugges ha in ype I cells 02 in e ac s wi h he K+ channels ei he di ec ly o h ough a si e closely associa ed wi h hem. In his espec ype I cells di e om ol ac o y o as e chemo ecep o cells in which na u al s imuli modula e ion channels h ough he ac ion o in acellula cyclic nucleo ides (15-17). In conclu- sion, ou indings demons a e ha en i onmen al 02 egu- la es he open p obabili y o a ype o K+ channel in chemo e- cep o cells o he ca o id body wi hou al e ing he single- channel conduc ance. 02 de ec ion by ype I cells seems o in ol e di ec in e ac ion wi h he K+ channels, pe haps h ough a heme-like p os he ic g oup bound o he channel molecule. This mechanism, wi hou p eceden in he li e a- u e, may no be es ic ed o he ca o id body bu i could also pa icipa e in o he physiological p ocesses- o exam- ple, he au o egula ion by local 02 ension o blood low in co ona y, ce eb al, and pulmona y a e ies. We hank D . J. U e ia o help in compu e p og aming, D s. G. Al a ez de Toledo and L. Taba es o commen s on he manusc ip , and J. R. L6pez-L6pez o his collabo a ion in he cons uc ion o he 02-sensing mic oelec ode. Resea ch was suppo ed by a g an om he Di ecci6n Gene al de In es igaci6n Cien ica y Tdcnica (PB-86/0250). Low P02 Reco e y I* Is. . ].I . . T" ----. 10"I Is 1%. .L-- --jo &a ,"- JL-A-A-.-A -.-I 'i V- qwy-wl yllw- i 44040 *%14104Y 11 ""O" Oa .Lo 09 #*Om"# 0- '. -- - , "mmi wo " LL -A& A .0 .j jq ww" I-M-1-1-1-1-- - - Neu obiology: Gan o nina and L6pez-Bameo "*Ab lw A- F II -P I MI' 1 -' i am --L PTMT WV'" 41. 2930 Neu obiology: Gan o nina and Lopez-Bameo A c Po 0.4 0.3 0.2 0.1 B 4- P02,mmHg 3- 2- 1 _ F150 1- b d L50~~~~~~~~0 2-b 30 s 2- a 2- 1-d C d 50 70 90 110 130 150 1-A j3pA P02,mmHg FIG. 3. Changes in 02 ension and single K+ channel ac i i y in a mul ichannel ou side-ou excised pa ch. (A) Time cou se o he a ia ions o Po2 in he eco ding chambe du ing a ansien exposu e o hypoxia. (B) Single-channel ac i i y eco ded du ing 1.3-s ol age s eps om -80 o +20 mV a he ime indica ed by he a ows and lowe case le e s in A. Consecu i e pulses we e applied e e y 30 s o allow o comple e eco e y om inac i a ion. A e age open channel p obabili y (p.) was calcula ed om p0 = (1/Ni )- Id , whe e N = numbe o channels in he pa ch, i = single-channel cu en ampli ude, = pulse du a ion, and I = ne cu en du ing he pulse. po alues we e 0.34 (a), 0.06 (b), 0.12 (c), 0.09 (d), and 0.25 (e). (C) Channel open p obabili y (p0, o dina e) as a unc ion o Po2 (abscissa) in he chambe . Solu ions and o he expe imen al condi ions we e as in Fig. 1 B-F. 1. Eyzagui e, C. & Zapa a, P. (1968) in A e ial Chemo ecep o s, ed. To ance, R. W. (Blackwell, Ox o d), pp. 213-251. 2. Fidone, S. J. & GonzAlez, C. (1986) in Handbook o Physiol- ogy, The Respi a o y Sys em II, ed. Fishman, A. (Am. Physiol. Soc., Washing on), pp. 247-312. 3. L6pez-Ba neo, J., L6pez-L6pez, J. R., U e ia, J. & GonzAlez, C. (1988) Science 241, 580-582. 4. U e ia, J., L6pez-LUpez, J. R., GonzAlez, C. & L6pez-Ba neo, J. (1989) J. Gen. Physiol. 93, 979-999. 5. Duchen, M. R., Caddy, K. W. T., Ki by, G. C., Pa e son, D. L., Pon e, J. & Biscoe, T. J. 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