Carotid body chemosensory responses in mice deficient of TASK channels

A icle The Rocke elle Uni e si y P ess $30.00 J. Gen. Physiol. Vol. 135 No. 4 379–392 www.jgp.o g/cgi/doi/10.1085/jgp.200910302 379 INTRODUCTION Oxygen- egula ed K+ channels, ini ially desc ibed in he abbi ca o id body (CB) glomus cell (López-Ba neo e al., 1988; Gan o nina and López-Ba neo, 1991), a e belie ed o play a undamen al ole in chemosenso y ansduc ion. I is b oadly accep ed ha educ ion o glomus cell K+ conduc ance in hypoxemia is he majo e en leading o depola iza ion and Ca2+ channel open- ing, ise o cy osolic [Ca2+], and ansmi e elease. These ansmi e s s imula e a e en ne e ibe s ac ing on b ains em espi a o y neu ons o e oke hype en ila ion (López-Ba neo e al., 1993; Buckle and Vaughan-Jones, 1994; U eña e al., 1994; Mon o o e al., 1996; o ecen e iews see P abhaka , 2006; López-Ba neo e al., 2008). Di e en unc ional sub ypes o O2- egula ed K+ chan- nels ha e been epo ed in glomus cells om se e al mammalian species (Pee s, 1990; S ea and Nu se, 1991; Gan o nina and López-Ba neo, 1992; Wya and Pee s, 1995; Buckle , 1997; Pé ez-Ga cía e al., 2004) as well as P. O ega-Sáenz and K.L. Le i sky con ibu ed equally o his pape . Co espondence o José López-Ba neo: [email p o ec ed] Abb e ia ions used in his pape : 4-AP, 4-aminopy idyne; CB, ca o id body; RT, e e se ansc ip ion; TH, y osine hyd oxylase. in o he neu osec e o y cell classes acu ely esponding o hypoxia ( o e iew see López-Ba neo e al., 2001; Nu se e al., 2006). Al hough he unde s anding o he cellula bases o CB chemo ansduc ion has ad anced conside ably, he p ecise molecula na u e o he O2 senso (s) and he e ec o K+ channel(s) is unknown (see Kemp, 2006). P og ess in his ield is hampe ed by me hodological limi a ions de i ed om he gaseous na u e o he s imulus and he delicacy o he O2-sensing appa a us, which can be al e ed du ing cell dissocia ion (O ega- Sáenz e al., 2007). Addi ionally, he small size o he CB has p ecluded la ge-scale biochemical analyses. These limi a ions can be pa ially o e come by he use o gene ically modi ied mice, in which he unc ional con- sequences o a ge ed molecula abla ion can be un- ambiguously demons a ed (e.g., O ega-Sáenz e al., 2006; Mulkey e al., 2007). To his end, we de eloped Ca o id body chemosenso y esponses in mice de icien o TASK channels Pa icia O ega-Sáenz, Kons an in L. Le i sky, Ma ía T. Ma cos-Alma az, Vic o ia Bonilla-Henao, Albe o Pascual, and José López-Ba neo Ins i u o de Biomedicina de Se illa (IBIS) and Cen o de In es igación Biomédica en Red sob e En e medades Neu odegene a i as (CIBERNED), Hospi al Uni e si a io Vi gen del Rocío, Consejo Supe io de In es igaciones Cien i icas, Uni e sidad de Se illa, 41013 Se illa, Spain Backg ound K+ channels o he TASK amily a e belie ed o pa icipa e in senso y ansduc ion by chemo ecep o (glomus) cells o he ca o id body (CB). Howe e , s udies on he sys emic CB-media ed en ila o y esponse o hy- poxia and hype capnia in TASK1- and/o TASK3-de icien mice ha e yielded con lic ing esul s. We ha e cha ac- e ized he glomus cell pheno ype o TASK-null mice and s udied he esponses o indi idual cells o hypoxia and o he chemical s imuli. CB mo phology and glomus cell size we e no mal in wild- ype as well as in TASK1/ o double TASK1/3/ mice. Pa ch-clamped TASK1/3-null glomus cells had signi ican ly highe memb ane esis- ance and less hype pola ized es ing po en ial han hei wild- ype coun e pa . These elec ical pa ame e s we e p ac ically no mal in TASK1/ cells. Sensi i i y o backg ound cu en s o changes o ex acellula pH was d as i- cally diminished in TASK1/3-null cells. In con as wi h hese obse a ions, esponsi eness o hypoxia o hype - capnia o ei he TASK1/ o double TASK1/3/ cells, as es ima ed by he ampe ome ic measu emen o ca echolamine elease, was appa en ly no mal. TASK1/3 knockou cells showed an enhanced sec e o y a e in ba- sal (no moxic) condi ions compa ible wi h hei inc eased exci abili y. Responsi eness o hypoxia o TASK1/3-null cells was main ained a e pha macological blockade o maxi-K+ channels. These da a in he TASK-null mouse model indica e ha TASK3 channels con ibu e o he backg ound K+ cu en in glomus cells and o hei sensi i - i y o ex e nal pH. They also sugges ha , al hough TASK1 channels migh be dispensable o O2/CO2 sensing in mouse CB cells, TASK3 channels (o TASK1/3 he e ome s) could media e hypoxic depola iza ion o no mal glo- mus cells. The abili y o TASK1/3/ glomus cells o main ain a powe ul esponse o hypoxia e en a e blockade o maxi-K+ channels, sugges s he exis ence o mul iple senso and/o e ec o mechanisms, which could con e upon he cells a high adap abili y o main ain hei chemosenso y unc ion. © 2010 O ega-Sáenz e al. This a icle is dis ibu ed unde he e ms o an A ibu ion– Noncomme cial–Sha e Alike–No Mi o Si es license o he i s six mon hs a e he publi- ca ion da e (see h p://www. up ess.o g/ e ms). A e six mon hs i is a ailable unde a C ea i e Commons License (A ibu ion–Noncomme cial–Sha e Alike 3.0 Unpo ed license, as desc ibed a h p://c ea i ecommons.o g/licenses/by-nc-sa/3.0/). The Jou nal o Gene al Physiology on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 h p://jgp. up ess.o g/con en /suppl/2010/03/22/jgp.200910302.DC1.h ml Supplemen al Ma e ial can be ound a : 380 Glomus cell O2/CO2 sensing in TASK1/3-null mice ano he labo a o y has obse ed in TASK1, bu no in TASK3, knockou mice dec eased en ila ion and a e en sinus ne e discha ges in esponse o hypoxia and hype capnia (T app e al., 2008). He ein, we epo he basic elec ophysiological p ope ies and in insic chemosenso y ac i i y o indi idual glomus cells om TASK-de icien animals. We show ha glo- mus cells om TASK1-null animals appea o be no - mal, bu TASK1/3 knockou cells exhibi cha ac e is ic elec ophysiological al e a ions and dec eased sensi- i i y o ex e nal pH. None heless, he sec e o y e- sponses o TASK1/3/ cells o hypoxia as well as o he chemosenso y s imuli emain essen ially unal e ed. The implica ions o hese indings o CB O2 sensing a e discussed. MATERIALS AND METHODS Animals Fo he expe imen s, we used young adul (2–6-mo-old) TASK- null mice (ei he TASK1/, TASK3/, o double TASK1/3/) and he co esponding wild- ype li e ma es p o ided by M. Alle (Ins i u o de Neu ociencias, Alican e, Spain). The non unc ional alleles we e gene a ed as desc ibed in de ail p e iously (Alle e al., 2005; B ickley e al., 2007). Mice we e geno yped as desc ibed p e iously (Alle e al., 2005; B ickley e al., 2007). Animal ca e and expe imen a ion we e pe o med acco ding o he ins i u- ional animal ca e commi ee guidelines. RNA analysis Fou animals o e e y geno ype we e killed by sodium pen oba bi al o e dose (in ape i oneally [i.p.]), and he CBs we e dissec ed, pooled, and s o ed in liquid ni ogen. mRNA was ex ac ed using Dynabeads mRNA DIRECT mic o ki (In i ogen). Fi s -s and cDNA was syn hesized om o al mRNA ex ac ion using he Supe sc ip i s -s and syn hesis sys em o e e se ansc ip ion (RT)-PCR (In i - ogen). PCR ampli ica ions o TASK1, TASK3, and GAPDH mRNAs we e pe o med using he ollowing p ime s: TASK1 (Kcnk3; 515 bp): 5-CACCGTCATCACCACAATCG-3 and 5-TGCTCTGCATCAC- GCTTCTC-3; TASK3 (Kcnk9; 413 bp), 5-ATGAGATGCGCGAG- GAGGAGAAAC-3 and 5-ACGAGGCCCATGCAAGAAAAGAAG-3; and GAPDH (255 bp): 5-CAAAATGGTGAAGGTCGGTGTG-3 and 5-TTTGATGTTAGTGGGGTCTCGC-3. Fo quan i a i e RT-PCR analysis, ou g oups o h ee young adul double TASK1/3/ o con ol mice we e killed by pen oba bi al o e dose (i.p.), and he CBs we e p ocessed as desc ibed abo e. Real- ime PCR was pe o med in an ABI P ism 7500 Sequence De ec ion Sys em (Applied Biosys ems) using SYBR G een PCR Mas e mix (Applied Biosys ems) and he he mocycle condi ions ecommended by he manu ac u e . Each sample was analyzed o cyclophilin o no malize o RNA inpu amoun s and o pe o m ela i e quan i ica ions. To no malize mRNA le els in knockou mice o hose in con ol samples, we calcula ed an a e age cycle h eshold o he con ol samples and p ocessed all he samples in he expe imen ela i e o his a e age cycle h eshold. P ime s we e designed using he compu e p og am P ime Exp ess (Applied Biosys ems). The ollowing p ime s we e used: maxi-K+ channel  subuni (Kcnma; 76 bp): 5-CATGGCTTTCAACGT- GTTCTTC-3 and 5-GCCAGAACCACAGCTTATCATTG-3; TASK5 (Kcnk15; 53 bp): 5-GCCTACTACTACTGCTTCATCACTCTCA-3 and 5-ACGAAGTCGCCGAAGCCT-3; and cyclophilin A (Ppia; 75 bp): 5-GCACTGGTGGCAAGTCCAT-3 and 5-GCCAGGACCT- GTATGCTTCAG-3. Mel ing cu e analysis showed a single sha p peak wi h he expec ed Tm o all samples. he mouse CB hin slice p epa a ion, whe e ep oducible esponses o glomus cells o chemosenso y s imuli can be ou inely ob ained (Pi ua e al., 2004; O ega-Sáenz e al., 2007). He e, we ha e e alua ed he chemosensi i i y o CB glomus cells om mice de icien o TASK channels. These belong o he andem po e domain (K2P) amily o channels and con ibu e o he leak o backg ound K+ conduc ance in a b oad a ie y o cells. TASK1 (Kcnk3 o K2P3.1) and TASK3 (Kcnk9 o K2P9.1), he ele an membe s o he TASK channel class (Dup a e al., 1997; Kim e al., 2000; Rajan e al., 2000), can o m he e ome s (Czi ják and Enyedi, 2002) and ha e been p oposed o be in ol ed in pe iphe al and cen al chemo ecep ion (Bayliss e al., 2001; Feldman e al., 2003; Mulkey e al., 2004). Recombinan TASK1 channel ac i i y is educed upon exposu e o low O2 ension (Kemp e al., 2004; Lee e al., 2006; howe e , o con as ing esul s see Johnson e al., 2004), and hese channels appea o media e he hypoxic depola iza ion o ce ebella g anule cells (Plan e al., 2002). In a CB glomus cells, an O2-sensi i e TASK-like s anding K+ cu en wi h weak ou wa d ec i- ica ion (in physiological asymme ical K+) and blocked by ex acellula Ba2+ bu esis an o he classical K+ channel blocke s TEA and 4-aminopy idyne (4-AP), has been e- po ed (Buckle , 1997). The CB s anding K+ cu en sha es o he pha macological p ope ies, such as ac i a ion by he ola ile anes he ic halo hane and inhibi ion by anan- damide, wi h cu en s media ed by TASK1 channels (Buckle e al., 2000). Mo eo e , backg ound single K+ channel cu en ac i i y in glomus cells shows licke ing kine ics and slope conduc ance compa ible wi h hose epo ed o ecombinan , he e ologously exp essed TASK1 channels (Williams and Buckle , 2004). The e o e, a popula iew is ha TASK1 channels may be unda- men al o CB O2 sensing (Dup a e al., 2007). Howe e , a de ailed ecen s udy by Kim e al. (2009) has shown ha he e ome ic TASK1/TASK3 a e he majo O2-sensi- i e backg ound K+ channels in a CB glomus cells. In ecen yea s, TASK1- and/o TASK3-de icien mice ha e been independen ly gene a ed in wo labo- a o ies ha epo ed he animals o be heal hy and wi h no mal li espan (Alle e al., 2005; B ickley e al., 2007; Mulkey e al., 2007). The absence o TASK1 and/o TASK3 esul s only in a mino pheno ype in cen al neu ons, despi e he loss o acid sensi i i y in some neu onal g oups and he comple e disappea ance o halo hane e ec on memb ane cu en s o conduc ance. In some mice s ains, in alida ion o TASK1 channels dis up s ad enal gland zona ion and p oduces hype - aldos e onism (Hei zmann e al., 2008). Howe e , he impac o TASK de iciency on pe iphe al chemo ecep- ion is a subjec o con o e sy. Al hough one g oup has epo ed no mal en ila o y esponses o hypoxia and hype capnia in he double TASK1 and TASK3 (TASK1/3/) knockou animals (Mulkey e al., 2007), on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 O ega-Sáenz e al. 381 70 K2SO4, 30 KCl, 2 MgCl2, 1 EGTA, and 10 HEPES, pH 7.2. The s anda d ba h solu ion con ained (in mM): 140 NaCl, 2.5 KCl, 10 HEPES, 10 glucose, 2.5 CaCl2, and 4 MgCl2, pH 7.4. Fo he pH expe imen s, he ex e nal solu ions con ained (in mM): 3 KCl, 118 NaCl, 1 MgCl2, 1.5 CaCl2, 25 HEPES, and 10 glucose, wi h he pH adjus ed o he desi ed le el using NaOH o HCl. We also added 10 mM TEA and 5 mM 4-AP o he solu ion o block ol age- dependen K+ channels. Es ima ed alues o es ing po en ial a e gi en a e co ec ion o junc ion po en ials. Mac oscopic Ca2+, Na+, and K+ cu en s we e eco ded in dialyzed glomus cells. The solu ions used o he eco ding o whole cell Na+ and Ca2+ cu - en s con ained (in mM): ex e nal: 140 NaCl, 9 BaCl2, 1 CaCl2, 10 HEPES, and 10 glucose; pH 7.4 and osmolali y 300 mOsm/kg; and in e nal: 110 CsCl, 30 CsF, 10 EGTA, 10 HEPES, and 4 ATP-Mg; pH 7.2 and osmolali y 285 mOsm/kg. The solu ions used o he eco ding o whole cell K+ cu en s con ained (in mM): ex e nal: 140 NaCl, 2.5 KCl, 10 HEPES, 10 glucose, 2.5 CaCl2, and 4 MgCl2, pH 7.4); and in e nal: 80 po assium glu ama e, 50 KCl, 1 MgCl2, 10 HEPES, 4 MgATP, and 5 EGTA, pH 7.2. Ampe ome ic eco ding o single-cell ca echolamine sec e ion in slices CB slices we e used because he mos ep oducible single–glomus cell esponses o hypoxia a e ob ained in his p epa a ion (Pa dal e al., 2000). Mice CB dissec ion, slicing, and cul u e, as well as he measu emen o ca echolamine sec e ion, we e pe o med ol- lowing he same p ocedu es desc ibed p e iously (O ega-Sáenz e al., 2003, 2006). CBs we e esec ed, cleaned o connec i e issue, and included in aga ose. A e moun ing he piece on he s age o a ib a ome, 150-µm hick slices we e cu , placed in a Pe i dish wi h cul u e medium (Dulbecco’s modi ied Eagle’s medium wi h 10% e al bo ine se um, 1% penicillin/s ep omycin, 1% l-glu amine, and 84 U o insulin pe ml), and main ained a 37°C in a 5% CO2 in- cuba o o 24–48 h. Slices we e ans e ed o a eco ding chambe and con inuously pe used wi h a solu ion con aining (in mM): 117 NaCl, 4.5 KCl, 23 NaHCO3, 1 MgCl2, 2.5 CaCl2, 5 glucose, and 5 suc ose. The osmolali y o he solu ion was 280 mOsm/kg. The “no moxic” solu ion was bubbled wi h a gas mix u e o 5% CO2, 20% O2, and 75% N2 (O2 ension, 145 mm Hg). Immunocy ochemis y and mo phological s udies Fo e e y geno ype, h ee animals we e killed by sodium pen o- ba bi al o e dose (i.p.), and he ca o id bi u ca ions we e dissec ed, washed wi h PBS, ixed 2 h a 4°C in 4% pa a o maldehyde, and equilib a ed o 12 h in a 30% suc ose solu ion. Bi u ca ions we e included in OCT (op imal cu ing empe a u e; Tissue Tek; Saku a) and snap- ozen by quenching in d y ice. 10-µm hick slices we e cu wi h a c yos a . Sec ions we e s ained wi h he an i– y osine hy- d oxylase (TH) polyclonal an ibody (1:1,000; Pel-F eez Biologi- cals). The En ision+ ki (Dako) was used o immunohis ochemis y acco ding o he manu ac u e ’s ecommended p o ocol. The sig- nal was de eloped wi h DAB (Dako). Images we e acqui ed unde a mic oscope (BX-61; Olympus). Es ima ion o CB olume was pe o med ac oss he en i e CB pa enchyma using he CAST G id Sys em. A Ca alie i size o 912.7 µm2 was used. Pa ch clamp eco dings Mac oscopic cu en s we e eco ded om dispe sed mouse glomus cells using ei he he pe o a ed pa ch o he whole cell con igu- a ions o he pa ch clamp echnique as adap ed o ou labo a o y (Muñoz-Cabello e al., 2005; Ga cía-Fe nández e al., 2007). P ep- a a ion o dispe sed mouse CB cells was pe o med as desc ibed p e iously (Pi ua e al., 2004; O ega-Sáenz e al., 2006). Pa ch elec odes (1.5–2.5 MΩ) we e pulled om capilla y glass ubes (1.5–1.6 mm OD; Kimax; Kimble P oduc s), i e polished on a mic o o ge MF-830 (Na ishige), and coa ed wi h silicone elas ome (Sylga d 184; Co ning) o dec ease capaci ance. Vol age clamp eco dings we e ob ained wi h an EPC-8 pa ch clamp ampli ie (HEKA) using s anda d ol age clamp p o ocols designed wi h Pulse so wa e (HEKA). Unless o he wise no ed, holding po en- ial was 80 mV. Da a we e il e ed a 10 kHz, digi ized a a sam- pling in e al o 20 µs wi h an ITC-16 A/D con e e (HEKA), and s o ed on a Macin osh compu e . O line analysis o da a was pe o med using cus om so wa e and Pulse Fi (HEKA). All ex- pe imen s we e conduc ed a oom empe a u e, 23–26°C. Expe i- men s designed o es ima e he cell’s es ing po en ial and inpu esis ance, as well as he pH dependence o he backg ound K+ cu en s, we e made using pe o a ed pa ches wi h ampho e icin B in he pipe e solu ion. This solu ion also con ained (in mM): Figu e 1. Molecula and his ological cha ac e iza ion o TASK1- and double TASK1/3–de icien mice. (A) RT-PCR analysis showing he absence o Task1 o Task3 mRNA exp ession in he CB o TASK1- o TASK1/3-null mice. (B) CB ana omy in young TASK1- o TASK1/3- de icien animals. Rep esen a i e sec ions o con ol (wild ype; le ), TASK1/ (middle), and TASK1/3/ ( igh ) ca o id bodies. CB glomus cells a e s ained wi h an an ibody agains TH. IC, in e nal ca o id a e y. (C) G aph ep esen ing he CB olume occupied by TH-posi i e cells. Da a a e n = 3 o each expe imen al condi ion. on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 382 Glomus cell O2/CO2 sensing in TASK1/3-null mice sum o he ime in eg al o successi e ampe ome ic e en s (Pa dal and López-Ba neo, 2002). Sec e ion a e (in ei he em o- o pico- coulombs/min) was calcula ed as he amoun o cha ge ans- e ed o he eco ding elec ode du ing a gi en ime pe iod. S a is ical analysis Unless o he wise speci ied, da a a e exp essed as mean ± SE, wi h he numbe (n) o expe imen s indica ed. S a is ical analysis was pe o med by unpai ed S uden ’s es . A alue o P < 0.05 was conside ed as s a is ically signi ican . Online supplemen al ma e ial Con ocal luo ime ic eco dings o in acellula Ca2+ concen a- ion in dispe sed mouse glomus cells a e shown in Fig. S1. The da a indica e ha in bo h wild- ype and TASK1/3/ glomus cells, hypoxia induces an inc ease o in acellula Ca2+ concen a ion. In Fig. S2, we show in dispe sed ch oma in cells he inhibi ion o The “hypoxic” solu ion was bubbled wi h 5% CO2 and 95% N2 o each an O2 ension in he chambe o 15 mm Hg. To pe o m dose– esponse cu es, he solu ions we e also bubbled wi h 12 and 6% O2, keeping CO2 a 5%. When hese solu ions we e used, he app oxima e alues o O2 ension in he chambe we e, e- spec i ely, 90 and 50 mm Hg. Expe imen s o sec e ion induced by hype capnia we e done wi h he same con ol solu ion bubbled wi h 10% CO2, 20% O2, and 70% N2 o 20% CO2, 20% O2, and 60% N2 (10 o 20% hype capnia, espec i ely). In hese condi- ions, ex acellula pH dec eased om 7.3 in 5% CO2 o 7.1 in 10% CO2 o 6.8 in 20% CO2. All he expe imen s we e made a a empe a u e in he chambe o 36°C. Sec e o y e en s we e e- co ded wi h a pola ized (+750 mV) 10-µm ca bon ibe elec ode posi ioned nea a cell unde isual con ol and connec ed o he cu en - o- ol age con e e o an EPC-8 pa ch clamp ampli ie . Ampe ome ic cu en s we e il e ed a 100 Hz and s o ed on a compu e . The cumula i e sec e ion signal was ob ained by he TABLE I Elec ophysiological pa ame e s o wild- ype and TASK-null mouse glomus cells Pa ame e Wild ype Task1/Task1/3/ Cell capaci ance (pF) 2.9 ± 0.1 (25) 2.7 ± 0.1 (29) 2.9 ± 0.1 (28) Inpu esis ance (GOhm) 7.8 ± 0.9 (25) 8.4 ± 0.8 (29) 10.8 ± 0.9* (31) Memb ane po en ial (mV) 57.0 ± 1.2 (24) 54.5 ± 1.8 (29) 51.5 ± 0.8* (33) Cu en densi y (20 mV, pH 8.2, pA/pF) 7.9 ± 2.7 (13) — 1.6 ± 0.5* (14) Peak K+ cu en (+30 mV, pA/pF) 362 ± 17 (13) 390 ± 21 (11) 262 ± 17** (21) Peak Na+ cu en (+10 mV, pA/pF) 26.2 ± 5.0 (11) 24.5 ± 5.2 (12) 27.5 ± 3.4 (22) Peak Ca2+ cu en (+20 mV, pA/pF) 7.5 ± 0.9 (21) 9.1 ± 1.9 (12) 3.3 ± 0.4** (33) Reco dings we e pe o med using pe o a ed pa ches wi h he excep ion o hose done o measu e mac oscopic ol age-dependen K+, Na+, and Ca2+ cu en densi y, in which we used whole cell (dialyzed) pa ch-clamped cells. Values a e gi en as mean ± SE, wi h he numbe o expe imen s in pa en heses. As e isks indica e s a is ical signi icance (*, P < 0.05; **, P < 0.01) wi h espec o co esponding alues in wild- ype cells. Figu e 2. Sensi i i y o ex e nal pH o backg ound po assium cu - en s om wild- ype and TASK- null mouse glomus cells. (A; op) Vol age amp p o ocol applied o dispe sed glomus cells s udied wi h he pe o a ed pa ch echnique. 10 mM TEA and 5 mM 4-AP we e added o he ex e nal solu ion o block ol age-dependen K+ chan- nels. (Middle) Cu en s eco ded in wild- ype glomus cells exposed o ex e nal solu ions wi h pH 6.3 and 8.2. (Bo om) Cu en s eco ded in double TASK1/3–null glomus cells exposed o he same ex e nal pH shi . (B) Quan i a i e analysis o he po assium cu en densi y (20 mV) a di e en pH o wild- ype (n = 13) and TASK1/3/-de icien (n = 14) cells. *, P < 0.05. (C) Quan i a i e analysis o he e ec o pH on inpu cell esis ance o wild- ype (n = 13) and TASK 1/3/ (n = 14) cells. **, P < 0.01. on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 O ega-Sáenz e al. 383 expe imen s we e pe o med on glomus cells om he TASK1/3 double knockou , al hough, when neces- sa y, expe imen s we e also done on single TASK1-null cells because hese las channels a e hose p oposed o pa icipa e mo e speci ically in CB O2 sensing (see Dup a e al., 2007; T app e al., 2008). In ou expe i- men al condi ions, he es ima ed es ing memb ane po- en ial o dispe sed wild- ype cells eco ded wi h he pe o a ed pa ch echnique was 57 ± 1.2 mV (n = 24); his alue was only sligh ly changed in TASK1-de icien cells (54.5 ± 1.8 mV; n = 29) bu dec eased signi ican ly ( o 51.5 ± 0.8 mV; n = 33; P < 0.05) in TASK1/3-null cells. Glomus cells om TASK1/3 knockou mice also had a s a is ically signi ican inc ease o inpu esis ance compa ed wi h con ols (Table I). Recombinan TASK1 and TASK3 channels a e highly sensi i e o ex acellula pH due o a p o on-sensing his idine esidue loca ed a he ex e nal mou h o he channels (Rajan e al., 2000). Based on pha macological expe imen s, i has been p oposed ha TASK1 channels con ibu e o pH sensing in glomus cells (Buckle e al., 2000). In cells ba hed in ex e nal solu ion con aining TEA and 4-AP o minimize he ion luxes h ough ol - age-dependen K+ channels, cu en s e oked by depo- la izing amps we e inc eased by pH alcaliniza ion (swi ching om pH 6.3 o 8.2) and, as expec ed, his e - ec was small o e en negligible in TASK1/3-null cells (Fig. 2, A and B). Upon exposu e o pH 8.2, ou wa d K+ cu en densi y a 20 mV in wild- ype cells was on a e - age i e old la ge han in cells om TASK1/3-de icien animals (Fig. 2 B and Table I). In pa allel wi h he e ec on K+ cu en densi y, abla ion o he TASK1/3 channel Ca2+-dependen mac oscopic K+ cu en s by paxilline, a selec i e blocke o maxi-K+ channels. Figs. S1 and S2 a e a ailable a h p://www.jgp.o g/cgi/con en / ull/jgp.200910302/DC1. RESULTS Mo phology o TASK-null ca o id bodies In wild- ype animals, we con i med by PCR he exp es- sion o TASK1 and TASK3 mRNAs in he CB and hei comple e disappea ance in TASK-de icien animals (Fig. 1 A). Immunocy ochemical analyses wi h an i-TH an ibodies demons a ed in all he animal ypes ha he CBs we e no mal and appea ed o ganized in he cha - ac e is ic clus e s (glome uli) o TH-posi i e glomus cells (Pa dal e al., 2007) (Fig. 1 B). The olume occupied by he CB pa enchyma was simila in TASK1 o TASK1 and TASK3 double (TASK1/3) knockou mice compa ed wi h wild- ype li e ma es (Fig. 1 C). The size o indi id- ual CB glomus cells, as indica ed by he alue o o al capaci ance measu ed in pe o a ed pa ch-clamped cells, was also simila in he h ee animal s ains (see Table I). Elec ophysiological pa ame e s and pH sensi i i y o TASK-de icien glomus cells TASK1 and TASK3 channels a e exp essed in nume ous neu al and non-neu al issues, as well as in he CB. These channels a e open o e a b oad ange o memb ane ol ages and con ibu e o se he cell’s es ing po en ial and memb ane esis ance. Hence, we sough o see whe he he absence o TASK channels esul ed in mod- i ica ions in he elec ophysiological pa ame e s o glo- mus cells. Fo he sake o simplici y, some o hese Figu e 3. Vol age-dependen K+ cu en s in mouse glomus cells e- co ded wi h he whole cell con igu- a ion o he pa ch clamp echnique. (A) Rep esen a i e amily o K+ cu - en s eco ded a a ious memb ane po en ials. The holding po en ial was 80 mV, and he depola iza ion ol ages (mV) a e indica ed nea each ace. (B) Po assium cu en – ol age ela ionship ob ained om wild- ype (n = 13), TASK1/ (n = 11), and TASK1/3/ (n = 21) glomus cells. (C) Le el o exp ession o he mRNA o maxi-K+ channel  sub- uni es ima ed by quan i a i e PCR om CB issue o wild- ype and TASK1/3-null animals. Da a a e scaled wi h espec o alues in wild- ype animals (n = 4 expe imen s). on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 384 Glomus cell O2/CO2 sensing in TASK1/3-null mice channels, he closes ela i e o TASK1 and TASK3 wi hin he TASK amily (Dup a e al., 2007), we e no signi i- can ly exp essed in he CB issue. The maxi-K+ channel  subuni , unc ionally exp essed in mouse CB (Yamaguchi e al., 2004; O ega-Sáenz e al., 2006), appea ed o be down- egula ed in TASK1/3/ animals (Fig. 3 C). Dispe sed mouse glomus cells exhibi ed ela i ely la ge Na+ and/o Ca2+ inwa d cu en s (Fig. 4 A). The cu en – ol age ela ion o he Na+ cu en was unal e ed in TASK-de icien cells, and TASK1/ cells also had Ca2+ cu en s o no mal ampli ude (Table I and Fig. 4, B and C). Howe e , in TASK1/3/ cells, he peak Ca2+ cu - en densi y dec eased o 50% o con ol alues, al- hough he ol age dependence o he cu en emained unchanged (Table I and Fig. 4 C). As hei abbi coun- e pa s (U eña e al., 1989), mouse glomus cells ha e wo well- ep esen ed dis inc popula ions o Ca2+ chan- nels ha we e easily sepa a ed by hei deac i a ions genes also abolished he dec ease o memb ane esis- ance induced by alcaliniza ion (Fig. 2 C). Collec i ely, hese da a sugges ha he lack o TASK channels in CB glomus cells (pa icula ly TASK3) does indeed esul in consis en al e a ions o hei elec ophysiological pa- ame e s, as well as he esponsi eness o changes in ex acellula pH. To u he in es iga e he TASK-null CB pheno ype, we measu ed he densi y o ol age-dependen K+, Na+, and Ca2+ cu en s in wild- ype and TASK1 o TASK1/3 knock- ou glomus cells. La ge ol age-dependen ou wa d K+ cu en s we e eco ded in he h ee glomus cell ypes (Fig. 3 A). The ampli ude o he cu en was unchanged in TASK1/ cells, bu a clea educ ion o K+ cu en densi y (30%) was obse ed in TASK1/3/ p epa a- ions (Fig. 3 B and Table I). We ha e es ed by quan i a- i e RT-PCR whe he he mRNA exp ession o o he K+ channel genes is al e ed in TASK1/3/ CB cells. TASK5 Figu e 4. Vol age-dependen Na+ and Ca2+ cu en s in mouse glomus cells eco ded wi h he whole cell con igu- a ion o he pa ch clamp echnique. (A) Rep esen a i e mac oscopic so- dium (INa) and calcium (ICa) cu en s eco ded in a cell du ing a depola iza- ion o +20 mV om a holding po en- ial o 80 mV. The decay o he ail cu en gene a ed on epola iza ion o 70 mV e lec s he closing ime cou se o he channels open du ing he pulse. (B) Peak sodium cu en – ol - age ela ionship o wild- ype (n = 11), TASK1/ (n = 12), and TASK1/3/ (n = 22) glomus cells. (C) Peak cal- cium cu en – ol age ela ionship o wild- ype (n = 18), TASK1/ (n = 12), and TASK1/3/ (n = 24) glomus cells. (D) Single-exponen ial unc ions i ed o he Ca2+ ail cu en (da k g ay, as deac i a ing componen ; ligh g ay, slowly deac i a ing componen ). The as and slow ime cons an alues in his ex- ample a e, espec i ely, 0.11 and 1.21 ms. (E and F) Quan i a i e analysis o he cu en densi y o as and slowly deac i- a ing componen s o Ca2+ ail cu en s om wild- ype (n = 25), TASK1/ (n = 12), and TASK1/3/ (n = 34) glo- mus cells. *, P < 0.05; **, P < 0.01. on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 O ega-Sáenz e al. 385 he cellula le el a e almos simila o he co ela ion be ween a e ial PO2 and he a e en discha ges o he CB sinus ne e eco ded in i o o in he explan ed CB in i o (Fig. 6, A and B). kine ics (a 70 mV:  as = 0.123 ± 0.009 ms and  slow = 1.60 ± 0.12 ms; n = 25; Fig. 4 D). These co espond o high h eshold, o as deac i a ing, and low h eshold, o slowly deac i a ing, Ca2+ channels, which in neu o- sec e o y cells can con ibu e o ansmi e elease (see Ca abelli e al., 2007; Le i sky and López-Ba neo, 2009). The deac i a ion ime cons an s o hese channels ypes we e also indis inguishable be ween wild- ype and TASK- null cells (TASK1/:  as = 0.093 ± 0.006 ms and  slow = 1.47 ± 0.09 ms; n = 12; TASK1/3/:  as = 0.106 ± 0.005 ms and  slow = 1.66 ± 0.09 ms; n = 34). The dec ease o Ca2+ cu en densi y obse ed in TASK1/3/ glomus cells was mainly due o dec ease o he high ol age– ac i a ed cu en , he mos p edominan componen in glomus cells (Fig. 4 E). Ne e heless, he densi y o he low ol age–ac i a ed cu en was also signi ican ly e- duced in he TASK1/3 knockou cells (Fig. 4 F). Chemosenso y esponses o hypoxia o glomus cells om TASK-null mice Responsi eness o CB glomus cells o hypoxia in TASK- de icien mice was s udied using hin CB slices, whe e he in insic O2 sensi i i y o in ac glomus cells can be e alua ed sepa a ely om he o he s eps along he chemo- senso y pa hway in ol ed in he hypoxic en ila o y esponses (Pi ua e al., 2004; O ega-Sáenz e al., 2006). Rep esen a i e eco dings o ca echolamine elease om indi idual glomus cells subjec ed o low O2 en- sion (PO2, 15 mm Hg) a e illus a ed in Fig. 5 A, and a quan i a i e summa y o he sec e ion a e in no moxic and hypoxic condi ions is shown in Fig. 5 (B and C). Sec e ion a e induced by hypoxia in wild- ype glomus cells (5,133 ± 1,010 C/min; n = 7) was simila o he al- ues obse ed in cells de icien o ei he TASK1 (5,175 ± 719 C/min; n = 12) o TASK1/3 (5,939 ± 1,595 C/min; n = 5) channels (Fig. 5 B). Basal sec e ion in no moxic condi ions was, howe e , o e wo old highe in TASK1/3- null cells han in he wo o he glomus cell ypes (Fig. 5 C). As he Ca2+ channel densi y was educed in TASK1/3/ cells, we also es ed ha , simila o o he oden glomus cells (Pa dal e al., 2000; Pi ua e al., 2004), he sec e- o y esponse o hypoxia was abolished by blockade o Ca2+ channels wi h Cd2+ (Fig. 5 A, inse , bo om), hus sugges ing ha i was igge ed by ansmemb ane Ca2+ in lux. P elimina y expe imen s pe o med on dispe sed glomus cells also indica e ha hypoxia induces simila ises o cy osolic [Ca2+] in wild- ype and TASK1/3-null cells (Fig. S1). Sensi i i y o hypoxia o TASK-null glomus cells was u he s udied by dose– esponse expe imen s in which mouse CB slices we e exposed o a ious PO2 le els. Glomus cells showed a g aded esponse upon exposu e o p og essi ely highe hypoxia wi h a cha ac e is ic hype bolic co ela ion ha was p ac ically simila in all he animal s ains s udied (wild ype, TASK1 null, and TASK1/3 null). These dose– esponse ela ionships a Figu e 5. Responsi eness o acu e hypoxia o CB glomus cells om wild- ype, TASK1-, and double TASK1/3–null mice. (A) Am- pe ome ic eco dings and co esponding cumula i e sec e ion signals (in pC) o ca echolamine elease induced by hypoxia (O2 ension, 15 mm Hg) in CB glomus cells om he di e en mice s ains s udied. The inse a he bo om o he panel shows ha he sec e o y esponse induced by hypoxia in TASK1/3-null glo- mus cells was inhibi ed by blockade o Ca2+ channels wi h 0.2 mM Cd2+. (B) Quan i ica ion o he inc ease in sec e ion a e (pC/min) induced by hypoxia. S a is ical signi icance (P < 0.05) wi h espec o basal alues in no moxia (O2 ension, 145 mm Hg; n = 7, 12, and 5 o wild- ype, TASK1/, and TASK1/3/ cells, espec- i ely). (C) Basal sec e ion a e o glomus cells om he a ious mice s udied in no moxic condi ions. Basal sec e ion a e (in C/min) in TASK1/3-null cells was signi ican ly di e en (P < 0.05) wi h espec o alues in wild- ype cells. on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 386 Glomus cell O2/CO2 sensing in TASK1/3-null mice s udied whe he in he mu an animals he O2-sensi i e maxi-K+ cu en was necessa y o compensa e o he lack o TASK channels. In hese expe imen s, maxi-K+ chan- nels we e blocked wi h paxilline, a b oadly used selec i e an agonis (G ibko e al., 1996; Sheehan e al., 2009) ha in ou expe imen al condi ions ully inhibi ed maxi-K+ channels (Fig. S2). In he p esence o high con- cen a ions o paxilline, he sec e o y esponse o hypoxia o TASK1/3-de icien cells emained unal e ed (Fig. 7 B). Chemosenso y esponses o hype capnia and hypoglycemia o glomus cells om TASK-null mice The CB is a polimodal chemosenso y ecep o ha be- sides hypoxia o pH is also ac i a ed by hype capnia and hypoglycemia (Pa dal and López-Ba neo, 2002; Ga cía- Fe nández e al., 2007; Zhang e al., 2007; Fi zge ald e al., 2009). The esponsi eness o wild- ype and TASK1/3 double knockou glomus cells o hype capnia was ana- lyzed keeping cons an O2 ension and changing CO2 concen a ion om 5 o 10 and 20%. Exposu e o high CO2 ension e oked a su ge o ansmi e elease om glomus cells ha , al hough weake han he esponse o hypoxia, was also concen a ion dependen (Fig. 8 A). As no iced in a p e ious se o expe imen s, basal sec e- ion a e was highe in TASK1/3-de icien han in con ol glomus cells; howe e , sensi i i y o hype capnia (ei he 10 o 20%) was unal e ed by abla ion o he TASK genes (Fig. 8 B). TASK1/3-de icien glomus cells exhib- i ed a sec e o y esponse o emo al o glucose quali a- i ely simila o ha desc ibed p e iously in he a CB (Pa dal and López-Ba neo, 2002), which was also po en- ia ed upon concomi an exposu e o hypoxia (Fig. 9). DISCUSSION Mo phological and elec ophysiological p ope ies o TASK1- and TASK1/3-null glomus cells In his pape , we show ha TASK1- o TASK1/3-null mice ha e mo phologically no mal CB, wi h TH-posi i e glomus cells a anged in clus e s (glome uli) ypical o he CB pa enchyma (Pa dal e al., 2007). TASK1-de icien glomus cells had no mal passi e elec ophysiological pa- ame e s. In con as , glomus cells lacking bo h TASK1 and TASK3 channels showed a clea elec ophysiological pheno ype cha ac e ized by an inc ease o memb ane esis ance and cell depola iza ion. These obse a ions sugges ha TASK3 channels (o he e ome s o TASK1 and TASK3 channels) con ibu e o se he es ing po en- ial o no mal mouse glomus cells (see Kim e al., 2009). Backg ound K+ cu en s eco ded om TASK1/3-null glomus cells we e less sensi i e o changes in ex acellu- la pH han hose in con ol cells. This inding i s well wi h he p oposal ha TASK-like channels pa icipa e in ex acellula acid sensing in he CB (Buckle e al., 2000; o discussion on CB acid sensing see López-López To gain u he insigh in o he ole o TASK channels in CB O2 sensing, we pe o med ampe ome ic expe i- men s in no mal (wild- ype) mouse glomus cells exposed o acidic pH o minimize he ac i i y o pH-sensi i e backg ound channels. Low pH, which p esumably inhibi s TASK channels (see Fig. 2), induced sec e o y ac i i y in glomus cells bu did no p e en a ull esponse upon subsequen exposu e o hypoxia. Indeed, low pH appea ed o po en ia e he e ec o hypoxia (Fig. 7 A). Al hough TASK1/3/ glomus cells showed down- egu- la ion o bo h mac oscopic K+ cu en ampli ude (Fig. 3 B) and maxi-K+ channel mRNA exp ession (Fig. 3 C), we Figu e 6. Responsi eness o CB glomus cells o di e en le - els o hypoxia in wild- ype, TASK1-, and double TASK1/3–null mice. (A) Ampe ome ic eco dings o ca echolamine sec e ion induced by di e en le els o O2 ension in CB glomus cells om he indica ed mouse s ains (con ol, 21% O2, 145 mm Hg; 12% O2, 90 mm Hg; 6% O2, 50 mm Hg; hypoxia, 15 mm Hg). (B) Dose– esponse cu es es ima ed om glomus cells o he a ious mouse s ains exposed o solu ions equilib a ed wi h a ious le els o O2 (150, 90, 50, and 15 mm Hg). Da a a e om h ee o se en expe i- men s. The a e age sec e ion a e alues in wild- ype, TASK1/, and TASK1/3/ glomus cells a each O2 ension we e no signi i- can ly di e en (P > 0.05). on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010 O ega-Sáenz e al. 387 ni ican popula ion o low h eshold T- ype Ca2+ channels ha , as desc ibed in neona al o hypoxic adul ch oma in cells (Ca abelli e al., 2007; Le i sky and López-Ba neo, 2009), could be ac i a ed by small memb ane depola - iza ions o induce exocy osis. Indeed, we ha e obse ed an inc eased basal sec e ion a e in TASK1/3-null cells (see below), which is compa ible wi h he sus ained de- pola iza ion and inc eased exci abili y o his cell ype. Vol age-dependen (Na+, K+, and Ca2+) cu en densi- ies we e no mal in TASK1 knockou glomus cells; how- e e , K+ and Ca2+ cu en s we e ma kedly educed in TASK1/3-de icien cells. These obse a ions u he sup- po he iew ha al hough TASK1 channels migh be dispensable, TASK3 channels (ei he as homome s o TASK1/TASK3 he e ome s) a e absolu ely equi ed o he main enance o he physiological pheno ype o glo- mus cells. The dec ease o Ca2+ and K+ cu en densi y in TASK1/3 knockou cells could be he esul o an elec o- physiological emodeling induced by he pe sis en depola iza ion o hese cells. In his ega d, i is known ha L- ype Ca2+ channel  subuni is down- egula ed by ch onic depola iza ion in PC12 and smoo h muscle cells and Pé ez-Ga cía, 2007). Ou da a also ag ee wi h p e i- ous s udies epo ing ha he absence o TASK1 in ce - ebella g anule neu ons has no e ec on any o he pa ame e s (memb ane po en ial and esis ance, as well as densi y o ol age-dependen channels) analyzed (Alle e al., 2005; Mulkey e al., 2007). Abla ion o he TASK1 gene does no seem o induce up- egula ion o o he TASK channels; howe e , some cen al neu ons lose hei sensi i i y o pH o halo hane, hus sugges ing ha o he K+ channels (o eplacemen o TASK1/TASK3 he e ome s by TASK3 homome s) compensa e o he lack o TASK1 (Alle e al., 2005; Mulkey e al., 2007). In con as , neu ons wi hou TASK3 (o TASK1/3) chan- nels exhibi inc eased exci abili y and al e a ion in hei i ing equency due o Na+ channel inac i a ion (B ickley e al., 2007). These esul s, in acco d wi h he elec o- physiological pheno ype obse ed in TASK1/3-null glomus cells, sugges ha in some neu ons, TASK3 chan- nels ( ha ha e la ge conduc ance han TASK1) a e ab- solu ely equi ed o he main enance o hei no mal es ing po en ial. In he case o mouse glomus cells, his could be o c i ical impo ance because hey ha e a sig- Figu e 7. Main enance o he sec e- o y esponse o hypoxia in wild- ype and TASK1/3/ glomus cells a e blockade o di e en po assium chan- nels. (A) Ampe ome ic eco ding and co esponding cumula i e sec e ion signal (in pC) o ca echolamine e- lease induced by hypoxia (15 mm Hg) in wild- ype CB glomus cells be o e and a e blockade o TASK channels wi h acidic pH. The composi ion o he con- ol solu ion (pH 7.4) is indica ed in Ma e ials and me hods and con ained 23 mM HCO3 . In he acidic (pH 6.5) solu ion, his was eplaced wi h 10 mM HCO3  plus 12 mM NaH2PO4 and 1 mM Na2HPO4. Sec e ion a e du ing hypoxia and acidic hypoxia we e 3,694 and 5,888 C/min, espec i ely. (B) Se- c e o y esponse o hypoxia and co e- sponding cumula i e sec e ion signal (in pC) in TASK1/3-null mice glomus cells in he p esence o he maxi-K+ chan- nel blocke paxilline (500 nM). Sec e ion a e du ing hypoxia was 3,193 C/min and changed o 4,095 C/min in he p esence o paxilline. on Sep embe 22, 2014jgp. up ess.o gDownloaded om Published Ma ch 29, 2010