A Small Domain in the N Terminus of the Regulatory a-Subunit Kv2.3 Modulates Kv2.1 Potassium Channel Gating

A Small Domain in he N Te minus o he Regula o y a -Subuni K 2.3 Modula es K 2.1 Po assium Channel Ga ing Ma ı´a Dolo es Chia a, F ancisco Monje, An onio Cas ellano, and Jose´Lo´ pez-Ba neo Depa amen o de Fisiologı´a Me´ dica y Bio ı´sica, Facul ad de Medicina, Uni e sidad de Se illa, E-41009 Se illa, Spain Recen wo k has demons a ed he exis ence o egula o y K 1 channel a -subuni s ha a e elec ically silen bu capable o o ming he e o e ame s wi h o he po e- o ming subuni s o modi y hei unc ion. We ha e in es iga ed he molecula de- e minan o he modula o y e ec s o K 2.3, a silen K 1 chan- nel a -subuni speci ic o b ain. This subuni induces on K 2.1 channels a ma ked decele a ion o ac i a ion, inac i a ion, and closing kine ics. We cons uc ed chime as o he K 2.1 and K 2.3 p o eins and analyzed he K 1 cu en s esul ing om he coexp ession o he chime as wi h K 2.1. The da a indica e ha a egion o 59 amino acids in he N e minus, adjacen o he i s ansmemb ane segmen , is he majo s uc u al elemen esponsible o he egula o y unc ion o K 2.3. The sequence o his domain o K 2.3 is highly di e gen compa ed wi h he same egion in he o he channels o he K 2 amily. Replace- men o he egula o y agmen o K 2.3 by he equi alen o K 2.1 leads o loss o modula o y unc ion, whe eas gain o modula o y unc ion is obse ed when he K 2.3 agmen is ans e ed o K 2.1. Thus, his s udy iden i ies a N- e minus domain in ol ed in K 2.1 channel ga ing and in he modula ion o his channel by a egula o y a -subuni . Key wo ds: molecula di e si y; b ain po assium channels; egula o y a -subuni ; s uc u e– unc ion ela ionships; ga ing; modula ion; he e ome ic channels Vol age-ga ed K 1 channels (K channels) a e mul i-subuni ansmemb ane p o eins necessa y o ac ion po en ial epola - iza ion and egula ion o epe i i e i ing (Hille, 1992). Al hough hese channels a e unc ionally di e se, hey sha e a common s uc u e ha consis s o ou homologous a -subuni s, each one wi h six ansmemb ane segmen s lanked by in acellula N- and C- e minal domains (Rudy, 1988; Pongs, 1992; Jan and Jan, 1994). Di e si y o K 1 channels a ises om he exis ence o mul iple genes encoding po e- o ming a -subuni s g ouped in se - e al amilies (Chandy and Gu man, 1993). Molecula di e si y is u he inc eased by he abili y o di e en a -subuni s o coas- semble as he e o e ame s (Isaco e al., 1990; Ruppe sbe g e al., 1990; Co a ubias e al., 1991; Li e al., 1992; Sheng e al., 1993; Wang e al., 1993). A new mechanism o gene a e K 1 channel di e si y has e- cen ly been p oposed a e he cloning o “ egula o y a -subuni s,” which canno p oduce unc ional channels by hemsel es bu a e able o coassemble wi h o he K a -subuni s o o m he e ome s wi h speci ic unc ional cha ac e is ics. One o he i s silen a -subuni s ecognized as egula o y was iden i ied in ou labo a- o y and designa ed as K 2.3 because o i s high sequence simi- la i y and unc ional in e ac ion wi h K 2.1 channels (Cas ellano e al., 1996, 1997). Hugno e al. (1996) independen ly cloned he same p o ein and called i K 8.1. Coexp ession o K 2.3 (o K 8.1) and K 2 channels (K 2.1 o K 2.2) esul s in mac oscopic K 1 cu en s wi h slowed kine ics and al e ed ol age dependence (Cas ellano e al., 1996, 1997; Salinas e al., 1997a). Besides K 2.3, o he silen a -subuni s wi h a possible egula o y unc ion ha e been iden i ied (Pos e al., 1996; Pa el e al., 1997; Salinas e al., 1997b; K ame e al., 1998). Mos o he egula o y subuni s s udied so a seem o in e ac speci ically wi h K 2 channels. These channels a e b oadly dis ibu ed in he mammalian b ain, and he K 2.1 ype appea s o be pa icula ly impo an in egu- la ing neu onal exci abili y because i is exp essed in i ually e e y ne e cell, being a majo con ibu o o he delayed ec i ie K 1 cu en in hippocampal neu ons (T imme , 1991; D ewe e al., 1992; Hwang e al., 1992, 1993; Mu akoshi and T imme , 1999). K 2.3 is speci ically exp essed in he b ain and, like K 2.1, i is ound a high le els in he hippocampus and neoco ex (T imme , 1991; Hugno e al., 1996; Cas ellano e al., 1997). Hence, he selec i e coexp ession o K 2.3 and K 2.1 in indi id- ual neu ons could be a mechanism in ol ed in he ine egula ion o hei in insic elec ophysiological p ope ies. Because K 1 channel modula ion by egula o y a -subuni s is a no el concep o b oad unc ional in e es , he p esen wo k was unde aken o iden i y he molecula de e minan o he e ec o K 2.3 on K 2.1. We show ha he modula o y ac ion o K 2.3 depends on a domain o 59 amino acids loca ed a he N e minus ha pa icipa es in he no mal ga ing o K 2.1 channels. The sequence o his agmen o K 2.3 is highly di e gen wi h espec o he same egion in K 2.1 and K 2.2 channels. A p elimina y accoun o hese da a has appea ed in abs ac o m (Chia a e al., 1998). MATERIALS AND METHODS Plasmid cons uc ions All cDNAs encoding wild- ype o chime ic a -subuni s we e cloned in o he p513 euka yo ic exp ession ec o (a de i a i e o pSG5; S a agene, La Jolla, CA) using s anda d cloning echniques (Samb ook e al., 1989). The cons uc ion o he a ious K 2.1/K 2.3 chime as was as ollows. Chime a Ch1. App op ia e p ime s we e used o ampli y he 176 C- e minal amino acids o K 2.3 and o c ea e a silen BamHI si e in he Recei ed Ma ch 25, 1999; e ised June 1, 1999; accep ed June 4, 1999. This esea ch was suppo ed by g an s o he Spanish Minis y o Educa ion and o Fundacio´n La Caixa. F.M. is a ecipien o a ellow/c edi om Colciencias (Colombia). We hank Rica do Pa dal and Emilio Fe na´ndez Espejo o help in he analysis o Zn 21 blockade o ecombinan K 1 channels. D s. Chia a and Monje con ibu ed equally o his wo k. Co espondence should be add essed o D . Jose´Lo´pez-Ba neo, Depa amen o de Fisiologı´a Me´dica y Bio ı´sica, Facul ad de Medicina, A enida Sa´nchez Pizjuan, 4 E-41009 Se illa, Spain. Copy igh © 1999 Socie y o Neu oscience 0270-6474/99/196865-09$05.00/0 The Jou nal o Neu oscience, Augus 15, 1999, 19(16):6865–6873 sequence encoding he K 2.3-S4 segmen . The PCR-ampli ied p oduc was diges ed wi h BamHI and KpnI, and he isola ed DNA agmen was used o eplace he BamHI-KpnI segmen o K 2.1 cDNA, which encodes he 549 C- e minal amino acids o his channel. In his cons uc , he 121 N- e minal amino acids o K 2.1 we e dele ed by diges ion wi h EcoRI and ClaI and eplaced by an EcoRI-ClaI agmen o he K 2.3 cDNA (con aining amino acids 1–146). Chime a Ch2. We sequen ially used in ame he sequences encoding he i s 314 amino acids o K 2.3, ollowed by amino acids 293–415 o K 2.1 and he 65 C- e minal amino acids o K 2.3. The coding sequences we e ampli ied by PCR om he p513-K 2.1 o p513-K 2.3 plasmids using he app op ia e p ime s ha in oduced silen mu a ions o c ea e BglII and EcoRI si es on he sequences encoding he K 2.1/K 2.3 u- sions, i.e., he beginning o he S4 and he end o he S6 segmen s, espec i ely. Chime a Ch3. The p513-Ch3 plasmid was ob ained using speci ic p ime s con aining EcoRI (59p ime ) and BglII (39p ime ) o syn hesize by PCR he sequence encoding he i s 314 amino acids o K 2.3. The ampli ied agmen was diges ed wi h EcoRI and BglII and cloned in o equally diges ed p513 plasmid. This cons uc was hen linea ized wi h BglII and liga ed o a BglII agmen con aining he sequence encoding he 560 C- e minal amino acids o K 2.1. Subsequen ly, an EcoRI-ClaI agmen encoding he i s 146 amino acids o K 2.3 was eplaced by he equi alen agmen o K 2.1, which encodes i s i s 121 amino acids. Chime a Ch4. A silen poin mu a ion, which gene a es an EcoRI si e, was in oduced in o he nucleo ide 666 o he K 2.1 cDNA by using he Al e ed Si es II in i o Mu agenesis Sys ems (P omega, Madison, WI) acco ding o he manu ac u e ’s ins uc ions. This cons uc , named p513-K 2.1/RI, was diges ed wi h EcoRI and A lII o dele e he amino acids 217–803 o K 2.1. The esul ing 5.7 Kb agmen was gel pu i ied and liga ed o an EcoRI/A lII PCR agmen con aining amino acids 244–314 o K 2.3, ollowed by amino acids 293–803 o K 2.1. The PCR-ampli ied p oduc was syn hesized using he Ch3 cDNA and p im- e s ha ca y EcoRI and A llII si es. Chime a Ch5. This cons uc was made by eplacing he ClaI-EcoRI agmen o he p513-K 2.1/RI plasmid, which encodes amino acids 122–214 o K 2.1, wi h a ClaI-EcoRI PCR agmen con aining he sequence encoding amino acids 147–241 o K 2.3. Chime a Ch6. The p513-Ch6 plasmid was made by eplacing he ClaI-EcoRI agmen o Ch5 cDNA wi h a ClaI-EcoRI PCR agmen con aining he K 2.1 coding sequence om amino acid 122 o 178, ollowed by an EcoRI-EcoRI PCR agmen con aining amino acids 208–241 o K 2.3. Chime a Ch7. The p513-Ch7 cons uc was made as he p513-Ch6 plasmid, excep ha he ClaI-EcoRI and he EcoRI-EcoRI PCR ag- men s con ain he K 2.3 coding sequence om amino acid 147 o 206 and he K 2.1 coding sequence om amino acid 179 o 214, espec i ely. Chime a Ch8. To make his cons uc , we syn hesized by PCR he sequence encoding amino acids 122–179 o K 2.1 using p ime s con ain- ing ClaI(59p ime ) and EcoRI (39p ime ) si es. The ampli ied agmen was diges ed wi h ClaI and EcoRI and cloned in o equally diges ed pBluesc ip SK 1/2 plasmid. This cons uc was hen diges ed wi h EcoRI and SmaI and liga ed o an EcoRI/SmaI-diges ed PCR agmen con- aining he 295 C- e minal amino acids o K 2.3. Then, he K 2.1-K 2.3 used coding sequences we e ob ained om his cons uc by diges ion wi h ClaI and No I and used o eplace he ClaI-No I agmen (357 las amino acids o K 2.3) in he p513-K 2.3 plasmid. The sequences o he chime ic cDNAs we e e i ied by es ic ion enzyme analysis and DNA sequencing In i o ansc ip ion and ansla ion In i o ansla ions we e pe o med using 0.5 m g o he indica ed plasmids in 12.5 m l o TNT-coupled ansc ip ion– ansla ion eac ion (P omega) as pe he manu ac u e ’s ins uc ions. [ 35 S]Me hionine- labeled p o eins we e esol ed in a 9% SDS-polyac ylamide gel and isualized by au o adiog aphy. All he chime as s udied (Ch1–Ch8) we e ansc ibed in i o in o p o eins o he p edic ed molecula weigh . Func ional exp ession o ion channels and elec ophysiological measu emen s Func ional exp ession o he a ious K 1 channel a -subuni s was done using Chinese hams e o a y (CHO) cells g own in McCoy’s 5A cul u e medium (BioWhi ake , Walke s ille, MD) supplemen ed wi h L-glu amine and an ibio ic solu ions. CHO cells we e ansien ly ans- ec ed wi h 1–6 m go heK 1 channel cDNAs by elec opo a ion using a Gene Pulse appa a us (Bio-Rad, He cules, CA). In all expe imen s, 2 m g o he g een luo escen p o ein cDNA was co ans ec ed wi h he a -subuni cDNAs o iden i y by luo escence he cells ha had been e icien ly ans ec ed. K 1 cu en s we e eco ded 24–48 h a e elec- opo a ion using he whole-cell con igu a ion o he pa ch-clamp ech- nique as adap ed o ou labo a o y (Hamill e al., 1981; Cas ellano and Lo´pez-Ba neo, 1991). We used low- esis ance elec odes (1–3 MV), capaci y compensa ion, and sub ac ion o linea leakage and capaci y cu en s. Se ies esis ance compensa ion (up o 50%) was sys ema ically used. The holding po en ial was 280 mV in all he expe imen s. Inac i- a ion and closing a es we e es ima ed by i ing he ime cou ses o he cu en s wi h a single exponen ial unc ion. Ac i a ion kine ics we e es ima ed by he ime in e al elapsed be ween 20 and 80% o maximal cu en ampli ude (20–80% ise ime). Inhibi ion by ex e nal Zn 21 was calcula ed om he cu en ampli ude measu ed a he end o 200 msec pulses. K 2.1 channels a e ela i ely esis an o inhibi ion by ex e nal Zn 21 in he millimola ange (De Biasi e al., 1993a; Cas ellano e al., 1997), bu sensi i i y o Zn 21 blockade inc eases wo o h ee imes in he e ome ic K 2.31K 2.1 channels (Cas ellano e al., 1997). In p elim- ina y expe imen s, we es ed he po ency o a ious concen a ions o ex e nal Zn 21 (0.1–5 mM) o inhibi he mac oscopic K 1 cu en s me- dia ed by ei he homome ic K 2.1 o he e ome ic K 2.31K 2.1 channels. In he p esen wo k, we used ou inely 1 mMZn 21 because, in hese condi ions, educ ion o cu en ampli ude was ai ly e e sible, hus allowing a quan i a i e compa ison o Zn 21 blockade o he a ious channels s udied. Reco e y o cu en ampli ude a e Zn 21 washou was slow and in many cases incomple e when highe concen a ions o he ca ion we e used. S anda d composi ion o he ex e nal solu ions we e (in mM): 140 NaCl, 2.7 KCl, 2.5 CaCl 2 , 4 MgCl 2 , and 10 HEPES, pH 7.4. In some expe imen s, 1 mMZnCl 2 was added o his solu ion. To s udy deac i a ion kine ics, 70 mMNaCl was eplaced by 70 mMKCl. Pipe e solu ion was (in mM): 80 KCl, 30 K-glu ama e, 20 K- luo ide, 4 ATP-Mg, 10 EGTA, and 10 HEPES, pH 7.2. In he ables and ex , a e age alues o he kine ic pa ame e s o he po assium cu en s a e gi en by mean 6 SD and in pa en heses he numbe o obse a ions. S a is ical analysis o Zn 21 blockade was done using he nonpa ame ic Mann–Whi ney U es . RESULTS K 2.3 in e ac s selec i ely wi h K 2.1 channels K 2.3 (o K 8.1) p oduce p o ound unc ional changes o K 2.1 o K 2.2 channels exp essed in CHO (Cas ellano e al., 1996, 1997) and COSm6 (Salinas e al., 1997a) cells, espec i ely. In con as , K 2.3 does no modi y he K 1 cu en kine ics o Rbk1 (K 1.1) o Shake B channels (Cas ellano e al., 1997). To u he e alua e he speci ici y o he in e ac ion o K 2.3 wi h o he K a -subuni s, we ex ended ou p e ious s udy o a ious channels ep esen a i e o ou gene ic amilies o ol age-dependen K 1 channels. Figu e 1 shows ypical eco ds o po assium cu en s elici ed by sho - and long-las ing depola izing pulses applied o ol age-clamped cells ans ec ed wi h he indica ed K 1 channel a -subuni s. The cu en aces a e scaled o he same peak am- pli ude o acili a e he compa ison o he ac i a ion (Fig. 1, le ) and inac i a ion ( igh ) kine ics in he a ious expe imen al con- di ions. No e ha coexp ession o K 2.3 wi h K 1.2 (A), K 3.3 (C), and K 4.2 (D) esul ed in cu en s wi hou app eciable al e a ions compa ed wi h hose ob ained when he channels we e exp essed alone. Deac i a ion ime cou ses o hese h ee channel ypes we e also unal e ed by coexp ession wi h he egula o y a -subuni (da a no shown). As desc ibed p e iously (Cas ellano e al., 1997), coexp ession o K 2.3 wi h K 2.1 leads o ma ked decele a ion o ac i a ion and inac i a ion (Fig. 1B), as well as channel closing (see Fig. 3). These esul s indica e ha , as sug- ges ed in ou p e ious wo k (Cas ellano e al., 1997), K 2.3 in e ac s selec i ely wi h channels o he K 2 amily. 6866 J. Neu osci., Augus 15, 1999, 19(16):6865–6873 Chia a e al. •K 1 Channel Ga ing Al e ed by a Regula o y a -Subuni Iden i ica ion o he s uc u al domain esponsible o he modula o y ac ion o K 2.3 To iden i y he molecula de e minan o K 2.3 modula ing K 2.1 channel ga ing, we cons uc ed chime ic p o eins by swapping di e en egions be ween K 2.3 and K 2.1 a -subuni s. Each o hese chime ic p o eins was coexp essed wi h K 2.1 in CHO cells o s udy he esul ing mac oscopic K 1 cu en s. Rep esen a i e cu en aces eco ded om cells ans ec ed wi h K 2.1 alone o co ans ec ed wi h K 2.1 plus ei he K 2.3 o a chime a a e shown in Figu e 2. Cu en s eco ded om co ans ec ed cells a e su- pe imposed on scaled K 2.1 cu en eco ds o acili a e compa - ison. We i s c ea ed chime as Ch1 and Ch2 by eplacing ei he o he wo hal es o he K 2.3 co e egion (S1–S6) by he co e- sponding agmen s o K 2.1. Coexp ession o Ch1 plus K 2.1 ga e ise o a po assium cu en wi h ime cou se simila o he cu en s p oduced by K 2.1 alone, whe eas coexp ession o Ch2 and K 2.1 esul ed in cu en s wi h clea decele a ion o ac i a- ion and inac i a ion kine ics. These modula o y e ec s on K 2.1 channels we e e ained in chime a Ch3, which di e s om Ch2 in ha i s N e minus (121 amino acids) and C e minus (560 amino acids) a e om K 2.1. Ch2 and Ch3, bu no Ch1, also induced a clea slowing o closing ime cou se as s udied by measu ing ail cu en s in cells exposed o high ex e nal K 1 (Fig. 3). A e age alues o he ac i a ion and inac i a ion pa ame e s ( ise ime and ime cons an , espec i ely) o cells exp essing K 2.1 o a ious ypes o he e ome ic K 1 channels a e summa ized in Table 1. Mean alues o closing kine ics a e gi en in Figu e 3 legend. The e ec s o Ch2 and Ch3 on K 2.1 appea ed o be quali a i ely simila o hose p oduced by wild- ype K 2.3 (Figs. 2, 3), hus sugges ing ha he modula o y ole o K 2.3 depended on a egula o y elemen loca ed in he egion spanning om amino acid a posi ion 147 in he N e minus (adjacen o S1) o he beginning o he S4 segmen o he K 2.3 p o ein. Ex e nal Zn 21 is known o block mo e s ongly K 2.31K 2.1 he e ome s han K 2.1 homome s (Cas ellano e al., 1997) (Fig. 4) and, al hough wi h quan i a i e di e ences, high sensi i i y o Zn 21 was main ained in he channels esul ing om he coex- p ession o K 2.1 and hose chime as conse ing any o he ex acellula domains o K 2.3 (Table 1). The mechanism o K 2.1 channel blockade by ex e nal Zn 21 is unknown, and i s cha ac e iza ion was no an objec i e o he p esen wo k; how- e e , sensi i i y o ex e nal Zn 21 was used as a ool o check whe he chime ic a -subuni s we e e ec i ely o ming oligome s wi h K 2.1. I is ob ious ha his Zn 21 block assay was pa icu- la ly impo an in he s udy o chime as ha did no al e he kine ics o K 2.1 o demons a e ha hey we e able o coas- semble wi h he K 2.1 a -subuni . Figu e 4 shows ha , ega dless o he kine ic o he cu en s, ex e nal Zn 21 blocked he e ome ic channels o med by chime as Ch1, Ch2, o Ch3 plus K 2.1 wi h Figu e 1. E ec o K 2.3 on K 1.2 (A), K 2.1 (B), K 3.3 (C), and K 4.2 (D) channels. In each case, no malized K 1 cu en aces o cells ans- ec ed wi h a K 1 channel a -subuni alone (K 1.2, K 2.1, K 3.3, and K 4.2) o wi h a 50% mix u e o each subuni and K 2.3 a e shown supe imposed. In all cases, he sho - and long-las ing depola izing pulses we e applied o 120 mV om a holding po en ial o 280 mV. Figu e 2. Compa ison o he ime cou se o K 1 cu en s eco ded om cells ans ec ed wi h K 2.1 o co ans ec ed wi h K 2.1 and K 2.3, Ch1, Ch2, o Ch3. The p oposed ansmemb ane opology o K 2.1, K 2.3, and he chime ic a -subuni s a e ep esen ed by schemes close o each se o aces. The p o ein sequences a e ep esen ed by hick lines and illed cylinde s (K 2.1) o hin lines and open cylinde s (K 2.3). Ac i a ion and inac i a ion ime cou ses o he cu en s a e shown by aces in he le and igh columns supe imposed in all cases on he same scaled K 2.1 eco ds o acili a e compa ison. In all he expe imen s, depola izing pulses we e applied o 120 mV om a holding po en ial o 280 mV. Chia a e al. •K 1 Channel Ga ing Al e ed by a Regula o y a -Subuni J. Neu osci., Augus 15, 1999, 19(16):6865–6873 6867 almos pe ec e e sibili y and signi ican ly highe po ency han ha obse ed o homome ic K 2.1 channels (Table 1). Hence, hese obse a ions sugges ed ha , like he pa en al K 2.3 p o ein, he chime ic a -subuni s we e able o o m he e ome ic channels wi h K 2.1. These he e ome s e ained he high sensi i i y o blockade by ex e nal Zn 21 independen ly o hei kine ic pa am- e e s, which appea ed modi ied only in hose channels con aining he egula o y domain o K 2.3. To de ine mo e p ecisely he loca ion o he K 2.3 egula o y domain, we cons uc ed se e al de i a i es o Ch3 (Ch4–Ch6), conse ing di e en pa s o K 2.3. Al hough wi h a iable e ec s on K 2.1, hese chime as we e also able o o m he e ome ic channels wi h K 2.1 as e idenced by he high sensi i i y o he cu en s o ex e nal Zn 21 (Fig. 5, Table 1). Fo he sake o simplici y, in s udying he e ec s o chime as de i ed om Ch3, we ocused ou analysis on ac i a ion and inac i a ion ime cou ses, al hough simila quali a i e e ec s we e obse ed on channel closing. Ch5, bu no Ch4, induced modi ica ions in he kine ic pa ame e s o he K 1 cu en simila o hose elici ed by K 2.3 (Fig. 5, Table 1). Ch51K 2.1 he e ome s appea ed o ha e a pa icula ly slow ac i a ion ime cou se, bu his was no s udied in de ail. The modula o y e ec o Ch5 was abolished by eplac- ing he agmen o he N e minus close o S1 (Fig. 5, as e isks) by he equi alen segmen o K 2.1. Coexp ession o his new chime a (Ch6) wi h K 2.1 esul ed in cu en s wi h as ac i a ion and inac i a ion ime cou ses (indis inguishable om K 2.1 cu - en s) bu high sensi i i y o ex e nal Zn 21 (Fig. 5, Table 1). The e o e, he da a sugges ed ha he N- e minal egion adjacen o S1 con ained he egula o y s uc u e o K 2.3. Fu he e i- dence suppo ing his idea was ob ained by s udying he e ec s o chime a Ch7, which only di e s om K 2.1 in ha he N- e minus agmen p oximal o S1 belongs o K 2.3 (Fig. 5). Ch71K 2.1 cu en s exhibi ed he decele a ion o ac i a ion and inac i a ion cha ac e is ic o K 2.31K 2.1 he e ome s (Fig. 5). As expec ed, because Ch7 does no con ain any o he ex acel- lula domains o K 2.3, ex e nal Zn 21 had a small inhibi o y e ec on Ch71K 2.1 channels, simila o ha o he ca ion on K 2.1 cu en s (compa e Fig. 4, op eco ds, wi h Fig. 5, igh column; Table 1). A de ini i e es in a o o he egula o y ole o he N- e minal agmen was ob ained by s udying chime a Ch8, which is almos iden ical o K 2.3, wi h he sole modi ica ion ha he N- e minal agmen adjacen o S1 is eplaced by he equi alen sequence o K 2.1. In con as o wild- ype K 2.3, coexp ession o Ch8 wi h K 2.1 ga e ise o K 1 cu en s simila o he K 2.1 cu en s. Ch81K 2.1 he e ome s we e, howe e , highly sensi i e o ex e nal Zn 21 (Fig. 5). Compa ison o he esul s ob ained wi h Ch7 and Ch8 indica ed ha he same agmen o K 2.3 ha con e ed egula o y unc ion o K 2.1 was necessa y o a oid he loss o unc ion in K 2.3 (Fig. 5). The e o e, a s e ch o 59 amino acids adjacen o S1 (Ch5,Ch7, Ch8,as e isks) cons i u es an N- e minus egula o y domain (NRD) esponsible o he modula o y ac ion o K 2.3 on K 2.1 channels. Kine ic p ope ies o chime ic (K 2.3/K 2.1) homome s The ac ha he coexp ession o K 2.3, o he chime as ha had he NRD o K 2.3, wi h K 2.1 esul ed in he e ome ic channels wi h al e ed kine ics led us o pos ula e ha he same changes should be p esen in homome ic channels o med by he a ious chime ic p o eins. Among all he chime as s udied, only Ch3– Ch7 we e capable o o ming unc ional channels by hemsel es. Rep esen a i e cu en aces ob ained om cells ans ec ed wi h cDNAs o hese chime as a e shown in Figu e 6A.Asin p e ious igu es, each se o cu en s ob ained wi h sho - and long-las ing depola izing pulses a e shown supe imposed on scaled K 2.1 cu en s o acili a e compa ison. A e age alues o he ac i a ion and inac i a ion pa ame e s a e gi en in Table 2. As expec ed, only he chime as con aining he NRD o K 2.3 (Ch3, Ch5, and Ch7) o med channels exhibi ing he decele a ion o kine ics cha ac e is ic o K 2.31K 2.1 he e ome s. Cu en s esul ing om he exp ession o Ch4 and Ch6 we e p ac ically simila o hose media ed by K 2.1. All he chime ic cu en s we e blocked by ex e nal Zn 21 wi h excellen e e sibili y bu di e - en ial sensi i i y, depending on he p esence o ex acellula domains o K 2.3 (Fig. 6B). The po ency o Zn 21 o block he chime ic cu en s was quali a i ely simila o he e ec o he ca ion on K 2.11chime a he e ome ic channels. In gene al, ho- mome ic channels made o chime ic a -subuni s exhibi ed mo e Figu e 3. Compa ison o he closing ime cou se o K 2.1 channels and he he e ome ic channels esul ing om he coexp ession o K 2.1 wi h K 2.3 o chime as Ch1, Ch2, and Ch3. Inwa d K 1 ail cu en s we e eco ded a he ins an o epola iza ion ( e ical a ow) o ei he 260 (A) o 280 (B) mV a e depola izing pulses o 120 mV. Tail cu en s a e supe imposed on he same scaled K 2.1 cu en s o acili a e compa ison. The ex e nal solu ion con ained 70 mMK 1 . Closing ime cons an s (in milliseconds) we e, a 260 mV, as ollows: K 2.1, 5.3 61.1 (6); K 2.31K 2.1, 22.6 64 (6); Ch11K 2.1, 5.2 61.1(4); Ch21K 2.1, 23 6 3 (6); and Ch31K 2.1, 21.4 66.1(6). Closing ime cons an s (in millisec- onds) we e, a 280 mV, as ollows: K 2.1, 3.5 61 (6); K 2.31K 2.1, 11.1 61.3 (6); Ch11K 2.1, 3.6 61.1(4); Ch21K 2.1, 12.3 61.1(6); and Ch31K 2.1, 11.6 62.7(6). Values a e gi en by mean 6SD, and he numbe o expe imen s is gi en in pa en heses. 6868 J. Neu osci., Augus 15, 1999, 19(16):6865–6873 Chia a e al. •K 1 Channel Ga ing Al e ed by a Regula o y a -Subuni p onounced kine ic modi ica ions and sensi i i y o ex e nal Zn 21 han he e ome ic channels esul ing om he coexp ession o he co esponding chime a and K 2.1 (compa e Tables 1, 2). Howe e , hese di e ences we e no oo la ge, sugges ing ha he p esence o one o wo mu a ed subuni s in he e ome ic channels is enough o p oduce almos ull egula o y e ec . This is con- sis en wi h p e ious obse a ions in o he he e ome ic channels in which a single subuni can impose new unc ional p ope ies (Monye e al., 1992; Waldmann e al., 1995). Thus, hese da a indica e ha he NRD is o c i ical impo ance o he ga ing o K 2.1 channels. The aligned amino acid sequences o he N- e minal egions o K 2.3 and he wo known unc ional channels o he K 2 amily (K 2.1 and K 2.2) a e shown in Figu e 7. The NRD o K 2.3 spans om esidue 148 (a ew amino acids a e he end o he B box) o nea he beginning o he i s ansmemb ane segmen S1 ( esidue 206). Gi en ha he las ew amino acids o he NRD egion a e conse ed in he h ee channel ypes, i is mos likely ha i s egula o y ole depends on he agmen be ween amino acids 148 and 196, whose sequence in K 2.3 is highly di e gen compa ed wi h he equi alen egions in K 2.1 and K 2.2 channels. The NRD amino acid sequence is almos iden ical in K 2.1 and K 2.2 channels, bu he pe cen age o iden i y in his egion o he channels wi h K 2.3 alls o ,20%. In e es - ingly, o he agmen s o he N e minus, such as he B box, in ol ed in p o ein e ame iza ion ha e an amino acid sequence much mo e conse ed among he h ee channel ypes (Cas ellano e al., 1997). DISCUSSION The majo inding in his pape is he iden i ica ion o he s uc- u al domain de e mining he egula o y e ec s o K 2.3 on K 2.1 channels. This domain is wi hin a agmen o 59 amino acids loca ed a he N e minus adjacen o he i s ansmemb ane segmen . Ou esul s s ongly indica e ha his N- e minal egion has a c i ical ole in ga ing o K 2.1 channels. Modula ion o K 2 channels by he egula o y a -subuni K 2.3 We show he e ha K 2.3 exe s a selec i e ac ion on K 2.1 channels, lea ing unal e ed he kine ics o K 1.2, K 3.3, and K 4.2 channels. In p e ious wo k, we also demons a ed ha K 2.3 does no modi y he unc ion o K 1.1 and Shake B channels (Cas ellano e al., 1996, 1997). Hugno e al. (1996) epo ed ha K 8.1 cRNA ( he hams e clone equi alen o a K 2.3) injec ed in Xenopus oocy es blocked comple ely he ex- p ession o Shab (K 2) and Shaw (K 3) channels, sugges ing ha K 8.1 was egula ing he unc ion o hese channels. Howe e , he same au ho s ha e shown ha , in mammalian cells (COSm6), K 8.1 only modula es K 2.2 wi hou a ec ing K 3.4 cu en s (Salinas e al., 1997a). Reduc ion o cu en ampli ude when Table 1. Compa ison o he kine ic pa ame e s o K 2.1 a -subuni exp essed alone o coexp essed wi h ei he K 2.3 o chime ic a -subuni s Rise ime (msec) Inac i a ion (sec) % o inhibi ion o cu en ampli ude (1 mMZn 21 ) 0mV 120 mV 140 mV 120 mV 0 mV 120 mV 140 mV K 2.1 22 66 (10) 13 63 (10) 11 62 (10) 3 61 (11) 21 64 (7) 16 64 (8) 13 68 (9) K 2.11K 2.3 51 69 (14) 21 63 (10) 19 63 (22) 12 63 (26) 40 65 (9)* 34 64 (24)* 29 64 (28)* K 2.11Ch1 21 66 (8) 13 65 (8) 10 63 (8) 5 61 (10) 39 67 (5)# 24 64 (4)§ 20 65 (4) K 2.11Ch2 86 66 (8) 43 68 (8) 24 64 (8) 13 63 (11) 66 64 (4)& 45 61 (4)# 33 64 (4)# K 2.11Ch3 100 613 (9) 58 612 (9) 34 69 (9) 19 61 (8) 50 68 (5)# 43 66 (5)# 38 66 (5)* K 2.11Ch4 21 65 (13) 12 64 (13) 10 63 (13) 4 61 (7) 56 68 (5)# 41 69 (5)# 30 66 (5)# K 2.11Ch5 97 68 (10) 78 615 (11) 49 614 (11) 14 63 (9) 66 69 (9)* 55 68 (9)* 42 66 (9)* K 2.11Ch6 22 64 (19) 12 63 (15) 10 62 (15) 2 61 (10) 42 67 (5)# 32 66 (5)# 26 65 (5)& K 2.11Ch7 106 69 (10) 42 615 (6) 14 63 (7) 21 610 (6) 15 65 (4) 13 63 (4) 11 65 (4) K 2.11Ch8 24 64 (8) 15 63 (8) 13 62 (8) 3 61 (6) 37 63 (7)* 27 63 (7)* 21 64 (7) Values a e gi en by mean 6SD, and he numbe o obse a ions is gi en in pa en heses. Inhibi ion by Zn 21 o each channel cons uc is compa ed wi h he po ency o Zn 21 o block K 2.1 channels a he co esponding memb ane po en ial. S a is ical signi icance (Mann–Whi ney U es ) is indica ed by he ollowing symbols: *p,0.001; #p,0.005; &p,0.01; and §p,0.05. Figu e 4. Blockade by ex e nal Zn 21 o K 1 cu en s eco ded om cells ans ec ed wi h K 2.1 o co ans ec ed wi h K 2.1 and K 2.3, Ch1, Ch2, o Ch3. All eco ds illus a e he e e sible (c& , con ol and eco e y) educ ion o he a ious ypes o cu en s by applica ion o 1 mMZn 21 o he ex e nal solu ion. In all expe imen s, depola izing pulses we e applied o 120 mV om a holding po en ial o 280 mV. Chia a e al. •K 1 Channel Ga ing Al e ed by a Regula o y a -Subuni J. Neu osci., Augus 15, 1999, 19(16):6865–6873 6869 K 2.3 is coexp essed wi h o he K 1 channels does no necessa ily indica e he exis ence o unc ional in e ac ion be ween he di e en a -subuni s because simila nonspeci ic educ ions in cu en ampli ude a e obse ed when K 1 channels a e coex- p essed wi h o he p o eins, such as g een luo escen p o ein o b galac osidase (Cas ellano e al., 1997; Salinas e al., 1997a). Toge he , hese da a sugges ha he physiological ole o K 2.3 (o K 8.1) is o modula e he ac i i y o he unc ional K 2 (2.1 and 2.2) channels. Exp ession o K 2.3 is es ic ed o speci ic a eas o he b ain in which he e a e also high le els o K 2.1 o K 2.2 mRNAs (T imme , 1991; D ewe e al., 1992; Hwang e al., 1992, 1993; Hugno e al., 1996; Cas ellano e al., 1996, 1997). K 2.1 and K 2.2 ha e, in gene al, a dis inc nono e lapping dis ibu ion in mammalian cen al neu ons (Hwang e al., 1992, 1993) and, hus, i is unlikely ha hese channels o m he e ome ic complexes (Blaine and Ribe a, 1998). Howe e , K 2.3 could o m he e o- me s wi h K 2.1 o K 2.2 channels o modula e hei unc ion. In ac , we ha e p elimina y indica ions ha K 2.3 and K 2.1 mRNAs can coexis in he same neu on. The exis ence o egu- la o y a -subuni s, such as K 2.3, wi h a modula o y ole on K 2.1 channels migh ha e special physiological signi icance because K 2.1 is abundan ly exp essed in he mammalian b ain (T imme , 1991; D ewe e al., 1992). K 2.1 is a majo con ibu o o he delayed K 1 cu en in hippocampal neu ons (Mu akoshi and T imme , 1999) and is localized uniquely among b ain K 1 chan- nels o la ge clus e s on he soma and on he e y p oximal po ions o dend i es (T imme , 1991; Scanne in e al., 1996; Du e al., 1998; Mu akoshi and T imme , 1999). I is possible ha K 2.1 has a majo ole in egula ing he ansmission o elec ical signals in o and ou o he neu onal soma a (Mu akoshi and T imme , 1999); hus, selec i e coexp ession o K 2.3 and K 2.1 could con e plas ici y o neu onal in eg a ion and p ocessing. Ano he modula o y e ec o K 2.3 on K 2.1 migh esul om he inc eased sensi i i y o ex e nal Zn 21 o K 2.31K 2.1 he - e ome s because in hippocampal ne e e minals, Zn 21 is highly en iched and i can each concen a ions nea he millimola ange in he synap ic cle (Huang, 1997). This ype o modula- Figu e 5. Compa ison o he ime cou se o K 1 cu en s eco ded om cells ans ec ed wi h K 2.1 o co ans ec ed wi h K 2.1 and Ch4–Ch8. The p oposed ansmemb ane opology o he chime ic a -subuni s a e ep esen ed by schemes close o each se o aces. The p o ein sequences a e ep esen ed by hick lines and illed cylinde s (K 2.1) o hin lines and open cylinde s (K 2.3). As e isks in Ch5,Ch7, and Ch8 indica e he loca ion o he egula o y domain o K 2.3. Ac i a ion and inac i a ion ime cou ses o he cu en s a e shown by aces in he le and middle columns supe imposed in all cases on he same scaled K 2.1 eco ds o acili a e compa ison. Reco ds in he igh column illus a e he e e sible (c& , con ol and eco e y) educ ion o he a ious ypes o cu en s by applica ion o 1 mMZn 21 o he ex e nal solu ion. In all expe imen s, depola izing pulses we e applied o 120 mV om a holding po en ial o 280 mV. 6870 J. Neu osci., Augus 15, 1999, 19(16):6865–6873 Chia a e al. •K 1 Channel Ga ing Al e ed by a Regula o y a -Subuni ion has a p eceden in he NMDA ecep o inhibi ion by Zn 21 (Wes b ook and Maye , 1987), which depends on he molecula subuni composi ion o he channels (Chen e al., 1997). N- e minal egula o y domain o K 2.3 and ga ing o K 2 channels We ha e iden i ied a egula o y domain (NRD) in he N e minus o K 2 channels ha de e mines he unc ional e ec s o K 2.3 on K 2.1. The sole p esence o he NRD o K 2.3 in K 2.1 con e s o he chime ic p o ein (Ch7) he abili y o modula e na i e K 2.1 channels in he same way as K 2.3. In con as , eplacemen o he NRD o K 2.3 by he same agmen o he K 2.1 p o ein esul s in a chime a (Ch8) wi h almos comple e loss o egula o y unc ion. Salinas e al. (1997a) ha e epo ed ha he e ec s o K 8.1 on K 2.1 channels a e media ed by amino acids in he S6 segmen based on he ac ha a chime a con aining om he N e minal o he po e o K 8.1 and he S6 segmen and ca boxyl end o K 1.3 (K 8/K 1) is unable o al e he p ope ies o K 2.1 cu en s. Howe e , in hese expe imen s, o ma ion o he e o- me ic channels by K 2.1 and he chime a K 8/K 1 was no di- ec ly es ed, so i is possible ha he chime a was unable o o m he e ome s wi h K 2.1 channels. Salinas e al. (1997a) ha e also shown ha a mu a ed K 8.1 subuni wi h wo amino acid eplace- men s in S6 is less e ec i e han he na i e K 8.1 o decele a e inac i a ion o K 2.1 channels. Mu a ions o S6 esidues a e known o modi y inac i a ion a e (Hoshi e al., 1991); howe e , hese changes canno ully explain he e ec o K 2.3 on K 2 channels (decele a ion o ac i a ion, closing, and inac i a ion). Ou s udy shows ha a ious chime as (e.g., Ch1 and Ch8), conse ing in ac la ge egions o he na i e K 2.3 p o ein includ- ing he S6 segmen bu lacking he NRD agmen o K 2.3, a e unable o al e he kine ics o K 2.1 cu en s. On he con a y, chime as wi h he S6 segmen o K 2.1 bu con aining he NRD o K 2.3 ha e ull egula o y e ec s on K 2 channels. Thus, he inescapable conclusion is ha he p esence o he NRD sequence in K 2.3 is he majo cause o he modula o y ac ion o he K 2.3 subuni on he K 2.1 channel. Ou expe imen s also indica e ha he NRD agmen is o pi o al impo ance o he ga ing o K 2.1 because he decele a- ion o kine ics imposed by K 2.3 in he e ome ic (K 2.31K 2.1) channels was obse ed in unc ional homome s o med by hose chime as ha ing he NRD o K 2.3 (Ch3, Ch5, and Ch7). The in acellula N- e minal egion is known o con ain conse ed, amily-speci ic, sequences (such as he A and B boxes o T1 domain) ha pa icipa e in ecogni ion and assembly o ol age- ga ed po assium channels (Li e al., 1992; Shen and P a inge , 1995; Yu e al., 1996). Howe e , he p ecise ole o he N e minus in channel ga ing is poo ly unde s ood. The e a e epo s indi- ca ing ha he N e minus de e mines he ol age-dependen ga ing beha io o eag (Scho¨nhe and Heinemann, 1996; Spec o e al., 1996; Te lau e al., 1997) and KAT amilies o channels (Ma en and Hoshi, 1998). Fo example, dele ions in he N e minus o eag channels can p oduce ol age shi s in he ac i a- ion pa ame e s and ma ked slowing o closing (Te lau e al., 1997). In addi ion, dele ions in he N and C e mini o K 2.1 channels a e also known o esul in p onounced modi ica ions o Table 2. Compa ison o he kine ic pa ame e s o K 2.1 and unc ional K 2.1/K 2.3 chime ic channels Rise ime (msec) Inac i a ion (sec) % o inhibi ion o cu en ampli ude (1 mMZn 21 ) 0mV 120 mV 140 mV 120 mV 0 mV 120 mV 140 mV K 2.1 22 66 (10) 13 63 (10) 11 62 (10) 3 61 (11) 21 64 (7) 16 64 (8) 13 68 (9) Ch3 98 615 (5) 67 610 (5) 61 615 (6) 21 63 (4) 88 68 (4)& 82 68 (3)& 59 67 (3)# Ch4 21 65 (14) 13 63 (14) 10 62 (14) 4 61 (6) 65 64 (8)* 52 67 (8)* 37 64 (8)* Ch5 110 616 (11) 91 614 (17) 68 613 (17) 18 67 (8) 72 68 (10)* 60 66 (10)* 50 68 (10)* Ch6 22 65 (16) 14 63 (16) 12 63 (16) 2 60.4 (16) 63 62 (4)& 51 64 (5)# 44 65 (5)* Ch7 110 69 (7) 62 611 (6) 18 65 (5) 13 63 (6) 14 65 (4) 12 64 (4) 10 63 (4) Values a e gi en by mean 6SD, and he numbe o obse a ions is gi en in pa en heses. Inhibi ion by Zn 21 o each channel cons uc is compa ed wi h he po ency o Zn 21 o block K 2.1 channels a he co esponding memb ane po en ial. S a is ical signi icance (Mann–Whi ney U es ) is indica ed by he ollowing symbols: *p,0.001; #p,0.005; &p,0.01; and §p,0.05. Figu e 6. Po assium cu en s media ed by he K 2.3/K 2.1-de i ed chi- me as capable o o ming unc ional homome ic channels. A,Le and middle columns, Ac i a ion and inac i a ion ime cou ses o he a ious ypes o po assium cu en s supe imposed in all cases on he same scaled K 2.1 eco ds o acili a e compa ison. B,Righ column, Re e sible (c& , con ol and eco e y aces) educ ion o he a ious ypes o cu en s by applica ion o 1 mMZn 21 o he ex e nal solu ion. In all expe imen s, depola izing pulses we e applied o 120 mV om a holding po en ial o 280 mV. Chia a e al. •K 1 Channel Ga ing Al e ed by a Regula o y a -Subuni J. Neu osci., Augus 15, 1999, 19(16):6865–6873 6871 ac i a ion and closing kine ics (VanDongen e al., 1990). Al- hough eplacemen o cys eine esidues in he N e minus o K 2.1 channels can p oduce slowing o ac i a ion (Pascual e al., 1997), he e ec s o K 2.3 on K 2.1 ac i a ion and closing mus depend on di e en esidues because he wo cys eines p esen in he NRD agmen o K 2.1 (C128 and C129) a e conse ed in K 2.3 (Fig. 7). Apa om he e ec s on ac i a ion and closing, he NRD o K 2.3 also induces a ma ked slowing o inac i a ion ime cou se. Because inac i a ion o K 2.1 channels is much slowe han ac i a ion, he a e o mac oscopic inac i a ion is no ol age-dependen and is una ec ed by mode a e decele a ion o ac i a ion kine ics. Thus, he slowing o inac i a ion induced by K 2.3 on K 2.1 cu en s is no a mani es a ion o he coupling be ween ac i a ion and inac i a ion, bu i is mos likely a esul o p ima y modi ica ion o he inac i a ion mechanism. This obse a ion is in e es ing because he mechanism o inac i a ion in K 2.1 channels is unknown, and i is belie ed o be o he C- o P- ype a he han o he N- ype and, in p inciple, independen o he N- e minal domain (Choi e al., 1991; Hoshi e al., 1991; De Biasi e al., 1993b; Lo´pez-Ba neo e al., 1993). Pa icipa ion o N and C e mini o K 2.1 in inac i a ion was al eady sugges ed by VanDongen e al. (1990), who showed ha la ge dele ions in he N e minus ( i s 139 amino acids) p oduced a slowing o inac i- a ion, which was e e ed by addi ional dele ions in he C e - minus. Howe e , he NRD egion, which de e mines he egula- o y e ec o K 2.3 on K 2.1 channels, spans om amino acid 148 o 196 and is loca ed close o S1 han he 139 amino acid agmen dele ed by VanDongen e al. (1990). In conclusion, ou esul s show ha K 2.1 channel ac i a ion and closing, as well as inac i a ion, a e egula ed by a domain (NRD) in he N e minus be ween he B box and he S1 segmen . 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