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 .
Sequence di e gence o he NRD agmen o K 2.3 wi h espec
o he same egion in K 2.1 and K 2.2 may explain he modula-
o y ole o he egula o y
a
-subuni on hese channels. Elucida-
ion o he mechanisms unde lying he in e ac ion o he NRD
wi h he main co e o he channel p o ein and cha ac e iza ion o
he p ocesses egula ing coexp ession o K 2.3 and K 2.1 chan-
nels in indi idual cen al neu ons mus be he subjec o u u e
expe imen al wo k.
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Cas ellano A, Molina A, Mells o¨m B, Na anjo JR, Lo´pez-Ba neo J
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1
channel ansc ip encodes o a egula o y
a
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