regulation of strex exon large conductance,calcium

N euroscience 149 (2007)789–803

R EG U LA TIO N O F STR EX EXO N LA R G E C O N D U C TA N C E, C A LC IU M A C TIVA TED PO TA SSIU M C H A N N ELS B Y TH E 4 A C C ESSO R Y SU B U N IT D .PETR IK A N D R .B R EN N ER *

Large-conductance,calcium -activated (so-called BK-type) potassium channels are activated by intracellularcalcium and by depolarization. In the brain BK channels are expressed in a large variety ofneurons (Tseng-C rank etal., 1996)and have diverse roles.Such roles include controlling action potential shape, regulating firing frequency (Shao etal.,1999;Faberand Sah,2002,2003)and regulating neurotransm itterrelease (H u etal.,2001;R affaelli etal.,2004).Tailoring ofBK channelbiophysicalproperties for these diverse functions is partially m ediated by alternative splicing of the pore-form ing subunit, Slo gene (Atkinson etal.,1991;Butleretal.,1993;Tseng-C rank et al.,1996;Xie and M cC obb,1998;Fettiplace and Fuchs, 1999;R am anathan etal.,1999;Yu etal.,2006;Adelm an etal.,1992).In addition a fam ily oftissue-specific accessory ( ) subunits m odulates BK channelproperties (M cM anus etal.,1995;Xia etal.,1999;Behrens etal.,2000; Brenner et al., 2000; M eera et al., 2000; W eiger et al., 2000;Lippiatetal.,2003). The brain-specific beta4 accessory subunit( 4)inhibits BK channels in sub-and low m icrom olarcalcium (Brenneretal.,2000;W ang etal.,2006).The 4 subunitconfers resistance to peptide blockers,charybdotoxin and iberiotoxin (Behrens etal.,2000;M eera etal.,2000),and slow s activation and deactivation kinetics (Brenneretal.,2000; H a etal.,2004).Finally, 4 subuniteffects on BK channels are dependenton phosphorylation status (Jin etal.,2002). W hereas the 4 subunitinhibits channelactivation,alternatively splicing ofthe BK channelSlo gene to include the so-called stress axis horm one-regulated exon (STR EX)dram atically enhances BK channelopenings (Saito etal.,1997; Xie and M cCobb,1998).The STREX nam e derives from its splicing regulation by stress-axis horm ones (Xie and M cC obb, 1998). The STR EX exon is the m ost thoroughly studied site ofalternative splicing ofthe Slo gene due to its num erous effects on BK channels. Such effects include changes in channel biophysical properties (Saito et al., 1997; Fettiplace and Fuchs, 1999; R am anathan et al., 1999), regulation by horm ones (Xie and M cC obb, 1998; M ahm oud and M cC obb, 2004; Laiand M cC obb, 2006), response to cyclic AM P (cAM P)–dependentphosphorylation (Tian etal.,2001;C hen etal.,2005)and response to oxidation status (Erxleben etal.,2002;M cC artney etal., 2005;also review ed in Shipston,2001;Fury etal.,2002). The STR EX exon resides in the C 2 site of the carboxyl term inaldom ain of the channelpore-form ing subunit. Tw o offoursplice isoform s atthe C 2 site are expressed in the brain (C hen et al., 2005; M acD onald et al., 2006). These are the cysteine-rich STR EX (Saito etal.,1997;Xie

D epartm entofPhysiology,U niversity ofTexas H ealth Science C enter atSan Antonio,7703 Floyd C urlD rive,San Antonio,TX 78229,U SA

A bstract— Large conductance (B K -type) calcium -activated potassium channels utilize alternative splicing and association w ith accessory subunits to tailor B K channelproperties to diverse celltypes.Tw o im portant m odulators of B K channel gating are the neuronal-specific beta4 accessory subunit ( 4) and alternative splicing at the stress axis horm one-regulated exon (STR EX). Individually, these m odulators affectthe gating properties ofthe B K channelas w ellas its response to phosphorylation.In this study,the com bined functionalconsequences ofSTR EX and the m ouse 4 subunit on m ouse B K channelbiophysicalproperties w ere investigated in transfected H EK 293 cells.Surprisingly,w e found that the com bined effects ofSTR EX and 4 are non-additive and even opposite for som e properties. At high calcium , 4 and the STR EX individually share properties thatprom ote BK channel opening via slow ing of deactivation. How ever, the com bined effects are a speeding of deactivation and a decreased open probability. 4 also inhibits B K channelopening by a slow ing of activation.This effectoccurs across calcium concentrations in the absence ofSTR EX,butpredom inates only atlow calcium forSTR EX containing channels.BK channelresponses to phosphorylation status are also altered by the com bination ofthe 4 subunit and STR EX. 4/STREX channels show a slow ing of activation kinetics follow ing dephosphorylation w hereas 4 channels lacking STR EX do not. In contrast, 4 confers a speeding of activation in response to cyclic A M P–dependent phosphorylation in channels lacking STR EX,butnot in channels containing STR EX.These results indicate thatthe com bination ofthe 4 subunitand STR EX confers non-additive and unique properties to BK channels.A nalysis of expression in brain slices suggests thatSTR EX and 4 m R NA overlap expression in the dentate gyrus ofthe hippocam pus and the cerebellar Purkinje cells,suggesting that these unique properties of BK channels m ay underlie B K channelgating in these cells.© 2007 IBR O .Published by ElsevierLtd.A llrights reserved. K ey w ords:hippocam pus,calcium ,potassium channel,ion, voltage clam p,phosphatase. *C orresponding author.Tel: 1-210-567-4343;fax: 1-210-567-4410. E-m ailaddress:brennerr@ uthscsa.edu (R .Brenner). Abbreviations:AP,alkaline phosphatase;BK channel,large conductance, calcium -activated potassium channel; cAM P, cyclic AM P; eG FP,enhanced green fluorescentprotein;G –V,conductance–voltage relationship;ISH ,in situ hybridization;PBS,phosphate-buffered saline; PC R , polym erase chain reaction; PFA, paraform aldehyde; pKA, protein kinase A; Q , effective gating charge of conductance– voltage relations; R C K, regulator of potassium conductance; SSC , sodium citrate sodium chloride; STR EX, stress axis horm one-regulated exon;TBS,blocking solution (pH 7.5)containing 0.9% N aC l,50 m M Tris base; V 1/2, voltage for half-m axim al activation; 4, beta4 accessory subunit.

0306-4522/07$30.00 0.00 © 2007 IBR O .Published by ElsevierLtd.Allrights reserved. doi:10.1016/j.neuroscience.2007.07.066

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and M cC obb,1998) and a second isoform thatlacks an insertion called ZER O (Xie and M cC obb,1998).R elative to ZER O BK channels,STR EX BK channels speed activation and slow deactivation kinetics resulting in a dram atic enhancem entofBK channelopenings (Saito etal.,1997;Xie and M cC obb,1998). Surprisingly,STR EX BK channels w ere proposed to enhance rather than reduce excitability ofadrenalchrom affin cells by sustaining the afterhyperpolarization ofaction potentials and rem oving sodium channelinactivation (Solaro etal., 1995;Lovelland M cC obb,2001).M ore recently ithas been show n thata gain-of-function m utation (D 434G ) ofthe Slo gene in hum ans causes epilepsy (D u etal.,2005).Sim ilarto STREX BK channels,the D 434G m utation speeds activation and slows deactivation ofBK channels (D iez-Sam pedro et al.,2006).The conceptthatBK channelgain-of-function is pro-epilepticunderscores the im portance oftightregulation of BK channel expression and biophysical properties in the C N S. Indeed deletion of the 4 subunit, w hich otherw ise inhibits BK channelactivation atallbuthigh calcium ,also enhances excitability in neurons and causes an epileptic phenotype in knockoutm ice (Brenneretal.,2005). The finding that the D 434G gain-of-function m utations resultin pathologicaleffects on neuronalexcitability suggests thatSTR EX channelexpression and gating properties should be tightly regulated to the needs ofindividualneuronaltypes. Though both STREX and the 4 subunitare expressed in the brain,the com binations ofthese tw o regulatory effectors of BK channels have not been studied.So-called type II BK channels,w hich are now know n to be a productofBK channel / 4 subunits (Brenneretal.,2005),have diverse properties in their gating kinetics and responses to kinase and phosphatase (Reinhart and Levitan, 1991). Such channel properties m ay be m ediated by a com bination ofalternative splicing ofBK channels and association w ith auxiliary subunits.Therefore,we investigated w here STR EX and 4 subunitexpression overlap in the C N S,and the consequence these tw o m odulatory elem ents have on gating properties of the channels.

EXPER IM EN TA L PR O C ED U R ES Expression ofcD N A s W e used the m ouse ZER O subunit cD N A expression vector in pcD N A3 (G enBank/EM BL/D D BJ accession no. M M U 09383), and m ouse 4 in the Invitrogen (C arlsbad, CA, U SA) vector pcD N A3.1H ygro( ). W e subcloned the m ouse STR EX from an EST C lone (# EM M 1002-4705604,O pen Biosystem s,H untsville, AL,U SA w hich is sim ilarto previously published AF295094)into m ouse ZER O subunit cD N A in pcD N A3 vector. The STR EX constructw as verified by sequencing.M ouse BK channel alone (ZER O orSTR EX splice isoform )or 4 (m olarratio 1:10)w as transfected into H EK 293T cells using 2–3 g totalD N A and 10 l Lipofectam ine R eagent(Invitrogen)per35 m m dish ofcells.After 4–5 h of incubation, the cells w ere w ashed and replated on G erm an glass coverslip (BioindustrialProducts,San Antonio,TX, U SA) and analyzed by electrophysiology for the follow ing 1–3 days. eG FP expression from cotransfection (0.2 g) of the EG FP-N 1 plasm id (C lontech Laboratories,Inc., M ountain View , C A,U SA)w as used to identify channel-expressing cells.

Patch-clam p electrophysiology M acropatch recordings w ere m ade using the patch-clam p technique in the inside-outconfiguration.Experim ents w ere perform ed at room tem perature. D ata w ere sam pled at 10-30- s intervals and low -pass filtered at8.4 kH z using the H EKA EPC 9 four-pole Besselfilter. D ata w ere analyzed w ithout further filtering. Leak currents w ere subtracted after the testpulse using P/5 negative pulses from a holding potentialof 120 m V ( 140 m V for120 M calcium ).Patch pipettes (borosilicate glass,W orld Precision Instrum ents,Sarasota,FL,U SA)w ere coated w ith Sticky W ax (Kerr C orporation, R om ulus, M I, U SA) and fire polished to 3–5 M resistance. The external recording solution (electrode solution) w as com posed of20 m M H epes,140 m M KM eSO 3,2 m M KC l, 2 m M M gC l2,pH 7.3.Internalsolutions w ere com posed ofa pH 7.3 solution of20 m M H epes,140 m M KM eSO 3,2 m M KC l,and buffered w ith 5 m M H ED TA and added C aC l2 (VW R ,W estC hester,PA,U SA) to the appropriate concentrations to give 1.0 and 5.3 M estim ated free C a2 concentrations.H igherC a2 solution (estim ated as 120 M ) w as buffered w ith 5 m M N TA.Although attem pts w ere m ade to collectdata atdifferentC a2 (Figs.1-3) from the sam e patch,the patches w ere notalw ays stable enough to collectallsolution changes.The estim ated free calcium concentration w as confirm ed using a calcium -sensitive electrode (O rion,Therm o Electron C orporation,Beverly,M A,U SA)thatw as calibrated by defined free-calcium solutions ranging from 0.01 M to 100 m M (C ALBU F1,W PI,Sarasota,FL,U SA). C onductance–voltage (G –V)relationships were obtained using a testpulse to positive potentials follow ed by a step to a negative voltage ( 80 m V) and then m easuring instantaneous tailcurrent am plitudes 200 s afterthe testpulse.V 1/2 and Z w ere determ ined by fitting G –V curves to a Boltzm ann function (G G m ax{1/ (1 e (V V 1/2)ZF/R T)})and norm alizing to the m axim um ofthe fit.At 1.0 M C a2 ,w here m axim um conductance could notbe obtained in the presence of 4,conductance w as norm alized to m axim alconductance at higher C a2 . M easurem ents of activation rates w ere obtained from currents elicited from 80 m V to m ore positive potentials.Activation and deactivation tim e constant( )w as obtained by fitting ofcurves to a single exponentialfunction,I I0e( t/).Tailcurrents w ere evoked by a 10-m s step to 160 m V (for120 M Ca2 )or 200 m V (for5.3 and 1.0 M free calcium ) and then m easured by stepping dow n to various voltages fordurations atleastgreaterthan three tim es the deactivation tim e constants.KaleidaG raph (Synergy Softw are,R eading,PA,U SA),Excel(M icrosoft,R edm ont,W A,USA) and IgorPro (W aveM etrics,Portland,O R ,U SA)w ere used forcalculations and presentation ofthe data.

Phosphorylation experim ents M acroscopic currents were recorded in buffered 5.3 M Ca2 solution foratleast5 m in before application ofalkaline phosphatase (AP, 10 D EA U/m l,Sigm a,St.Louis,M O ,U SA).Follow ing dephosphorylation,the inside-outpatches w ere perfused by buffered 5.3 M C a2 solution w ith 1 m M ATP,1 m M cAM P and 10 nM okadaic acid (allfrom Sigm a),pH 7.3.D ata w ere obtained 5 m in afterapplication of each solution change and represent m ean S.E.M . Statistical analysis used paired t-test.U n-paired t-testwas used for V 1/2 and for activation and deactivation kinetics ifthe data w ere com pared w ith or without co-expression of 4 subunit. Activation and deactivation kinetics representsubtraction ofindividualpaired data points (before vs.phosphatase,orphosphatase vs.cAM P treatm ent) ofactivation ordeactivation tim e constants ata given potential.In the figures,one asterisk represents P 0.05,two representP 0.01,and three representP 0.001.

In situ hybridizations (ISH)on free-floating brain sections Tem plates for riboprobes w ere synthesized from BK channel cD N A-specific polym erase chain reaction (PC R ) fragm ents that


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Fig.1. The 4 subunitinhibits STR EX and activates ZER O BK channels in 120 M calcium .R epresentative currenttraces forSTR EX alone (A)and STR EX 4 BK channels (B).(C )Plotofconductance versus voltage (G –V)relationship forSTR EX and ZER O BK channels w ith orw ithoutthe 4 subunit.(D )Plotofaverage V 1/2 forSTR EX,STR EX 4,ZER O ,and ZER O 4 BK channels.(E)Plotofactivation tim e constants as a function of voltage.(F)Average activation tim e constants at 80 m V.(G )G raph ofdeactivation tim e constants as a function ofvoltage.(H )Average deactivation tim e constantat 80 m V.In C ,E,F,solid sym bolrepresentBK channels w ithoutthe 4 subunit,w hereas open sym bolrepresentBK channels co-expressed w ith the 4 subunit.Squares show STR EX and triangles ZER O BK channels.In D ,F,H ,black colum ns show values forSTR EX alone, lightgray forSTR EX 4,dark gray forZER O alone,and w hite forZER O 4 BK channels.O ne asterisk represents P 0.05,tw o representP 0.01, and three representP 0.001;unpaired t-test.

w ere subcloned into the pC R -BluntVector(Invitrogen)according to m anufacturer’s instructions. cR N A for sense or anti-sense probes to a non-alternatively spliced region (representing total m RN A) and to the STR EX exon w as transcribed from linearized plasm ids using the Am pliScribe T7 Transcription Kit(EpicenterBiotechnologies, M adison, W I, U SA) w ith digoxigenin-U TP (Roche,

M annheim ,G erm any)in the ratio of6.5 m M UTP to 3.5 m M digoxigenin-U TP.For totalBK channels,residues encom passing 1659– 2093 (ofcD N A M M U09383)were used.ForSTREX BK channels, the com plete STREX insertw as used to generate a probe. Brain slice preparation for ISH w as m odified from previous m ethods (Strassle etal.,2005;O w ens etal.,2006).BK channel

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Fig.2. In 5.3 M calcium ,the 4 subunitinhibits both STR EX and ZER O BK channels by differenteffects on kinetics.R epresentative currenttraces forSTR EX alone (A)and STR EX 4 BK channels (B).(C )Plotofconductance versus voltage (G –V)relationship forSTR EX and ZER O BK channels w ith orw ithoutthe 4 subunit.(D )Plotofaverage V 1/2 forSTR EX,STR EX 4,ZER O and ZER O 4 BK channels.Activation (E)and deactivation (G )tim e constants are plotted as a function ofvoltage.(F)Activation tim e constantat 80 m V.(H )D eactivation tim e constantat 80 m V.In C ,E, F,solid sym bols representBK channels w ithoutthe 4 subunit,w hereas open sym bols are forBK channels co-expressed w ith the 4 subunit.Squares show STR EX and triangles show ZER O BK channels.In D ,F,H ,black colum ns show values forSTR EX alone,lightgray forSTR EX 4,dark gray for ZER O alone,and w hite for ZER O 4 BK channels.O ne asterisk represents P 0.05,tw o representP 0.01,and three representP 0.001; unpaired t-test.


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Fig.3. Inhibitory effects ofthe 4 subuniton STR EX BK channels are dram atically increased in low calcium .R epresentative currenttraces m easured in 1.0 M calcium forSTR EX alone (A)and STR EX 4 BK channels (B).(C )G –V relationships are plotted as a function ofvoltage.(D )Plotofaverage V 1/2 forSTR EX,STR EX 4,ZER O and ZER O 4 BK channels.Plotofactivation (E)and deactivation (G )tim e constants as a function ofvoltage. (F)Average activation tim e constants taken at 180 m V.(H )Average deactivation tim e constantat 80 m V.In C ,E,F,solid sym bols representBK channels w ithoutthe 4 subunit,w hereas open sym bols are forBK channels co-expressed w ith the 4 subunit.Squares show STR EX and triangles show ZER O BK channels.In D ,F,H black colum ns show values forSTR EX alone,lightgray forSTR EX 4,dark gray forZER O alone,and w hite forZER O 4 BK channels.O ne asterisk represents P 0.05,tw o representP 0.01,and three representP 0.001;unpaired t-test.

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4 subunitknockoutm ale m ice (Brenner etal.,2005) or control C 57BL/6J anim als from 4 to 5 m onths w ere used. All anim al procedures w ere review ed and approved by the U niversity of Texas H ealth Science C enteratSan Antonio InstitutionalAnim al C are and U se C om m ittee and carried outin accordance w ith the N ationalInstitutes ofH ealth and InstitutionalAnim alC are and U se G uidelines. Every effort w as m ade to m inim ize the num ber of anim als used and their suffering.Anim als w ere deeply anesthetized w ith isoflurane and transcardially perfused w ith an initial 50 m lsolution ofsterile,D EPC -treated 0.9% saline and then w ith 60 m lice-cold 4% paraform aldehyde in 0.1 M phosphate buffer saline (pH 7.4,PFA solution).Brains w ere rem oved and post-fixed for3 h at4 °C in 4% PFA solution.The brains w ere then placed in 0.1 M D EPC -treated phosphate buffered saline (PBS) w ith 20% nuclease-free sucrose (Sigm a)overnightin 4 °C untilthey sank. The follow ing day the brains w ere cryosectioned at40 m thickness under nuclease-free conditions. The sections w ere postfixed in 4% -PFA for15 m in and w ashed tw ice for10 m in in 0.1 M D EPC -treated PBS. Brain sections w ere pre-hybridized for3 h at42 °C in hybridization buffercontaining 5 sodium citrate sodium chloride buffer, SSC (pH 7.5) in D EPC -treated w ater, 50% form am ide, salm on sperm D N A (ssD N A,100 M ,Sigm a),transfer R N A,type X-SA (100 M ,Sigm a)and 0.1% Tw een-20 (FisherScientific,H ouston, TX, U SA). The pre-hybridized slices w ere incubated w ith the D IG -labeled riboprobes (400 ng/m lhybridization buffer)overnight at45 °C .Follow ing hybridization,the sections w ere w ashed tw ice for25 m in in 2 SSC w ith 0.2% Tw een-20 atroom tem perature, tw ice for 25 m in in 2 SSC w ith 0.2% Tw een-20 at50 °C and tw ice in 0.2 SSC w ith R N ase A (50 g/m l,Sigm a)at55 °C .The sections w ere equilibrated for2 h atroom tem perature in blocking solution (pH 7.5) containing 0.9% N aC l,50 m M Tris base,TBS (FisherScientific),0.2% Tw een-20 and 2% bovine serum album in (IC N Biom edicals,Aurora,O H ,U SA).Afterblocking,the sections w ere incubated for 1.5 h atroom tem perature w ith an anti-D IG antibody conjugated to AP (1:4000, R oche). Post-antibody w ashes included three w ashes for 25 m in of TBS w ith 2 m M levam isole (Sigm a)atroom tem perature and one 30-m in w ash in staining buffer(pH 9.5)containing 0.1 M N aC l,50 m M Tris,0.2% Tw een-20 and 2 m M levam isole for30 m in.The section w as then stained in the dark w ith nitro blue tetrazolium /5-brom o-4-chloro3-indoxyl phosphate (7.5 l/m l SB, R oche) for 1–2 h at room tem perature.The reaction w as stopped by w ashing in PBS (pH 7.5,tw ice for15 m in).The sections w ere post-fixed in 4% PFA for 15 m in follow ed by tw o briefPBS w ashes.Finally,the sections w ere dehydrated in a PBS/glycerolseries (10% ,20% ,40% ,60% and 80% glycerol)and m ounted.

Im m unohistology 4 m R N A expression can be detected utilizing a knock-in ofthe enhanced green fluorescent protein (eG FP) reporter in the 4 gene locus (Brenner et al., 2005) utilizing free floating section techniques (Strassle et al., 2005). Briefly, brain sections from eG FP/ 4 knock-in m ice w ere obtained as described above, stained w ith rabbit anti-G FP antibody (ab6556, Abcam , 1:5000 dilution)and detected w ith biotinylated goatanti-rabbitsecondary antibody at1:200 dilutions.The staining w as visualized using the Peroxidase Substrate Kit(Vector Laboratories,Burlingam e,C A, U SA).

R ESU LTS To com pare 4 subunit m odulation of BK channels, w e co-expressed ZER O orSTR EX BK channels w ith orw ithoutthe 4 subunitin H EK 293T cells.To controlinternal calcium , recordings w ere m ade of cell-excised m acropatches in the inside-out configuration. Previously, w e have show n that the effects of the 4 subunit on BK channels are calcium dependent.The 4 subunitprom otes BK channel activation at high intracellular calcium , but inhibits channelactivation atlow calcium (Brenner etal., 2000; W ang et al., 2006). Therefore, w e com pared 4 effects on BK channel splice isoform s at relatively high (120 M ),m oderate (5.3 M )and very low (1 M )calcium concentrations (Fig.1–Fig.3 respectively).These data are also sum m arized in Table 1. A thigh calcium , 4 has opposite effects on G –Vs of ZER O and STR EX channels R epresentative current traces are show n for STR EX BK channels alone (Fig.1A)and forSTR EX 4 BK channels (Fig.1B) in 120 M C a2 .M ean steady-state G –V relations are show n in Fig.1C .The voltage for half-m axim al activation (V 1/2)w as derived from individualG –V curves to quantify the effects of the 4 subunit (Fig. 1D ). W hen co-expressed w ith ZER O BK channels athigh calcium ,the 4 subunitsignificantly shifts V 1/2 tow ard negative potentials (Behrens etal.,2000;Brenneretal.,2000;W ang et al.,2006).The 4 subunitshifts V 1/2 from 5.41 9.19 m V

Table 1.Sum m ary data V 1/2 (m V) 120 M C a2 ZER O ZER O 4 STR EX STR EX 4 5 M C a2 ZER O ZER O 4 STR EX STR EX 4 1 M C a2 ZER O ZER O 4 STR EX STR EX 4

Q (e)

Act.tim e (m s)

D eact.tim e (m s)

N

5.41 29.20 62.98 31.98

9.19 6.70 6.28 6.35

1.95 1.56 4.78 2.05

0.07 0.18 1.23 0.21

0.48 1.69 0.55 0.56

0.05 0.32 0.17 0.07

4.34 16.7 6.36 3.7

1.07 5.07 1.72 0.71

10 13 8 9

31.21 61.63 18.24 2.64

8.45 5.43 3.85 5.08

1.35 1.09 1.34 1.21

0.08 0.06 0.06 0.05

1.71 8.15 2.07 1.13

0.30 1.14 0.26 0.18

1.69 5.41 11.32 2.1

0.23 0.63 3.72 0.26

9 8 8 9

91.22 166.78 70.26 183.28

3.40 6.61 5.42 9.24

1.59 1.07 1.28 1.06

0.13 0.11 0.09 0.11

2.70 87.68 2.79 234.71

0.49 6.65 0.31 78.7

0.38 1.39 1.11 3.21

0.04 0.13 0.08 0.62

16 12 11 13


(ZER O alone)to 29.20 6.70 m V (ZER O 4,Fig.1D ). H ow ever,co-expression ofthe 4 subunitw ith STR EX BK channels has opposite effects. 4 dram atically shifts STR EX V 1/2 tow ard positive potentials from 62.98 6.28 m V (STR EX alone)to 31.98 6.35 m V (STR EX 4,Fig. 1D ). Interestingly, the presence of the 4 subunit elim inates the greaterthan 50 m V difference in V 1/2 ofthe tw o splice form s,m aking theirsteady-state openings very sim ilar(Fig.1D ). An additional property of 4 and other subunits is that they reduce the slope (effective gating charge, Q ) of the G –V relations forZERO channels (W ang and Brenner, 2006;W ang etal.,2006).Sim ilarly,w e found here that 4 significantly reduces the Q forboth ZER O and STR EX channels across allcalcium concentrations (Table 1).Forexam ple,at5.3 M calcium 4 reduces Q from 1.35 0.08e to 1.09 0.06e forZER O (P 0.007),and from 1.34 0.06e to 1.21 0.05e forSTR EX channels (P 0.04).Sim ilareffects w ere seen in 1 M calcium (see Table 1). O ne distinct effectofSTR EX channels in 120 M calcium w as a significantincrease in Q thatw as notseen in ZER O channels (Fig.1C ,Q is 4.78 1.23e forSTR EX versus 1.95 0.07e for ZER O channels, P 0.026) nor in STR EX channels alone atlow er calcium (1.34 0.06e for STR EX alone in 5.3 M calcium and 1.28 0.09e forSTR EX alone in 1 M calcium ).N evertheless, coexpression of 4 w ith STR EX channels in 120 M calcium reduced Q to values sim ilar to ZER O alone or ZER O / 4 channels (STR EX alone, 4.78 1.23 e; STR EX/ 4, 2.05 0.21 e; ZER O alone, 1.56 0.18 e;ZER O / 4,1.95 0.07 e). A thigh calcium ,effects on deactivation underlie differences betw een ZER O and STR EX channels w ith 4 Fig. 1E show s average activation kinetics plotted as a function ofvoltage for both ZER O and STR EX BK channels w ith orw ithoutthe 4 subunit.The data are sum m arized in Fig.1F foractivation tim e constants taken at 80 m V.W e did notobserve a significantdifference in activation kinetics betw een STR EX alone (0.55 0.17 m s) and STR EX 4 (0.56 0.07 m s) BK channels in 120 M C a2 .H ow everactivation kinetics ofZER O BK channels w ere significantly slow ed by the presence ofthe 4 subunit (from 0.48 0.05 m s to 1.69 0.32 m s). Both STR EX and BK channel subunits slow deactivation kinetics (R am anathan etal.,1999;Brenner etal., 2000).This is show n atvarious voltages in Figs.1G and 2G , and sum m arized for 80 m V in Figs. 1H and 2H . Interestingly, w hereas both the 4 subunit and STR EX individually slow deactivation kinetics to prom ote channel openings in high calcium ,theircom bination speeds deactivation ofBK channels (Figs.1H and 2H ).W ith STR EX BK channels, the 4 subunit significantly speeds the deactivation tim e constantfrom 6.36 1.72 m s (STR EX alone) to 3.7 0.71 (STR EX 4) w hereas w ith ZER O BK channels it slow s the deactivation tim e constant (P 0.05) from 4.34 1.07 m s (ZERO alone) to 16.7 5.07 m s (ZERO 4).Thus,the opposing effects of 4 subuniton

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STREX and ZERO BK channeldeactivation kinetics underlie opposing effects on steady-state conductance. A tinterm ediate and low calcium , 4 dram atically inhibits both ZER O and STR EX conductance W hile the 4 subunit conferred opposite effects on V 1/2 w ith ZER O versus STR EX BK channels in 120 M C a2 , in 5.3 M C a2 4 inhibits both ZER O and STR EX isoform s (Fig.2A–D ).Sum m ary data in Fig.2D show thatthe 4 subunit significantly shifts V 1/2 of STR EX and ZER O m ore positive by 20 m V ( 18.24 3.85 m V, STR EX alone;to 2.64 5.08 m V,STR EX 4) and 30 m V,respectively ( 31.21 8.45 m V, ZERO alone; 61.63 5.43 m V,ZER O 4).Although the positive shifts ofSTREX and ZER O V 1/2 are sim ilar,they are m ediated by different 4 effects on channelgating kinetics.Atthis C a2 concentration,the 4 subunitinhibits STR EX BK channels by speeding deactivation,butinhibits ZER O BK channels by slow ing activation. Activation tim e constant at 80 m V for STR EX is reduced slightly (2.07 0.26 m s,STR EX alone; to 1.13 0.18 m s,STR EX 4) butis increased dram atically forZER O (1.71 0.3 m s,ZER O alone;8.15 1.14 m s, ZER O 4).O pposite effects ofthe 4 subuniton ZER O versus STR EX BK channels are also observed in deactivation kinetics (Fig. 2G ). D eactivation tim e is dram atically decreased by the 4 subunit for STR EX (Fig. 2H , 11.32 3.72 m s for STR EX alone; 2.1 0.26 m s for STR EX 4) but increased for ZER O BK channels (1.69 0.23 m s,ZER O alone;5.41 0.63 m s,ZER O 4). An interesting effectunique to STR EX channels is that deactivation tim e is actually slow erat5 M calcium than 120 M (Table 1).A sim ilareffectis seen forSTR EX 4 channels at1 M calcium as com pared w ith 5 M calcium . These findings w ere further confirm ed by paired recordings ofSTR EX channels during solution changes from 120 to 5 M calcium .W e indeed found thatSTR EX patches (seven ofeight)show ed a speeding ofdeactivation w ith an average 5.5 3.9 m s (P 0.09)increase in tim e constantat 80 m V and a 0.75 0.28 m s (P 0.015)increase in tim e constantat 120 m V as calcium w as reduced.In contrast, reducing calcium alw ays speeds deactivation tim es for ZER O and ZER O 4 (Table 1). To investigate the effects ofthe 4 subunitin calcium concentrations closer to resting cytoplasm ic levels, w e com pared STR EX BK channels alone to STR EX 4 BK channels in buffered 1.0 M C a2 solution (Fig.3). 4 In low calcium shifts BK channelG –V curves to very positive potentials w here itis notfeasible to achieve the m axim um steady-state conductance. Therefore, recordings from each patch in low calcium w ere subsequently exposed to high C a2 (120 M )to norm alize to the m axim alconductance.R epresentative currenttraces are show n forSTR EX alone (Fig. 3A) and STR EX 4 BK channels (Fig. 3B). Fig.3C show s G –V curves forallfourvariants as a function of voltage. Plots of V 1/2 values (Fig. 3D ) show that 4 dram atically shifts both STR EX and ZER O BK channels to positive potentials, w ith a greater effect on STR EX BK channels.The 4 subunitshifts V 1/2 ofSTR EX BK channels from 70.26 5.42 m V to 183.28 9.24 m V. V 1/2 of

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ZER O BK channels is also significantly shifted by the 4 subunit to m ore positive values from 91.22 3.40 m V to 166.78 6.61 m V.Taken together,these results suggest thatthe 4 subunitreduces the difference in conductance thatarises by alternatively splicing to include the STR EX. In addition, 4 dram atically inhibits BK channelactivation atlow C a2 (1.0 M ),regardless ofthe splice isoform . A tlow calcium , 4 inhibits both STR EX and ZER O B K channels by slow ing activation Atlow calcium 4 effects on activation kinetics underlie the large positive shiftofthe V 1/2.Fig.3E and 3F sum m arizes activation tim e constants as a function ofvoltage.The 4 subunit slow s activation by orders of m agnitude, from 2.79 0.31 m s to 234.71 78.7 m s for STR EX or from 2.70 0.49 m s to 87.68 6.65 m s forZER O (at 180 m V). The 4 subunitalso slow s deactivation kinetics,albeitto lesserextents atthis C a2 concentration (Fig.3G ).D eactivation tim e ofBK channels is significantly slow ed by the 4 subunit;from 1.11 0.08 m s to 3.21 0.62 m s (STR EX) orfrom 0.38 0.04 m s to 1.39 0.13 m s (ZER O )(Fig.3H ). Interestingly,although 4 slow s ZER O BK channeldeactivation across calcium concentrations,only atlow calcium concentrations does itslow STR EX BK channeldeactivation. The 4 subunitconfers sensitivity to dephosphorylation ofSTR EX butnot ZER O B K channels Ithas been reported thatthe 4 subunit(Jin etal.,2002) and STR EX (Tian etal.,2001)each alterthe response of BK channels to dephosphorylation and cAM P-dependent phosphorylation status. H ere w e m easured the com bined effectofboth STREX and the 4 subunit.The experim ental approach was to m easure responses to dephosphorylation by AP followed by cAM P ATP application to m easure effects ofprotein kinase A (pKA)–dependentphosphorylation (BK channels in H EK cells have pKA pre-tethered to the channeland therefore do notrequire application ofthe catalytic enzym e (Tian et al., 2001, 2003)). Because all data w ere paired, w e could m easure both effects on steady-state values (Fig. 4 and Fig. 5, left panels) and relative changes from controlvalues (Fig. 4 and Fig. 5, right panels). R esults are show n for effects on V 1/2 and gating kinetics in 5.3 M C a2 w hen STR EX and ZER O BK channels,respectively,are expressed w ith and w ithout the 4 subunit. R epresentative G –V relations forSTR EX illustrate that application ofAP to STR EX channels tends to shiftV 1/2 to positive potentials (Fig.4A,C –D ,from 18.24 3.85 m V to 9.38 6.17 m V), although this shift is not statistically significant(P 0.086).In contrast,STR EX 4 BK channels show a greater response to dephosphorylation (Fig. 4B–D ). D ephosphorylation causes a significant positive shiftofV 1/2 from 1.6 6.22 m V ofcontrolto 19.14 4.89 m V (Fig. 4C –D ). This is m ediated predom inantly by a slow ing of activation kinetics (Fig. 4E–F, STR EX 4 is 1.1 0.17 versus STR EX 4 phosphatase 2.1 0.11 m s, P 0.002).

Activation ofpKA has been show n to inhibitSTR EX BK channels in H EK293 cells (Tian etal.,2001).C onsistent w ith this,w e found thatactivation ofpKA w ith 1 m M cAM P and 1 m M ATP caused a sm allbutsignificantspeeding of deactivation kinetics (Fig.4G and H )forboth STR EX (from 7.16 1.22 m s to 5.53 0.92 m s,P 0.03)and STR EX 4 BK channels (from 2.30 0.21 m s to 1.66 0.19 m s, P 0.02).H ow ever,the sm allchange in kinetics conferred a relatively sm allpositive shiftofthe V 1/2 thatdid notreach statisticalsignificance (Fig.4C –D from 9.38 6.17 m V to 5.07 7.73 m V,P 0.09 forSTR EX and from 19.14 4.89 m V to 22.49 6.08 m V,P 0.33 forSTR EX 4).Interestingly, the com bined effects of dephosphorylation and cAM P-dependentphosphorylation show ed a significantinhibition ofSTR EX BK channels (Fig.4C ,P 0.011 fornet difference betw een controland cAM P). ZER O B K channels are activated by cA M P-dependentphosphorylation w hen co-expressed w ith the 4 subunit C ontrary to STR EX 4 BK channels thatare inhibited by dephosphorylation (Fig.4),ZER O and ZER O 4 BK channels are notsignificantly affected by dephosphorylation (Fig. 5). The application of cAM P after dephosphorylation,how ever,produces a sm allbutstatistically significant shift of the V 1/2 to m ore negative potentials for ZER O 4 BK channels (from 52.27 5.02 m V to 38.07 5.54 m V).This negative V 1/2 shiftis m ediated by faster activation (Fig.5E,from 8.11 1.5 m s for dephosphorylation to 5.88 0.81 m V for cAM P application). W e did notobserve a significantV 1/2 shiftforZER O BK channels (Fig. 5C –D ). In sum m ary, the 4 subunit confers a significantly greater sensitivity ofSTR EX BK channels to dephosphorylation, and ZER O BK channels to pKA-dependentphosphorylation. The 4 subunitand STR EX B K channels are coexpressed in the hippocam pus and cerebellum O urfindings using an in vitro expression system suggest thatthe 4 subunitdifferentially affects alternative splice products ofthe BK channeland their response to phosphorylation status. U sing quantitative PC R , it has been previously show n that the STR EX is relatively abundant in pituitary m R N A but w eakly expressed in w hole brain m R N A (C hen et al., 2005). U tilizing an eG FP knock-in reporterfor 4 gene expression,itw as found thatthe 4 subunitalso has abundantexpression in the anteriorand posteriorpituitary (Brenneretal.,2005).To betterunderstand the physiologicalrelevance of 4 subunit/STR EX BK channelinteractions in othercentralbrain regions,w e used m R N A ISH com bined w ith eG FP im m unohistochem istry to revealifSTR EX BK channels overlap in their m R N A expression w ith the 4 subunitin the brain.W e m ade tw o specific cR N A riboprobes labeled w ith digoxigenin-U TP for STR EX and totalBK channels.The antisense strand riboprobes w ere tested forspecific cR N A–m R N A hybridization tow ard m R N A of STR EX and total BK channels w hen com pared w ith negative controls ofcorresponding sense riboprobes (Fig.6).C oronalsections ofthe hippocam pus


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Fig.4. STR EX 4 butnotSTR EX BK channels are inhibited by dephosphorylation.R ecordings w ere m ade in 5.3 M internalcalcium .R epresentative G –V relationships forSTR EX (A)and STR EX 4 BK channels (B)forpre-treatm entcontrol(filled squares),dephosphorylation by AP (em pty squares)and cAM P-dependentphosphorylation (cAM P,triangles).(C )Sum m arized V 1/2.(D ) V 1/2 ofcontrolversus dephosphorylation (w hite bars), and dephosphorylated versus cAM P (dark bars).(E)M ean ofactivation tim e constanttaken at 80 m V.(G )M ean ofdeactivation tim e constanttaken at 80 m V. Activation (F)and deactivation (H )show m eans ofpaired subtraction ofactivation and deactivation tim e constants at 80 and 80 m V respectively.In C ,E,G ,controlvalues (black) are com pared w ith AP treatm ent(gray) and subsequentcAM P application (w hite).In D ,F,H subtraction ofcontrolversus dephosphorylated values (w hite)is com pared w ith subtraction ofdephosphorylated versus cAM P-treated values (black). Paired t-testis used for statisticalanalysis (STR EX n 8;STR EX 4 n 11).U n-paired t-testis used only for com parison of V 1/2.O ne asterisk represents P 0.05,tw o representP 0.01,and three representP 0.001.

show thatsignals fortotalBK channels (Fig.6A upper)and forSTR EX BK channels (Fig.6B upper)are absentw hen

controlsense riboprobes are used (low erpanelofFig.6A and 6B).

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Fig.5. cAM P-dependentphosphorylation activates ZER O 4 BK channels.R ecordings w ere m ade in 5.3 M internalcalcium .R epresentative G –V relationships forZER O (A)and ZER O 4 BK channels (B)forpre-treatm entcontrol(filled squares),dephosphorylation by AP (AP,em pty squares) and cAM P-dependent phosphorylation (cAM P, triangles). (C ) Sum m arized V 1/2. (D ) V 1/2 of controlversus dephosphorylation (w hite bars), and dephosphorylated versus cAM P (dark bars).(E)M ean ofactivation tim e constanttaken at 80 m V.(G )M ean ofdeactivation tim e constanttaken at 80 m V (G ). Activation (F)and deactivation (H )show m eans ofpaired subtraction ofactivation and deactivation tim e constants at 80 m V and 80 m V,respectively.In C ,E,G ,controlvalues (black)are com pared w ith AP treatm ent(gray)and subsequentcAM P application (w hite).In D ,F, H subtraction of controlversus dephosphorylated values (w hite) is com pared w ith subtraction of dephosphorylated versus cAM P-treated values (black).Paired t-testis used forstatisticalanalysis (ZER O n 7;ZER O 4 n 11).U n-paired t-testis used only forcom parison of V 1/2.O ne asterisk represents P 0.05,tw o representP 0.01,and three representP 0.001.


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Fig.6. R iboprobes forSTR EX only and totalBK channels are specific.Antisense strand cR N A probe againsttotalBK channels independently ofa splice isoform (A,top panel)orantisense strand cR N A againstSTR EX only (B,top panel)w as tested on coronalsections ofthe hippocam pus from 4-m onth-old m ale C 57BL/6J m ice.As negative controls,sense strands ofcR N A ofthe sam e cD N A tem plates thatw ere used forantisense probes w ere transcribed.(A)Bottom panelshow s sense strand riboprobe fortotalBK channels.(B)Bottom panelshow s sense strand forSTR EX BK channels. Insets show the dentalgyrus area (solid line)and the C A3 area (dashed line)ofthe hippocam pus.The scale barin the panels corresponds to 100 m and to 10 m in the insets.Incubation ofthe brain sections w ith the sense strand riboprobes results in alm osttotalloss ofsignalw hen com pared w ith the antisense strand riboprobes suggesting very low background hybridization.

Figs.7 and 8 com pare the expression pattern ofthe 4 subunit(top panels)w ith STR EX (m iddle panels)and total BK channels (low er panels) in sagittal sections of the hippocam pus (Fig. 7A), the frontal lobe neocortex (Fig. 7B),the cerebellum (Fig.8A),and in the olfactory bulb (Fig. 8B). In general, the expression of STR EX BK channels represented a sm allerfraction oftotalBK channels.In the hippocam pus, w e observed expression of both the 4 subunit and STR EX in the granule cells of the dentate gyrus and in the C A3 pyram idalneurons (insets in Fig.7A). Because eG FP is able to diffuse throughoutneuronalprocesses,the 4 reporter is observed in both the dendritic layers ofthe granule cells (lined inset)and the m ossy fiber projections (dashed inset)(Fig.7A upperpanel).The ISH forSTR EX and totalBK channels is instead localized in the cellbody layers ofthe dentate gyrus and C A3 regions (Fig. 7A m iddle and low er panels). The expression of the 4 subunitin the C A1 region w as m uch w eakeralthough both STR EX and totalBK channels w ere hom ogeneously expressed in the w hole hippocam pus including C A1 pyram ids.In the neocortex (frontallobe)expression ofboth 4 and total BK channels predom inates, w hereas STR EX channels are very w eakly expressed (Fig.7B).O n the other hand,STREX and 4 show overlapping expression in the Purkinje neurons ofthe cerebellum (insets ofFig.8A).Interestingly, 4 subunitim m unohistochem istry show ed m ore w idespread expression in the ventralolfactory bulb (insets ofFig.8B)than BK channels,w hich w ere m ore confined to a very narrow layerofthe granule neurons.

D ISC U SSIO N The STR EX exon show s relatively w eak C N S expression as m easured by PC R ofw hole brains (C hen etal.,2005). In contrast, STR EX appears to show greatest neuronal expression in early C N S developm entand in neurosecretory tissues such as adrenal gland and pituitary (M acD onald etal.,2006).C onsistentw ith this,w e found thatthe STR EX show s significant expression only in few brain regions.D espite relatively w eak expression ofSTR EX in som e brain regions,the finding thata single STR EX containing subunitdom inates the properties ofa BK channel tetram er (Tian etal.,2004) suggests thateven w eak expression m ay be functionally im portant. It has been recently reported that gain-of-function of BK channels in hum ans and m ice results in epilepsy phenotypes (D u etal., 2005; Brenner et al., 2005). Thus, the ability of the 4 subunitto inhibitSTR EX BK channels in a w ide range of calcium concentrations m ay have protective effects in som e centralneurons w here STR EX has lim ited expression.These m ay include the hippocam pus dentate gyrus and the cerebellum Purkinje cells w here STR EX and 4 expression overlaps. The m echanism used by 4 to inhibitSTR EX BK channels appears to be calcium dependent.Atlow calcium 4 reduces channelopenings by slow ing activation.Atinterm ediate and high calcium ( 5.3 M C a2 ), 4 reduces BK channelopening by opposing the slow deactivation conferred by the STR EX exon. Effects on deactivation are

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Fig.7. STR EX BK channels are expressed in the hippocam pus and very w eakly in the neocortex.Expression ofSTR EX versus totalBK channels is com pared w ith the expression ofthe 4 subunit.Specific antisense strand riboprobes w ere used fordetecting STR EX and totalBK channels in w ild-type m ale C 57BL/6J m ice.Anti-G FP im m unohistochem istry w as em ployed to detectthe 4 expression pattern in the m ale 4 / C 57BL/6J m ice since the 4-specific prom oterdrives expression ofG FP as a transgenic m arkerforthe 4 subunit.Panels show serialsections ofthe hippocam pus (A)and the frontallobe (B)stained forthe 4 subunit(top panels),STR EX (m iddle panels),and totalBK channels (bottom panels).In the hippocam pus, insets show the granule neurons ofthe dentalgyrus (solid line fram e) and the pyram idalneurons ofthe C A3 region (dashed line fram e).In the neocortex ofthe frontallobe,insets show pyram idalneurons ofthe layerII/III.W e detected distinguishable signalforSTR EX overlapping w ith the 4 subunitexpression in the hippocam pus butfairly w eak signalforSTR EX in the neocortex ofthe frontallobe.Scale bars 100 m .In the insets,scale bars 10 m .

som ew hat surprising given that 4, for channels lacking STR EX,prom otes BK channelopening by slow ing deactivation.Yetthe com bined effects ofSTR EX and 4 athigh C a2 speed deactivation relative to eitheralone.This is in contrast to the avian beta subunit that creates a further slow ing ofdeactivation kinetics w hen STR EX and subunits are com bined (R am anathan etal.,1999).The m echanism s thatunderlie differences betw een the avian subunitand the m ouse 4 are notunderstood.G iven thatthe avian subunithas highersim ilarity to the related 1 and 2 subunitfam ily m em bers (Xia etal.,1999),these differences m ay be related to functionaldivergence betw een 1/ 2 and 4 paralogues.In contrastto STR EX BK channels, 4 slow s both activation and deactivation ofZER O channels.In high C a2 ,this causes a neteffectthatshifts the G –V relationship to negative potentials.As calcium is reduced (1 M C a2 )the slow activation conferred by 4 predom inates to strongly inhibit both STR EX and ZER O BK channels. This is from m illisecond tim es, in the absence of 4,to hundreds ofm illiseconds forBK channels w ith 4.Thus,atlow calcium ,the 4 subunitslow s activation kinetics to w elloutside norm alphysiologicaltim escales for processes such as action potentialrepolarization orregulation ofneurotransm itterrelease. Intracellular calcium concentrations in m icrodom ains can indeed approach 20–30 M during calcium sparks

(ZhuG e etal.,2000)and up to severalhundred M in the presynaptic sites near calcium channels (O heim et al., 2006).In neurons,BK channels have been found in close vicinity of m icrodom ain calcium (Francioliniet al., 2001; Parsons et al., 2002) or in presynaptic sites (H u et al., 2001).W e can hypothesize thatBK channels m ay experience briefbutvery robustelevations ofcalcium .Therefore, the localcalcium environm entin com bination w ith ZER O / STR EX splicing m ay determ ine w hether BK/ 4 channels can contribute to shaping ofaction potentials orregulation ofneurotransm itterrelease.Forexam ple,given the significantly faster activation rates at interm ediate calcium concentrations (such as 5 M ,Fig.2F) the 4 subunit m ay perm itSTR EX channels butnotZER O BK channels to contribute to action potentialrepolarizations.In addition,the speeding ofdeactivation ofSTR EX channels by 4 w ould lim itBK channelcontribution to the afterhyperpolarization. As calcium concentrations rise to higher levels,activation rates are m uch fasterand deactivation m uch slow er(independentofSTR EX orZER O splicing, Fig.1)so thateitherBK channelisoform m ay contribute to action potential repolarization and dam pen firing rates. M odulation of BK channels involves m any m echanism s (W eiger etal.,2002;Lu etal.,2006) including reversible phosphorylation.An additionaldistinction betw een


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Fig.8. STR EX BK channels are expressed in the cerebellum and w eakly in the olfactory bulb.As in Fig.7,expression ofSTR EX versus totalBK channels is com pared w ith the expression ofthe 4 subunit.Panels show serialsections ofthe cerebellum (A)and the olfactory bulb (B)stained for the 4 subunit(top panels),STR EX (m iddle panels),and totalBK channels (bottom panels).In the cerebellum ,insets show signalforSTR EX and the 4 subunitin the Purkinje pyram idalneurons.In the olfactory bulb,the expression ofboth totalBK channels and STR EX is lim ited to a restricted neuronalpopulation w hereas the 4 subunitdisplays w idespread distribution.Scale bars 100 m .In the insets,scale bars 10 m .

STR EX and ZER O BK channels is their response to cAM P-dependentphosphorylation.STR EX containing BK channels are frequently inhibited by cAM P dependent phosphorylation in H EK293 cells w hereas ZER O isoform s are activated in the m ajority ofchannelpatches (Tian etal., 2001). O ur results indicate that the ZER O / 4 channels have a greater sensitivity to cAM P-dependent activation than ZER O channels alone.The factthatZER O BK channelw ere notsignificantly activated by cAM P m ay be due to pretreatm ent of the channels w ith AP. BK channel response to phosphorylation has been show n to be quite variable in neurons (R einhartetal.,1989,1991;R einhart and Levitan,1995;W idm eretal.,2003)and even in transfected H EK293 cells (Tian et al., 2001). For exam ple, application ofcAM P to ZER O BK channels expressed in H EK293 cells has been show n to activate 10 of 16 BK channelpatches (Tian etal.,2001).In part,this m ay arise from cell-to-cellvariation in basallevels ofphosphorylation of channels. Therefore, to produce a m ore reproducible response to pKA-dependent phosphorylation, w e pretreated allpatches w ith phosphatase.Although phosphatase m ay hom ogenize the channel’s response to cAM Pdependentphosphorylation,itm ay affectotherphosphorylation sites thatalterthe response ofZER O channels but notZER O / 4 channels to cAM P-dependentphosphorylation. For exam ple, in cerebellar Purkinje cells cAM P-dependentchannelactivation ofBK channels appears to be conditionalon the previous phosphorylation state.Protein

kinase C and protein phosphatase-2A occlude cAM P-dependent activation, w hereas protein phosphatase-1 prom otes pKA activation (W idm eretal.,2003). W hereas our results indicate thatBK channels alone are not significantly affected by dephosphorylation, dephosphorylation of 4 containing channels confers a slow eractivation ofBK channels along w ith a positive shift ofthe G –V relations.Surprisingly,this effectw as dependenton the presence ofSTR EX.In H EK cells 4 subunits are tonically phosphorylated (Jin etal.,2002).These results suggestthatthe inhibition ofBK STR EX/ 4 channels by dephosphorylation m ay be attributed to the phosphorylation state ofthe 4 subunit.Itw ould also indicate that tonic phosphorylation of the 4 subunit reduces its full ability to inhibitSTR EX BK channels. The non-additive effects ofSTR EX and the 4 subunit suggestthat 4 som ehow perturbs interactions ofSTR EX w ithin BK channels. The physicalbasis of this is only a m atterofspeculation.M any effects of 1 are m ediated by theirintracellulardom ains (O rio and Latorre,2005;O rio et al.,2006;W ang and Brenner,2006).To date,the subunit dom ains found to interactw ith subunits are the N -term inal,transm em brane S0 dom ain (W allneretal.,1996)and the carboxylterm inaltailregion (Q ian etal.,2002).The STR EX coding region lies w ithin a variable region (Schreiberand Salkoff,1997)ofthe carboxylterm inus thatlinks tw o conserved functionaldom ains ofthe channel.Am ino term inal to STR EX is the so-called R C K1 (regulator of

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potassium conductance)calcium and m agnesium sensing dom ain (Shietal.,2002;Xia etal.,2002).C arboxylterm inal to STR EX is a putative second R C K-like dom ain (R C K2).Although the R C K2 sequence show s w eak evidence ofR C K hom ology (Fodor and Aldrich,2006),itis required forchannelfunction and m ay provide a hydrophobic interface for calcium -dependent gating (Kim et al., 2006).Positioned betw een the tw o dom ains,itis indeed possible thatSTR EX m odulates R C K dom ain interactions. W hetherornotthe 4 intracellulardom ains directly interactw ith this region is yetto be determ ined. Early characterization ofBK channelsubtypes in neuronalsynaptosom alm em brane preparations identified tw o classes ofBK channels.These w ere the fast-gated,iberiotoxin sensitive (type I)BK channels and the slow -gated, iberiotoxin resistant (type II) BK channels (R einhart and Levitan,1991).Type IIBK channels are now realized to encode BK/ 4 subunit channels due to the iberiotoxin resistance they confer(Behrens etal.,2000;M eera etal., 2000). H ow ever, type I and type II BK channels show diverse properties in response to kinase and phosphatasedependentm odulation (R einhartand Levitan,1991).O ur findings suggestthatthe line betw een type Iand type II channels m ay notbe so distinct.STR EX/ 4 BK channels, w hich m ay be classified as type IIBK channels based on iberiotoxin resistance,could nevertheless appearas fastgated type I BK channels due to their fast deactivation kinetics at high calcium and activation by phosphatase treatm ent(R einhartand Levitan,1991).Thus,the com bination ofalternative splicing and association w ith accessory subunits m ay create a greaterdiversity in BK channelproperties than previously expected. Acknow ledgm ents— W e thank Bin W ang and R achelJ.Brenner forcriticalreading ofthe m anuscript.This w ork w as supported by a N ationalInstitutes ofH ealth grantN S052574,a N ationalAm erican H eartAssociation grant0335007N and an Epilepsy Foundation ofAm erica grant.

R EFER EN C ES Adelm an JP,Shen KZ,Kavanaugh M P,W arren R A,W u YN ,Lagrutta A, Bond C T,N orth RA (1992) C alcium -activated potassium channels expressed from cloned com plem entary D N As.N euron 9:209–216. Atkinson N S, R obertson G A, G anetzky B (1991) A com ponent of calcium -activated potassium channels encoded by the D rosophila slo locus.Science 253:551–555. Behrens R ,N olting A,R eim ann F,Schw arz M ,W aldschutz R ,Pongs O (2000)hKC N M B3 and hKC N M B4:cloning and characterization oftw o m em bers ofthe large-conductance calcium -activated potassium channelbeta subunitfam ily.FEBS Lett474:99–106. Brenner R ,C hen Q H ,Vilaythong A,Toney G M ,N oebels JL,Aldrich R W (2005) BK channelbeta4 subunitreduces dentate gyrus excitability and protects againsttem porallobe seizures.N atN eurosci 8:1752–1759. BrennerR ,Jegla TJ,W ickenden A,Liu Y,Aldrich R W (2000)C loning and functionalcharacterization ofnovellarge conductance calcium -activated potassium channel beta subunits, hKC N M B3 and hKC N M B4.J BiolC hem 275:6453–6461. Butler A,Tsunoda S,M cC obb D P,W eiA,SalkoffL (1993) m Slo,A com plex m ouse gene encoding “m axi” calcium -activated potassium channels.Science 261:221–224.

C hen L,Tian L,M acD onald SH ,M cC lafferty H ,H am m ond M S,H uibant JM , R uth P, Knaus H G , Shipston M J (2005) Functionally diverse com plem entoflarge conductance calcium - and voltageactivated potassium channel(BK)alpha-subunits generated from a single site ofsplicing.J BiolC hem 280:33599–33609. D iez-Sam pedro A,Silverm an W R ,Bautista JF,R icherson G B (2006) M echanism ofincreased open probability by a m utation ofthe BK channel.J N europhysiol96:1507–1516. D u W , Bautista JF, Yang H , D iez-Sam pedro A, You SA, W ang L, Kotagal P, Luders H O , Shi J, C uiJ, R icherson G B, W ang Q K (2005)C alcium -sensitive potassium channelopathy in hum an epilepsy and paroxysm alm ovem entdisorder.N atG enet37:733–738. Erxleben C ,EverhartAL,R om eo C ,Florance H ,Bauer M B,Alcorta D A,R ossie S,Shipston M J,Arm strong D L (2002) Interacting effects ofN -term inalvariation and strex-exon splicing on slo potassium channelregulation by calcium , phosphorylation and oxidation.J BiolC hem 277:27045–27052. FaberES,Sah P (2002)Physiologicalrole ofcalcium -activated potassium currents in the ratlateralam ygdala.J N eurosci22:1618–1628. FaberES,Sah P (2003)C a2 -activated K (BK)channelinactivation contributes to spike broadening during repetitive firing in the rat lateralam ygdala.J Physiol552:483–497. Fettiplace R ,Fuchs PA (1999)M echanism s ofhaircelltuning.Annu R ev Physiol61:809–834. FodorAA,Aldrich R W (2006)Statisticallim its to the identification ofion channeldom ains by sequence sim ilarity.J G en Physiol127:755–766. FrancioliniF,H ogg R ,C atacuzzeno L,Petris A,TrequattriniC ,Adam s D J (2001)Large-conductance calcium -activated potassium channels in neonatalratintracardiac ganglion neurons.Pflugers Arch 441:629–638. Fury M ,M arx SO ,M arks AR (2002)M olecularBKology:the study of splicing and dicing.SciSTKE 2002:PE12. H a TS,H eo M S,Park C S (2004)Functionaleffects ofauxiliary beta4subuniton ratlarge-conductance C a(2 )-activated K( )channel. Biophys J 86:2871–2882. H u H ,Shao LR ,C havoshy S,G u N ,Trieb M ,Behrens R ,Laake P, Pongs O ,Knaus H G ,O ttersen O P,Storm JF (2001)Presynaptic C a2 -activated K channels in glutam atergic hippocam palterm inals and theirrole in spike repolarization and regulation oftransm itterrelease.J N eurosci21:9585–9597. Jin P,W eiger TM ,W u Y,Levitan IB (2002) Phosphorylation-dependent functional coupling of hSlo calcium -dependent potassium channeland its hbeta 4 subunit.J BiolC hem 277:10014–10020. Kim H J, Lim H H , R ho SH , Eom SH , Park C S (2006) H ydrophobic interface betw een tw o regulators ofK conductance dom ains criticalforcalcium -dependentactivation oflarge conductance C a2 activated K channels.J BiolC hem 281:38573–38581. LaiG J,M cC obb D P (2006)R egulation ofalternative splicing ofSlo K channels in adrenaland pituitary during the stress-hyporesponsive period ofratdevelopm ent.Endocrinology 147:3961–3967. LippiatJD ,Standen N B,H arrow ID ,Phillips SC ,D avies N W (2003) Properties ofBK(C a)channels form ed by bicistronic expression of hSloalpha and beta1–4 subunits in H EK293 cells.J M em br Biol 192:141–148. LovellPV,M cC obb D P (2001)Pituitary controlofBK potassium channel function and intrinsic firing properties of adrenal chrom affin cells.J N eurosci21:3429–3442. Lu R ,Alioua A,Kum arY,EghbaliM ,StefaniE,Toro L (2006)M axiK channelpartners:physiologicalim pact.J Physiol570:65–72. M acD onald SH , R uth P, Knaus H G , Shipston M J (2006) Increased large conductance calcium -activated potassium (BK)channelexpression accom panied by STR EX variant dow nregulation in the developing m ouse C N S.BM C D ev Biol6:37. M ahm oud SF,M cC obb D P (2004)R egulation ofSlo potassium channel alternative splicing in the pituitary by gonadal testosterone. J N euroendocrinol16:237–243. M cC artney CE,M cC lafferty H ,H uibantJM ,R owan EG ,Shipston M J, R ow e IC (2005) A cysteine-rich m otifconfers hypoxia sensitivity to

D .Petrik and R .Brenner/N euroscience 149 (2007)789–803 m am m alian large conductance voltage- and C a-activated K (BK) channelalpha-subunits.Proc N atlAcad SciU S A 102:17870–17876. M cM anus O B,H elm s LM ,Pallanck L,G anetzky B,Sw anson R ,Leonard R J (1995)Functionalrole ofthe beta subunitofhigh conductance calcium -activated potassium channels.N euron 14:645–650. M eera P,W allnerM ,Toro L (2000)A neuronalbeta subunit(KC N M B4) m akes the large conductance, voltage- and C a2 -activated K channelresistantto charybdotoxin and iberiotoxin.Proc N atlAcad SciU S A 97:5562–5567. O heim M ,KirchhoffF,Stuhm er W (2006) C alcium m icrodom ains in regulated exocytosis.C ellC alcium 40:423–439. O rio P, Latorre R (2005) D ifferential effects of beta 1 and beta 2 subunits on BK channelactivity.J G en Physiol125:395–411. O rio P,Torres Y,R ojas P,C arvacho I,G arcia M L,Toro L,Valverde M A,Latorre R (2006) Structuraldeterm inants for functionalcoupling betw een the beta and alpha subunits in the C a2 -activated K (BK)channel.J G en Physiol127:191–204. O w ens N C , H ess FM , Badoer E (2006) In situ hybridization using riboprobes on free-floating brain sections. M ethods M ol Biol 326:163–171. Parsons R L,Barstow KL,Scornik FS (2002) Spontaneous m iniature hyperpolarizations affectthreshold for action potentialgeneration in m udpuppy cardiac neurons.J N europhysiol88:1119–1127. Q ian X,N im igean C M ,N iu X,M oss BL,M agleby KL (2002)Slo1 tail dom ains,butnotthe C a2 bow l,are required forthe beta 1 subunit to increase the apparentC a2 sensitivity ofBK channels.J G en Physiol120:829–843. R affaelli G , Saviane C , M ohajerani M H , Pedarzani P, C herubini E (2004)BK potassium channels controltransm itterrelease atC A3C A3 synapses in the rathippocam pus.J Physiol557:147–157. R am anathan K, M ichaelTH , Jiang G J, H ielH , Fuchs PA (1999) A m olecular m echanism for electricaltuning of cochlear hair cells. Science 283:215–217. R einhartPH ,C hung S,Levitan IB (1989)A fam ily ofcalcium -dependentpotassium channels from ratbrain.N euron 2:1031–1041. R einhartPH,C hung S,M artin BL,Brautigan D L,Levitan IB (1991)M odulation of calcium -activated potassium channels from rat brain by protein kinase A and phosphatase 2A.J N eurosci11:1627–1635. R einhart PH , Levitan IB (1995) Kinase and phosphatase activities intim ately associated w ith a reconstituted calcium -dependentpotassium channel.J N eurosci15:4572–4579. Saito M ,N elson C ,SalkoffL,Lingle C J (1997)A cysteine-rich dom ain defined by a novelexon in a slo variantin ratadrenalchrom affin cells and PC 12 cells.J BiolC hem 272:11710–11717. SchreiberM ,SalkoffL (1997)A novelcalcium -sensing dom ain in the BK channel.Biophys J 73:1355–1363. Shao LR ,H alvorsrud R ,Borg-G raham L,Storm JF (1999)The role of BK-type C a2 -dependentK channels in spike broadening during repetitive firing in rathippocam palpyram idalcells.J Physiol521 (Pt1):135–146. ShiJ,Krishnam oorthy G ,Yang Y,H u L,C haturvediN ,H arilalD ,Q in J,C uiJ (2002) M echanism ofm agnesium activation ofcalcium activated potassium channels.N ature 418:876–880. Shipston M J (2001) Alternative splicing of potassium channels: a dynam ic sw itch ofcellularexcitability.Trends C ellBiol11:353–358. Solaro C R , Prakriya M , D ing JP, Lingle C J (1995) Inactivating and noninactivating C a(2 )- and voltage-dependentK currentin rat adrenalchrom affin cells.J N eurosci15:6110–6123.

803

Strassle BW ,M enegola M ,R hodes KJ,Trim m erJS (2005)Lightand electron m icroscopic analysis ofKC hIP and Kv4 localization in rat cerebellargranule cells.J C om p N eurol484:144–155. Tian L,C oghillLS,M acD onald SH ,Arm strong D L,Shipston M J (2003) Leucine zipperdom ain targets cAM P-dependentprotein kinase to m am m alian BK channels.J BiolC hem 278:8669–8677. Tian L,C oghillLS,M cC lafferty H ,M acD onald SH ,AntoniFA,Ruth P, Knaus H G ,Shipston M J (2004)D istinctstoichiom etry ofBKC a channeltetram erphosphorylation specifies channelactivation and inhibition by cAM P-dependentprotein kinase.Proc N atlAcad SciU S A 101:11897–11902. Tian L,D uncan R R ,H am m ond M S,C oghillLS,W en H ,R usinova R , C lark AG , Levitan IB, Shipston M J (2001) Alternative splicing sw itches potassium channelsensitivity to protein phosphorylation. J BiolC hem 276:7717–7720. Tseng-C rank J, G odinot N , Johansen TE, Ahring PK, Strobaek D , M ertz R , Foster C D , O lesen SP, R einhart PH (1996) C loning, expression, and distribution of a C a(2 )-activated K channel beta-subunit from hum an brain. Proc N atl Acad Sci U S A 93:9200–9205. W allnerM ,M eera P,Toro L (1996)D eterm inantforb-subunitregulation in high-conductance voltage-activated and C a21-sensitive K1 channels:An additionaltransm em brane region atthe N term inus. Proc N atlAcad SciU S A 93:14922–14927. W ang B,Brenner R (2006) An S6 m utation in BK channels reveals beta1 subunit effects on intrinsic and voltage-dependent gating. J G en Physiol128:731–744. W ang B,R othberg BS,Brenner R (2006) M echanism of 4 subunit m odulation ofBK channels.J G en Physiol127:449–465. W eiger TM , H erm ann A, Levitan IB (2002) M odulation of calcium activated potassium channels.J C om p PhysiolA N euroetholSens N euralBehav Physiol188:79–87. W eigerTM ,H olm qvistM H ,Levitan IB,Clark FT,Sprague S,H uang W J, G e P, W ang C , Lawson D, Jurm an M E, G lucksm ann M A, SilosSantiago I,D iStefano PS,C urtis R (2000)A novelnervous system beta subunitthatdow nregulates hum an large conductance calcium dependentpotassium channels.J N eurosci20:3563–3570. W idm erH A,R ow e IC ,Shipston M J (2003)C onditionalprotein phosphorylation regulates BK channelactivity in ratcerebellarPurkinje neurons.J Physiol552:379–391. Xia XM ,D ing JP,Lingle C J (1999)M olecularbasis forthe inactivation ofC a2 -and voltage-dependentBK channels in adrenalchrom affin cells and ratinsulinom a tum orcells.J N eurosci19:5255–5264. Xia XM , Zeng X, Lingle CJ (2002) M ultiple regulatory sites in largeconductance calcium -activated potassium channels. N ature 418: 880–884. Xie J,M cC obb D P (1998)C ontrolofalternative splicing ofpotassium channels by stress horm ones.Science 280:443–446. Yu JY,U padhyaya AB,Atkinson N S (2006)Tissue-specific alternative splicing of BK channel transcripts in D rosophila. G enes Brain Behav 5:329–339. ZhuG e R , Fogarty KE, Tuft R A, Lifshitz LM , Sayar K, W alsh JV Jr (2000)D ynam ics ofsignaling betw een C a(2 )sparks and C a(2 )activated K( )channels studied w ith a novelim age-based m ethod fordirectintracellularm easurem entofryanodine receptorC a(2 ) current.J G en Physiol116:845–864.

(Accepted 7 August2007) (Available online 12 Septem ber2007)