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Cloning and functional expression of a TEA-sensitive A-type potassium channel from rat brain Klaus-Flasso S c h r f t e d , ' J o h a n n Peter Ruppersl~rg ~, Frank Wundet a. Jens R e t t i ~ , Martm S t o c k e d and Olaf Pongx a ) Afax. Plmtck.htxr#ut fiTr mrdt:htl~rh¢ For~rhung. Juht~trajJ¢ 29, dgO0 Heht¢lbcr~, Germmu" and J Ruhr Univers~tdt B.rhmn, Lrhrzttthl fiir lhoeheml¢, .I630 gotham, r;¢rmanv

R¢ceiwd 2? Novcmbw 1990 A rat brain e D N A (Raw.l} related to the Dra.fophgaSha. K= channel l'Amllyhas been character)xed, Raw3 c R N A leads to the formation of TEA. in~nsltt,,'¢, ras! inactivating (A-tyI~) K ' ,:hannels whefl )njecled into ,Wnapus laevl~ o¢¢ytes Raw3 ch{tnnels have markedly different prolztrttes from the prcv)ously cloned rat A,|yp¢ K" channel RCK4, Raw3 channels ol~rate m the positive voltztge range ~DNA cloninlil, ~.RNA exprasslon, Potaslium channel, A.tyr~ K" chtmnel, I^ channel

1, I N T R O D U C T I O N

2. MATERIALS A N D M E T H O D S

Voltage.gated ,~." channels play an =mportant role m the regulation of membrane potential and cell excltabdity [1], A-Type potassium channels regulate the firmg frequency of neurons because they are usually active in the potentml range negatwe to the threshold of excitat=on. A-Currents often have been distinguished from other K+ currents by their sensitivity to 4-AP [2]. Many different mammahan cDNAs encoding K + channel forming proteins have been cloned [3,4]. Functional expressrun of the derived cRNAs led to K+ channels having delayed rectzfter properues except tn the case of RCK4 cRNA encoding R.CK4 is, so far, the only one that leads to the formation of fast inacti~atmg (A-type) K + channels [5] when rejected into Xenopus oocytes AChannels descrtbed m the hterature vary widely m their pharmacologtcal profiles and kinetics [2]. Some of them resemble RCK4 channels, other do not. RCK4-mediated A-currents are resistant to TEA and are not very senstove to blockage by 4-AP. Thu~ it is most likely that the rat genome encodes other A-type K+ channel proterns different from RCK4, Here we report the tsolauon and functional expression of a new A-type K" channel eDNA (Raw3).

A rat cortex eDNA library (a gift from P Seeburg, Heidelberg) has beenscreened w=th a )=P labelled NGK2 [18] eDNA probe under con. ehtionsoflow stringency[6, 171 TwoovcrlappmgcDNAswer¢isolated and sequenced (7,8] The combined e D N A sequence ~as 2858 bp long The composite raw3 c D N A was cloned into tileexpression see tot pAS2, Th~s vector was constructed of Bluescrlpt KS" (Stratagene) and pAKSI8 18] After filhng m with DNA-polymeraz¢ [ (Klenow) an Azel fragment (nt 3320-352) of pAS 18, containing the SP6 promoter, the mtaltlnle cloning s~teand the polyA regton (266 bp) was cloned into Pvull cut Bluescript, KS' Raw2 pAKS2 was generated byclonmg the 5'Petl/S/ul fragmen~ of cDNA 76 (at - 155 to 749) together with the 3' Stu [/AspTI 8 fragment of cDNA 3 (nt 749-2437) into lastI/Asp7l 8 cut pAKS2 The resuhmg raw3.pAKS2 recombinant was hneanzed by cutting at the EcoR[ slte Capped run off c R N A for injectioninto Xenopus oocytes v,a~ synthes.,cd by a standard protocol [9] Xenopus/news oocytes were rejected wsth cRNA and incubated for 2-3 days at 19*C [10] The kmeuc properties of Raw3 channels were determined from macro.patch recordings lit] in the cell-attacl~cd conhguratlon of the patch clamp technique I'h¢ pharmacologtcal profile was estabhsl~ed ushtg a conventional two.mlcroelectrode voltage-clamp Microelectrodes were filled with I M KCI and had a resistance of 200-500 k.q Single channels were recorded m the cellattached configuratton All experiments were done at 20°C m normal frog Rnlger solution of the following composltmn (m raM) NaCI I IS, KCI2, CaCl= 1 8, Hepes 10, pH 7 2 In some exper,mentssodlum wa~ replaced by 4-AP or TEA (Sigma) or DTX (gift from Dr F, Dreyer, Glessen, Germany) was added Patch p~pettes were filled with normal frog Ringer solutmn Leak and capacitive currents were subtracted digitally using the P/4 method [12]

Correspondence address J P Ruppcrsberg, Abtedung Zeliphys,ologle, Max-Planck-innt)tut fdr medlzmlsche Forschung, Jahnstral3e 29, 6900 He~delber~;, Germany

Abbrev4atlotl~, K *, potassium, C a ; ' , calcium, cRNA, cDNA derived mRNA, TEA, tetraethylammontum, 4-AP, 4-ammopyrldme, DTX, dendrotoxin

Pubhshed bb ElsevJer S.tence Pubhshers B V fBtamedwal Dwtswn)

3. R E S U L T S 3.1. P r t m a r y sequence o f raw3 K ÷ channel protein

Two overlapping cDNAs, encoding Raw3, were isolated from a rat cortex ~.DNA hbrary. The longest open reading frame corresponds to an amino aczd sequence of 625 residues (Fig. 1). The hydropathy analysis [13]

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F=g, I Amino acld s~qaence dealt=ted from Raw3 ~ D N A , P~l|allVe membrane spannlnll sellmenls ($1 [o S6) are untlerllned Polenllal N.

glycosylatton sites are marked with an asterisk, Residues $}?-$41 (KRADS) encode ;t putati~,e PKA phosphorylation silt The open reading frame was d¢rived from t~o oserlappmg cDNAs The composil¢ Raw3 nu¢leotlde sequence has been submitted to the EMBL data ha.k.

( n o t shown) reveals six p u t a t t v e m e m b r a n e spanning segments (underlined in Fig. 1) s=mdar to other cloned K * channel proteins, The segments consist of' f i v e h y d r o p h o b i c ( S I . $2, $3, $5 and S6) and one p o s i t i v e l y charged

segment (S4), possibly the voltage sensor of the channel, A leucine ztpper m o t i f w h i c h may be r e v o l v e d in channel gating connects segments S4 wtth SS, as for m o s t other K" channels [14]. T h e charge d~str,but=on in s e g m e n t s S 2 and S3 ~s s~mflar to that previously f o u n d m other K" channel protein sequences [15], L i k e other K" channel prote=ns Raw3 protein is p r o b a b l y posttranslationally modified by N-glycosylation between segments SI and $2 and by p h o s p h o r y l a t l o n in the c a r b o x y - t e r m m u s (Ftg. 1). T h e most r e m a r k a b l e difference between the Raw3 protein sequence and other K* channel sequences ts that the two highly conserved sequence motifs N E Y F F D m the N.terminal sequence and M T T V G Y between segments $5 and S6 [16] are m u t a t e d to C E F F F D and to M T T L G Y , respectively. Since the Raw3 protein sequence ts htghly conserved in the t r a n s m e m b r a n e spanning region ($1-$6) and m the residues preceding segment S1, Raw3 protein appears as a n o t h e r m e m b e r o f the K + channel protein superfamily. When c o m p a r e d with other K ÷ channel sequences, that o f Raw3 protein is most closely related (78o7o identity) to those o f Shaw [17], N G K 2 [18] and R K S h I I I A [4] proteins. Obviously, R a w 3 is a m e m b e r o f the Shaw K ÷ channel f a m d y .

3 2. Functwna/ and pharmacologtcal propemes of currents mediated by Raw3 channels C u r r e n t s mediated by R a w 3 K " channels were characterized in the Xenopus laews oocyte expression syst e m . Oocytes expressing Raw3 specific c R N A were first 212

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brane po~enual was stepped from a holding potentml of -80 mV to test potentlah ranging from -70 mV to +SO mV, in 10 mV increments Intervah between pulses were 25 s The current was low pass filtered at I $ kHz (B) Voltage dependence ot conductance and steady state tnactwat~on (stars) The data points were fitted with single Boltzmann isotherms to get Vn,, (13,2 mV) and an (7 4 mV) for the activation curveand V.,.=(-29 3 mY)and a, (8.3 mV) for the macuvat~on curve

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F~g 3 Effect of blockers on Raw3 channels measured m two.mlcroelectrode voltage.clamp Current responses to depolarizing pulses from - 8 0 mV to +20 mV test potential The current was low pass filtered at 1 5 kHz (A) Sensltlvlty to TEA and 4.AP The control trace shows the current response before the apphcatton o f drugs and the following four traces dlustrate the mcreasmg block of current by increasing concentrat)ons of TEA, The concentratlon of 50~a block (ICs0) was about 0 3 mM TEA The sixth trace shows that the acuon of TEA is reverslble The block by 4-AP was tested on the sanl¢ oocyt¢ after wash-out of TEA. The traces 7-9 show the effect of 4-AP m concentratlons between 0 I mM to 10 mM [Cso was 0 5 rnM 4.AP and the block was only to 30% reverslble (not shown) (B) Sensltlsny to DTX tested m another oocyte The first trace shows the current response before addltmn of DTX The following three traces illustrate that up to the concentratlon of 100 nM DTX has no effect on the current The last trace show~ the unchanged current after wash-out of DTX 213

Vs=turn¢ 27m, humor 2

tested with two.mieroelectrod¢ voltage,,clamp, Dope. lad~inll step,= to positive tes~ potentials elicited transien| outward currents with a peak amplitude of 5-20 ~A, To determine Ih©ir exact time course and vollaie depend. ence of activation, ensemble K ° outward currents were measured in cell.att~hed macro-patches, Fig. 2A shows a family ot" current traces in response to depolarizing test pulses ranging from -'70 mV to +50 mV. The threshold of activation was at -. 10 inV, The time course of activation was rapid, rising from I0~'= to 9 0 ~ of peak amplitude in 3,4 = 1,2 ms (n =. 8) at +~$0 mV test potential. Saturation of the current peak amplitude occurred at test potentials positive to +30 mV, The peak current amplitudes or the experiment shown in Fig, 2A were divided by the driving force to obtain the condue|ante, assuming a K" equilibrium potentml of -100 inV. The normalized conductance-voltage relation [G/Gr~,,(V)] is shown in Fig, 2B (crosses), A single Boltzmann isotherm was fitted to the data points to obtain the voltage of half-maximal activation Vn, ~= 14.0 ± 9.2 m V (n = 13) and the voltage change for an efold increase in conductance an = 9.7 ± 3,0 mV, Inactivation of the current mediated by R a w 3 channels was almost complete during the test pulses of I00 ms duration as shown in Fig, 2A, The time constant of inacttva|ton was 15.2 ± 3,2 ms (n = 5) at +50 mV, A steadystate =nacttvatlon curve is shown in F~g. 2B (ctrcles). It was measured by responses to test pulses to +20 m V from different prepuise potentials (prepulse duration of 25 s) ranging from -100 mV to +30 mV in l0 mV increments. The data points were fitted with a Boltz. m a n n isotherm from which half-mac|we|ion was at -29.7 ± 6.S m V (n = 5), wlth an e-fold decrease of peak current per 12,2 ± 4,2 m V voltage change, R a w 3 channels recovered slowly from inactivation after a de. polarizing pulse. The time for 50°7o recovery from complete inactivation was found to be 1,9 ± 0.9 s (n = 6). The reversal potential o f K* currents medtated by Raw3 channels was tested by short activating pulses o f 20 ms duration to +20 mV, followed by a step back to various negative potentials (not shown). The currents measured following the r e p o l a n z m g step reversed their sign between - 8 0 mV and - 1 0 0 mV as expected for K* currents. T h e sensitivity o f Raw3 currents to 4-AP, T E A and DTX was tested m whole-cell current measurements (Ftg. 3). Raw3 currents are rather sensmve to 4-AP (ICso at 0.5 raM) and T E A (ICs0 at 0.3 raM), but insensttlve to D T X up to 100 nM, These results contrast with those for RCK4 currents which are a b o u t 20-fold less sensitive to 4-AP, and are insensitive to T E A as well as to D'I'X. 3 ~ Single-channel properttes o f Raw3 channels Single channel currents were recorded m cell-attached patches. Fig. 4A shows a transient current recorded m a patch contaimng about 30 channels, on a slow time 214

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scale. The outward current was activated by a step f r o m -80 m V to +20 inV. The current m Ftg. 4 A shows rapid inactivation with a time course similar to the m a c r o - p a t c h currents m Fig. 1. After 200 ms o f depolarization the current is sufficiently reactivated that single channel openings can be distinguished. Single channel openings are shown on a faster time scale m Fig. 4B. T h e trace represents the single channel activity in a patch after a long lasting depolarization to 0 inV. T h e amplitude histogram constructed from the digitized

V~lume ~.71~,numl~r 2

FI~I~S LF.TTER~

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raw data of thi, rccordln| is shown In FiB.4C, The single channel amplitttde was computed by fitting ~ sum of two Oausdans to the data ~olld curve In Fill, 4C). The amplitude was calculated as the difference between the two peaks (I,09 pA at 0 mY), Single channel ¢urrent-voltaile relations if.V) were established semi. automatically. The amplitudes were measured from channels that remained open for at le~t 10 ms, The llraph in Fill. 4D summarizes the data obtained from three patches in the vohalle ranlle from -S0 mV to +60 mY, Ea¢h point represents the mean amplitude of at least 30 determinations of the unitary current level at each voltage, The I- V is almost linear in the range from -S0 mV to - l0 mV, In this range the slope conductante is 14 pS, However, the sinllle channel current reaches a maximum at about +20 mV and the slope conductance becomes negative at more positive potentials.

nels have a ne|aliv¢ slope conductance The n~$atlv¢ slope condolence is possibly caused by a magnesium block, and will be l'unher Investlilated, Finally, the pharma¢oloilical profile is remarkably different between the two K ' channels. K" channel proteins are e=pable of formlnll heteromuitlmers with other K" channel subunifs, The forms. tier of heteromultlmers ehanje~ conductance, inactive. tier and pharmacological properties of K ° ch=nnels [2},24], Therefore, RCK4. and Raw."l-containinil heteromultlmeric g " channels millht show the proper. ties of a number or different A.type channels reported for excitable cells [2], A-Type K" channels like RCK4 start to activate negative to the threshold of sodium channel activation, Raw3 K ° channels, on the other hand, have a more positive threshold of activation. They app=trently operate in the voltage range of Ca =* current activation, Thus, Raw3 K ° channels might be involved in the modulation of Ca =.. inward currents.

4. DISCUSSION

Acl¢l=c~wltdlemel=lJI, We thank Dr D, Colquhoun, Dr T, Verdoorn and B. Warner for readina the manuscrlph Dr B. $&kmann for help. ful dlscuss=ons and Dr F Dreyer for the air| of dendrotoxln This

K* channels are particularly diverse, and are probably present in all excitable and nonexcitable ceils [2]. The first K" channel gene cloned was the Shaker R* channel 8¢ne in Drosophila [15; 19-21]. The transcripuon of this gene leads to the syntheses of several mRNAs expressing either rapidly or slowly inactivating K* channels when injected into Xenopusoocytes. Subsequently, the Shaker.related K + channel cDNAs Shah, Shaw, and Shal were identified m Drosophila [17]; these code for slowly- or noninactLvating K ~" channels [22], Similar vertebrate K* channel cDNAs were ~denttfied in the mammalian brain, most notably the Shaker.ranted RCK g ÷ channel family in rat brain [5], Expression of RCK cRNA in Xenopus oocytes produces several types of voltage.gated K* channels, of both the delayed rectifier (slowly-reactivating) or A-type (rapidly.inactivating). Members of the Shaw-related K* channel family m rat brain also resemble delayed-rectifler type K + channels (NGK2, RKShlIIA) [4, 18] or A-type K ÷ channels (Raw3). These observations suggest that A-type and delayed-rectifier K + channels were both drayed from a prototype voltage-gated K" channel and that within a g=ven K" channel family small sequence varlatmns obviously suffice to convert an A-type K ~ channel into a delayed rectifier type K" channel. The first mammalian A-type K* channel to be described was the RCK4 K ~ channel [5]. Here, we have descrlbed the properties of a second A-type K" channel. The Raw3 K + channel differs in several ways from the RCK4 K ÷ channel. Raw3 currents activate in a more positive potential range, inactlvatc more rapxdly and the steady state inactivation curve has 35 mV more positive. The single channel conductance of Raw3 K ÷ channels is about 3-fold higher than of RCK4 RCK4 channels do not saturate at positive potentials whereas Raw3 ct~an-

work was supported by grants or the Deutsche Forschunsssemeln. sehaft,

REFERENCES [I] l.[die, B, (1984) Ionic Channels or Excitnbl© Membranes, Smauer, Sunderland, MA, [2] Rudy, O, (1988) ~euroscience 25,729-750 [3] Jan, Y J and Jnn, Y N (1990) Trends Neurosch I3, 4l$-419 [4] McCormack, R, Vega.Sacnz, E C and Rudy, B, (1990) Proc Nail Acad Scl U S A 87, 5227-5231, [5] Stdhmer, W., Ruppersberg, J.P., Schr~ter. g H , Sakmann, B. Sleeker. M , Gies¢, K P . Perschk¢, A . Baumann. A and Pongs, O (1989) E M B O J 8, 3235-3244 [6] Benton. W D and Davis. R W (1977) Science 196. 180-182 [7] Maniatis. T . Pntsch, E F and Sambrook. J. (1982) Molecular Cloning. A Laborarory Manual. Cold Spring Harbor taboratory Press, Cold Spring Harbor, NY, i8] Sanger, F,, Nlcklen, S and Coulson, A R (1977) Pro~. N.tl, Acad Sck U S A 74, 5463-5467 [9] Kneg. P and Melton, D A (1984) Nucleic Acids Res 12. 705%7070 [I0] Methfessel, C , Wttzemann, V , Takahashi, T, M=shma, M , Numa, S and Sakmann, B (1986) Pfltlgers Arch, 407,577-588, [II] StLlhmer, W, Methfesse], C , Sakmann, B , Node, M and Numa, S (1987)Eur B,ophys J 14, 131-138 [12] Bezamlla, F and Armstrong, C M, (1977).l Gen Phystol 70, 549-566. [13] Kyte, J and Doohttle, R.F (1982) I Mol Bml 157, I0S-132. [14] McCormack, K , Campanelh, J T.. Ramaswamh M , Mathew, M K , Tanouye, M A , lverson, L.E. and Rudy, B (1989) Nature 3,;0. 103-104 [15] Pongs, O,, Kecskemethy, N., M~ller, R, Krah-Jentgens, I , Baumann, A , Ktltz, H H , Canal, 1, Llamazares, S, and Ferrus, A (1988)EMBO J 7, 1087-1096. [16] Froth, G C , V~n Dongen. A,M,, Schuster, G., Brown, A,M and Joho, R H (1989) Nature 340. 642=64~ [1"/] Butler, A., We=, A., Baker, K and Salkoff, L (1989) Science 243,943-947

215

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