Cloning and expression of cDNA and genomic clones encoding three delayed rectifier potassium channels in rat brain

Neuron,

Vol. 4, 929-939,

June, 1990, Copyright

0 1990 by Cell Press

Cloning and Expression of cDNA and Cenomic Clones Encoding Three Delayed Rectifier Potassium Channels in Rat grain Richard Swanson: John Marshall,* Jeffrey 5. Smith: Jacinta 6. Williams: Mary B. Boyle,*5 Kimberly Tolander: Christopher J. Luneau: Joanne Antanavage: Carlos Oliva: Susan A. Buhrow: Carl Bennett,+ Robert B. Stein: and Leonard K. Kaczmarek* *Department of Pharmacology +Department of Biological Chemistry Merck Sharp and Dohme Research Laboratories West Point, Pennsylvania 19486 *Department of Pharmacology Yale University School of Medicine New Haven, Connecticut 06510

Summary Rat brain cDNA and genomic clones encoding three K+ channels, K,l, K,2, and K,3, have been isolated by screening with Shaker probes and encode proteins of 602,530, and 525 amino acids. Each of the deduced protein sequences contains six hydrophobic domains (including an S4-type region characteristic of many voltage-gated channels) and are 689672% identical to each other overall. Transcripts of m3.5, m6.5, and -9.5 kb encode K,l, K,2, and K,3, respectively. The K,2 mRNA is expressed only in brain, whereas the K,l and K,3 transcripts are found in several other tissues as well. There is a marked increase in the amount of K,l mRNA in cardiac tissue during development and a similar, but less pronounced, increase of both this mRNA and the K,2 transcript in brain. RNAs synthesized in vitro from the three clones induce voltage- and time-dependent, delayed rectifier-like K+ currents when injected into Xenopus oocytes, demonstrating that they encode functional K+ channels. Introduction K+ channels control both the repolarization phase of action potentials and the pattern of firing of neurons and other cells. Electrophysiological studies have shown that K+ currents are much more diverse than Na+ or Ca2+ currents (Hille, 1984). This diversity plays an important role in allowing different cells to display a variety of modes of electrical behavior, such as spontaneous pacing or bursting, and accounts for some of the diversity in the shapes of action potentials. K+ channels also play a central role in determining the way a cell responds to an external stimulus. For example, the rate of adaptation or the delay with which a neuron responds to synaptic input is strongly determined by the presence of different classes of K+ channels. Furthermore, K+ channels are subject to 5 Present University

address: Department of Iowa, Iowa City,

of Iowa

Physiology 52242.

and

Biophysics,

short-term and long-term modulation by neurotransmitters and hormones, allowing cells to alter their electrical properties (Kaczmarek and Levitan, 1987). Until recently, knowledge of the function and regulation of K+ channels had resulted largely from electrophysiological investigations. The cloning of a family of K+ channels from the Shaker locus of Drosophila (Schwarz et al., 1988; Pongs et al., 1988; Kamb et al., 1988) has provided the basis for a molecular understanding of these proteins. A number of K+ channel genes have now been cloned from both invertebrates (Butler et al., 1989) and vertebrates (Tempel et al., 1988; Baumann et al., 1988; Takumi et al., 1988; McKinnon, 1989; Frech et al., 1989; Stuhmer et al., 1988, 1989b; Christie et al., 1989; Yokoyama et al., 1989; Chandy et al., 1990; Douglass et al., 1990). Expression of these channels in Xenopus oocytes indicates that they encode components of either inactivating or delayed rectifier-like channels (Iverson et al., 1988; Timpe et al., 1988; Stuhmer et al., 198913). We now report the cloning, expression, developmental regulation, and tissue distribution of three delayed rectifier-type K+ channels from rat brain. Furthermore, we show that these proteins are each encoded by single exons, suggesting that the functional diversity of KC channels in the mammalian brain arises, at least in part, from the expression of different genes. Results Isolation of cDNAs and Cenomic Clones Oligonucleotides derived from the fourth (S4) and fifth (S5) hydrophobic domains of the Shaker sequence hybridized to a 3-3.5 kb transcript on Northern blots of rat brain mRNA. A mixture of the two oligonucleotides was therefore used to screen sizeselected cDNA libraries constructed from poly(A)+ mRNA isolated from the brains of 12-day-old rats. Two clones, K41 and PI, were isolated. Nucleic acid sequence analysis demonstrated that they could encode the carboxy-terminal regions of two proteins with homology to the Shaker channels. To isolate the cDNAs encoding the amino-terminal region of the proteins, a specifically primed cDNA library was constructed and screened as described in Experimental Procedures. Clone 18, isolated from this library, overlaps K41 and extends -1 kb in the Sdirection. A variation of the polymerase chain reaction (PCR) was then used to isolate a cDNA (clone ISRACE) that overlaps clone 18 and extends it farther 5’. K,l, a cDNA containing the entire open reading frame, was constructed in vitro by ligating the three partial clones together at overlapping restriction sites (Figure IA). While screening the specifically primed library for sequences upstream of K41, cDNAs encoding part of a homologous, but nonidentical, protein were also isolated. All had the same 3’ end, and clone 15 ex-

Neuron 930

A

x

E ,C

H

Kvl K41

18 -l&RACE

B 15 15PCR

0

I Figure

1. Partial

1

,

I Restriction

3 1 kbp

2

,

I

Maps

of the

, K,l

and

K,2 cDNAs

The solid boxes represent the putative protein coding regions; the thin lines, the Sand 3’ untranslated regions. Sites for restriction enzymes used to construct the full-length coding region are shown. Abbreviations: X, Xhol, E, EcoRI; C, Clal; H, Hindlll; A, Aocl; S, Stul. The bars below K,l and K,2 represent the positions of the individual cDNAs isolated from hgtl0 libraries or by the PCR as described above.

tended the farthest 5’. Sequence analysis of this clone showed that it encodes the amino-terminal region of a protein homologous to K,l. A genomic DNA clone containing the entire open reading frame encoding this protein was isolated in parallel (see below). Using sequence information from the genomic clone, a cDNA encoding the carboxy-terminal region (clone 15-PCR) was isolated from poly(A)+ mRNA using the PCR (Kawasaki and Wang, 1989). K,2, a cDNA encoding the entire protein, was then assembled in vitro from the two partial cDNAs (Figure 16). K41 and PI were also used to screen a rat genomic DNA library, and the EcoRl fragment of each genomic clone that hybridized to the probes was identified by Southern blotting and sequenced. Two classes of clones, differing in their restriction maps and in the intensity of their hybridization signals, were isolated with the K41 probe. Sequencing revealed that they represent the genomic counterparts of the K,l and K,2 cDNAs. Similarly, a genomic K,3 clone was isolated using the PI probe. No introns were found within the genomic DNA sequences representing the entire open reading frames of K,l, K,2, or K,3. DNA sequence upstream and downstream of that represented by the cDNAs has been determined from each of the genomic clones. Potential introns within these noncoding regions and the Sends of the mRNAs have not yet been mapped. Sequence Analysis and Primary Structures The assembled K,l cDNA is 2825 bp long and has a single long open reading frame encoding a protein that is homologous to members of the Shaker family of K+ channels (Figure 2). The cDNA contains neither a poly(A) tail nor a poly(A) addition signal sequence

(AATAAA or ATIAAA; Nevins, 1983). The ATG at nucleotides l-3 was assigned as the translation initiation codon because it encodes the first methionine following an in-frame stop codon (at -201) and is contained within a strong consensus sequence for initiation (Kozak, 1989). The deduced protein is 602 amino acids long (M, = 66.6K) and has six hydrophobic regions (labeled Sl-S6). S4 contains a positive charge at every third residue, a sequence motif found in many VOEtage-gated channels and demonstrated to be involved in sensing voltage changes across the membrane (Stuhmer et al., 1989a). A potential leucine zipper is found between the S4 and S5 domains (Leu*2-Leu430), as has been previously noted for several other K+ channels (McCormack et al., 1989). The deduced protein sequence has four potential sites for phosphorylation by CAMP-dependent protein kinase (R/K-RI K-X-(X)-S/T; Krebs and Beavo, 1979); three are in the carboxy-terminal region and one is in the aminoterminal region (Figure 2). The amino-terminal phosphorylation site (Seral) also lies within a casein kinase II recognition sequence (Kuenzel et al., 1987). There are five potential sites for N-linked glycosylation (N-X-S/T; Hubbard and Ivatt, 1981); four are iocated in the amino-terminal region of the protein, and one (Asn2g0) is within the putative Sl-52 loop (Figure 2). The cDNA encoding the second K+ channel, K,2, is 2000 bp long (Figure 3). Like K,‘i, it has neither a poly(A) tail nor a poly(A) addition signal. The single long open reading frame encodes a protein of 530 amino acids (M, = 58.8K). The ATG at position l-3 was assigned as the initiation codon since it encodes the first methionine downstream of an in-frame stop codon (at -30) and is within a strong initiation consensus sequence. The deduced protein also has six hydrophobic regions, including the characteristic S4type sequence, and a potential leucine zipper (Leu3s6Leu3B4). The protein is homologous to K,l, differing predominantly in the aminoand carboxy-terminal regions and in the loop linking the Sl and S2 domains. In K,2, this region is highly acidic, whereas in K,l, it is enriched in proline. K,2 has two potential sites for phosphorylation by CAMP-dependent protein kinase (both in the carboxy-terminal region), one potential casein kinase fl phosphorylation site within the Sl-S2 loop (Ser**z), and one potential N-linked glycosylation site in the amino-terminal domain (Figure 3). The deduced amino acid sequence of the third KC channel, K,3, was determined from the DNA sequence of a genomic clone, since the corresponding cDNA clone, PI, encodes only part of the coding sequence (Ala 143-Vals25). The predicted protein contains 525 amino acids (M, = 58.4K) and is homologous to both K,l and K,2, again differing predominantly in the aminoand carboxy-terminal regions and in the Sl-S2 loop. The protein has six potential N-linked glycosylation sites, one in the amino-terminal region, two in the Sl-S2 loop, and three within the carboxyterminal domain. In addition, there are two potentia!

Cloning 931

and Expression

of Rat Brain K+ Channels

AGGCCGGGW\GCTCAGCCAGAGAGGGGCTGCTGGAGGTTG Figure 2. Nucleotide and Deduced CACTGAGAGGGAW\GAGAGGCAGGGAGCAGGGGCAGCAGCTTC~GACGTCAGGACCAGCC~G~TCGGGCCAGCTACCCCGGCCAGAcid Sequences of the K,l cDNA CCTAGAGTCAGCGGGGCCCTCGGCTGWIGAGW\GACCTACGCGAGGCTTG~GCGTGAGTTGGGGGTGTGG~CCGGTTGTCTGGGGCG -25

Amino Clone

The nucleotide sequence is numbered from the 5’to 3’direction. The first residue of the initiator methionine codon is +I, GASCVQTPRGECGCPPTSGLN?QSKETLLR and nucleotides in the 5’ untranslated reGGGGCAAGCTGTGTGCAGACCCCCAGGGGA~GTGTGGGTGCCCTCCGACGTCTGGACTC~T~TCAGTCC~G~CACTG~~GG 1:: 0 gion are indicated by negative numbers. GRTTLEDANQGGRPLPPl4AQELPQPRRLSA GGGCGCACAACGCTCWIG6ATGCGAACCAGGGTGGACGGCCTTTGCCCCCTATGGCTCAGGAGCTGCCAC~CCTAG~GGCTATCTGCT 2:: The deduced amino acid sequence is shown above the nucleotide sequence, EDEEGEGDPGLGTVEEDQAPQDAGSLHHQR 112 GAGGATGAGGAGGGAGMGGCGACCCTGGCCTGGGCACAGTGGAG~GGACCAGGCTCCTCAG~TGCAGGGTCACTCCATCACCAGCGA 336 numbered from amino to carboxyl terminus. The six hydrophobic domains of the VLl%SGLRFETQLGTLAQFPNTLLGDPAK 142 GTCCTCATAAACATCTCCGGGTTGCGmCGAW\CGCAGCTGGGCACCCTGGCACAGTTTCCC~CACCCTCCTGGGG~CCCAGCC~G 426 protein (Sl-S6), determined using the algorithm of Kyte and Doolittle (1982) with a RLHYFDPLRNEYFFORNRPSFDGILVYVQS 172 CGCCTGCACTACTTCGACCCC~~G~T~TACTTCTTC~CCGC~CCGGCCCAGCTTCGATGGCA~TTGTACTACTACCAGTCT 516 window of20 amino acids, are indicated by the horizontal bars. Asparagines within GGRLRRPV?VSLDVFADEIRFYQLGDEANE 202 GGGGGCCGCCTGCGCAGGCCCGTCAATGTCTCCCTGGATGTG~TGCAGATGAGATCCGCTTTTACCAGCTGGGG~CGAGGCCATG~G 606 the consensus N-linked glycosylation sequence are indicated with arrows. Serines RFREDEGFIKEEEKPLPRNEFQRQVULIFE 232 CGCTTCCGGGAGGATGAGGGCTTCATCAAGGAAGAGGAGA 696 and threonines within a consensus CAMPI Sl 1 dependent protein kinase recognition site YPESSGSARAIAIVSVLVlLISIITFCLET 262 TACCCAGAAAGCTCTGGGTCCGC~~GC~TCGCCATAGTGTCGGTCCTGGTCATTCTCATCTCTATCATCACC~CTGCCTG~GACT 706 are indicated by filled circles. This and the other nucleotide sequences reported in LPEFRDERELLRHPPVPPQPPAPAPGl?GS 292 CTGCCTGAGTTCAGG6AT~GCGGGAGCTGCTACGCCATCCCCCAGTGCCGCCCCAGCCCCCAGCCCCTGCCCCTGG~TC~TGGCAGC 076 this paper will appear in the EMBL, CenI Bank, and DDBJ Nucleotide Sequence VSGALSSGPTVAPLLPRTLADPFFIVETTC 322 GTCTCTGGAGCACmCCTCTGGCCCTACffiTGGCTCCACTC~GCCTAG~CACTGGCC~TCCA~C~CATCGTGGA~CCACATGT 966 Databases under the accession numbers 52 I r M27158, M27159, and M31744. VINFTFELLVRFFACPSKAEFSRNIMNlID 352 MElSLVPLE?GSA"TLRGGGEA CAGCATGCCCCCTGCCCCCGGAtCATGGAW\TCTCCCTGGTGCCCCTG~~TGGCAGTGCCAT~CCClCAGAG~GGAGGG~GGCA

GTWITCTGGTTCAC~GAGTGCTCGTGCGCTTCmGttTC 1056 53 I VVAIFPYFITLGTELAEQQPGGGGQNGQQA 382 GTCGTGGCCATC~CCCCTACmATtACCCTGGG~C~GCTGGCA~GC~C~CCAGGGG~GGGGGTCA~TGGGCAGCAGGCC 1146 , S4 I NSLAILRVIRLVRVFRIFKLSRHSKGLQIL 412 ATGTCCCTAGCCATCCTCAGGGTW\TCCGCCTGGTGC~TG~CG~TC~C~GCTCTCCCGCCACTCC~GG~CTGCA~TCCTG 1236 I S5 GKTLQASNRELGLLIFFLFIGVILFSSAVY 442 GGT~~CCTTGC~GCATCCATGCGGW\ACTCGGGCTACTCATCTTC~CCTC~CATTG~GTCATCCTC~CTCCAGCGCTGTCTAC 1326 7 472 FAEADNHGSHFSSIPDAFYNAVVTMTTVGV TTCGCAW\GGCAGACAATCACGGGTCCCATTTtTCTCTAGTATCCCA~TGCC~CTGGTGGGCAGTAGTCACTAT~CCACTGTAGGCTAT 1416 I 56 GDMRPITVGGKIVGSLCAIAGVLTIALPVP 502 GW\GACATGAGACCCATCACTGTAGGGGGC~GATCGTGGG~CACTGTGCGCCATAGCTGGGGTCCTCACCATTGCCCTGCCTGTCCCC 1506 VIVSNFNVFYHRETDHEEQAALKEEQGNQR 532 GTCATCGTCTCCAACTTTAATTACTTCTATCATCGGGAGAGG 1596 0 562 R E ? GLDTGGQRKVSCSKASFCKTGGSLESS CGG~GTCTGGGCTG6ACACA66656TCMC66M66TCAGCTGCAGC~GGCCTCC~GC~~CTGGGGG~CCCTG~~6~CT 1686 0 592 DSIRRGSCPLEKCHLKAKSNVDLRRSLVAL 1776 ~CAGTATW\WGGGGTAGCTGTCCTCT~GTGTtAGCCCTC 602 CLDTSRETDL TCCAGGCAGACTGGCACCAGTGAAGCTGGCCACAGGGGTGCCCCTTGAGC 1866 TGTCTGGACACTAGCCGTMMCAGATTTGTAWiAGAU 1956 CTGGGWITCTGCmACACCACtWGTAmMGCCCACtTGGTtACCTGCC 2046 CTCTAAC~CCCCAmTMCTCCTtmCtATMCCCCCAGGGTCGCCTA~M~GTAT~~~CCAT~CGC~GCCG~ 2136 GAAGTGCTGAGCCCTCACTGGAAGATGGATGCATTtATAGCC~~CTACACCCAGCA~GG~TMTC~C~~~C~~ 2226 AAGCTTAWTCCC~~TCCATAGCA~CCCTACCCGTG~CC~T~AC~ACATGG~~A~~GTGTAT~~TA~ 2316 ATTmATGGCCGATWICTGCA~GTACAGTGtAWTDIT 2406 GTTGGGGGGGTCACCA~CCTGGMCACT~GGMC~~CCCT~~G~G~CAGG~CTGTG~CT~GC~C~A~A~ 2496 CTGGOilGTACTATTGGTGCTTCTGGTCTAG~~~TG~CTA~GACCAGC~TCT~TC~GT~CTGTC~CACA~GCA 2531 TTTTAAGGATGTTGGAAGAAGGATTTGGAGAATTC

sites for phosphorylation by CAMP-dependent protein kinase, both in the carboxy-terminal domain of the protein. The nucleotide sequence of K,3 is essentially identical to the RCK3 sequence that has been reported by Stuhmer et al. (1989b). It differs from RCK3 at only 3 nucleotides (positions +135, +297, and +316), resulting in a single amino acid difference (Phelo6 [UUC] in K,3; Leulo6 [CUC] in RCK3). Regulation of Expression in Different Tissues during Development Restriction fragments that are specific to each of the three clones were used to probe Northern blots of rat poly(A)+ mRNAs. Probes derived from K,l hybridize to a transcript of ~3.5 kb, those from K,2 to an RNA of ~6.5 kb, and those from K,3 to an RNA of w9.5 kb (Figure 4). As expected, probes from each of the clones hy-

bridize to transcripts in mRNA isolated from adult rat brain (Figure 48). K,2 transcripts were not detected in any other tissue analyzed. K,l, however, was also found in heart, kidney, lung, and skeletal muscle, and K,3 was found in lung and spleen. The expression of K,l mRNA changes significantly during development in brain and heart (Figure 4A). In brain, the levels of this mRNA increase gradually from the neonate to the adult. In heart, the amount of K,l mRNA increases abruptly in the first 2 weeks after birth and thereafter remains relatively constant. The levels of K,2 mRNA in brain, like those of K,l mRNA, increase gradually during development. In contrast, the expression of K,3 in brain remains relatively constant postnatally. Expression and RNA transcribed was translated

Functional Assay of the Three Clones in vitro from each of the three clones in a rabbit reticulocyte lysate, and pro-

Neuron 932

TTGCGGGTTCCAGGCATCTCAGAAATCTTGAGCACGGAGGCGCGGCTACTGAGAGCCAGAGCCACATCCCAGACCTAGCCTGGCAGAGAG 154 Figure ACCAGCTGCAGGGTTCACCGACCTAACCGCCAGGTCAGAGCACGGGCCCCACCCT~GGAGGGCGCAGCCGGAGCTGGG~GCCGGTGC -64

Acid

3. Nucleotide and Deduced Sequences of the K,2 cDNA

Amino Clone

MRSEKSLTL 9 The 21 CGCGCTCCGGAGCTCGTGTCGTGGGCGCCGTCCTAGTGGCGGGGAGCGCACCGCCGAGGTGACATGAGATCGGAG~TCCCTGACGCTG

nucleotide sequence is numbered the S’to 3’ direction. The first residue methionine codon is +I, and nucleotides in the 5’ untranslated re69 SERLVI%SGLRYETQLRTLSLFPDTtLGD gion are indicated by negative numbers. 207 AGTW\GAGGCTGGTGATCAACATCTCTGGGCTGCGCTACGAGACGCAGCTGCGCACCTTGTCGCTGTTCCCTGACACGCTGCTAGGAGAC The deduced amino acid sequence is PGRRVRFFDPLRNEYFFDRNRPSFDAILYY 99 shown above the nucleotide sequence, CCTGGCCGCAGAGTCCGCTTCTTTGACCCCTTGAGGAATGAGTACTTCTTTGACCGC~CCGACCCAGCTTCGACGCTATCCTTTATTAC 297 numbered from amino to carboxyl termiYQSGGRLRRPVNVPLDIFMEEIRFVQLGDE 129 nus. The six hydrophobic domains of the TACCAGTCGGGGGGTCGCCTGCGCAGGCCGGTT~CGTGCCCCTTGACATCTTTATGG~GAGATTCGCTTCTATCAGTTGGGAGATG~ 387 protein (Sl-S6), determined using the al159 ALAAFREOEGCLPEGGEDEKPLPSQPFQRQ gorithm of Kyte and Doolittle (1982) with a GCCCTGGCGGCCTTCCGGGAGGATGAGGGTTGCCTGCCCG~GGTGGTGAGGATGA~GCCACTCCCCTCCCAGCCTTTCCAGCGACAG 471 I window of 20 amino acids, are indicated by V Y L L F E Y P E S 5 G P A R G I A I V 5 V LslV I L I S I V ies the horizontal bars. Asparagines within the GTCTGGCTCCTCTTTGAGTATCCGGAGAGTTCTGGGCCCGCCCGAGGCATTGCCATCGTCTCAGTGTTGGTCATCCTCATCTCCATTGTC 567 I consensus N-linked glycosylation seIFCLETLPQFRADGRGGSNEGSGTRMSPAS 219 quence are indicated with arrows. Serines ATCTTTTGCCTGGAGACCTTGCCTCAGTTCCGTGCAGATGGGCGCGGTG~GC~CGAGGGGAGTGGGACCCGCATGTCCCCGGCCTCC 657 and threonines within a consensus CAMPRGSHEEEDEDEDSYAFPGSIPSGGLGTGGT 249 dependent protein kinase recognition site AGGGGGAGCCACO\GGAGGAAW\TW\AWICGAGGATTCCTATGCAT~CCTGGTAGCA~CCCTCTGGGGGGTTGGG~CCG~G~CT 747 I are indicated by filled circles. SSFSTLGGSFFTDPFFLVETLCl';lfFTFEL from

AAPGEVRGPEGEQQDAGEFQEAEGGGGCCS of the initiator GCGGCGCCGGGGGAGGTCCGTGGGCCGGAGGGGGAGCAACAGGATGCGGGTGAGTTCCAGGAGGCCGAGGGCGGCGGCGGCTGCTGTAGT

279 T~~CA~AGTA~TCT~GGGGGTTC~~~TT~ACAGA~~~~TT~~~~TGGTG~CT~TGTGTAT~GT~TGGTTCA~C~~GCTC 037 1 I LVRFSACPSKAAFFRNlNNIIDLVi31FP"F 309 CTGGTGCGCTTCTCTGCCTGTCCCAGCAAGGCGGCCTTCT~CGC~TATCAT~CATCA~~C~GGTGGCCATC~CCCCTACTTT 927 1 ITLGTELVQRHEQQPVSGGSGQNGQQAUSL 339 ATCACCCTGGGCACCW\GCTAGTGCAACGTCACGAGCAGCAGCCTGT~GTGGTGGCAGTGGTCAG~TGGGCAGCAGGCCATGTCCCTA 1017 I t A I L R V I R L V R V Fs4R I F K L S RH 5 KG L Q I L G KT 369 GCCATCCTCAGGGTGATCCGCCTGGTCCGGGTGmCGW\TC~C~GCTCTCCCGCCACTCC~GGGG~GCAGATCCTGGGT~GACC 1107 I I LQASNRELGLLIFFLFIG'ZILFSSAVVFAE 399 TTGCAAGW\TCCATGCGGGCTCGGGCTACTCATC~C~CCTCTTCA~GGAGTCATCCTC~CTCCAGCGCTGTCTAC~CGCA~G 1197 ADDVDSLFPSIPDAFNUAVVTNTTVGYGDH 423 GCAGATGACGTTGACTCGCTC~CCCTAGCATCCCAGATGCATG 1287 I I VPMTVGGKIVGSLCAIAGV?TIALPVPVIV 459 ATACCCCATWCGGTGG~G6M~~GTGGGCTCACTGTGTGCCATTGCTGGGGTCCTCACCA~GCA~ACCGGTACCGGTCA~GTC 137, SNFNYFVHRETEQEEQGQVTHVTCGQPTPD TCCAAmCMCTACrrCTACCACC~~GACGGAGCAG~G~C~GGCCAGTATACCCACGTCAC~GTGGGCAGCCTACACCG~C I",:; 0 LKATDNGLGKPDFAEASRERRSSVLPTPHR CTGAAGGC~CGCGGACAATGG~~~C~CCT~CmGCGGAGGC~CACGG~CGGCGGTC~GCTACC~CC~CTC~CATCGAI::; AVAEKRMLiEV GCTTATGCAGAGAMAGMTGTCACCGAGGTTTW\T66ATG 1% GCTTCCTTCTCATGCTCACTACTCCCGCCTTAGCTCCAMG~CCTC~CCCCCGCCCCGCCCCCTCCCGTACACAGTACACGGCATCC 1737 TGGACCAAATATCTGGACT 1756

of ~67, ~56, and m55 kd were synthesized (K,l-K,3, respectively; Figure 5), consistent with the sizes of the proteins predicted from the DNA sequences (M, = 66.6K, 58.8K, and 58.410. To test whether the cloned sequences encode functional K+ channel proteins, Xenopus oocytes were injected with RNA transcribed from the K,l, K,2, and K,3 cDNAs. The cells were voltage-clamped 1-3 days after injection, and large outward currents could be measured upon depolarization from a holding potential of -80 mV (Figure 6A-6C). The currents displayed delayed rectifier-type kinetics, showing little inactivation during 200 ms pulses. The three channels have a similar voltage dependence of activation (Figure 6D) with a threshold of approximately -40 mV. Half-maximal activation occurred at -3 mV for K,l, -13 mV for K,2, and -10 mV for K,3. The ionic selectivity of the three channels was determined by measuring the reversal potential of tail currents in bathing solutions containing various KC concentrations. Figure 6E shows the dependence of the reversal potential on the external K’ concentration for K,2 tail currents. The straight line through the data points is a least squares linear regression fit teins

to the Nernst equation with a slope of 58 mV per IOfold change in K’ concentration. Similar results were obtained for K,l and K,3 (data not shown), which is consistent with the interpretation that all three cloned channels are highly selective for K+. Pharmacological Characterization of the Expressed Currents Several K+ channel blockers and toxins were tested for blocking activity on the expressed currents (Table 1). 4-Aminopyridine was found to block the three currents half-maximally at less than 500 PM. Tetraethylammonium was an effective blocker of K,2, exhibiting half-maximal block at 4 mM. In contrast, K,1 and K,3 were only partially blocked by 40 mM tetraethylammonium. The polypeptide Ki channel toxins (Moczydlowski et al., 1988), dendrotoxin and mast cell degranulating peptide, were potent blockers of K,2 currents, with half-maximal blocking concentrations of 9 nM and 20 nM, respectively. K,l and K,3 currents were insensitive to dendrotoxin and mast cell degranulating peptide at concentrations as high as 200 nM. Two scorpion peptide toxins, charybdotoxin and noxiustoxin, were also tested for blocking activity on the

Cloning 933

and Expression

rat brain

of Rat Brain

K+ Channels

rat heart

rat brain

+

18s

rat heart

*

rat brain

4

18s

rat heart

*

28s

4

18s

ACTIN

.‘

Figure

4. Northern

Blot Analysis

of K,l,

K,2, and

K,3 mRNAs

(A) Developmental expression of the three mRNAs in brain and heart. Poly(A)+ mRNAs, isolated from the to adult rats, were resolved by electrophoresis, blotted to nylon membranes, and probed with K,l-, K,2, scribed in Experimental Procedures. K,l, K,2, and K,3 are encoded by transcripts of approximately 3.5,65, insets at the bottom of each blot are the signals obtained by reprobing for actin transcripts. (B) Tissue distribution of the K,l-K,3 mRNAs. Poly(A)+ mRNAs isolated from various tissues of adult rat that the K,l blot was probed with aZP-labeled, random hexamer-primed K41. Since this probe contains found in K,2, both the K,l (3.5 kb) and K,2 (6.5 kb) mRNAs hybridize. RNA was isolated from brain (BR), (LV), lung (LG), skeletal muscle (SM), and spleen (SP).

expressed currents. Charybdotoxin has been previously shown to block high conductance K(Ca2+) channels (Miller et al., 1985) as well as cloned Shaker(MacKinnon et al., 1988) and rat brain (Stuhmer et al., 1989b) K+ channels expressed in oocytes. Noxiustoxin is a low-affinity (300 nM I&,) blocker of squid axon

hearts and brains of neonatal or KJ-specific probes as deand 9.5 kb, respectively. The were probed as above except some sequences that are also heart (HT), kidney (KD), liver

K+ channels (Carbone et al., 1982). Charybdotoxin blocked K,3 currents half-maximally at 1 nM; K,l and K,2 currents were insensitive at concentrations as high as 200 nM. Noxiustoxin was found to block K,3 currents with an I& of 0.2 nM and K,2 currents with an I& of 400 nM, but had no effect on K,l currents

Neuron 934

acid homology between K,l, sentatives of other mammalian The three sequences reported

K,2, and K,3 and repreK+ channel sequences. here represent new

mammalian members of the Drosophila Shaker family, which now includes six distinct channels (K,lK,3, RCKl, RCK4, and RCKS). Homologs of RCKI have been cloned from both mouse genomic DNA (MK7; Chandy et al., 1990) and mouse (MBKI; Tempel et al., 1988) and rat (RBKI; Christie et al., 1989) brain cDNAs. A mouse genomic DNA clone of RCK5 (MK2; Chandy et al., 1990) and cDNAs of another allele of this gene (BK2; McKinnon, 1989; NGKI; Yokoyama et al., 1989) have also been isolated. Similarly, mouse (MK3; Chandy et al., 1990) and rat (RGK5; Douglass et al., 1990) genomic homologs of the K,3 (RCK3) cDNA have been isolated. Significantly less homology exists between these Shaker-type channels and three other cloned K+ channels, DRKI (Frech et al., 1989), NCK2 (Yokoyama et al., 1989), and isK (Takumi et al., 1988). DRKI and NGK2 are mammalian homologs of the K+ channels encoded by the Drosophila Shab and Shaw loci (Butler et al., 1989), respectively. The isK channel is structurally unique and has been cloned from rat kidney, heart, and uterine cDNAs (Takumi et al., 1988;

29 Figure

5. In Vitro

Translation

Products

of the

Three

RNAs

RNA transcribed in vitro from the K,l, KJ, and K,3 clones was translated in a rabbit reticulocyte lysate, and the [35S]methionine-labeled proteins were resolved by SDS-PAGE and visualized by fluorography. Proteins of approximately 67,56, and 55 kd are synthesized in the presence of KJ, K,2, or K,3 RNA, respectively. The band at -45 kd is produced nonspecifically by the lysate in the absence of any exogenous RNA.

at concentrations as high as 2.5 PM. Noxiustoxin blockade of K,3 and K,2 currents was fully reversible by washing the chamber with several volumes of toxinfree buffer. Discussion We have identified sequences encoding three functional voltage-dependent K+ channels in rat brain. A variant of one (K,3) has previously been reported by Stuhmer et al. (198913) and was called RCK3. The latter sequence differs from K,3 by 3 nucleotides and 1 amino acid, suggesting that they represent two alleles of the same gene. Table 2 compares the overall amino

Table

1. Pharmacological

Characteristics

of Cloned

mRNA

TEA (mM)

4.AP fmM)

W

>40 4 >40

0.4 0.3 0.4

W

K,3

Numbers in this table refer to I& concentrations at which no effect cell degranulating peptide; NTX,

K+ Channels DTX

wm

Folander et al., 1990) and human genomic DNA (Murai et al., 1989). Members of the Shaker family can apparently encode channels that produce either inactivating currents of the A-current type or re!atively noninactivating delayed rectifiers (Stuhmer et al., 198913). RNAs transcribed from the KJ, K,2, and K,3 sequences all induce the expression of delayed rectifier currents in Xenopus oocytes. There are, however, distinct differences in these three K+ channels with respect to tissue distribution, expression during development, and pharmacological sensitivity. This diversity does not appear to arise from alternative splicing of a single large transcript, as is the case in the Drosophila Shaker locus (Schwarz et al., 1988; Pongs et al., 1988; Kamb et al., 1988). For each of these three channels, we isolated a separate genomic DNA clone containing the entire coding sequence on a single, uninterrupted exon. It is likely, therefore, that these three clones represent separate gene products. Three genomic DNA clones encoding K+ channel genes in the mouse (MKI-MK3) and one rat genomic K+ channel clone (RCK5) have also recently been reported to contain coding sequences that have no introns (Chandy et al., 1990; Douglass et al., 1990).

in Xenopus

(nM)

9

600)

values (50% inhibition of peak current), was measured. TEA, tetraethylammonium; noxiustoxin; ChTX, charybdotoxin.

MCDP

(600) 20 (600)

Oocytes (nM)

NTX

(nM)

(2500) 400 0.2

measured at 40 mV. Numbers 4.AP, 4.aminopyridine;

ChTX

(nM)

v.00) (200) 1 in parentheses DTX, dendrotoxin;

indicate highest MCDP, mast

Cloning 935

and Expression

of Rat Brain

K+ Channels

0.0

0.2

0.0 - 100

-I?0

-80

-00

Membrane Figure

6. Expression

of K,l,

-20

0

potentlal

imV;

K,2, and

-1

20

-2

-I

‘-og I [Kb

K,3 in Xenopus

/

Ooocytes

Cells were voltage-clamped at -80 mV, and currents were elicited with 200 ms depolarizing pulses in 10 mV increments to +40 mV. Current records from K,l are shown in (A), from K,2 in (B), and from K,3 in (C). Time scale is 30 ms. Current scale is 0.5, 3, and 1 PA for (A), (B), and (C), respectively. (D) Normalized conductance-voltage relations for K,l (closed squares), K,2 (closed circles), and K,3 (open triangles). Conductance values were obtained by dividing the current by the driving force assuming a K+ equilibrium potential of -100 mV. Each data point represents the mean value from 6 oocytes (standard deviation is no more than 0.07 for any point). Lines drawn are nonlinear least squares fits of the Boltzman isotherm: G = C,,,I[l + exp(V - V&a)]. C,,, values obtained from the fit were used for normalization of the data. Half-activation values are -3, -13, and -10 mV for K,l, K,2, and K,3, respectively. Slope factors (a) are 15, 19, and 17 mV for K,l, K,2, and K,3, respectively. (E) Tail current reversal potential as a function of the external K+ concentration for K,2. Data represent the mean and standard deviation of 8 determinations. The line is the linear least squares regression fit to the Nernst equation and has a slope of 58 mV per IO-fold change in K+ concentration.

One interesting aspect of the diversity of the K+ channels described thus far is the existence of variable numbers of putative phosphorylation sites. In common with other members of this family of pro-

Table

2. Amino

Acid Kvl

L1

KJ K,3 RCKI RCK4 RCK5 DRKI NGK2 5K

88 83 80 76 78 61 63 49

Sequence

Homology

between

Cloned

teins, the three clones we have described all encode proteins with a conserved substrate sequence for the CAMP-dependent protein kinase about 40 amino acid residues after the S6 region. K,l, however, contains

Mammalian-K+

Channels

KJ

K,3

RCKI

RCK4

RCK5

DRKI

NGK2

IrK

68

69 72

66 67 77

60 67 68 70

66 70 76 80 68

37 40 35 38 37 3%

40 41 39 40 41 41 38

24 25 21 22 22 23 19 18

86 81 80 83 62 63 46

88 80 85 58 63 45

84 88 59 65 48

80 58 64 46

59 63 49

63 44

46

Values above the diagonal represent percent amino acid identity between each two proteins. Values below the diagonal cent homology (including identical and conserved residues) between each pair of proteins. One unique representative of channel is listed in the table. Alleles and species variants that have been reported are described in the text.

indicate of each

pertype

NellrOn 936

additional sites at both the carboxyand aminoterminal ends. It is known that phosphorylation regulates many types of K+ channels in intact cells (Kaczmarek and Levitan, 1987). The function of these sites could therefore be to modify the electrical characteristics of the channel in response to second messengers. Phosphorylation could also play a role in the organization of channel-forming subunits. There is reason to believe that a functional K+ channel may not be composed of a single polypeptide chain. The homology of an entire K+ channel protein to a single internal repeat of a Na+ (Noda et al., 1986) or Ca2+ (Tanabe et al., 1987) channel a subunit suggests that K+ channels could be composed of at least four subunits. Moreover, although the injection of a single RNA species is sufficient to induce functional K+ channels in oocytes, it is possible that, as expressed in mammalian tissues, these polypeptides may form heterooligomeric complexes or complexes with additional structurally unrelated subunits (Rehm and Lazdunski, 1988; Parcej and Dolly, 1989; Rudy et al., 1988). Several mechanisms exist to generate the diversity of K+ currents observed in the mammalian central nervous system. Three distinct structural classes of K+ channels (the Shaker-type, DRKI, and NGK2) have now been cloned from rat brain (Stuhmer et al., 1989b; Frech et al., 1989; Yokoyama et al., 1989). The expression of members of these different classes of channels can result in diverse currents. Moreover, the different members of a given class of structurally homologous channels can also elicit diverse currents, with differences in their activation and inactivation kinetics, voltage dependence, and pharmacological sensitivity (Stuhmer et al., 198913; MacKinnon and Miller, 1989). The Shaker-type K+ channels, for example, despite showing greater than 70% sequence homology, display remarkably dissimilar functional responses to pharmacological probes. The tissue of origin within the brain, e.g., neural or glial, and the neuroanatomical distribution of these various K+ channels are also currently unresolved and may contribute to the functional diversity. The evolutionary maintenance of this genetic diversity, the tissue-specific and developmental regulation of expression, and the marked differences in pharmacological responsivity all imply that these channels subserve different, defined functions. The functional implications of the structural diversity of mammalian voltage-gated K+ channels remain to be fully elucidated. Experimental

Procedures

Materials and General Methods AMV reverse transcriptase, terminal deoxynucleotidyl transferase, and the Klenow fragment of DNA polymerase were purchased from Pharmacia, restriction enzymes from New England Biolabs, and a rat genomic DNA library (partial EcoRl cut in Charan 4A) from Clontech. Reagents for cDNA synthesis, RNasin, and pCEM-9Zf(-) were from Promega, oiigo(dT) cellulose (type 3) from Collaborative Research, and radioactive dNTPs and Hybond-N filters from Amersham. Zetaprobe membranes and

Biogel A-5M were purchased from Bio-Rad, collagenase (type 11) from Worthington, and female Xenopus !aevis from NASCO. Reagents for the PCR were purchased from Cetus, reagents foor in vitro transcription and Bluescript vectors from Stratagene, and reagents foor DNA sequencing from US Biochemicals. Standard molecular biological techniques were carried out as described (Sambrook et al., 1989; Ausubel et al., 1987). Oligonucleotides were synthesized on an Applied Biosystems Model 380B DNA synthesizer using B-cyanoethylphosphoramidite chemistry. Full-length products were purified, when required, by denaturing polyacrylamide gel electrophoresis and C18 chromatography (Ferretti et al., 1986). RNA Isolation and Analysis Total cellular RNA was isolated by the method of Chirgwin et al. (1979), and the poly(A)’ fraction was purified by oligo(dT) chromatography (Aviv and Leder, 1972). For Northern blot analysis, 6 PLg aliquots of poly(A)+ mRNA were resolved by electrophoresis through a 2.2 M formaldehyde, 0.8% agarose gel and electroblotted onto Hybond-N membranes in 25 mM Na+ phosphate (pH 6.5) at 400 mA for 8 hr. Probes were generated from restriction fragments isolated from each cDNA: K,l with the fragment from the 5’ end to the EcoRl site at 1025 bp, K,2 with the fragment 5’ to the Narl site at nucleotide -39, and K,3 with the entire PI cDNA fragment. Fragments were labeled with [aJ2P] dCTP to >lO’ cpm per pg of DNA by reaction with the Klenow fragment of DNA polymerase using random hexanucleotides as primers (Feinberg and Vogelstein, 1983). Blots were probed for actin transcripts using a 32P-end-labeled oligonucleotide complementary to the sequence encoding amino acids 82-91 of the human skeletal muscle a actin sequence (Hanauer et al., 1983). The blots were hybridized to the probes (>2.5 x IO6 cpmimlj in 50% formamide, 5x SSPE (20x SSPE = 3.6 M NaCI, 0.2 M Na+ phosphate, 0.02 M EDTA [pH ZO]), 5x Denhardt’s solution (50x Denhardt’s = 1% Ficoll, 1% polyvinylpyrrolidone, 1% BSA), 0.5% SDS, and 50 Kg/ml E. coli tRNA at 42’C for 18 hr. Blots were washed stepwise to a final stringency of 0.2x SSC (20x SSC = 3 M NaCI, 0.3 M Na+ citrate), 03% SDS at 65’C. Kodak X-Omat AR film was exposed to the blots at -7O“C for 4-6 days using two intensifying screens. Isolation of cDNA Clones Encoding K,I Poly(A)+ mRNA, prepared from the brains of 12-day-old Sprague-Dawley rats, was used to construct a size-selected (>1 kb), oligo(dT)-primed hgtl0 cDNA library. Recombinants (2 x 105) were screened with a mixture of two 32P-labeled oligonucleotides (oligo 1 sequence: 5’.CAGCrrCAAGATCCGGAAGACCCGCACCACCCCGATCACCCGCAGGAT3’; oligo 2 sequence: 5’-CAACAGCACCACGCCAATGAACACGAAGAAGATCACCACGCCCAGCTCCCGCAT-3’). These are complementary to sequences encoding the fourth and fifth hydrophobic segments of the Drosophila Shaker A sequence (Schwarz et al., 1988), but with changes in codon usage for some amino acids. Two positive clones were isolated, and one of these (K41) was found to hybridize independently to both probes. To extend K41 in the 5’ direction, a specifically primed hgtl0 library was constructed. Reverse transcription was primed with an oligonucleotide located -180 bp from the Send of K41 (positions 1193-1212 in the K,l sequence). Recombinants (2 x IOj) were screened with a probe derived from the 5’end of K41 (positions 1034-1133 in K,l). The probe (>108 cpm/pmol) was synthesized from two oligonucleotides, partially overlapping at their 3 ends, using the Klenow fragment of DNA polymerase in the presence of all four [a-32P]dNTPs. The plaque lifts were hybridized in 5x SSPE, 5x Denhardt’s, 0.5% SDS, 10% formamide, 100 pg of salmon sperm DNA at 42’C and washed to a final stringency of 01x SSC, 0.1% SDS at 65OC. Eleven positive clones were isolated, 1 of which, clone 18, overlaps K41 and extends it ~1 kb in theYdirection.Theother IOclonesarederivedfroma homologous, but nonidentical, K’ channel (K,2; see below). Clone 18 was further extended in the Sdirection using a variation of the PCR, the RACE technique (Frohman et al., 1988). First strand cDNA was primed with an oligonucleotide located -400

Cloning 937

and Expression

of Rat Brain K+ Channels

bp from the 5’ end of clone 18. The cDNA was separated from excess primers and nucleotides by chromatography on a Biogel A-5M column and tailed with terminal deoxynucleotidyl transferase and dATP, and the Send was amplified using the PCR. The primers used for the PCR were an oligo(dT)/adapter (Frohman et al., 1988) that hybridized to the synthetic poly(A) tail and a specific primer derived from the sequence of clone 18 (complementary to nucleotides 228-245 in the K,l sequence). The PCR products were resolved by electrophoresis, transferred to Zetaprobe membranes, and probed with a 32P-labeled oligonucleotide derived from the Send of clone 18 (positions 67-163 in K,l). The blots were hybridized to the probe (>108cpmlpmol; ~5 x IO6 cpmiml) at 50°C overnight in 0.25 M Na+ phosphate (pH 7.2), 0.5 M NaCI, 7% SDS, 1 mM EDTA, 1% BSA, 100 up of salmon sperm DNA. Washes were to a final stringency of 0.1x SSC, 0.1% SDS at 65OC. Two bands, of ~500 and ~250 bp, hybridized to the probe. Sequence analysis of a subclone of each showed that the shorter DNA was a truncated form of the longer. The 515 bp band, clone I&RACE, encodes the putative initiation codon of K,l and 5’ untranslated sequence. Isolation of cDNA Clones Encoding K,2 While screening the specifically primed lgtl0 library with a randomly primed K41 probe, a second class of cDNAs encoding a homologous channel (K,2) was isolated. Ten clones of this class were isolated; clone 15 extends the farthest 5’. The sequence of the 3’ region of K,,2, not represented in clone 15, was determined from a genomic clone of this channel that was isolated In parallel (see below). A cDNA containing this 3’ region was subsequently cloned from braln poly(A)+ mRNA using the PCR (Kawasaki and Wang, 1989). The oligonucleotide used to prime first strand cDNA synthesis was derived from the genomic DNA sequence of this clone and was complementary to the coding sequence 145-163 nucleotides downstream of the stop codon. First strand cDNA synthesis was carried out at 42’C for 60-90 min In a volume of 40 ul containing 2 ug of poly(A)+ mRNA, 1 uM primer, 2 mM each dNTP, 50 mM Tris-HCI (pH 8.3), 8 mM MgCIL, 30 mM KCI, 1 mM DTT, 40 U of RNasin, and 30-40 U of AMV reverse transcriptase. The first strand cDNA was then used directly in the PCR. The upstream oligonucleotide primer was derived from the 3’ end of clone 15 (nucleotides 630-648 in the K,2 sequence). The PCR was carried out in a volume of 100 PI containtng the first strand cDNA, 1 PM each oligonucleotide primer, 10 mM Tris-HCI (pH 8.3), 50 mM KCI, 1.5 mM MgCI,, 0.01% gelatin, 200 ftM each dNTP, and 2.5 U of Thermus aquaticus DNA polymerase. Amplification was performed for 40-50 cycles of 1 min strand separation at 94OC, 2 min annealing at 56’C, and 3 min extension at 72°C. Ten microliters of each PCR was analyzed by electrophoresis through a 1% agarose gel and ethidium bromide staining. Positive identification of the 3’ cDNA, clone 15.PCR, was accomplished by Southern blotting (as described above) and probing the blots with an oligonucleotide derived from the sequence of the genomic clone (nucleotides 1512-1587 in the K,2 sequence). Isolation of Pl cDNA Another hgtl0 cDNA library, constructed from 12-day-old rat brain poly(A)+ mRNA was screened with the two oligonucleotides described above for the isolation of K41. A clone (PI) containing an insert of ~1200 bp was isolated and shown, by sequence analysis, to encode the carboxy-terminal region of a protein homologous to that encoded by K41. Isolation of Cenomic Clones Encoding K,l, K,2, and K,3 Approximately IO6 plaques from a commercial rat genomic DNA library (partial EcoRl cut in Charon 4A) were screened with 3*P-labeled (>lOa cpm/bg), randomly primed K41 and PI probes. Hybridization was in 5x SSPE, 5x Denhardt’s, 0.5% SDS, 50% formamide, 100 ug of salmon sperm DNA at 42OC, and washes were to a final stringency of 0.1x SSC, 0.1% SDS at 65OC. Two classes of clones (the genomic counterparts of the K,l and K,2 cDNAs) were purified in the K41 screen, and one class (the K,3

genomics) in the PI screen. The genomic clones were digested with EcoRI, and each fragment was subcloned into Bluescript vectors. The EcoRl fragments containing the exons hybridizing to K41 or PI were identified by Southern blotting as described above. Nucleotide Sequencing and Analysis cDNAs and genomic EcoRl fragments were subcloned into the EcoRl site of Bluescript vectors, and nucleotide sequences were determined using a modified T7 DNA polymerase (Sequenase; US Biochemicals) and the dideoxynucleotide chain termination method (Sanger et al., 1977). Each cDNA was sequenced in its entirety on both strands. The genomic clone encoding K,l was sequenced from 670 nucleotides upstream of the initiation codon to 293 bp downstream of the stop codon. Similarly, the genomic clones encoding K,2 and K,3 were sequenced from 673 bp upstream of the coding region to 431 bp downstream and from 513 bp upstream to 180 bp downstream, respectively. DNA sequences were assembled and analyzed using the University of Wisconsin Genetics Computer Group software package (Devereux et al., 1984).

Construction and Expression of cDNA and Cenomic Clones Containing the Entire Protein Coding Regions of K,l, K,2, and K,3 pGEM-9Zf(-) was modified by ligation of the following synthetic duplex into the Not1 site: S-pGCCCA4aCC-3 3’T,0CGCCCGp-5 The sequence of the synthetic region of the resulting vector, pGEMA, was confirmed by DNA sequencing. The plasmid contains a unique Notl site downstream of a T7 promoter, a polylinker, and a poly(A) tail and enables in vitro synthesis of polyadenylated RNA transcripts. The full-length coding regions of the two K+ channels were assembled by sequential ligations of the individual cDNAs into pGEMA (Figure 1). To construct K,l, K41 was first inserted into the polylinker as an EcoRI-Hindlll fragment and then extended in the Sdirection by ligation to clone 18 at the overlapping Clal site. Clone I&RACE was ligated to the 18/K41 construct at the Xhol site to complete the 5’ coding sequence. For the construction of K,2, clones 15 and 15-PCR were ligated at their Aocl site and inserted into pCEM-A. The final constructs were sequenced in their entirety on both strands to confirm their structure. The Bgll-Sty1 fragment of the genomic DNA clone that contains the entire coding region of K,3 was excised and isolated, blunted by reaction with T4 DNA polymerase, ligated to EcoRl adaptors, and cloned into EcoRI-digested pGEMA using standard procedures. Plasmids were linearized with Notl and RNA was synthesized in vitro by reaction with T7 RNA polymerase using conditions described for transcription with SP6 RNA polymerase (Kreig and Melton, 1984). Transcripts were capped at the Send by including diguanosine triphosphate in the transcription reaction (Nielsen and Shapiro, 1986). Oocytes were obtained from female Xenopus, defolliculated by collagenase digestion, and injected with RNA (50 nl, 0.5-2 mg/ml) as described (Folander et al., 1990). Injected cells were incubated for l-3 days at room temperature in ND96 (96 mM NaCI, 2 mM KCI, 1.8 mM CaCla, 1 mM MgCla, 5 mM HEPESNaOH [pH 7.61) supplemented with penicillin (100 IUlmL), streptomycin (100 IUlml), and gentamicin (IO @ml) and then screened electrophysiologically for the expression of K+ currents using a two microelectrode voltage clamp (Folander et al., 1990). Currents were measured in OR2-Ca2+ (82.5 mM NaCI, 2.5 mM KCI, 1 mM MgClz, 5 mM HEPES-NaOH [pH 761) at room temperature (22OC-24°C). Microelectrodes were filled with 3 M KCI and had tip resistances of 0.5-2 MD. Currents were sampled at 2 kHz, lowpass-filtered at 300-1000 Hz, and stored for analysis. Leakage currents were removed using a P/4 subtraction routine.

Acknowledgments We thank Maria Garcia and Chris Miller for samples of charybdotoxin, Lourival Possani for noxiustoxin, Mordecai Blaustein for dendrotoxin, and Angeliki Buku for mast cell degranulating peptide. This work was supported, in part, by a grant (HL38156) to L. K. K. from the National Institutes of Health. Received

February

16, 1990;

revised

March

27, 1990.

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K+

Timpe, L., Schwarz, T., Tempel, B., Papazian, D., Jan, Y., and Jan, L. (1988). Expression of functional K+ channels from Shaker cDNA in Xenopus oocytes. Nature 337, 143-145. Yokoyama, S., Imoto, K., Kawamura, T., Higashida, H., Iwabe, N., Miyata, T., and Numa, S. (1989). Potassium channels from NG108-15 neuroblastoma-glioma hybrid cells: primary structure and functional expression from cDNAs. FEBS Lett. 259, 37-42. Note

Added

in Proof

Using the polymerase mRNA isolated from

chain reaction, K,3 was also cloned rat peripheral leukocytes.

from