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A Component of Calcium-Activated Potassium Channels Encoded by the Drosophila slo Locus Nigel S. Atkinson; Gail A. Robertson; Barry Ganetzky Science, New Series, Vol. 253, No. 5019. (Aug. 2, 1991), pp. 551-555. Stable URL: http://links.jstor.org/sici?sici=0036-8075%2819910802%293%3A253%3A5019%3C551%3AACOCPC%3E2.0.CO%3B2-D Science is currently published by American Association for the Advancement of Science. Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at http://www.jstor.org/about/terms.html. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at http://www.jstor.org/journals/aaas.html. Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. The JSTOR Archive is a trusted digital repository providing for long-term preservation and access to leading academic journals and scholarly literature from around the world. The Archive is supported by libraries, scholarly societies, publishers, and foundations. It is an initiative of JSTOR, a not-for-profit organization with a mission to help the scholarly community take advantage of advances in technology. For more information regarding JSTOR, please contact [email protected]. http://www.jstor.org Tue Jul 17 19:59:42 2007 J. Bowman, C. M. Yentsch, W. T. Peterson, Eds. (Springer-Verlag,Berlin, 1986), pp. 193-223. 19. The processes responsible for the shape of the curves in Fig. 2 are not known. 20. P. F. Sale, Enoiron. Biol. Fish. 3, 85 (1978); P. K. Dayton, in Erological Procwec in C o m l and Marine System, R. J. Livingston, Ed. (Plenum Press, New York 1979),pp. 3-18; M. A. Kendall, R. S. Bowman, P. Williamson, J. R. Lewis, Neth. J. Sea Res. 16, 119 (1982); A. J. Underwood andE. J. Denley, inEcobgual Communities: Conrep~ualIrnw and the Evidence, D. R. Strong, Jr., D. Simberloff, L. G. Abele, A. B. Thistle, Eds. (Princeton Univ. Press, Princeton, NJ, 1984), pp. 151-180; J. Roughgarden, S. Gaines, H. Possmgham, ScMlre 241, 1460 (1988). 21. P. M. Yoshioka, Ecology 63, 457 (1982). 22. Because of the obviously nonnormal distribution of the data a nonparameaic test was used. Each daily settlement value consisted of the average of two or three settlement plates per site. One site was observed from 29 April until 8 August 1989; the Spearman correlation coefficients between settlement and water temperature anomaly were -0.258 for Chthamalus and -0.223 for Polliripes (overall), whereas those from 1 June to 29 August were -0.266 and -0.400. Coefficients from the other site, observed from 3 June until 27 July, were 23. 24. 25. 26. 27. -0.274 for Chthamalw and -0.482 for IJollicipes. All correlation c d c i e n ~ are signilicantly different from 0 at a = 0.025 (one tail). Small magnitude of correlation values might reflect uncertainty of the offshore larval reservoir, differential behavior of internal bores at the Scripps Pier and Dyke R& unrecorded large-event bores, or other phenomena affecting settlement. L. R. Haury, M. G. Briscoe, M. H. Orr, Nature 278, 312 (1979). A. M. Barnett and A. E. Jahn, Cont. SheFRes. 7, 1 (1987). P. E. Smith and R. W. Eppley, Limnol. Oceanogr. 27, 1 (1982). J. Pineda, unpublished observations. I thank P. Dayton, J. Enright, L. Haury, M. Tegner, E. Vetter, and C. Wlnant, and two reviewers for consauctive comments on the manuscript. I also thank many divers who helped sample. Temperame data were supplied by the ScrippsInstitutionof Omography. E. del Castillo supported me in every stage of the research. I especially wish to thank the people of Mexico who supported this research through a CONACYTgraduate fellowship which also covered research expenses. I also arknowledge grants from NSF m P. K. Dayton and from MMS to C. D. Winant. 4 February 1991; accepted 16 May 1991 A Component of Calcium-Activated Potassium Channels Encoded by the Drosophila slo Locus Calcium-activated potassium channels mediate many biologically important functions in electrically excitable cells. Despite recent'progress in the molecular analysis of voltage-activated K+ channels, Ca2+-activated K+ channels have not been similarly characterized. The Drosophila slowpoke (slo) locus, mutations of which specitically abolish a Ca2+-activated K+ current in muscles and neurons, provides an opportunity for molecular characterization of these channels. Genornic and complementary DNA clones &om the slo locus were'isolated and sequenced. The polypeptide predicted by slo is similar to voltage-activated K+ channel polypeptidesin discrete domains known to be essential for function. Thus, these results indicate that slo encodes a structural component of Ca2+-activated K+ channels. P OTASSIUM CHANNELS COMPRISE A large and diverse group of integral membrane proteins that determine the level of excitability and the repolarization properties of neurons and muscle fibers (1). On the basis of gating mechanisms K+ channels can be subdivided into at least two distinct classes: voltage-activated K+ channels respond to changes in membrane potential, whereas the gating of Ca2+-activated KC channels is dependent on intracellular Ca2+, although members of this class are also sensitive to voltage. Ca2+-activatedK+ channels occur in neurons, muscles, and secretory cells of vertebrates and invertebrates and govern action potential repolarization, the rate and pattern of repetitive firing, and Ca2+ entry ( 2 ) . Laboratory of Genetics, University of Wisconsin, Madison, WI 53706. *Present address: Department of Zoology, University of Texas, Austin, TX 78712. 2 AUGUST 1991 The molecular analysis of KC channels was initiated by the cloning of the Drosophila Shaker ( ~ h locus, ) a gene encoding one class of voltage-activated KC channels (3). Subsequently, many KC channel genes were cloned from various organisms on the basis of sequence similarity with Sh, enabling elucidation of important structural features essential to K+ channel activity (4). However, none of these genes appears to encode a K+ channel gated by Ca2+ In Drosophila, a fast, Ca2+-activatedKC current, I,,, has been identified in adult and larval muscles and in 'larval neurons (5-7). Like the BK class of Ca2+-activated K+ channels in mammalian muscle (8), the Drosophila channels that conduct I,, are sensitive to charybdotoxin and have a relatively large single channel conductance (6, 7). Mutations of the slo locus specifically eliminate I,, (6, 7, 9). To determine if slo encodes a structural component of Ca2+activated K+ channels or perhaps affects their function by some other means, we undertook a molecular analysis of this locus (10). We first pinpointed the cytological location of the "gene., defined on the basis of a single, cytologically normal mutation, slol (6). We mapped this mutation by meiotic recombination to position 90.0 on the third chromosome between bar-3 (map position 79.1) and taxi (map position 91), corresponding to polytene chromosome region 95-96 (11).To refine this location, we isolated three y-ray induced slo mutations (slo3, slo4, and slog) on the basis of their failure to complement the slol behavioral phenotype (6, 12) (Table 1).The slo phenotype of these new mutations was confirmed electrophysiologically (13). &ological examination of these mutations revealed that all contained chromosome rearrangements with a common breakpoint at polytene band 96A17 (Table 1). These results together with the cytological and genetic characterization of other chromosome lesions in this region, originally isolated by other criteria, identify band 96A17 as the cytological location of slo (Table 1). One of the breakpoints in the slo4 inversion is at the slo locus and the other is at 96F5-8 close to the previously cloned E(sp1) locus (14). We were thus able to k e this invirsibn'to clone genomic DNA from the slo locus by chromosome walking and jumping (15). Chromosomal in situ hybridization o f t w o sets of phage clones from existing chromosome walks in the E(sp1) region revealed that one set was proximal and the other set distal to the 96F5-8 breakpoint. Consequently, we extended the proximal walk by isolating overlapping cosmids from a wild-type library until the 96F5-8 break~ o i n t&as reached. To identifv a cosmid spanning the inversion breakpoint (Fig. lA), we assayed each cosmid from the walk > , Table 1. Summary of chromosome aberrations used in cytological mapping of the slo locus. - -- Name - Cytological lesion Normal Deletion (95E6-7 to 96A18) do4 Inversion (96A17 and 96F5-8) slo8 Deletion (96A2-9 to 96D2-4) Df(3R)S87-5 Deletion (95F7-9 to 96A17-18) Df(3R)S87-4 Deletion (95E8-F1 to 95F15-96A2) T(Y;3)AI 17 Translocation (broken at 96A10-17) slo' S ~ O ~ slo phenotype Mutant Mutant Mutant Mutant Mutant Normal Mutant REPORTS 551 by chromosomal in situ hybridization to slo4 homozygotes: a cosmid spanning the breakpoint should hybridize to both ends of the inversion, whereas cosmids that stop short of this break should hybridize to a single site. DNA disrupted by the inversion was further delimited by in situ hybridization and Southern blot analysis to a 3-kb Eco RI restriction fiagrnent within this cosmid. In slo4, the 3-kb Eco RI genomic fragment at 96F5-8 is broken into two parts, each juxtaposed to DNA sequences from the slo locus at 96A17. A phage containing one of these junction fiagments was isolated by probing a slo4 genomic library with the 3-kb wild-type Eco RI fragment. This junction fragment was subsequently used to initiate a chromosome walk in a wild-type cosmid library to obtain genomic DNA that induded the slo locus (Fig. 1B). An approximate location of the slo transcription unit was defined by the 96A breakpoint of slo4, which mapped to a 3-kb Eco RI-Hind 111 genomic fiagment. We used this fragment to screen cDNA libraries from - -10 I I p yl $? Y 1 1 1 1 I 1 1 1 1 1 I I - 53-' 1 I 2p 3p -L I I ;; ;'I Fig. 1. Cloning of genomic DNA from the slo locus. (A) Hybridization of a cosmid from the genomic wak to polytene chromosomes of slo4 homozygotes (34). (8) An EEO RI map of the cloned region. The position of the slo4 inversion breakpoint is indicated. H, the Hind I11 site that marks the rightward limit of the resmction fragment containing the breakpoint; black bars, the genomic resmction fragments that hybridize with slo CDNAs 254, 28,and CH4 (see text and Fig. 3). (The presence of a black bar indicates only that the corresponding resmction fragment above the bar contains one or more exons of unknown size. Because the CDNAs lack internal Eco RI sites, a break berween bars indicates the presence of an intron in genomic DNA that is spliced out in the cDNAs.) Broken lines, regions where the order of the resmction fragments has not been determined so the distance between exons is not known. The entire slo locus must extend beyond the limits shown because the cDNAs hybridize to genomic resmction fragments not contained in the cloned region and because the T(Y;3)A117 breakpoint lies outside the limits of the chromosome walk. The direction of transcription relative to the chromosome wak was determined from Northern blots by using single-stranded RNA probes. Drosophila heads. One DNA, 28, hybridizcd to both sides of the slo4 inversion and also to both parts of the T(Y;3)A117translocation (Fig. 2, A and B). The fact that 28 spans two different slo mutations demonstrates that this cDNA is derived fiom the slo transcript. Two transcripts of 5.8 kb and 11kb are detected by 28 probes on North- ern blots of polyadenylated [poly(A)+] RNA isolated fiom heads (Fig. 2C). The absence of these transcripts in slo4 homozygotes (Fig. 2C) identifies them as slo transcripts. We do not know whether the two wild-type transcripts encode different slo polypeptides. Because 28 is incomplete (see below), we Fig. 2. Identification of the slo transcription unit. IA) Hvbridization of d l N A 28 to wlvtene chro;ndsokes homozygous for the slo4*&ersion. Arrowheads, hybridization signals at inversion end points. (8) Hybridization of CDNA 28 to polytene chromosomes heterozygous for the T(Y;3)A117 translocation and a normal third chromosome. A hvbridization s i d (arrowhead) is seen at the &tact sfo locusYon ;he normal chromosome. In addition, both the proximal (double arrowhead) and distal parts ( 0 6 arrow) of the translocated chromosome contain sites that hybridize. In this preparation part of the distal piece of the translocation has asynapsed from the normal chromosome. (C) Northern blot analysis of slo transcripts. Approximately 10 pg each of poly(A)+ RNA isolated from heads of Canton S (CS) and homozygous slo4 adults was loaded into individual lanes. The filter was probed with 32Plabeled cDNA 28 (left). S i results were obtained when blots' w&e probed with either *m . I I I purified fragments of 28 lacking any intron sequenccs or single-stranded RNA transcribed from the T7promoter of the pBluescript SK- (Stratagene) plasmid that carried cDNA ZS.Single-stranded probes transcribed in the reverse orientation from the SP6 promoter of 28 did not detect any nanscripts (not shown). As a control, the blot was stripped and reprobed with an eag cDNA (right). Experimental conditions were as described (26). Composite CDNA ' 0 S1' S2S3 S4SSWS8 254 I exonA exonB y - t h d i n g ? I Z8 I I V I 500 Amlm .cldmldw A M M A M A A CH4 loo0 Fig. 3. Diagrammatic representation and hydrophobicity plot of overlapping slo cDNAs that were assembled to yield a composite sequence. (A) The three CDNAs drawn to scale. Dashed underlines, CDNA segments that were assembled into the composite shown below; open boxes, open reading frames; hatched boxes, introns that were not spliced out of some CDNAs (17). The open reading frame of 254 is preceded by an untranslated segment (not shown) that may also represent an unspliced intron (17). ALI the regions shown have been completely sequenced on both strands (16), with the exception of the central segment of 254 (interrupted bar), which has not been sequenced. For those regions that have been fully sequenced, the nucleotide sequences in overlapping segments are identical except for the unspliced i n m and the two sites of alternative splicing indicated. At site A an exon encoding 14 amino acids (Al) that is present in CH4 is replaced by a different exon encodmg 25 amino acids (A2) in 28.At site B an exon 22 amino acids long is present in 254 and 28 but lacking in CH4.28, which does not end in a poly(A)+ tail, extends beyond the poly(A)+ tail present in CH4 suggesting the presence of alternative polyadenylationsites. (8) Hydropathy plot for the deducedslo polypeptide (Fig. 4) encoded by the composite cDNA. The plot has been aligned with the composite cDNA sequence above. Hydrophobicity values were calculated accordmg to Kyte and Doolittle (19) with a window size of nine amino acids. The seven shaded hydrophobic peaks were named according to the corresponding domains of other K+ channels after aligning sequences of the fourth and sixth hydrophobic peaks with S4 and H5, respectively. SCIENCE, VOL. 253 hydrophobic domains near the NH,-terminus of the slo polypeptide (Slo) similar to the structure of other known K+ channel polypeptides (4). The fourth hydrophobic segment of Slo is similar to the S4'domainin voltage-gated channels, which is thought to mediatevoltage sensitivity. The S4 m&f is characterized by a positively charged residue at every third position separated by two hydrophobic residues (20). Out of 26 positions in the alignment of S4 domains from Slo and Shaker, there are six amino acid 1 m G C D Q S T V ESLADDPTDS PFDADDCLKV RmWCFLLSS IFTFLAGLLV Fig' Deduced acid se- identities and four conservative substituSI - quence encoded by a composite slo 51 VLLWRAFAFV CCRKEPDLGP NDPKQKEQKA SRNKQEFEGT FMTEAKDWAG A cDNA. The hydrophobic domains tions. This region of Slo shows a slightly 101 ELISGQTTTG RILVVLVFIL SIASLIIYFV DASSEEVERC QXWSNNITQP are underlined. &sensus sites are better alignment with an S4 domain of rat -aindicated for CAMP phosphoryla- brain Na+ channels, yielding seven identi151 IDLAFNIFFM VYFFIRFIAA SDKLWFMLEM YSFVDYFTIP PSFVSIYLDR . Q I tion (W) (35), PKC-dependent ties and four conservauvesubstitutions (Fig. 201 TWIGLRFLRA LNNTVPDIL QYLNVLKTSS SIRLAQLVSI FISVWLTAAG phosphorylation (#) (35), a potenS 4 - S S # ' 251 IIRLLENSGD PLDFDNAHRL SYWTCVYFLI VTMSTVGYGD VYCETVLGRT tial ATP-bindingsite (e)(31), and 5A). The Slo S4 domain contains fewer I B ' L a region containing a potential positive charges than S4 domains in voltage301 FLVFFLLVGL AMFASSIPEI IELVGSGNm GGELKREHGK RAIWCGRIT s 6 Ca2+-bindingloop (*) (30). Below gated channels but the hnctional signifi351 YESVSAFLKD FLREDREDW VEVVFLRRKP PDLELEGLFK RAFTTVEFFQ are the deduced amino acid se- cance of this is not known. The presence of 401 GTIMNPIDLQ RWNREADAC L W C Q D PDAEDRANIM RVISIKNYSD quences of two alternative exons # not included in the composite se- an S4 domain in a guanosine 3',5'-mono451 DIRVIIQW YRNKAYLLNI PSWDWKQGDD VICLAELKLG FIAQSCLAPG quence. Alternative exon A2 is phosphate (cGMP)-gated channel has led to 501 FSTMMANLFA MRSFKTSPDM QSWTNDYLRG TGMEMYTETL SPTFIGIPFA found in cDNA 28 in the place of the suggestionthat the S4 sequence contrib# 551 QATELCFSKL KLLLLhIEIK GAEEGADSKI SINPRGAKIQ ANTWFFIAQ exon Al. Alternative exon B is utes to the basic architecture of various found in cDNAs 28 and 254 but channels in addition to its function as a 601 SADEVRRAWF Y C W H E D I K DETLIKKCKC KNLTVQPRSK FDDLDEAAPA L N t k A l 2 missing in CH4. The single letter 651 PTFTPPELPK RVRVRGSVSG DITRDREDTN LLNRNVRReN GTGNGTGGMH abbreviations for amino acids are voltage sensor (21) . 701 HbUWTAAAAA AAAAAGKQVN KVKPTVNVSR QVEGQVISPS QYNRPTSRSS The greatest similarity between Slo and A, Ala; C, Cys; D, Asp; E, Glu; F, NLEmaB f # 751 GTGTQNQNGG VSLPAGIADD QSKDFDFEKT EMRlDSTGMF HWSPAKSLED Phe; G, Gly; H , His; I, Ile; K, Lys; K+ channel polypeptides is in the H5 do** * L, Leu; M, Met; N, Asn; P, Pro; Q, main, an integral part of the ion conduction 801 CILDRNQAAM T V L N G W C LFADPDSPLI GLRNLVMPLR ASNFRYRELK Gln; R, Arg; S, Ser; T, Thr; V, Val; 851 H W I V G S W Y IRREWi+%QN LPKISVLNGS PLSRADLRAV NVNLCDMCCI W, Trp; and Y, Tyr. The nucleotide pore (22-25). When aligned with an H 5 901 LSAKVPSNDD PTLADKEAIL A S L N I M F DDTIGVLSQR GPEFDNLSAT sequence of the composite cDNA consensus sequence for the Sh superfamily, # # has been submitted to GenBank Slo shares 11identities and five conservative 951 AGSPIVLQPR GSVYGANVPM ITELVNDGNV QFLDQDDDDD PDTELYLTQP under accession number M69053. substitutions out of 25 positions (Fig. 5B). 1001 FACGTAFAVS VLDSLMSTTY FNPNALTLIR SLITGGATPE LELILAEGAG This alignment is centered on a core of 11 1051 LRGGYSTVES LSNRDRCRVG QISLYDGPLA QFGECGKYGD LFVAALKSYG # amino acids in which there are nine identi1101 MLCIGLYRFR DTSSSCDASS KRYVITNPPD DFSLLPTDQV FJTNQFDPGL ties and two conservative substitutions. ## 1151 EYXPPAVRAP AGGRGTNTQG SGVGGGGSNK DDNS Moreover, at five positions in the H 5 do# main where Slo deviates from the consensus, Alternative exon A2 it shares identities with Eag, another Dro632 ATFRKGVPAVQMVGRAKDDEYSLSN sophila K+ channel polypeptide identified by means of genetic strategies (26, 27). These Alternative exon B 745 PPENDANPYAGYQLAYEW relations indicate that slo and other K+ channel genes have evolved from a common ancestor. One of the conservative changes within Fig. 5. Alignment of hydrophobic ., domains S4 and H5 encoded by slo x c m the H5 domain of Slowith respect to the Sh with corresponding segments of family consensus is a substitution of serine D I L Q other channel polypeptides. (A) for threonine at wsition 284. Creation of P G L K Alignment of S4 domains of Sh K+ the same replacement in Sh by site-directed channel (top) (6, 25), rat brain Na+ channel type I (R,) (bottom) (36), mutagenesis results in a tenfold decrease in and slo (middle).Amino acid idenB sensitivity to internal tetraethylammonium tities are shaded and conservative sh T ~ c V F w 8 (TEA) blockade (23). This result is consissubstimtions are boxed. The posi- ~ K I. , ". D A F ~ W A V V S Y T T V G Y tent G Dwith H the Y Pobservation VFIF G in vivo, I,, is that, tive gating charges in the S4 doBE2 ID$ S-UW,,A V V, S M T T .V G Y G D H V P T T I G G! relatively insensitive to internal TEA main of Sh and rat Na+ channel are indicated. The charges in parenthe- RCKS D " A . F ~ R . W - A ? V , T H T T V G x G D H B P V T I G G (K,= 100 rnM) (28). Site-directed muta,-I' . ses are those present in the Sh and RCIM tions in Sh that alter ionic selectivity and +% w ?:A v v:"' M ~ V GT- Y - G D M K P I T v G G Na+ channel sequence but lacking to external TEA also drkl ~ ~ F W . W ~ T I ~ T ~ T T V G Y G D I Y P - increase K ~ T Lsensitivity G G in slo. Members of the following occur naturally at the equivalent positions in groups of amino acids were considS h d E@FHWAGI;~.MT~T,V.GY G D I C P T T A L G k ered to be conserved: [M, I, L, V]; lsl slo sequence (24, 25). For example, the the sham L ~ L ' w . w ; ~ -L -.~ ~ ~ ~ - ~ s . v + G Y G D N A P ~ ~ T . Y I G [A, GI; [S, TI; [Q, Nl; [K, Rl; [E, presence of tyrosine at position 292 would s h d A -, ~- W T YI V ~ ~ ~ T T L G Y ( Dl; [F, Y, W]. (B) Alignment of I I T I A G . be predicted to result in a high sensitivityof H5 domains of K+ channels in the I,, to external TEA blockade (25). These Sh superfamily (4, 21), eag (27), properties have not yet been empirically and slo. used it as a probe to isolate the overlapping slo cDNA (Fig. 3) is 4232 nucleotides (16) cDNAs, 254 and CH4. Although none of with an open reading frame (between posithese cDNAscontains a completeopen reading tions 1 and 3552) that encodes a deduced frame (ORF), together they yield a composite polypeptide at 1184 amino acids (Fig. 4). sequence capable of speafylng a polypeptide This sequence appears incomplete at the 5' over 1100 amino acids in length (Fig. 3A). end (17) but complete at the 3' end because These cDNAs represent a spliced transcript the untranslated tail of CH4 terminates in a encompassingover 40 kb of genornic DNA as run of adenine residues preceded by the polyadenylation signal AATAA (18). indicated by Southern analysis (Fig. 1B). Hydropathy analysis (19) revealed seven The nucleotide sequenceof the composite - '' .. . I - $ r ~ I'i ST 2 s - 2 AUGUST 1991 1 r J REPORTS 553 determined in Drosophila. Outside these two discrete regions of rimilarity, the Slo sequence is distinct from all known K+ channels (29). One unusual feature of Slo is its length of 1184 amino acids, including a stretch of about 850 amino acids between the S6 domain and the COOHterminus, which contains two additional POtentid membrane-spanning domains. Among other K+ channels, only Eag (27) has a similar length (1174 amino acids). Across the entire hydrophobic core of the polypeptide, Slo shares less than 20% amino acid identity with known K+ channel sequences. In contrast, members of the extended Sh family share at least 35% identity in the core region (4). These define Slo as a type P ~ ~ Y P Because the K+ current affected by slo requires intracellular for we searched the Slo sequence for an EF hand that would bind Ca2+ (30). A candidate domain is present near the COOH-terminus (Fig. 4). Although this segment deviates significantly from the consensus EF hand sequence overall, there is very good agreement with the consensus sequence for those residues required for coordinating Ca2+ (30). A sequence that matches the adenosine triphosphate (ATP)-binding site of tyrosine (31) is located in a neighboring region (Fig. 4). Although its effect in Drosohas not been characterized, ATP may directly modulate ca2+-activated K+ thannels in mammalian smooth muscle (32). sequence comparisonsamong slo c ~ indicates that the slo transcript is alternatively spliced (Figs. 3 and 4). Alternative splicing of the Sh generates a number of gene products with different functional properties (33). whether alternative splicing has similar significance is unknown. The mutant phenotype that results when the slo polypeptide is absent or altered allows us to infer the in viva function of the w i l d - t ~ ~gene e product' Mutations such as do4 cause the selective and complete elimination of I ~ , , indicating that the polypeptide is essential for the expression or function of the channels mediating this current' We 'Ioned the 'lo locus On the basis of the chromosomal location of these mutations and found that it encodes a polypeptide with sequence similarity to known K+ channels. Therefore, on the basis of both functional and structural evidence, we propose that Slo is a strtlctural component of Ca2+-activatedK+ channels. Together with s h (3) and eag (26, 27), the locus defines a third type of K+ channel structural gene identified and cloned on the basis of its mutant phenotype. Although the organization of Slo resem554 bles that of other K+ channel polypeptides, its overall sequence differs extensively from these polypeptides, indicating that the divergence of genes encoding voltage-activated and Ca2+-activatedK+ channels is ancient. The strong conservation of the H5 domain in otherwise divergent channels provides additional support for the fundamental importance of this segment in K+ channel function. It should now be possible to use slo to test emerging models for K+ selectivity and permeation and to explore the molecular basis for Ca2+-dependentgating. R. Coulson, Proc. Natl. Acad. Sci. U . S.A. 74, 5463 ~ ~ ] , ] ~ ~ ~ N ~ Regions showing GC compression were sequenced with deox~inosinetri~hos~hate ( D m ) . Sequence analyses were performed with software from the Genetics Computer Group [J, Devereux, P, Haeberli, 0 . Smithies, Nudeic Acids Res. 12, 387 q ~ ~~ ~ ~~ ~ Biolok?' ~ e ~ 17, c ~2 8 and ~ 254hcontain segments judged to be unspliced introns (see Fig. 3A) because of the ~ ;$ : :i~ g ~ ei c ~ R ~ Sn.10, ~459 w quences [s. M O W ~ ~, ~ c l e Acids (1982)], and sequence comparisons with overlapping cDNAs from which these segments were missing. The untranslated segment at the 5' end of 254 preceding the ORF could also be part of an unspliced intron because the first methionine of the open reading frame is not precededrby a consensus REFERENCES AND NOTES translation start site [D. Cavener, Nucleic Acids Res. 1. B. Hille, Ionic Channels ofExcitable Membranes (Sin15, 1353 (1987)l. Furthermore, unlike lo, other auer, Sunderland, MA, 1984). K+ channel polypeptides generally have a stretch of ~ 2.PB.~Rudy, ~ ~Neuroscience . 25, 729 (1988); R. Latorre, 100 to 250 amino acids between the initiating A. Oberhauser, P. Labarca, 0 . Alvarez, Annu. Rev. methionine and the first hydrophobic domain (7). If Physiol. 51, 385 (1989). an unspliced intron is present at the 5' end of 254, 3. A. Kamb, L. E. Iverson, M. A. Tanouye, Cell 50, it may include some of the initial sequence present in 405 (1987); D. M. Papazian, T. L. Schwarz, B. L. the conceptual translition (Fig. 4); several . Temple, Y. N. Jan, L. Y. Jan, science 237, 749 splice sites occur between the start of the O m and (1987); 0. et E M B O J . 7, 1087 (1988). the S1 domain. A high frequency of unspliced 4. B. Rudy, C Kentros, E. Vega-Saenz de Miera, J. introns in c ~encoding ~ ah ~ ~ ~N ~~than+ ~ Mol. Cell. Neurosci. 2, 89 (1991). nel has been noted [K. Loughney, R. Kreber, B. 5. L. SalkoK Nature 3023 249 (1983). Ganeaky, Cell 58, 1143 (1989)l. 6. T. Ekns, B. Ganemky, C.-F. Wu, Proc. Natl. h a d . 18. N. J. Proudfoot and G. G. Brownlee, Nature 263, Sci. U . S . A . 83, 8415 (1986). 211 (1976). 7. S. Singh and C.-F. Wu, Neuron 2,1325 (1989); A. 19, J. Kyte and R. F. Doolittle, J . M O ~Biol. . 157, 105 Komatsu, S. Singh, P. Rathe, C.-F. Wu, ibid. 4,313 (1982). (1990). 20. W. A. Catterall, Science 242, 50 (1988); W. Stuh8. B. S, Pallotta, K. L. Magelby, J. N. Barrett, Nature mer et al., Nature 339,597 (1989); D. M. Papmian, 293, 471 (1981); R. Latorre, C. Vergara, C. L. C. Timpe, Y. N. Jan, L. Y. Jan, ibid. 349, 305 Hidalgo, Proc. Natl. Acad. Sci. U . S . A . 79, 805 (1991). (1982); C. Miller, E. Moczydlowski, R. Latorre, M. 21. L. y . jan and y. N. J ~ ibid. , 345, 672 (1990). Phillips, Nature 313, 316 (1985). 22. R. MacKinnon, L. Heginbotham, T. Abramson, and C.-F. Wu, J. Neurosci. 11, 2135 9. M. Neuron 5, 767 (1990), H. A. ~ a m n a n net dl., (1991). Science 251, 942 (1991). 10. N. S. Atkinson, G. A. Robertson, B. Ganeuky, Soc. 23. G. Yellen, M. E. Jutman, T. Abramson, R. MacKinNeurosci. Abstr. 15, 541 (1989); G. A. Robertson, non, Science 251, 939 (1991). S. Atkbon, B. Ganemky, ibid. 16,670 (1990); 24. A. J. Yo01 and T. Schwarz, Nature 349,700 (1991). ~N. ~ s , Biophys. J . 59, 196a (1990). 25. R. MacKinnon and G. Yellen, Science 250, 276 11. D. L. Lidsley and E. H. Grell, ~ e ~ t Variations i c ~j (1990). Drosophh melawater (PublicationNo. 627, Camegie 26. R. Drysdale, J. Warrnke, R. Kreber, B. Ganetsky, Institution of Washington, Washington, DC, 1968). G e n e t b 127, 497 (1991). 1% To isolate new $0 mutations, wild-cype Canton-S 27, J. Warmke, R. Drysdale, B. Ganetzky, Science 252, males were mutagenized by y-irradiation (4500 R) 1560 (1991); Y. Zhong and C.-F. Wu, ibid., p. and mated to bar-3 do' females. Among 25,000 F1 1562. male offspring screened, three new mutations exhib- 28. M. Gorczyca and C.-F. WU, J . Mem. Biol. 121,237 iting the slo paralytic phenotype (6) were recovered. (1991). The presence of bar-3+ identified the mutagenized 29. One additional region of similarity is the alignment chromosome. of the S6 hydrophobic domain of Slo with S5 of 13. The electrophysiological phenotype of the new slo other Kt channels. For example, when aligned with mutations was assayed by recording action potenthe ~5 segment o f ~ there h are ten identities and five tials in adult flight muscles in response to stimulaconservative substitutions among 21 residues. In tion of the motor pathway and direct current injeccontrast, the S5 with S5 alignment yields five idention (6).[T. E k n s and B. Ganeuky, J. Neurosci. 8, tities and three conservative substitutions and the S6 428 (1988)l. The defect in I,, was confirmed by with S6 alignment yields four identities and four voltage-clamp analysis of larval muscles for each new conservative substitutions. The significance of this slo mutation either when homozygous or in hetalignment between hydrophobic domains that do eroallehc withs104, not occur at corresponding p~sitionsis not known. 14. E. Knust, K. Tietze, J. A. Campos-Ortega, E M B O 30. R. M. Tufty and R. Kretsinge?, s c i e p 187, 167 J . 6, 4113 (1987). (1975); R. H . Kretsinger, Cold SpringHnrb. Symp. 15. W. Bender, P. Spierrer, D. S. Hogness, J. Mol. Quant. Biol. 52, 499 (1987). Biol. 168, 17 (1983). 31. The consensus sequence for the ATP-binding site in 16. The composite cDNA was assembled as follows: tyrosine kinases is G-X-G-X-X-G-(X)15.20-K[M. P. nucleotide position 1to 744 (encoding amino acids Kamps, S. S. Taylor, B. M. Sefton, Nature 310, 589 1 to 248) is from cDNA 254; nucleotide position (1984)l. 745 to 1626 (encoding amino acids 249 to 542) is 32. C. H . Gelband, S. D. Silberberg, K. Groschner, C. from cDNA 28; nucleotide position 1626 to 4232 van Breemen, Proc. R . Soc. London Ser. B 242, 23 (encoding amino acids 543 to 1184) is from cDNA (1990). CH4. Nucleotide position 1is the first nucleotide of 33. T. L. Schwarz, B. L. Tempel, D. M. Papazian, Y. N. the open reading frame (ORF) in cDNA 254. The Jan, L. Y. Jan, Nature 331, 137 (1988); L. C. ORE of 254 is preceded by a nontranslatable segTimpe, Y. N. Jan, L. Y. Jan, Neuron 1,659 (1988). ment lacking a consensus translational start signal. 34. Chromosomal in situ hybridization was performed The ORF of C H i~s followed by an untranslated as described [W. Engels, C. Preston, P. Thompson, segment of 680 nucleotides terminating in a W. Eggleston, Focus 8, 6 (1986)] except that we poly(A)+ tail. DNA was sequenced in M13 and used sulfonated (Chemiprobe kit, FMC BioProdpBluescript (Stratagene) vectors by the dideoxy ucts) rather than biotinylated probes. chain termination method [F. Sanger, S. Nicklen, A. 35. Consensus recognition sequences were (R,K)- . SCIENCE, VOL. 253 ~ ~ h ~ (R,K)-X-S a (R,K)-(R,K)-X-XSfor adenosine 3', J. for & doacs;I 4 months of age) with a slower -ion, (CAMPT m T. Schwuq L.JW Y.N. JW B. HvniLaS allowe, he remaining E.b. D. 1. Gnva. E.Eeltamm udE.M~forawidioplikaria:udour G. ='I. id. dr;m '252. & 1 1 h 1 ;nd ~o~tl~ucs&dvicclod&mthc&-it~ sixfbmderanimak.whichwereasvmDaJmatc. (s,,T)-x-(& for protan &'c ( P K + ~ c & ~ I SUPF%UI ~ by NIH NS15390. ~ ~a&-t did y nat transgene (1;). phosphoryhnon R Woodgq K. L. Gould, T. nabk Tnag ud a Khguma EcIkdup to B.G. Ofthe Hunter, Eur. J. Biodwrr 161,177 (1986), A K d h N.SA raarvcd suppart 6um NIH gram T32 (RNA)-h m cr d. I. EM. Ckm 260,12492 (19891. GMOn31udchcM~mbrDystrophyAssochtioa OftheaarrseeneRvealedRNAsofsizescom.GAR. .& s u p p a t h . & d w , ~ e a t~ t o v a ~ ~ a n d t o ~ s 36. M.NO&-& d.,Nahtn 320; 188 (1986). ofW~NIHgrantT32GMOn31, 37. We dedicate this papa to the mcmory of Tom - , - O D U C . ~ Y ~ ~ ~ M - ~ mmicrnRNAon1yinthehebrainandinthe s n d i a provided the groundwork for this uulysi. mbcr3194~~LabornayofGaucia,Uniofd with disease .yqnp k t y dWWe thank C.Pemscn, and R Krebcr for tcdmid toms (Fig. 1B). usisancc;F.Blarmcr, L. Mur, and manbas of* In all animals expressing either construct, BLmKt Lb for hdp with ~cqutncing;E.bust and 3 May 1991; mcpDd 18 June l99l - :5 vlon slm IB. the u. * or& ~~~~Manhp~~w the pathological findings were resmcted to the central nervous system (CNS) and to the striated muxle. The disease phenotype was transmitted to the offspring with a penetrance of 100%. ~ n i m kG t h the c k d Progressive Encephalopathy and Myopathy in symptoms described above showed variable Transgenic HUIMll Foemy vhUSGenes dof selective nerve cel degeneration in the forebrain, with tissue atrophy and KATRIN BOTHE, ADRIANOAGUZZI,HANS LASSMANN, reactive gliosis. The areas affected most fieAXEL RETHWILM, IVANHORAK* quently were the CA3 layer of the hippocampus and the telencephalic cortex (Fig. Transgenic mice carrying the bel region of human foamy retrovirus (HFV) under 2). Some lesions exhibited a more abnormal tramaiptiond contrd of its own long terminal repeat expressed the transgene in their pattern, with incomplete cortical and subcentral nervous systems and in smooth and striated muscle tissues. The animals cortical necrosis and occasionally with macdeveloped a progressive degenerative disease of the central nervous system and of the rophages and mild lymphocytic infilaates in spaces striated musde. Because -ion of the transgene was closely correlated with the the meninges and in the periv& v c e of structuraldamage and ' " ztory reactions were scanty, the disease (13). In situ hybridization analysis revealed a is likely to be caused dkctiy by the HPV proteins. These unexpectd hdiags caLl for complex pattern of expression in the CNS, with groups of neurons, oligodendrocytes, a reevaluation of the pathogenic potential of HFV in humans. and amocytes containing high amounts of FV IS A RETROVIRUS ORIGINALLY form becaw of a frameshift mutation in the transcript in the brain and spinal cord. The isolated 20 years ago from individ- integrase domain that created a premature distribution of positive cells was bilaterally uals with various diseases (1, 2). stop codon in pol, and (ii) pAgpe, in which symmetrical. In animals 4 weeks old, most Although the prevalence of HFV was re- 2.3 kb of gagpd and 1.6 kb of env were neurons in the CA3 sector of the hippocamported to be high in certain geographical deleted (7) (Fig. 1A). In both conmuas, pus and a subpopulation of cortical neurons areas (3), it has not been possible to identify arpression is controlled by the HFV long ter- were strongly positive for HFV RNA, even HFV as the causative agent of any human mind repeat (LTR).Eight and nine fbunBer in animals in which onset of symptoms had disease (4). As with the human T cell lym- animals (11) were derived with p W M and not yet ocax~ed(Fig. 2A). In sick animals phoma virus (HTLV) and human immpAgpe, m q x d d y . F i pHFVM and six older than 6 weeks, most CA3 neurons had ficiency virus (HIV),the HFV genome has, in pAgpe mice developed a m l o g i c a l syn- degenerated (Fig. 2B), and the highest levels drome consisting of ataxia, spastic tetraparesis, of expression were seen in cortical neurons. addition to struaural reaoviral genes, a containing t h m open redmg frames, M-1 to and blindness. The symptoms in the pHFV, Two pHFVM and four pAgpe mice M-3. Bel-1 has some homology to HIV-2 Tat mice appeared as early as 5 to 6 we& after showed focal degeneration of striated mus, led to death proteinand,simibrfytoHIVTatandHTL.V-I birth, progressed r a p ~ U ~and Tax,fimaim as a tramaipional mmaaiw- within 4 to 6 we&. The pAgpe transgenic mice displayed a later onset ofthe disease (1 to (5-8). A Transgenic mice (9) have proved useful in P r ? i 4 = G 7 8 9 r O 12 kb the dissection of the pathogenic potential of 1. (A) or and mlgas IPO~ 1lenv be1 I I fl the regulatory genes of the human retrovi4 m~lwqaaon ma Expressing H won -- rusesHTLV-IandHIV(lO).Toanalyzethe ~ h a v c b e e n s h o w n m ~ H F V biological activity of HFV regulatory proteins we generated transgenic mice with two constructs: (i) p w ~ which h contains the complete HFV genome in a noninfectious - 362isaansgcnicforpHFVM.LVKs1~9~ fiornmousc75:lancl,brain;lant2,liva,lanc3, ~ . ~ a h g ~ ~ a h m ~ . ~ o n t . l n r n h& - o lfa n ~cif k r g l a n t 5 , % m u = ; l a n t 6 , and hmmnob~ u~vcrsityof wunburg,vaslane 7, musde, lane 8, lymph nodes, lane 7,% Wunburg, Federal Republic of 9, spkaL Lane 10, mousc 362 bnin; lane 11, -Y. w, w, ~~ mousc253e,lanc12,mousc253brain;lanc A. re^^ In*mte of Pat*, 267 +lane 14, 13, Dr. Bohr Gasse 7,1030 V i A& H. Lasmam, h n m of Neurology, Schw~fppanicr- lmarnatebrain;lane15,nontransgauclittamatc brain, lanc 16, mouse 881 forrbrain,h sassc 17,1090 Vienna, Austria. 17, mouse 881 p a k d arid , lane 18, *To whom a~tspondaKcshould be addrcsscd. mouse 881 meWhun and brain smn. 2 AUGUST 1991 AI in cuhmcd cellsAf, Afl E, Eco RI,H, Hind Hp, Hpa II,N,Nco I; Pv, n. (B) invrau ti^^ of aansgcnic micc. Micc 75, 253, 267, 11 0 k b and 881 harbor the p+ tramgene, arid morsc 7 1 k b p. 6 p 5 k b 1 1 v p PV PAgPe H-E t i !-Ias M**m 1 2 3 4 5 6 7 8 9 1011 12131415 161718 P-actln REPORTS 555 ~ b g e -