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Multiple Shaker Potassium Channels in a Primitive Metazoan

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Multiple Shaker Potassium Channels in a Primitive Metazoan The Journal of Neuroscience, December 1995, 15(12): 7989-7999 Multiple Shaker Potassium Channels in a Primitive Metazoan Timothy Jegla,’ Nikita Grigoriev,z Warren J. Gallin,* Lawrence Salkoff,’ and Andrew N. Spencer2 ‘Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110 and 2Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 and Bamfield Marine Station, Bamfield, British Columbia Canada VOR 1 BO Voltage-gated potassium channels are critical elements in be accounted for by a diverse, but highly conserved, set of Vg providing functional diversity in nervous systems. The di- potassiumchannel proteins that are present in most metazoans versity of voltage-gated K+ channels in modern triploblas- (Rudy, 1988; Salkoff et al., 1992). tic metazoans (such as mollusks, arthropods and vette- The molecular basis of K+ channel diversity is well under- brates) is provided primarily by four gene subfamilies stood in many triploblastic metazoans,including vertebrates(Jan (Shaker, Shal, Shab, and Shaw), but there has been no data and Jan, 1990; Rudy et al., 1991; Salkoff et al., 1992; Chandy from the ancient diploblastic metazoans until now. Diplo- and Gutman, in press), arthropods (Jan and Jan, 1990; Salkoff blasts, represented by jellyfish and other coelenterates, et al., 1992; Pongs, 1993) and mollusks (Pfaffinger et al., 1991; arose during the first major metazoan radiation and are the Quattrocki et al., 1994; Zhao et al., 1994) and has been exam- most structurally primitive animals to have true nervous ined in other triploblasts groups such as annelids (Johansenet systems. By comparing the K+ channels of diploblasts and al., 1990), nematodes(Wei et al., 1991), and flatworms (Kim et triploblasts, we may determine the fundamental set of K+ al., 1995). In these organisms,much of the K+ channel diversity channels present in the first nervous systems. We now re- appearsto be encoded by four closely related gene subfamilies port the isolation of two Shaker subfamily cDNA clones, of Vg K+ channels: Shaker, Shal, Shab, and Shaw. However, it jShak1 and jShak2, from the hydrozoan jellyfish Polyorchis has not been clear whether this set of Vg K+ channelsdiversified penicillafus (Phylum Cnidaria). JShakl and jShak2 express within the triploblastic lineage or whether it was, at least in part, transient outward currents in Xenopus oocytes most sim- present during the establishmentof the nervous system in more ilar to Shaker currents from Drosophila in their rates of primitive metazoans. inactivation and recovery from inactivation. The finding of The most ancient extant metazoansto have true nervous sys- multiple Shaker subfamily genes is significant in that mul- tems are the diploblasts, organismssuch as jellyfish, sea anem- tiple Shaker genes also exist in mammals. In Drosophila, ones,and comb-jellies.These organismshave only two germinal multiple Shaker channels are also produced, but by a layers, lacking a true mesoderm.Both the fossil record and mo- mechanism of alternative splicing. Thus, the Shaker K+ lecular phylogenies basedon ribosomal RNA sequencesplace channel subfamily had an established functional identity the emergenceand diversification of the extant diploblastic phy- prior to the first major radiation of metazoans, and multiple la well before the appearanceof modern triploblasts, during an forms of Shaker channels have been independently select- initial major radiation of metazoanspecies (Christen et al., 1991; ed for in a wide range of metazoans. Morris, 1993; Wainwright et al., 1993). Thus, the first nervous [Key words: Shaker, potassium channel, voltage-gate& systemsmost likely appearedin one of the latest common an- jellyfish, Cnidarian, diploblast, triploblast, polyorchis, in- cestorsof diploblastsand triploblasts. activation] Physiological evidence shows that the diploblasts, like tri- ploblasts, have a diverse set of Vg K+ currents (Anderson and Electrical excitability is a fundamental property of the neuro- McKay, 1987; Dunlap et al., 1987; Holman and Anderson, 1991; muscular systemsof metazoans.This property is conferred by a Meech and Mackie, 1993; Przysiezniak and Spencer, 1994), diverse set of voltage-gated (Vg) ion channel proteins that has hinting at an early diversification of Vg K+ channel genes.By evolved in concert with neurons and other excitable cells. Neu- examining the molecular diversity of Vg K+ channelsin diplo- robiologists have come to appreciatethat the surprisingly varied blasts such as jellyfish, we can gain an understanding of the responsesof neuronsto electrical excitation can in a large part molecular diversity of Vg K+ channelsin the first nervous sys- tems and how it has been conserved or specializedto meet the needs of modern triploblasts with their complex nervous and Received June 14, 1995; revised Aug. 3, 1995; accepted Aug. 7, 1995. effector systems. We thank Dr. Aguan Wei for his role in developing OUT PCR screen, as well as his helpful comments and enthusiasm. The Shaker H37 clone was kindly Using a PCR screeningprotocol, basedon degenerateprimers provided by Dr. Mark Tanouye, Shaker B by Dr. Tom Schwarz, and Kv1.2 by correspondingto the regions most highly conserved between the Dr. David McKinnon. We thank the Barnfield Marine Station for their assis- Shaker, Shal, Shab, and Shaw subfamilies,we have isolated two tance in collecting and holding jellyfish and maintenance of a Xenopus colony. Research was supported by grants from the NIH and MDA to Lawrence Salkoff Shaker subfamily homologs,jShak1 and jShak2, from the hy- and from the NSERC to W.J.G. and A.N.S. dromedusanjellyfish Polyorchis penicillatus. We report the se- Correspondence should be addressed to Timothy J. Jegla, Washington Uni- quencesof these K+ channel subunitsand demonstrateremark- versity School of Medicine, Department of Anatomy and Neurobiology, Box 8108, 660 South Euclid Avenue, St. Louis, MO 63 110. able similarity to their triploblastic homologs. These results Copyright 0 1995 Society for Neuroscience 0270-6474/95/157989-I 1$05.00/O show that the Shaker Vg K+ channel gene subfamily has been 7990 Jegla et al. * Jellyfish Shaker K+ Channels functionally differentiated from other Vg K+ channel lineages at initiation sequence (Kozak, 1987) and an internal primer 3’ of a unique internal Sac I site 5’-(AATCATAATCATTCATCG)-3’. The PCR prod- least since the appearance of the nervous system. Thus, the di- uct was then digested with Sac I and cloned into the jShakI-Bluescript versity of Vg K+ channels observed in triploblasts is likely to KS+ subclone digested with Sac I. The PCR amplified region was se- have arisen during or even prior to the earliest major radiation quenced in both directions to confirm correct orientation and lack of of metazoan species. PCR-introduced mutations. A truncated jShak1 construct (jShukl7’) that eliminates the first 23 predicted amino acids and replaces phenlyalanine Materials and Methods 24 with an initiator methionine preceded by a consensus translational initiation sequence was constructed using the sense primer 5’-(GGGA- Cloning ofjShak1 and jShak2. Amplification and isolation of fragments GCTCCACCATGGAAGACAATGCAGA)-3’. of jShuk1 and jShak2 from Polyorchis penicillatus genomic DNA was The jShak2 construct was produced in two steps. First, an incomplete performed as described in Jegla and Salkoff, 1995. Briefly, the degen- jShuk2 cDNA isolated in the original bank screen described above was erate primers used to generate the fragments were 5’.TCGGAATI- cut with Nhe I and Not I and subcloned into an Spe I/Not I cut p- CTATGACTACTGTTGG(A\C\G\T)TA(T\C)GG(A\C\G\T)GA-3’ BScMXT oocyte expression vector (Wei et al., 1994). The 5’ end was (sense) and 5’.ACTTCTAGAGGTAGTGCTAIT(GM)(‘DC)(A\C\G\T)- amplified with a sense oligonucleotide 5’.(TTACGAATTCCACCATG- AG(A\C\G\T)AC(A\C\G\T)CC-3’ (antisense) which are derived from the TTACCAGTTCTAACGCAAACG))3’ which replaces the 5’ UT se- consensus amino acid sequences of the pore region [MTTVGYG(D/E)] quences with a consensus translation initiation sequence and the pre- and sixth transmembrane domain [GVL(TN)TIAL] of Vg K+ channels. viously described pore region antisense oligonucleotide. This product The jShak1 and jShak2 fragments obtained in the amplification screen was cut with Eco RI and Nhe I and subcloned into the vector produced were used to probe oligonucleotide dT primed Polyorchis cDNA li- in the first step (Eco RI/Nhe I cut). The amplified region was sequenced braries under high stringency conditions (Butler et al., 1989). Purifica- in both directions to confirm that no PCR-introduced mutations existed. tion of mRNA from neuronally enriched P. penicillatus tissue samples Expression of cRNA in Xenopus oocytes. Capped cRNAs were pre- and construction of the cDNA libraries in the lambdaZAP II vector pared by run-off transcription with either T7 RNA polymerase (for j- (Stratagene, La Jolla) have been described previously (Gallin, 1991). Shukl and jShuklT), or with T3 RNA polymerase (jShak2, Shaker H37, Bluescript SK- subclones of each positive clone were obtained using Shaker B, and Kv1.2) using the mMessage mMachine kit (Ambion, Stratagene’s in viva excision protocol and sequenced in both directions. Austin). Mature stage IV Xenopus oocytes were prepared for injection This procedure yielded a complete cDNA clone of jShuk1. However, as described in Wei et al., 1990. Oocytes were injected with 50 nl of all jShuk2 cDNAs were truncated, containing only a region encoding cRNA at concentrations of 5-100 ng/pl (adjusted based on the expres- Sl through the 3’ end of the coding sequence. The missing 5’ region sion level of the particular construct), and incubated at 15°C for l-3 d was obtained by high stringency amplification (95°C 1 min, 60°C 3 min, in ND96 (96 mu NaCl, 2 mM KCl, 1.8 mu CaCI,, 1 mM MgCl,, 5 mM 30 cycles) of a cDNA bank with 0.5 pmol/pl of an oligonucleotide HEPES-NaOH, pH 7.5) supplemented with 100 U/ml penicillin, 100 matching the T7 region of Bluescript 5’.(GCGCGTAATACGACTCAC- pg/ml streptomycin and 2.5 mM sodium pyruvate.
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