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Mutational Analysis of the Shab-encoded Delayed Rectifier K+ Channels in Drosophila

1999; Elsevier BV; Volume: 274; Issue: 31 Linguagem: Inglês

10.1074/jbc.274.31.22109

1083-351X

Priti S. Hegde, Gang-Gou Gu, Dong Chen, Stephen J. Free, Satpal Singh,

Neurobiology and Insect Physiology Research

K+ currents inDrosophila muscles have been resolved into two voltage-activated currents (IA and IK) and two Ca2+-activated currents (ICF and ICS). Mutations that affect IA (Shaker) and ICF (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (IK) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K+ channel gene, we show that this gene encodes the delayed rectifier K+channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K+ current (IK). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivophysiological role for the Shab-encoded K+channels in Drosophila. The availability of mutations that affect IK opens up possibilities for studyingIK and its role in larval muscle excitability. K+ currents inDrosophila muscles have been resolved into two voltage-activated currents (IA and IK) and two Ca2+-activated currents (ICF and ICS). Mutations that affect IA (Shaker) and ICF (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (IK) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K+ channel gene, we show that this gene encodes the delayed rectifier K+channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K+ current (IK). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivophysiological role for the Shab-encoded K+channels in Drosophila. The availability of mutations that affect IK opens up possibilities for studyingIK and its role in larval muscle excitability. Voltage-activated K+ channels play a crucial role in repolarizing the membrane following action potentials, stabilizing membrane potentials and shaping firing patterns of cells (1Hille B. Ionic Channels of Excitable Membranes. 2nd Ed. Sinauer Associates Inc., Sunderland, MA1992Google Scholar). Many human diseases such as long QT syndrome, Jervell and Lange-Nielson syndrome, episodic ataxia, and epilepsy are associated with mutations in these channels (2Wang Q. Curran M.E. Splawski I. Burn T.C. Millholland J.M. VanRaay T.J. Shen J. Timothy K.W. Vincent G.M. de Jager T. Schwartz P.J. Toubin J.A. Moss A.J. Atkinson D.L. Landes G.M. Connors T.D. Keating M.T. Nat. Genet. 1996; 12: 17-23Crossref PubMed Scopus (1505) Google Scholar, 3Bulman D.E. Hum. Mol. Genet. 1997; 6: 1679-1685Crossref PubMed Scopus (63) Google Scholar, 4Tyson J. Tranebjaerg L. Bellman S. Wren C. Taylor J.F. Bathen J. Aslaksen B. Sorland S.J. Lund O. Malcolm S. Pembrey M. Bhattacharya S. Bitner-Glindzicz M. Hum. Mol. Genet. 1997; 6: 2179-2185Crossref PubMed Scopus (265) Google Scholar, 5Charlier C. Singh N.A. Ryan S.G. Lewis T.B. Reus B.E. Leach R.J. Leppert M. Nat. Genet. 1998; 18: 53-55Crossref PubMed Scopus (821) Google Scholar, 6Singh N.A. Charlier C. Stauffer D. DuPont B.R. Leach R.J. Melis R. Ronen G.M. Bjerre I. Quattlebaum T. Murphy J.V. McHarg M.L. Gagnon D. Rosales T.O. Peiffer A. Anderson V.E. Leppert M. Nat. Genet. 1998; 18: 25-29Crossref PubMed Scopus (1036) Google Scholar). Hence, it is important to understand how K+ channels function. With an excellent repertoire of available genetic tools, Drosophila provides a powerful system for such studies. The existence of distinct behavioral phenotypes that arise due to defects in neuromuscular pathways has aided in identifying mutations that affect ion channels. A functional voltage-gated K+ channel consists of four α-subunits, each with six transmembrane domains (S1–S6) flanked by cytoplasmic amino- and carboxyl-terminal regions. A number of genes coding for K+ channel α-subunits have been cloned. These include genes from six families, defined by six DrosophilaK+ channel genes: Shaker (Kv1.1–1.7),Shab (Kv2.1–2.2), Shaw (Kv3.1–3.4),Shal (Kv4.1–4.3), ether-a-go-go (HERG) and slowpoke (maxiK) (7Salkoff L. Baker K. Butler A. Covarrubias M. Pak M.D. Wei A. Trends Neurosci. 1992; 15: 161-166Abstract Full Text PDF PubMed Scopus (256) Google Scholar, 8Wu C.F. Ganetzky B. Narahashi T. Ion Channels. 3. Plenum Press, New York1992: 261-314Google Scholar, 9Jan L.Y. Jan Y.N. Annu. Rev. Neurosci. 1997; 20: 91-123Crossref PubMed Scopus (462) Google Scholar, 10Armstrong C.M. Hille B. Neuron. 1998; 20: 371-380Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). The Shaker,ether-a-go-go (eag), and slowpoke(slo) genes were identified on the basis of behavioral mutations that helped in the cloning and extensive molecular analysis of these genes and their encoded channels (11Kamb A. Iverson L.E. Tanouye M.A. Cell. 1987; 50: 405-413Abstract Full Text PDF PubMed Scopus (296) Google Scholar, 12Papazian D.M. Schwarz T.L. Tempel B.L. Jan Y.N. Jan L.Y. Science. 1987; 237: 749-753Crossref PubMed Scopus (535) Google Scholar, 13Pongs O. Kecskemethy N. Muller R. Krah-Jentgens I. Baumann A. Kiltz H.H. Canal I. Llamazares S. Ferrus A. EMBO J. 1988; 7: 1087-1096Crossref PubMed Scopus (315) Google Scholar, 14Atkinson N.S. Robertson G.A. Ganetzky B. Science. 1991; 253: 551-555Crossref PubMed Scopus (541) Google Scholar, 15Warmke J. Drysdale R. Ganetzky B. Science. 1991; 252: 1560-1562Crossref PubMed Scopus (282) Google Scholar, 16Adelman J.P. Shen K.Z. Kavanaugh M.P. Warren R.A. Wu Y.N. Lagrutta A. Bond C.T. North R.A. Neuron. 1992; 9: 209-216Abstract Full Text PDF PubMed Scopus (432) Google Scholar). On the other hand,Shab, Shal, and Shaw were identified in homology screens using Shaker cDNA as probe (17Butler A. Wei A.G. Baker K. Salkoff L. Science. 1989; 243: 943-947Crossref PubMed Scopus (205) Google Scholar). Expression of cRNAs of these genes results in the generation of K+ currents in Xenopus oocytes (18Wei A. Covarrubias M. Butler A. Baker K. Pak M. Salkoff L. Science. 1990; 248: 599-603Crossref PubMed Scopus (258) Google Scholar, 19Covarrubias M. Wei A.A. Salkoff L. Neuron. 1991; 7: 763-773Abstract Full Text PDF PubMed Scopus (221) Google Scholar). However, no mutations have been reported in any of these three genes, thus making it difficult to elucidate their in vivophysiological function. We describe the identification and molecular analysis of the first behavioral mutations that disrupt the Shab gene. These mutations were initially identified as causing a temperature-induced paralytic phenotype (20.Chopra, M., Ph.D. thesis, 1994, G. N. D. University, Amritsar, India.Google Scholar). 1M. Chopra, G.-G. Gu, and S. Singh, manuscript in preparation.. They selectively affect the delayed rectifier potassium current (IK), in larval body wall muscles, without affecting other K+ currents and reveal the in vivo functional role of the Shab gene inDrosophila. A mutation (z66) with the phenotype of temperature-induced paralysis had been earlier identified to specifically affect the delayed rectifier potassium current in larval muscles. 1The abbreviations used are: CS, Canton-S; OR, Oregon-R; PCR, polymerase chain reaction. 30 male flies (3 days old) carrying theebony (e s) marker on chromosome 3 were mutagenized with 3500 rads of x-irradiation and mated in batches of three males and 10 z66 virgin female flies (3–5 days old). The males were removed from the vials after 3 days. F1 progeny from the cross were tested for temperature-induced paralysis at 39 °C for 5 min. Of the 5673 F1 progeny tested, one male fly (9g) paralyzed within 3 min at 39 °C and recovered from paralysis within 4 min of being transferred to room temperature (25 °C). The mutant was mated to virgin females carrying the third chromosome balancer TM3, Sb p p e s/TM6B, Tb Hu red e. F2 males and virgin females having ebony body color (with the TM3 balancer) were then mated to render the mutation homozygous. All stocks were maintained at 21 °C (21Chopra M. Singh S. J. Neurobiol. 1994; 25: 119-126Crossref PubMed Scopus (15) Google Scholar). Body wall muscle 12 (22Crossley A.C. Ashburner M. Wright T.R.F. The Genetics and Biology of Drosophila. 2B. Academic Press, London1977: 499Google Scholar, 23Bate M. Development. 1990; 110: 791-804PubMed Google Scholar) of mature third instar larvae of wild-type (CS)2 and various mutant strains was used for the two-electrode voltage clamp experiments as described previously (24Kraliz D. Singh S. J. Neurobiol. 1997; 32: 1-10Crossref PubMed Scopus (41) Google Scholar). Ca2+-free recording solution contained 77.5 mm NaCl, 115 mm sucrose, 5 mm KCl, 0.5 mm EGTA, 20 mmMgCl2, 5 mm Trehalose, 2.5 mmNaHCO3, and 5 mm HEPES (pH 7.1) (25Stewart B.A. Atwood H.L. Renger J.J. Wang J. Wu C.F. J. Comp. Physiol. A. 1994; 175: 179-191Crossref PubMed Scopus (622) Google Scholar, 26Gu G.G. Singh S. J. Neurobiol. 1997; 33: 265-275Crossref PubMed Scopus (19) Google Scholar). Electrodes were pulled from thin walled 1.0-mm borosilicate glass capillaries (World Precision Instruments, Sarasota, FL) and had resistances of about 10 megaohms. Voltage electrodes contained 2.5m KCl, and current electrodes contained a 3:1 mixture of 2.5 m KCl, 2 m potassium citrate (27Wu C.F. Haugland F.N. J. Neurosci. 1985; 5: 2626-2640Crossref PubMed Google Scholar). A Macintosh IISi computer provided the voltage clamp command pulses through a 12-bit digital-to-analog converter using the MacADIOS II/16 board (GW Instruments, Somerville, MA). Data were acquired after a 16-bit analog-to-digital conversion. Analysis was performed with a program written in Think-C (Symantec Corp., Cupertino, CA). Test currents were digitally sampled every 500 μs and digitally corrected for linear leakage with respect to currents obtained at −40 mV. Current densities (nanoamperes/nanofarads) were calculated as described previously (28Gielow M.L. Gu G.G. Singh S. J. Neurosci. 1995; 15: 6085-6093Crossref PubMed Google Scholar, 29Kraliz D. Bhattacharya A. Singh S. J. Neurogenet. 1998; 12: 25-39Crossref PubMed Scopus (10) Google Scholar). All experiments were carried out at 4 °C. Genomic DNA was extracted from CS and 9g flies (30Ashburner M. Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The genomic DNA was digested withPstI restriction endonuclease, subjected to electrophoresis in an agarose gel, transferred to a nylon membrane, and hybridized with probe prepared from the Shab cDNA. Probe was generated using the DIG High Prime DNA labeling and chemiluminescent detection kit (Roche Molecular Biochemicals). Chemiluminescent detection was performed according to the instructions provided by the manufacturer. Total RNA was isolated from CS, Shab 1, Shab 2, and Shab 3 larvae and adult flies (1–2 days old) (30Ashburner M. Drosophila: A Laboratory Handbook. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). 5 μg of total RNA was reverse transcribed using Superscript II (Life Technologies, Inc.) according to the instructions of the manufacturer. PCR was performed using the UlTma DNA polymerase enzyme (Perkin-Elmer). RT-2 (5′-CGCCGAATTCCTCCTCGTCGCGCTGCCGCAGGGA-3′), RT-4 (5′-CGGATGCACGACATTTGTTTGCTTTCCTGTGTCGACAAT-3′), and RT-6 (5′-GGATGGGCAGT CCCTTGGCC-3′) were used for reverse transcription of RNA. Primers PCR-1 (5′-GAGGAAGCTTATGGTCGGGCAATTGCAAGGTGGACAGGCTGCTGG-3′) and RT-2 were used to amplify region 1 (base pairs 1–1231) of the cDNA templates (Fig. 3a). Primers PCR-3 (5′-GGAGGAATTCGGCGAAGGTAAATTCTCCGAGTACCA-3′) and RT-4 were used to amplify region 2 (base pairs 1215–2175) of the cDNA templates. Primers PCR-5 (5′- AATCAGATGCGCCGCGAAAAGGC-3′) and RT-6 were used to PCR amplify region 3 (base pairs 2000–2771) of the cDNA templates. PCR amplifications were performed as described by the manufacturer (Perkin-Elmer), using the hot start PCR technique. The PCR conditions were as follows: initial denaturation (93 °C, 2 min) followed by 35 cycles of denaturation at 95 °C for 1 min, annealing at 65 °C for 1.5 min, and extension at 72 °C for 2 min. Amplified DNA was run on a 0.8% agarose gel and extracted by centrifugal filtration using Ultrafree-MC filter (Millipore Corp.). DNA was sequenced by the Roswell Park Biopolymer Sequencing Facility. Several ethylmethanesulfonate-induced mutations had been previously isolated in our laboratory by using the conditional phenotype of temperature-induced paralysis (20.Chopra, M., Ph.D. thesis, 1994, G. N. D. University, Amritsar, India.Google Scholar). These mutations were identified with the use of compound chromosomes (31Chovnick A. Ballantyne G.H. Baillie D.L. Holm D.G. Genetics. 1970; 66: 315-329Crossref PubMed Google Scholar, 32Holm D.G. Ashburner M. Novitsky E. Genetics and Biology of Drosophila. 1b. Academic Press, London1976: 529-561Google Scholar, 33Williamson R. Drosophila Information Service. 1977; 51: 150Google Scholar, 34Singh S. Bhandari P. Chopra M.J.S. Guha D. Mol. Gen. Genet. 1987; 208: 226-229Crossref Scopus (2) Google Scholar). Compound chromosomes have homologous copies of a chromosomal arm attached at the centromere. The use of compound chromosomes facilitates the isolation of mutants, because this allows heterozygous mutations to become homozygous during meiotic recombination. They enable identification of recessive autosomal mutations without setting up individual fly lines. Initial characterization showed that the delayed rectifier current,IK, was reduced in two of the temperature-sensitive paralytic mutants, z66 and z4 (Fig. 1a). Genetic analysis demonstrated that the mutations do not complement each other and therefore reside in the same gene. The effect of thez66 mutation on various other currents expressed in larval muscles has been examined. No significant change was seen in the fast transient voltage-activated K+ current,IA; the fast transient calcium-activated K+ current, ICF; the slow sustained calcium-activated K+ current, ICS; and the total Ca2+ current, thus suggesting that the mutation selectively affects IK(20.Chopra, M., Ph.D. thesis, 1994, G. N. D. University, Amritsar, India.Google Scholar).1 As shown in Table I,IK was reduced by 46.0 ± 3.0% inz66 mutants. The z4 mutants showed a 43.6 ± 3.4% reduction in IK.Table IEffect of mutations on IK in larval musclesGenotypeCurrent amplitudenReduction inIKReduction in Shab componentnA/nFaNanoamperes/nanofarads.%%CS16.5 ± 0.41000Shab 29.3 ± 0.4943.6 ± 3.471.2 ± 7.0Shab 18.9 ± 0.31246.0 ± 3.075.2 ± 6.6Shab 36.4 ± 0.21061.2 ± 2.7100Experiments were performed as in Fig. 1. IK current amplitudes were calculated for a voltage pulse to +40 mV. nrefers to the number of larval muscle fibers assayed. To determine the percentage reduction in the Shab component, reduction inIK from the 9g (Shab 3) null alleles was taken as representing 100% of the Shabcurrent. All values are given as mean ± S.E.a Nanoamperes/nanofarads. Open table in a new tab Experiments were performed as in Fig. 1. IK current amplitudes were calculated for a voltage pulse to +40 mV. nrefers to the number of larval muscle fibers assayed. To determine the percentage reduction in the Shab component, reduction inIK from the 9g (Shab 3) null alleles was taken as representing 100% of the Shabcurrent. All values are given as mean ± S.E. To obtain additional alleles of the gene affected by the z66and z4 mutations, including some with chromosomal aberrations that would aid in identifying the gene affected by the mutations, we performed x-ray mutagenesis (35Brittnacher J.G. Ganetzky B. Genetics. 1983; 103: 659-673Crossref PubMed Google Scholar). Male ebonyflies were subjected to x-ray mutagenesis and mated with virginz66 females (see “Experimental Procedures”). Screening of 5673 F1 progeny for temperature-induced paralysis at 39 °C led to the identification of a new mutant, 9g. In paralysis tests performed on 9g/z66 flies, the two mutations did not complement each other and hence are alleles of the same gene. As in z66, IK was affected in 9g mutants (Fig. 1a). There was a 61.2 ± 2.7% reduction of IK in9g homozygotes (see Table I). Recombination and deletion mapping localized the z66 and z4mutations to the left arm of chromosome 3 at 63A1-B6 (20.Chopra, M., Ph.D. thesis, 1994, G. N. D. University, Amritsar, India.Google Scholar).1 Deletion analysis of 9g showed that the mutation mapped to the same position. Shab, a K+ channel gene cloned by homology to Shaker lies on chromosome 3 at position 63A (17Butler A. Wei A.G. Baker K. Salkoff L. Science. 1989; 243: 943-947Crossref PubMed Scopus (205) Google Scholar). By using deletions in this region (63A1-B9), Tsunoda and Salkoff (36Tsunoda S. Salkoff L. J. Neurosci. 1995; 15: 5209-5221Crossref PubMed Google Scholar) showed that IK is reduced in embryonic neurons and myotubes of these deletion strains and suggested that Shab may code for a delayed rectifier potassium channel. To determine if the 9g mutation lies in the Shabgene, Southern blot analysis was performed on genomic DNA from wild type (CS) and 9g flies using the entire ShabcDNA as probe. CS was used in the analysis because the mutants were obtained from CS-derived strains. As shown in Fig. 2, a comparison between thePstI-digested CS and 9g DNA revealed a restriction fragment length polymorphism between mutant and wild type DNAs, indicating that the 9g mutation disrupts theShab gene. The 9g mutation and its two noncomplementing alleles, z66 and z4, contain the first mutations to be identified in the Shab gene. The mutant alleles in z66, z4, and 9g will be referred to hereafter as Shab 1,Shab 2, and Shab 3 respectively. To investigate the underlying molecular defects in the mutants, ShabcDNA was prepared by reverse transcription-PCR fromShab 1, Shab 2,Shab 3, and wild type (CS) flies (Fig. 3a). PCR products were sequenced, and results were compared with the published sequence ofShab derived from the OR strain (17Butler A. Wei A.G. Baker K. Salkoff L. Science. 1989; 243: 943-947Crossref PubMed Scopus (205) Google Scholar). Comparison of the sequences revealed that the CS sequence has an insertion of a single base (G) at position 2708 of the published OR sequence. This produces a shift in the reading frame, resulting in the addition of 60 amino acids at the C terminus of the Shab channel in CS. In addition to the insertion, we found other nucleotide changes, seven of which alter the encoded amino acid sequence. These changes from OR to CS are as follows (the number system is as previously defined for theShab (OR) gene; GenBankTM accession no. M32659): 1) a T to A transversion at nucleotide 92 that changes amino acid 31 from a leucine to a glutamine; 2 and 3) two G to A transitions at nucleotides 658 and 1084 that change amino acids 220 and 362 from glycine to serine; 4) a T to G transversion at nucleotide position 1483 that changes amino acid 495 from serine to alanine; 5) a C to G transversion at nucleotide 2481 that changes amino acid 827 from an aspartic acid to a glutamic acid; 6) a G to C transversion at nucleotide 2482 that changes amino acid 828 from a glutamic acid to a glutamine, and 7) a G to C transversion at nucleotide position 2630 that changes amino acid 877 from glycine to alanine. Butler et al. (17Butler A. Wei A.G. Baker K. Salkoff L. Science. 1989; 243: 943-947Crossref PubMed Scopus (205) Google Scholar) reported the presence of a 90-nucleotide coding region (nucleotides 2151–2240) in the longest correctly splicedShab cDNA (Shab11) (GenBankTMaccession no. M32659) not found in other Shab cDNAs. In our experiments, the CS cDNA did not show the presence of these 90 nucleotides. The Shab gene sequence from CS has been entered in GenBankTM. Sequence comparison between CS and Shab 1 cDNAs revealed a single G to A transition at nucleotide position 1304, which changes an arginine at amino acid 435 to a glutamine (Fig. 3b). This arginine at position 435 is thought to be the last residue before the protein enters into the membrane as the first transmembrane segment (S1) (Fig. 4) and is an arginine or a lysine in most members of voltage-gated potassium channels (Fig. 5). In Shaker potassium channels, the NH2-terminal region and the S1 segment are essential for subunit interactions as well as expression of functional channels at the cell membrane (37Babila T. Moscucci A. Wang H. Weaver F.E. Koren G. Neuron. 1994; 12: 615-626Abstract Full Text PDF PubMed Scopus (98) Google Scholar, 38Xu J. Yu W. Jan Y.N. Jan L.Y. Li M. J. Biol. Chem. 1995; 270: 24761-24768Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar, 39Tu L. Santarelli V. Sheng Z. Skach W. Pain D. Deutsch C. J. Biol. Chem. 1996; 271: 18904-18911Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). The position of theShab 1 mutation is interesting and may suggest an important role for this region in the formation of a functionalShab channel.Figure 5Conservation of a positively charged amino acid immediately before S1. A sequence alignment of K+channels in the region of S1 shows conservation of basic residues (arginines and lysines) at the position immediately preceding the S1 transmembrane segment. The sequences are as follows: Shab,Drosophila melanogaster (Swiss-Prot P17970); Kv2.2 (CDRK),Rattus norvegicus (Swiss-Prot Q63099); Kv2.1 (DRK1),R. norvegicus (Swiss-Prot P15387); Kv1.5, Homo sapiens (Swiss-Prot P22460); Kv3.4, H. sapiens(Swiss-Prot Q03721); RCK1, R. norvegicus (Swiss-ProtP10499).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Analysis of the Shab 2 cDNA revealed a T to A transversion at nucleotide position 1823 (Fig. 3c). This missense mutation changes a valine to an aspartic acid at amino acid 608. This residue is found in the pore region of the ShabK+ channel (Fig. 4). The pore region consists of a turret, pore helix, and selectivity filter (40Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5769) Google Scholar). While the pore helix and the selectivity filter are highly conserved in their amino acid sequence, the turret, which lies at the external mouth of the pore, is less conserved. The amino acids in this region form high affinity binding sites for peptide inhibitors from scorpion venom (41MacKinnon R. Heginbotham L. Abramson T. Neuron. 1990; 5: 767-771Abstract Full Text PDF PubMed Scopus (190) Google Scholar). The V608D mutation falls at the junction of the turret and the pore helix in the toxin binding site of the pore region. The sequences of Shab 1 and Shab 2, both of which were isolated in the same mutagenesis, corroborate the sequence of the CS Shab gene and provide evidence that the mutations observed are not due to polymorphic differences between the parent strain used for the isolation of the Shab 1and Shab 2 mutations and the original CS strain. Sequencing of reverse transcription-PCR products fromShab 3 RNA revealed two deletions. The first removed 24 base pairs from nucleotide positions 508–531. The second was a 356-base pair deletion from nucleotide positions 656–1011 (Fig. 3d), which caused a shift in the reading frame, creating a stop codon 74 bases downstream of the mutation. TheShab 3-encoded protein, as defined by conceptual translation of the cDNA, is therefore truncated prior to the S1 segment and lacks channel function (Fig. 4). Unless alternate splicing removes the region containing the second deletion,Shab 3 defines a functionally null mutation. To look for the existence of introns in the region of the deletions, we amplified and characterized Shab gene sequences from CS genomic DNA. Sequencing of the region from −55 to 1200 nucleotides showed no difference between wild-type genomic and cDNA sequences, indicating that there were no introns within this region. Thus, theShab 3 deletions are present within the first exon. We conclude that the Shab 3 protein lacks the entire transmembrane region and defines a null allele. The Shab, Shal, and Shawsubfamilies of voltage-gated K+ channels were identified on the basis of homology to Shaker (17Butler A. Wei A.G. Baker K. Salkoff L. Science. 1989; 243: 943-947Crossref PubMed Scopus (205) Google Scholar). Subsequently, channels belonging to these families have been cloned from a number of other species. However, the absence of in vivo mutations in any of the three genes has made it difficult to elucidate the physiological role and significance of these channels. Lack of mutations has also limited molecular analysis of the function and regulation of these channels. We have now identified viable behavioral mutations in theShab channels and have analyzed them at a molecular level. Our data show that the Shab gene codes for delayed rectifier K+ channels in larval body wall muscles. Three mutant alleles of the Shab gene have been characterized. Two alleles, Shab 1 and Shab 2, contain missense mutations. The third allele,Shab 3, contains two deletions in the cytoplasmic amino-terminal region of the protein and is a null allele (Fig. 4). It is significant that the delayed rectifier current,IK, is only reduced by 61.2 ± 2.7% and not completely abolished in Shab 3 (Fig. 1; Table I). If Shab channels carried all of theIK, then a null mutation in the Shabgene would be expected to lack delayed rectifier current. Thus, our results indicate that IK consists of more than one component, with a large fraction of the current being carried byShab channels. Detailed pharmacological and physiological studies performed on Shab 3 mutants support this hypothesis. 3Singh, A., and Singh, S. (1999) J. Neurosci., in press.Electrophysiological studies performed using genetic aberrations that delete a large region of chromosome containing the Shablocus in Drosophila show a 77% reduction ofIK in embryonic myotubes (36Tsunoda S. Salkoff L. J. Neurosci. 1995; 15: 5209-5221Crossref PubMed Google Scholar). The gene that codes for the remaining current seen in the Shab 3mutants remains to be identified. One possible candidate is theShaw gene that codes for a noninactivating K+channel (18Wei A. Covarrubias M. Butler A. Baker K. Pak M. Salkoff L. Science. 1990; 248: 599-603Crossref PubMed Scopus (258) Google Scholar). The availability of mutations that affect the residual current in the Shab 3 mutants will be helpful in studying this current and the gene coding for the channels. In Shab 1, an R435Q mutation exhibits a 46.0 ± 3.0% reduction in IK (Fig. 1). SinceShab channels carry 61.2% of the delayed rectifier current,Shab 1 mutants show a 75.2 ± 6.6% reduction in the Shab component of IK (Table I). The Shab 1 mutation changes a highly conserved arginine to a glutamine (R435Q) at the last amino acid before the protein enters the membrane as the first transmembrane segment (S1). Most transmembrane proteins contain signal/anchor sequences prior to the first transmembrane segments. These sequences include positively charged arginines or lysines, which are essential for determining membrane topology (42Beltzer J.P. Fiedler K. Fuhrer C. Geffen I. Handschin C. Wessels H.P. Spiess M. J. Biol. Chem. 1991; 266: 973-978Abstract Full Text PDF PubMed Google Scholar, 43Parks G.D. Lamb R.A. J. Biol. Chem. 1993; 268: 19101-19109Abstract Full Text PDF PubMed Google Scholar, 44Gafvelin G. Sakaguchi M. Andersson H. von Heijne G. J. Biol. Chem. 1997; 272: 6119-6127Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Substituting the arginines or lysines with neutral or negatively charged residues results in a significant fraction of membrane-spanning proteins anchoring in reverse topological orientation in the membrane (42Beltzer J.P. Fiedler K. Fuhrer C. Geffen I. Handschin C. Wessels H.P. Spiess M. J. Biol. Chem. 1991; 266: 973-978Abstract Full Text PDF PubMed Google Scholar, 43Parks G.D. Lamb R.A. J. Biol. Chem. 1993; 268: 19101-19109Abstract Full Text PDF PubMed Google Scholar). Voltage-gated K+channels show similarly conserved arginines and lysines just before S1 (Fig. 5). It will be interesting to examine if the R435Q mutation inShab 1 results in a topological inversion. Mutations in the pore region affect various properties such as gating, channel activation, ion selectivity, and permeability (45Yool A.J. Schwarz T.L. Nature. 1991; 349: 700-704Crossref PubMed Scopus (320) Google Scholar, 46Kubo Y. Receptors Channels. 1996; 4: 73-83PubMed Google Scholar, 47D'Adamo M.C. Liu Z. Adelman J.P. Maylie J. Pessia M. EMBO J. 1998; 17: 1200-1207Crossref PubMed Scopus (68) Google Scholar). The V608D missense mutation in Shab 2 is in the pore region of the channel. This region is subdivided into the turret, pore helix, and selectivity filter (40Doyle D.A. Cabral J.M. Pfuetzner R.A. Kuo A. Gulbis J.M. Cohen S.L. Chait B.T. MacKinnon R. Science. 1998; 280: 69-77Crossref PubMed Scopus (5769) Google Scholar). The turret lies at the extracellular entryway of the pore and forms a high affinity binding site for various scorpion venom toxins such as charybdotoxin (41MacKinnon R. Heginbotham L. Abramson T. Neuron. 1990; 5: 767-771Abstract Full Text PDF PubMed Scopus (190) Google Scholar) and agitoxin (48Gross A. MacKinnon R. Neuron. 1996; 16: 399-406Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The amino acid composition in the turret is not highly conserved, but the overall structure of this region appears to be conserved (49MacKinnon R. Cohen S.L. Kuo A. Lee A. Chait B.T. Science. 1998; 280: 106-109Crossref PubMed Scopus (371) Google Scholar). Using agitoxin footprinting on mutant Shaker channels, Gross and MacKinnon (48Gross A. MacKinnon R. Neuron. 1996; 16: 399-406Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar) determined the spatial location of amino acids in the pore entryway. According to their studies, Lys427 in Shaker channels, which corresponds to Val608 inShab channels, lies away from the center of the pore but makes direct contact with agitoxin. Mutating Lys427 to a negatively charged glutamic acid does not alter single channel conductance in Shaker channels when expressed in Xenopusoocytes (41MacKinnon R. Heginbotham L. Abramson T. Neuron. 1990; 5: 767-771Abstract Full Text PDF PubMed Scopus (190) Google Scholar). However, in Shab 2 mutants,IK is reduced in amplitude by 43.6 ± 3.4% (a 71.2 ± 7.0% reduction in the Shab-encodedIK). Further functional studies will have to be performed to understand the mechanism for the reduction ofIK in Shab 2. In vivo mutations have been used extensively to characterize genes and their protein products. Due to a large repertoire of genetic mutations and the ease of performing electrophysiological analysis, Drosophila has provided a very useful system for ion channel study. Two voltage-activated K+ currents (IA and IK) and two Ca2+-activated K+ currents (ICF and ICS) have been reported in the larval muscles ofDrosophila (27Wu C.F. Haugland F.N. J. Neurosci. 1985; 5: 2626-2640Crossref PubMed Google Scholar, 50Gho M. Mallart A. Pflugers Arch. 1986; 407: 526-533Crossref PubMed Scopus (43) Google Scholar, 51Singh S. Wu C.F. Neuron. 1989; 2: 1325-1329Abstract Full Text PDF PubMed Scopus (85) Google Scholar). Mutations that affectIA (Shaker) and ICF (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect the delayed rectifier channels had made their genetic and functional identity difficult to elucidate. Availability of the Shab mutations that affectIK opens up many possibilities for studying this current and its role in larval muscle excitability. We thank Scott Chouinard and Barry Ganetzky for the generous gift of the Shab cDNA clone. We thank Maninder Chopra for identifying the Shab 1 and Shab 2 mutants and performing the initial characterization.