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

K+ currents inDrosophila muscles have been resolved into two voltage-activated currents (I A andI K) and two Ca2+-activated currents (I CF and I CS). Mutations that affect IA (Shaker) andI CF (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 (I K) 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 (I K). 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 I K opens up possibilities for studyingI K and its role in larval muscle excitability.

K ؉ currents in Drosophila muscles have been resolved into two voltage-activated currents (I A and I K ) and two Ca 2؉ -activated currents (I CF and I CS ). Mutations that affect I A (Shaker) and I CF (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 (I K ) 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 (I K ). Two of the mutant alleles contain missense mutations, one in the aminoterminal 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 vivo physiological role for the Shab-encoded K ؉ channels in Drosophila. The availability of mutations that affect I K opens up possibilities for studying I K and its role in larval muscle excitability.
Voltage-activated K ϩ channels play a crucial role in repolarizing the membrane following action potentials, stabilizing membrane potentials and shaping firing patterns of cells (1). Many human diseases such as long QT syndrome, Jervell and Lange-Nielson syndrome, episodic ataxia, and epilepsy are associated with mutations in these channels (2)(3)(4)(5)(6). Hence, it is important to understand how K ϩ 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 ϩ channel consists of four ␣-subunits, each with six transmembrane domains (S1-S6) flanked by cytoplasmic amino-and carboxyl-terminal regions. A num-ber of genes coding for K ϩ channel ␣-subunits have been cloned. These include genes from six families, defined by six Drosophila K ϩ channel genes: , ether-a-go-go (HERG) and slowpoke (maxiK) (7)(8)(9)(10). The Shaker, ether-ago-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 (11)(12)(13)(14)(15)(16). On the other hand, Shab, Shal, and Shaw were identified in homology screens using Shaker cDNA as probe (17). Expression of cRNAs of these genes results in the generation of K ϩ currents in Xenopus oocytes (18,19). However, no mutations have been reported in any of these three genes, thus making it difficult to elucidate their in vivo physiological 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 temperatureinduced paralytic phenotype (20). 1 They selectively affect the delayed rectifier potassium current (I K ), in larval body wall muscles, without affecting other K ϩ currents and reveal the in vivo functional role of the Shab gene in Drosophila.

EXPERIMENTAL PROCEDURES
Isolation of 9g Mutant-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. 1 30 male flies (3 days old) carrying the ebony (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. F 1 progeny from the cross were tested for temperature-induced paralysis at 39°C for 5 min. Of the 5673 F 1 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. F 2 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 (21).
Electrophysiology-Body wall muscle 12 (22, 23) 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 (24). Ca 2ϩ -free recording solution contained 77.5 mM NaCl, 115 mM sucrose, 5 mM KCl, 0.5 mM EGTA, 20 mM MgCl 2 , 5 mM Trehalose, 2.5 mM NaHCO 3 , and 5 mM HEPES (pH 7.1) (25,26). 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 2.5 M KCl, and current electrodes contained a 3:1 mixture of 2.5 M KCl, 2 M potassium citrate (27). 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 pro-* This work was supported by National Science Foundation Grants IBN-9011427 and MCB-9604457 and National Institutes of Health Grant GM-50779. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM  gram written in Think-C (Symantec Corp., Cupertino, CA). Test currents were digitally sampled every 500 s and digitally corrected for linear leakage with respect to currents obtained at Ϫ40 mV. Current densities (nanoamperes/nanofarads) were calculated as described previously (28,29). All experiments were carried out at 4°C.
Southern Blot Analysis-Genomic DNA was extracted from CS and 9g flies (30). The genomic DNA was digested with PstI 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.

Identification of Mutations
Affecting I K -Several ethylmethanesulfonate-induced mutations had been previously isolated in our laboratory by using the conditional phenotype of temperature-induced paralysis (20). These mutations were identified with the use of compound chromosomes (31)(32)(33)(34). 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, I K , 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 the z66 mutation on various other currents expressed in larval muscles has been examined. No significant change was seen in the fast transient voltage-activated K ϩ current, I A ; the fast transient calcium-activated K ϩ current, I CF ; the slow sustained calciumactivated K ϩ current, I CS ; and the total Ca 2ϩ current, thus suggesting that the mutation selectively affects I K (20). 1 As shown in Table I, I K was reduced by 46.0 Ϯ 3.0% in z66 mutants. The z4 mutants showed a 43.6 Ϯ 3.4% reduction in I K .
To obtain additional alleles of the gene affected by the z66 and z4 mutations, including some with chromosomal aberrations that would aid in identifying the gene affected by the mutations, we performed x-ray mutagenesis (35). Male ebony flies were subjected to x-ray mutagenesis and mated with virgin z66 females (see "Experimental Procedures"). Screening of 5673 F 1 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 muta-tions did not complement each other and hence are alleles of the same gene. As in z66, I K was affected in 9g mutants (Fig.  1a). There was a 61.2 Ϯ 2.7% reduction of I K in 9g homozygotes (see Table I).
The 9g Mutation Maps to the Shab Locus-Recombination and deletion mapping localized the z66 and z4 mutations to the left arm of chromosome 3 at 63A1-B6 (20). 1 Deletion analysis of 9g showed that the mutation mapped to the same position. Shab, a K ϩ channel gene cloned by homology to Shaker lies on chromosome 3 at position 63A (17). By using deletions in this region (63A1-B9), Tsunoda and Salkoff (36) showed that I K 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 Shab gene, Southern blot analysis was performed on genomic DNA from wild type (CS) and 9g flies using the entire Shab cDNA 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 the PstI-digested CS and 9g DNA revealed a restriction fragment length polymorphism between mutant and wild type DNAs, indicating that the 9g mutation disrupts  the Shab 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. Molecular Characterization of Shab Mutations-To investigate the underlying molecular defects in the mutants, Shab cDNA was prepared by reverse transcription-PCR from Shab 1 , Shab 2 , Shab 3 , and wild type (CS) flies (Fig. 3a). PCR products were sequenced, and results were compared with the published sequence of Shab derived from the OR strain (17). 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 the Shab (OR) gene; GenBank TM 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. (17) reported the presence of a 90-nucleotide coding region (nucleotides 2151-2240) in the longest correctly spliced Shab cDNA (Shab11) (GenBank TM accession 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 GenBank TM .
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 NH 2 -terminal region and the S1 segment are essential for subunit interactions as well as expression of functional channels at the cell membrane (37)(38)(39). The position of the Shab 1 mutation is interesting and may suggest an important role for this region in the formation of a functional Shab channel.
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 Shab K ϩ channel (Fig.  4). The pore region consists of a turret, pore helix, and selectivity filter (40). 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 (41). 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 1 and Shab 2 mutations and the original CS strain.
Sequencing of reverse transcription-PCR products from Shab 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. The Shab 3encoded 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 Ϫ55 to 1200 nucleotides showed no difference between wild-type genomic and cDNA sequences, indicating that there were no introns within this region. Thus, the Shab 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. DISCUSSION The Shab, Shal, and Shaw subfamilies of voltage-gated K ϩ channels were identified on the basis of homology to Shaker (17). 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 the Shab channels and have analyzed them at a molecular level. Our data show that the Shab gene codes for delayed rectifier K ϩ channels in larval body wall muscles.
Shab Mutations Affect the Delayed Rectifier Current, I K -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, I K , is only reduced by 61.2 Ϯ 2.7% and not completely abolished in Shab 3 ( Fig. 1; Table I). If Shab channels carried all of the I K , then a null mutation in the Shab gene would be expected to lack delayed rectifier current. Thus, our results indicate that I K consists of more than one component, with a large fraction of the current being carried by Shab channels. Detailed pharmacological and physiological studies performed on Shab 3 mutants support this hypothesis. 3 Electrophysiological studies performed using genetic aberrations that delete a large region of chromosome containing the Shab locus in Drosophila show a 77% reduction of I K in embryonic myotubes (36). The gene that codes for the remaining current seen in the Shab 3 mutants remains to be identified. One possible candidate is the Shaw gene that codes for a noninactivating K ϩ channel (18). 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 Ϯ 3.0% reduction in I K (Fig. 1). Since Shab channels carry 61.2% of the delayed rectifier current, Shab 1 mutants show a 75.2 Ϯ 6.6% reduction in the Shab component of I K (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 (42)(43)(44). 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 (42,43). Voltage-gated K ϩ channels show similarly conserved arginines and lysines just before S1 (Fig. 5). It will be interesting to examine if the R435Q mutation in Shab 1 results in a topological inversion.
Mutations in the pore region affect various properties such as gating, channel activation, ion selectivity, and permeability (45)(46)(47). 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 (40). 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 (41) and agitoxin (48). The amino acid composition in the turret is not highly conserved, but the overall structure of this region appears to be conserved (49). Using agitoxin foot- 3 Singh, A., and Singh, S. (1999) J. Neurosci., in press. FIG. 4. A schematic representation of the Shab potassium channel. The Shab 3 deletions are in the cytoplasmic amino terminus and result in a null allele. The R435Q missense mutation in Shab 1 occurs at the last amino acid before the protein enters into the membrane as S1. The V608D mutation in Shab 2 is in the entryway to the pore.
printing on mutant Shaker channels, Gross and MacKinnon (48) determined the spatial location of amino acids in the pore entryway. According to their studies, Lys 427 in Shaker channels, which corresponds to Val 608 in Shab channels, lies away from the center of the pore but makes direct contact with agitoxin. Mutating Lys 427 to a negatively charged glutamic acid does not alter single channel conductance in Shaker channels when expressed in Xenopus oocytes (41). However, in Shab 2 mutants, I K is reduced in amplitude by 43.6 Ϯ 3.4% (a 71.2 Ϯ 7.0% reduction in the Shab-encoded I K ). Further functional studies will have to be performed to understand the mechanism for the reduction of I K in Shab 2 .
Shab Mutations Will Facilitate Further Analysis of I K -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 ϩ currents (I A and I K ) and two Ca 2ϩ -activated K ϩ currents (I CF and I CS ) have been reported in the larval muscles of Drosophila (27,50,51). Mutations that affect I A (Shaker) and I CF (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 affect I K opens up many possibilities for studying this current and its role in larval muscle excitability.