Neuron, Vol. 5, 67-73,July,1990,Copyright© 1990by Cell Press
Alteration of Potassium Channel Gating: Molecular Analysis of the Drosophila Sh5 Mutation Medha Gautam and Mark A. Tanouye Division of Biology California Institute of Technology Pasadena, California 91125
Summary The Drosophila Shaker (Sh) 8ene encodes a family of voltage~ated K÷ channels. Mutant alleles of Sh alter the currents expressed from these channels in a variety of ways. To identify the molecular basis of these alterations, $h transcript sequences were amplified using the polymerase chain reaction after reverse transcription of mutant RNA. Amplified products from each mutant were cloned and sequenced. Two alleles, Sh I°2 and Shs, had single base substitutions in the central conserved region shared by all Sh channels. RNA synthesized in vitro from a cDNA construct carrying the S/~ mutation was injected into Xenopus oocytes. Currents expressed by the mutant RNA were altered in their voltage dependence of activation and inactivation, similar to the alterations in $h currents recorded from different preparations of Shs fly tissue. The changes in current properties and the location of the mutation are consistent with the participation of a novel region of the channel in voltage gating. Introduction
Several DrosophilaShaker(Sh) mutants have been isolated on the basis of abnormal leg-shaking behavior under ether anesthesia (Kaplan and Trout, 1969; Trout and Kaplan, 1973). $h mutants show several other defects, such as prolonged action potentials, altered muscle A-type K+ currents, and abnormal synaptic transmission; these defects appear to be due to alterations in voltage-gated K+ channels (Tanouye et al., 1981; Jan et al., 1977; Salkoff and Wyman, 1981; Wu et al., 1983; Wu and Haugland, 1985). Voltage-clamp experiments on flies carrying different allelic combinations of Sh have indicated the nature of these alterations. Functional Sh channels are absent in ShM flies and Sh Ka2aflies; these mutations are genetic nulls or amorphs (Wu and Haugland, 1985; Timpe and Jan, 1987). ShKsT~3and Sh 7°2 mutations behave genetically as antimorphs, apparently producing nonfunctional channel subunits that interfere with the function of normal subunits (Salkoff, 1983; Timpe and Jan, 1987; Haugland and Wu, 1990). Thus, these two alleles apparently destroy the ability to translocate ions across the membrane without eliminating the ability of subunits to interact. In larval and pupal muscle, ShE62 and ShrK°12° behave as leaky alleles or hypomorphs (Wu and Haugland, 1985; 13rope and Jan, 1987). Finally, the Shs mutation results in the production of Sh currents that are normal in amplitude, but are altered in properties such as voltage sensitivity of acti-
vation (Salkoff, 1983; Wu and Haugland, 1985). Genetically, Shs behaves as a gain-of-function mutation or neomorph. The isolation and characterization of the Sh gene demonstrated that it encodes a family of voltage-gated K+ channels (Kamb et al., 1987; Papazian et al., 1987; Baumann et al., 1987). Sh is a large gene with a complicated genomic organization and a variety of different transcripts generated by alternative splicing (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). All Sh K+ channels have six potential membrane-spanning segments, designated $1-$6 (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). Sh cDNAs that express K+ channels have a central conserved region that is identical in every transcript, spans most of the coding region, and includes the segments $1-$5 (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). The conserved region is flanked by variable 5' and 3' ends (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). The diverse phenotypic effects of Sh mutations are behaviorally, electrophysiologically, and genetically well characterized (Tanouye et al., 1986). Localization of mutations within the structure oLthe K+ channel will provide molecular explanations for the variety of allele types defined genetically (neomorph, antimorph, etc.). In addition, their molecular localization can be used to dissect apart various functional domains of the channel and provide a basis for subsequent sitedirected mutagenesis studies. To identify precisely the changes that cause the alterations in channel function, we analyzed Sh transcripts from some of these mutants. Transcript sequences were amplified using the polymerase chain reaction (PCR) after reverse transcription of mutant RNA. The combined use of RNAwith PCR had the advantage of high sensitivity and overcame problems associated with the low abundance of Sh transcripts and the presence of large introns within the gene (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). Results
The central constant region contains most of the coding sequence of Sh cDNAs and thus was the initial focus of our search for structural defects. The central region is depicted in a schematic representation of the $h cDNA H4 (Figure 1). Also shown are oligonucleotides used to prime PCRs and to identify amplified products. The constant region of transcripts from wild-type and mutant flies was amplified by PCR. Template was single-stranded cDNA generated by reverse transcription of RNA from the different stocks. The oligonucleotide pair PI/P2 was used to prime amplification of the5' portion of the constant region; P3/P4 primed amplification of the 3' portion. Amplification of $h M
Neuron
68
Sh C D N A H4 conserved
central
Sl
.qTART
P1 P5 I
$2
region
$3
$4
S6
$5
~rPNp
P2 587 b p
I I
595
bp
P3
P6 P4
587
-- 595 bp
12345678
12345678
A
B
Figure1. Positionsof Oligonucleotides Used for PCR and Identification of Amplified Products from Sh Transcripts Depicted is a schematic representationof the Sh cDNA H4 (Kambet al., 1988). Potentialmembrane-spanningsegments$1-$6 are indicated. Notethat we use the $1-$6terminologyfor membrane-spanningsegments(pongset al., 1988) ratherthan the HD1-HD6terminology used previouslyfor K+ channels (Kamb et al., 1988; Schwarzet al., 1988).Verticalarrows demarcatethe extent of the central conserved region. The horizontal arrows representthe relative positions of oli8onucleotides P1-P6and their 5' to 3' orientation. P1, P5, and P3are sense;P2, P6, and P4are antisense.Amplified products were of the expected sizes(587 bp for the 5' portion of the constant region; 595 bp for the 3' portion); however,transcripts from ShM and Sh"K°12° failed to give products in the amplification of the 5' portion. (A) Products from the amplification using P1 and 1>2primers probed with 32p-labeledoligonucleotide I)5.A 587 bp band was detected for all samplesexceptthose from Sh M, SWK°12°, and Df(1)B55d/W32P. (B) Products from the amplification using P3 and P4 primers probed with 32p-labeledoligonucleotide P6.A 595 bp band was detected for all samplesexceptthose from Df(1)B55d/W32P.Since RNA from Df(1)B55dNV32Pfailed to give any amplified products, Sh sequences, but not those from other homologous genes, were amplified. The RNA for PCR in (/%)and (B) was from Sh ~°z (lane 1), ShKs~ (lane 2), ShE62(lane 3), Sh M (lane 4), Sh rK0120(lane 5), Oregon-R (lane 6), Shs (lane 7), and Df(1)BS5dNV32P(lane 8).
and Sh rK0120 transcripts generated products with the oligonucleotide pair P3/P4, but not with P1/P2 (Figure 1A, lanes 4 and 5). All other amplifications gave prOducts of the expected size; no extra bands were detected for any of the mutants. The amplified products were isolated, cloned, and sequenced. Inserts containing Sh E62, Sh Ks133, Sh 102, and Sh s sequences between I'1 and P2 were identical to wild type. Among the products amplified with P3 and P4, Sh E62, Sh Ks133, Sh u, and Sh rK°~2° sequences were identical to wild type; two of the mutants, Sh 1°2 and Sh 5, had single base changes. In Sh 7°2 there was a single base change from G to A (Figure 2A). This results in the substitution of a stop codon (TAG) for a tryptophan codon (TGG) at amino acid 434 in the deduced amino acid sequence of cDNA H4 (Kamb et al., 1988). Thus, Sh ~°2 apparently forms a product that is truncated between segments $5 and $6. Since this region is common to all Sh transcripts, every K+ channel expressed from Sh should be altered bythis mutation. Our identification of Sh I°2 is an independent confirmation of another report describing its location (Gisselmann et al., 1989). In Sh s there was a single
base change from Tto A (Figure 2B). This results in the substitution of an isoleucine codon (ATT) for a phenylalanine codon (TI-F) at amino acid 401 in the deduced amino acid sequence of cDNA H4 (Kamb et al., 1988). Thus, Sh 5 apparently forms a product with a single amino acid substitution at the beginning of the $5 segment. Interestingly, this is part of the leucine zipper region, occurring 2 amino acid residues upstream of the fifth leucine in the repeat (Leu4°3; McCormack et al., 1989). Since this region is common to all Sh transcripts, every K+ channel expressed from Sh should be affected by this mutation. A mutant cDNA (29.4,Sh5) containing the altered Sh s sequence was constructed for electrophysiological analysis. The normal host cDNA (29-4) was a chimera composed of a 5' end from cDNA H29 and a 3' end from cDNA H4 (Iverson and Rudy, 1990). 29-4 and 29.4,Sh5 cDNAs were linearized and used as template to prepare RNA in vitro. Transient outward currents resembling A-currents were observed in Xenopus oocytes after injection of normal and mutant RNA (Figure 3). The normal 29.4 currents had properties similar to earlier reports and were completely blocked by
Structural Defects in Shaker Mutants 69
OR
AC GT
Sh 5
OR
S h 7° 2
ACG
ACG T
T
AC G T
A
TGG -->TAG trp stop
~-_TTT--~ ATT phe ile
$5 wild-type: S h I02
-ala-phe-trp-trp-ala-
: -ala-phe-stop
wild-type
: -gly-leu-leu-ile-phe-phe-leu--
Sh 5 : - g l y - l e u - l e u - i l e - i l e - p h e - l e u -
Figure 2. Sequence Analysis of Sh ;°2 and Sh s (A) The entire constant region for Sh I°2 was sequenced, and a single nucleotide substitution was detected. The sequence depicted corresponds to nucleotides 1282-1320 in the cDNA H4 (Kamb et al., 1988; Schwarz et al., 1988). The arrow designates the location of the single G to A base change in the Sh ~°2 sequence. In Sh I°2, this results in the substitution of a stop codon for a tryptophan codon at amino acid 434 (deduced cDNA H4 sequence; Kamb et al., 1988). (B) The entire constant region for Sh s was sequenced, and a single nucleotide substitution was detected. The sequence depicted corresponds to nucleotides 1176-1233. The arrow designates the location of the single T to A base change in the Sh s sequence. In Sh s, this results in a single amino acid change from phenylalanine to isoleucine at amino acid 401. At this location, the K+ channel heptad leucine repeat overlaps the $5 segment (McCormack et al., 1989).
5 mM 4-aminopyridine (1-impe et al., 1988; Zagotta et al., 1989; Iverson and Rudy, 1990). The Sh 5 mutation alters voltage dependence of activation and voltage dependence of steady-state inactivation of the currents induced in Xenopus oocytes. Figure 4 shows the conductance-voltage relation and the voltage dependence of steady-state inactivation from pooled data for currents expressed from normal and mutant cDNA transcripts. The conductance-voltage curve for 294,Sh5 was shifted about 18 mV toward more positive potentials (Figure 4A, open circles). The voltage for half-maximal conductance was - 6 mV for 29-4 currents (n = 5) and +12 mV for 29-4,Sh5 currents (n = 6). The voltage dependence of steady-state inactivation for the macroscopic currents expressed from normal and mutant transcripts is shown in Figure 4B. The currents for 29-4,Sh5 were shifted about +5 mV in their voltage dependence of inactivation compared with the currents for 29-4. The voltage for half-maximal inactivation was -30 mV for 29-4 currents (n = 5) and -25 mV for 29-4,Sh5 currents (n = 5). With prepulses between -20 and -30 mV, the slope of the inactivation curve was less steep for 29-4,Sh5 currents than for 29-4 currents. These functional changes in the voltage dependence of activation and inactivation and in the slope of the inactivation curve are similar to those described previously for Sh s mutant muscle (Wu and Haugland, 1985). Thus, the single amino acid
change from phenylalanine to isoleucine is adequate to account for the Sh 5 phenotype. Discussion PCR amplification followed by nucleotide sequence analysis provides a powerful method for the molecular localization of mutations (Wong et al., 1987; Cross et al., 1988). They are a good alternative to genomic sequencing or construction of mutant cDNA libraries. PCR methods were particularly useful in localizing Sh 7°2 and Sh 5 mutations because of the large size of the gene and the low abundance and diversity of transcripts. These methods were not completely revealing in every case we examined. We found no alterations in nucleotide sequence in the constant regions of Sh E62and Sh Ks733.The alterations in these mutants are either elsewhere in the gene or within the primer sequences, since Taql polymerase is known to accommodate a mismatch in the primer region during PCR amplification (Lee et al., 1988). The absence of Sh M and 5h rK012° products using the oligonucleotide pair P1/P2 suggests that their defects may lie in the 5' portion of the constant region. For Sh u, this is consistent with the presence of a 2.2 kb insertion in the corresponding genomic region (Kamb et aL, 1987). A large insertion in the region could disable the amplification; alternatively, sequences corresponding to the
Neuron 70
A
START
sl $2 $3 $4 $5 phe
$6
A
SOP 1.8
START
ile
STOP
0.8
29-4,Sh5
0.6 0.4 0.2 0.0 -70
B
-so
v
-30
-10
10
30
so
70
V m (mV)
1.8
0.8 o
B
~
: -
~
0.6 0.,4 0.2
,400 n A
C
0.8
-6o
i
-so
-4o
.so
.zo
-1o
o
lO
PrelpUlsoVoltaoo (mV) Figure 4. Comparison of Current Properties for 29-4 and 29-4,Sh5 (A) Conductance-voltage curve for 29-4 (closed circles) and 29-
Figure 3. K+ Currents Expressed from 294 and 294,Sh5 RNA (A) Schematic representation of the 294 and 29-4,Sh5 cDNAs showing the approximate location of the Shs substitution within the $5 segment. RNA samples were transcribed in vitro from these cDNAs, denatured, and sized by electrophoresis on agarose-formaldehyde gels. (B and C) Ion currents recorded from oocytes injected with 29.4 and 294,Sh5 RNAs using a two-microelectrode voltage clamp. Membrane potentials were held at -90 mV, hyperpolarized to -120 mV for 2 s, and stepped to test potentials (90 ms duration) ranging from -80 to +70 mV in 10 mV increments. The time between trials was 5 s~
primers could be altered or deleted. Interestingly, whatever the defects are, they do not eliminate mutant transcripts completely, since amplifications using the oligonucleotide pair P3/P4 showed no sequence alteration in Shm and Sh rK0120for the 3' portion of the constant region. We are c o n t i n u i n g to determine the exact defects associated w i t h these four Sh mutant alleles. Sh 1°2 causes the substitution of a stop codon for a tryptophan codon in the coding region of Sh between the $5 and $6 segments. O u r results are in agreement w i t h an i n d e p e n d e n t identification of this mutation using genomic sequencing (Gisselmann et al., 1989). Introduction of a c D N A carrying Sh 7°2 into wild-type flies by germline transformation caused an antimor-
4,5h5 (open circles). Relative peak conductance was determined by dividing the conductance at the indicated potential (G) by the conductance at +80 mV, taken as GB=. Conductance was determined by the equation G = I(%/,. - Vk), assuming Vk = --80 mV. Means ± SD are plotted. For 29-4, n = 5; for 29-4,Sh5, n = 6. (B) Steady-state inactivation of currents for 29-4 (closed circles) and 29.4,Sh5(open circles). Membrane potential was held at -90 mV, prepulsed for I s to the indicated voltages, and stepped to a test pulse of +50 mV (80 ms duration). Peak current (I) was recorded. The ratio I/Io, was plotted as a function of the prepulse potential, where Io = peak current during the test pulse after a prepulse of -120 mV. The voltage of half-maximal inactivation was -30 mV for 294 and -25 mV for 29-4,Sh5.Means + SD are plotted. For 294, n = 5; for 29-4,Sh5, n = 5.
phic Sh defect. Thus, the antimorphic behavior of Sh 7°2 in genetic and electrophysiological analyses can now be explained by the apparent formation of truncated products that associate w i t h normal subunits in a channel complex and interfere w i t h their function (Gisselmann et al., 1989). Sh 5 has been one of the most interesting alleles of $h, behaving as a genetic neomorph. Behaviorally, $h 5 is a vigorous leg-shaker, but unlike other alleles, shaking tends to occur in bursts; Sh 5 also causes abnormal wing-scissoring (Tanouye and Ferrus, 1985). Sh 5 action potentials also differ from those of other Sh mutants: instead of having abnormally long durations (all other Sh mutants), Sh s action potentials display incomplete repolarization leading to multiple spikes. Thus, typical Sh s activity consists of a burst of four to five spikes following delivery of a single stimulus (Tanouye and Ferrus, 1985). In voltage-clamp experiments of larval muscle, embryonic myotubes, and pupal neurons, Shs fails to show an alteration in inac-
Structural Defects in Shaker Mutants 71
tivation kinetics, but changes the voltage dependence of activation by about +20 mV and inactivation by +5 to +10 mV (Wu and Haugland, 1985; Zagotta and Aldrich, 1990; Baker and Salkoff, 1990). In voltage-clamp experiments of pupal muscle, $ h 5 currents are altered in inactivation kinetics (Salkoff, 1983). We have identified a single nucleotide change in transcripts from Sh 5 flies. This changes the a m i n o acid sequence gly-leu-ile-phe-phe-leu to gly-leu-ileile-phe-leu at the beginning of the $5 segment in the central region (Figure 2B). This is a surprisingly subtle change of one neutral a m i n o acid residue for another. Nevertheless, this substitution is sufficient to account for at least some of the observed Sh 5 phenotypes. That is, transcripts from the 29-4,Sh5 construct, w h e n injected into Xenopus o0cytes , induced currents that were altered in voltage dependence for activation and inactivation, similar to the currents recorded from Sh s larval muscle, embryonic myotubes, and pupal neurons (Wu and Haugland, 1985; Zagotta and Aldrich, 1990; Baker and Salkoff, 1990). We suspect that this one change w o u l d also mimic other Sh s phenotypes if examined under the appropriate experimental conditions. Recently, two conserved sequence motifs in Na +, K+, and Ca2+ channels have been postulated to mediate voltage-dependent gating. The transmembrane $4 segment appears to be involved in voltage sensing (Stuhmer et al., 1989a; Papazian et al., 1989, Soc. Neurosci., abstract). A strongly conserved leucine heptad repeat or leucine zipper, which lies between the $4 and $5 segments and partially overlaps them, appears to be involved in the channel gate (McCormack et al., 1989; Auld et al., 1990). Our results on Sh s are consistent w i t h these ideas of channel gating: the mutation lies w i t h i n the region where the leucine repeat overlaps the $5 segment and alters channel voltage dependence. The percentage of a m i n o acid identities in the region containing $4, the leucine repeat, and $5 segments is remarkably high among $ h homologs of different species. In the " $ h class" channels (Stuhmer et al., 1989b; M a t h e w et al., 1989, Soc. Neurosci., abstract), o n l y 3 a m i n o acid positions appear to support any variation. This is in contrast to the larger variations in a m i n o acid sequence that are present in the regions immediately upstream of $4 and downstream of $5. An extrapolation from o u r results on Sh 5 is that many of these a m i n o acid identities are critical for channel gating, and their analysis may reveal underlying features of the gating mechanism. ExperimentalProcedures Fly Stocks and RNA Extraction The wild-type strain used was Oregon-R(OR).The Sh stocks have all been described previously: Shs, Sh rK°Tz°, Sh Ks733, and Sh 1°2 are ethyl methane sulfonate-induced alleles; Sh ~ is a spontaneous mutation associated with a 2.2 kb insertion (Tanouyeet al., 1981; Salkoff, 1983; Wu and Haugland, 1985; Kamb et al., 1987; ~mpe and Jan, 1987). Df(1)B55d/W32pis deleted for most of the coding region of Sh (Tanouyeet al., 1981;Kambet al., 1987; Schwarz et al., 1988). Fly RNA was prepared from 0.5 g of frozen
flies with guanidine isothiocyanate and subjected to ultracentrifugation (Maniatis et al., 1982). The RNA preparations were adequate for PCR without selection of poly(A)+ RNA. Oligonucleotides for PCR Two pairs of oligonucleotides were used as primers to amplify the entire constant region of Sh products (Figure 1). Amplifications were between P1 (183)(5:GTCTI-I'GCCCAAAI-I'GAGCAG-3"; sense) and P2 (769) (5'-CCTTGTAATGCTTAAATIC-3';antisense); and between P3(751)(5"GAATTI'AAGCATTACAAGG-3';sense)and P4 (1346) (5'-GTCATGTCACCATATCCAACG-3";antisense). PCR-amplified sequences were identified using two oligonucleotides as hybridization probes: P5 (268) (5:CCTCACGATCATGATI-rCTG-3';sense)and P6 (895)(5'-GGAACCTGACAGTIAGTT-3'; antisense). The position of the first nucleotide for each of the above sequences in the H4 cDNA (or ShB1)is designated by the number in parentheses(Kambet al., 1988; Schwarzet al., 1988). PCR Amplification and Identification of Amplified Products Reversetranscription was performed in a mix (20 Is.I)containing 5 I~g of total RNA, 100 ng of P2 or P4 oligonucleotides, 0.5 mM each dATP, dCTP, dGTP, and d-I-IP, 20 U of RNAase inhibitor (Boehringer Mannheim), 50 mM Tris-HCI (pH &3), 75 mM KCI, 10 mM dithiothreitol, 3 mM MgCI2, and 400 U of MMLV reverse transcriptase (Bethesda Research Laboratories). Reverse transcription reactions were incubated at 37°Cfor 3 hr. After incubation, the reversetranscription mix was used directly for PCR in a solution (50 ILl)containing 300 ng of I>1or P3 oligonucleotides, 200 ng of P2 or P4 oligonucleotides, 25 mM Tris-HCI (pH &3), 50 mM KCI, 2 mM MgCI2,0.006%gelatin, and 4 U of Taql polymerase (Cetus, Promega).PCR amplifications were for 40 cycles in a Thermal Cycler (Perkin-Elmer-Cetus) using annealing, extension, and denaturation conditions of 45°C (2 rain), 72°C (4 rain), and 94°C (1 rain), respectively.At the end of the run, reactions were incubated at 45°Cfor 2 rain and then at 72°C for 7 rain. Ampliflcation products were evident after electrophoresis through 1.5% agarose gels. Southern blots of amplified products were probed with 32p-labeledP5 or P6 oligonucleotides. The probes were prepared using a protocol provided in the TaqTrackDNA sequencing kit (Promega);hybridization conditions were as previously described (Meinkoth and Wahl, 1984). PCR-amplified sequences (100 ng) were prepared for cloning by incubation with 2 U of Klenow polymerase(BoehringerMannhelm) and 1 mM each dATE dCTP, dGTP, and dl-IP for 1 hr at 22°C. T4 DNA polymerase (1 U; Boehringer Mannheim) was added for the final 5 rain of incubation. Inserts were gel-purified, precipitated in ethanol, and resuspended in water (10 I~1). The inserts were then ligated into Sinai-cleavedBluescript vector (Stratagene), keeping an insert to vector molar ratio of 10:1. Sequencing of cloned inserts was done using Sequenase(US Biochemicals) and KS and SK primers (Stratagene).At least six isolares of amplified products from the 5' and 3' portions of the constant region were sequenced entirely for each mutant. A cDNA carrying the Shs mutation in Bluescript vector was constructed for electrophysiological analysis.The host cDNA for the mutation was a chimera composed of a 5' end from 1-129and a 3' end from H4 called 29-4 (Iverson and Rudy, 1990).29-4 is the best-expressing cDNA in our collection and is identical to the deduced amino acid sequence for Shl3and ShD1 (Pongset al., 1988; Schwarz et al., 1988). The resulting mutant cDNA was called 29-4,Sh5.The construction took advantageof the fact that the Shs mutation is flanked by two unique restriction enzyme sites, Bsml and Nsil (at nucleotides 1094and 1256, respectively, in 29-4). A 29-4 cDNA and a plasmid carrying the Shs insert were digested with Bsml and Nsil. The appropriate fragments were then gel-purified and ligated to generate 29-4,Sh5cDNA. Isolates of 29-4,Sh5cDNA were identified by restriction digests. Sequence analysis confirmed that there were no additional changes in this region of the construct. Preparation of RNA for Electrophysiology Full-length, capped transcripts were generated using 1"7 RNA polymerase (Promega)by in vitro transcription of linearized plas-
Neuron 72
mids (Krie8 and Melton, 1984; Iverson et al., 1988). Xenopus oocytes (stage V or stage VI) were microinjected with RNA (1-5 ng) and incubated for 2-3 days at 18°C in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCI2, 1 mM MgCI2, 5 mM HEPES [pH 7.5]) supplemented with 100 U/ml penicillin, 100 p.8/ml streptomycin, and 2.5 mM sodium pyruvate (Krafte et al., 1988). Macroscopic currents were recorded with a standard two-microelectrode voltage clamp (Krafte et al., 1988). Bath solution was ND96; electrodes were filled with 3 M KCl and had a resistance of 0.7-1.5 MQ. All experiments were carried out at room temperature (20°C-22°C). Current signals were low-pass-filtered at 3 kHz. Voltage and current signals were digitized and analyzed using the pCLAMP package (Axon Instruments, Burlingame, CA) as previously described (Iverson et al., 1988). No significant K+ currents were observed in uninjected oocytes under these conditions. Leakage current was estimated from the average response to 10 mV steps from -90 mV in a voltage range in which time-dependent currents were not activated. Acknowledsments We thank K. McCormack, M. K. Mathew, and M. Ramaswami for valuable discussions and comments on the manuscript, R. McMahon for excellent technical assistance, and Dr. H. A. Lester for a generous gift of oocytes. This study was supported by USPHS grant NS 21327-06 to M. A. T. The Caltech Xenopus oocyte facility was supported by National Institutes of Health grant GM29836 to Dr. H. A. Lester. Received March 16, 1990; revised April 25, 1990.
References Auld, V.J., Goldin, A. L., Krafte, D.S., Catterall, W.A., Lester, H. A., Davidson, N., and Dunn, R. J. (1990). A neutral amino acid change in segment 1154dramatically alters the gating properties of the voltage-dependent sodium channel. Proc. Natl. Acad. Sci. USA 87, 323-327. Baker, K., and Salkoff, L. (1990). The Drosophila Shaker gene codes for a distinctive K+ current in a subset of neurons. Neuron 4, 129-140. Baumann, A., Krah-Jentgens, I., Mueller, R., Mueller-Holtkamp, F., Seidel, R., Kecskemethy, N., Casal, J., Ferrus, A., and Pongs, O. (1987). Molecular organization of the maternal effect region of the Shaker complex locus of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na + channel. EMBO J. 6, 3419-3429. Cross, N. C. P., Tolan, D. R., and Cox, T. M. (1988). Catalytic deftciency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation. Cell 53, 881-885. Gisselmann, G., Sewing, S., Madsen, B. W., Mallart, A., AngautPetit, D., Muller-Holtkamp, E, Ferrus, A., and Pongs, O. (1989). The interference of truncated with normal potassium channel subunits leads to abnormal behavior in transgenic Drosophila melanogaster. EMBO J. 8, 2359-2364. Haugland, F. N., and Wu, C.-E (1990). A voltage-clamp analysis of gene-dosage effects of the Shaker locus on larval muscle potassium currents in Drosophila. J. Neurosci. 10, 1357-~1371. Iverson, L.E., and Rudy, B. (1990). Both transient and nontransient potassium currents are expressed from RNAs derived from the Shaker gene of Drosophila. J. Neurosci., in press. Iverson, L. E., Tanouye, M. A., Lester, H.A., Davidson, N., and Rudy, B. (1988). Expression of A-type K+ channels from Shaker cDNAs. Proc. Natl. Acad. Sci. USA 85, 5723-5727. Jan, Y. N., Jan, L. Y., and Dennis, M. J. (1977). Two mutations of synaptic transmission in Drosophila. Proc. Roy. Soc. (Lond.) B 198, 87-108. Kamb, A., Iverson, L. E., and Tanouye, M. A. (1987). Molecular characterization of Shaker, a Drosophila gene that encodes a potassium channel. Cell 50, 405-413. Kamb, A., Tseng-Crank, J., and Tanouye, M. A. (1988). Multiple
products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron 1, 421-430. Kaplan, W. D., and Trout, W. E., III. (1969). The behavior of four neurological mutants of Drosophila. Genetics 61, 399-409. Kraftet D. S., Snutch, T., Leonard, J., Davidson, N., and Lester, H. A. (1988). Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes. J. Neurosci. 8, 2859-2868. Krieg, R, and Melton, D. A. (1984). Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucl. Acids Res. 12, 7057-7070. Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. G., Muzny, D. M., and Caskey, C.T. (1988). Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science 239, 1288-1291. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). McCormack, K., Campanelli, J.T., Ramaswami, M., Mathew, M. K., Tanouye, M. A., Iverson, L. E., and Rudy, B. (1989). Leucinezipper motif update. Nature 340, 103. Meinkoth, J., and Wahl, G. (1984). Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267-284. Papazian, D. M., Schwarz, T. L., Tempel, B. L., Jan, Y. N., and Jan, L. Y. (1987). Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science 237, 749-753. Pongs, 0., Kecskemethy, N., Muller, R., Krah-Jentgens, I., Baumann, A., Kiltz, H. H., Canal, I., Llamazares, S., and Ferrus, A. (1988). Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila. EMBO J. 7, 10871096. Salkoff, L. (1983). Genetic and voltage clamp analysis of a Drosophila potassium channel. Cold Spring Harbor Symp. Quant. Biol. 48, 221-231. Salkoff, L., and Wyman, R.J. (1981). Genetic modification of potassium channels in Drosophila Shaker mutants. Nature 293, 228-230. Schwarz, T. L., Tempel, B. L., Papazian, D. M., Jan, Y. N., and Jan, L. Y. (1988). Multiple potassium channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature 331, 137-142. Stuhmer, W., Conti, F., Suzuki, H., Wang, X., Noda, M., Yahagi, N., Kubo, H., and Numa, S. (1989a). Structural parts involved in activation and inactivation of the sodium channel. Nature 339, 597-603. Stuhmer, W., Ruppersberg, J. P., Schroter, K. H., Sakmann, B., Stocker, M., Giese, K. R, Perschke, A., Baumann, A., and Pongs, O. (1989b). Molecular basis of functional diversity of voltagegated potassium channels in mammalian brain. EMBO J. 8, 3235-3244. Tanouye, M. A., and Ferrus, A. (1985). Action potentials in normal and Shaker mutant Drosophila. J. Neurogenet. 2, 253-271. Tanouye, M. A., Ferrus, A., and Fujita, S. C. (1981). Abnormal action potentials associated with the Shaker complex locus of Drosophila. Prec. Natl. Acad. Sci. USA 78, 6548-6552. Tanouye, M. A., Kamb, C. A., Iverson, L. E., and Salkoff, L. (1986). Genetics and molecular biology of ionic channels in Drosophila. Annu. Rev. Neurosci. 9, 255-276. Timpe, L. C., and Jan, L. Y. (1987). Gene dosage and complementation analysis of the Shaker locus in Drosophila. J. Neurosci. 7, 1307-1317. Timpe, L. C., Jan, Y. N., and Jan, L. Y. (1988). Four cDNA clones from the Shaker locus of Drosophila induce kinetically distinct A-type potassium currents in Xenopus oocytes. Neuron 1, 659667. Trout, W. E., III, and Kaplan, W. D. (1973). Genetic manipulation
Structural 73
Defects
of motor output 4, 495-512.
in Shaker
in Shaker
Mutants
mutants
of Drosophila.
Wong, C., Dowling, C. E., Saiki, R. K., Higuchi, H. A., and Kazazian, H. H. (1987). Characterization saemia mutations using direct genomic sequencing single copy DNA. Nature 330, 384-386
J. Neurobiol. R. C., Erlich, of B-thalasof amplified
Wu, C.-F., and Haugland, F. N. (1985). Voltage-clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants. 1. Neurosci. 5, 2626-2640. Wu, C.-F., Ganetzky, B., Haugland, F., and Liu, A.-X. (1983). Potassium currents in Drosophila: different components affected by mutations of two genes. Science 220, 1076-1078. Zagotta, W. N., and Aldrich, R. W. (1998). Alterations gating of single Shaker A-type potassium channels mutation. 1. Neurosci., in press.
in activation by the Sh5
Zagotta, W. N., Hoshi, 1, and Aldrich, R. W. (1989). Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA. Proc. Natl. Acad. Sci. USA 86, 7243-7247.
Alteration of Potassium Channel Gating: Molecular Analysis of the Drosophila Sh5 Mutation Medha Gautam and Mark A. Tanouye Division of Biology California Institute of Technology Pasadena, California 91125
Summary The Drosophila Shaker (Sh) 8ene encodes a family of voltage~ated K÷ channels. Mutant alleles of Sh alter the currents expressed from these channels in a variety of ways. To identify the molecular basis of these alterations, $h transcript sequences were amplified using the polymerase chain reaction after reverse transcription of mutant RNA. Amplified products from each mutant were cloned and sequenced. Two alleles, Sh I°2 and Shs, had single base substitutions in the central conserved region shared by all Sh channels. RNA synthesized in vitro from a cDNA construct carrying the S/~ mutation was injected into Xenopus oocytes. Currents expressed by the mutant RNA were altered in their voltage dependence of activation and inactivation, similar to the alterations in $h currents recorded from different preparations of Shs fly tissue. The changes in current properties and the location of the mutation are consistent with the participation of a novel region of the channel in voltage gating. Introduction
Several DrosophilaShaker(Sh) mutants have been isolated on the basis of abnormal leg-shaking behavior under ether anesthesia (Kaplan and Trout, 1969; Trout and Kaplan, 1973). $h mutants show several other defects, such as prolonged action potentials, altered muscle A-type K+ currents, and abnormal synaptic transmission; these defects appear to be due to alterations in voltage-gated K+ channels (Tanouye et al., 1981; Jan et al., 1977; Salkoff and Wyman, 1981; Wu et al., 1983; Wu and Haugland, 1985). Voltage-clamp experiments on flies carrying different allelic combinations of Sh have indicated the nature of these alterations. Functional Sh channels are absent in ShM flies and Sh Ka2aflies; these mutations are genetic nulls or amorphs (Wu and Haugland, 1985; Timpe and Jan, 1987). ShKsT~3and Sh 7°2 mutations behave genetically as antimorphs, apparently producing nonfunctional channel subunits that interfere with the function of normal subunits (Salkoff, 1983; Timpe and Jan, 1987; Haugland and Wu, 1990). Thus, these two alleles apparently destroy the ability to translocate ions across the membrane without eliminating the ability of subunits to interact. In larval and pupal muscle, ShE62 and ShrK°12° behave as leaky alleles or hypomorphs (Wu and Haugland, 1985; 13rope and Jan, 1987). Finally, the Shs mutation results in the production of Sh currents that are normal in amplitude, but are altered in properties such as voltage sensitivity of acti-
vation (Salkoff, 1983; Wu and Haugland, 1985). Genetically, Shs behaves as a gain-of-function mutation or neomorph. The isolation and characterization of the Sh gene demonstrated that it encodes a family of voltage-gated K+ channels (Kamb et al., 1987; Papazian et al., 1987; Baumann et al., 1987). Sh is a large gene with a complicated genomic organization and a variety of different transcripts generated by alternative splicing (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). All Sh K+ channels have six potential membrane-spanning segments, designated $1-$6 (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). Sh cDNAs that express K+ channels have a central conserved region that is identical in every transcript, spans most of the coding region, and includes the segments $1-$5 (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). The conserved region is flanked by variable 5' and 3' ends (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). The diverse phenotypic effects of Sh mutations are behaviorally, electrophysiologically, and genetically well characterized (Tanouye et al., 1986). Localization of mutations within the structure oLthe K+ channel will provide molecular explanations for the variety of allele types defined genetically (neomorph, antimorph, etc.). In addition, their molecular localization can be used to dissect apart various functional domains of the channel and provide a basis for subsequent sitedirected mutagenesis studies. To identify precisely the changes that cause the alterations in channel function, we analyzed Sh transcripts from some of these mutants. Transcript sequences were amplified using the polymerase chain reaction (PCR) after reverse transcription of mutant RNA. The combined use of RNAwith PCR had the advantage of high sensitivity and overcame problems associated with the low abundance of Sh transcripts and the presence of large introns within the gene (Kamb et al., 1988; Schwarz et al., 1988; Pongs et al., 1988). Results
The central constant region contains most of the coding sequence of Sh cDNAs and thus was the initial focus of our search for structural defects. The central region is depicted in a schematic representation of the $h cDNA H4 (Figure 1). Also shown are oligonucleotides used to prime PCRs and to identify amplified products. The constant region of transcripts from wild-type and mutant flies was amplified by PCR. Template was single-stranded cDNA generated by reverse transcription of RNA from the different stocks. The oligonucleotide pair PI/P2 was used to prime amplification of the5' portion of the constant region; P3/P4 primed amplification of the 3' portion. Amplification of $h M
Neuron
68
Sh C D N A H4 conserved
central
Sl
.qTART
P1 P5 I
$2
region
$3
$4
S6
$5
~rPNp
P2 587 b p
I I
595
bp
P3
P6 P4
587
-- 595 bp
12345678
12345678
A
B
Figure1. Positionsof Oligonucleotides Used for PCR and Identification of Amplified Products from Sh Transcripts Depicted is a schematic representationof the Sh cDNA H4 (Kambet al., 1988). Potentialmembrane-spanningsegments$1-$6 are indicated. Notethat we use the $1-$6terminologyfor membrane-spanningsegments(pongset al., 1988) ratherthan the HD1-HD6terminology used previouslyfor K+ channels (Kamb et al., 1988; Schwarzet al., 1988).Verticalarrows demarcatethe extent of the central conserved region. The horizontal arrows representthe relative positions of oli8onucleotides P1-P6and their 5' to 3' orientation. P1, P5, and P3are sense;P2, P6, and P4are antisense.Amplified products were of the expected sizes(587 bp for the 5' portion of the constant region; 595 bp for the 3' portion); however,transcripts from ShM and Sh"K°12° failed to give products in the amplification of the 5' portion. (A) Products from the amplification using P1 and 1>2primers probed with 32p-labeledoligonucleotide I)5.A 587 bp band was detected for all samplesexceptthose from Sh M, SWK°12°, and Df(1)B55d/W32P. (B) Products from the amplification using P3 and P4 primers probed with 32p-labeledoligonucleotide P6.A 595 bp band was detected for all samplesexceptthose from Df(1)B55d/W32P.Since RNA from Df(1)B55dNV32Pfailed to give any amplified products, Sh sequences, but not those from other homologous genes, were amplified. The RNA for PCR in (/%)and (B) was from Sh ~°z (lane 1), ShKs~ (lane 2), ShE62(lane 3), Sh M (lane 4), Sh rK0120(lane 5), Oregon-R (lane 6), Shs (lane 7), and Df(1)BS5dNV32P(lane 8).
and Sh rK0120 transcripts generated products with the oligonucleotide pair P3/P4, but not with P1/P2 (Figure 1A, lanes 4 and 5). All other amplifications gave prOducts of the expected size; no extra bands were detected for any of the mutants. The amplified products were isolated, cloned, and sequenced. Inserts containing Sh E62, Sh Ks133, Sh 102, and Sh s sequences between I'1 and P2 were identical to wild type. Among the products amplified with P3 and P4, Sh E62, Sh Ks133, Sh u, and Sh rK°~2° sequences were identical to wild type; two of the mutants, Sh 1°2 and Sh 5, had single base changes. In Sh 7°2 there was a single base change from G to A (Figure 2A). This results in the substitution of a stop codon (TAG) for a tryptophan codon (TGG) at amino acid 434 in the deduced amino acid sequence of cDNA H4 (Kamb et al., 1988). Thus, Sh ~°2 apparently forms a product that is truncated between segments $5 and $6. Since this region is common to all Sh transcripts, every K+ channel expressed from Sh should be altered bythis mutation. Our identification of Sh I°2 is an independent confirmation of another report describing its location (Gisselmann et al., 1989). In Sh s there was a single
base change from Tto A (Figure 2B). This results in the substitution of an isoleucine codon (ATT) for a phenylalanine codon (TI-F) at amino acid 401 in the deduced amino acid sequence of cDNA H4 (Kamb et al., 1988). Thus, Sh 5 apparently forms a product with a single amino acid substitution at the beginning of the $5 segment. Interestingly, this is part of the leucine zipper region, occurring 2 amino acid residues upstream of the fifth leucine in the repeat (Leu4°3; McCormack et al., 1989). Since this region is common to all Sh transcripts, every K+ channel expressed from Sh should be affected by this mutation. A mutant cDNA (29.4,Sh5) containing the altered Sh s sequence was constructed for electrophysiological analysis. The normal host cDNA (29-4) was a chimera composed of a 5' end from cDNA H29 and a 3' end from cDNA H4 (Iverson and Rudy, 1990). 29-4 and 29.4,Sh5 cDNAs were linearized and used as template to prepare RNA in vitro. Transient outward currents resembling A-currents were observed in Xenopus oocytes after injection of normal and mutant RNA (Figure 3). The normal 29.4 currents had properties similar to earlier reports and were completely blocked by
Structural Defects in Shaker Mutants 69
OR
AC GT
Sh 5
OR
S h 7° 2
ACG
ACG T
T
AC G T
A
TGG -->TAG trp stop
~-_TTT--~ ATT phe ile
$5 wild-type: S h I02
-ala-phe-trp-trp-ala-
: -ala-phe-stop
wild-type
: -gly-leu-leu-ile-phe-phe-leu--
Sh 5 : - g l y - l e u - l e u - i l e - i l e - p h e - l e u -
Figure 2. Sequence Analysis of Sh ;°2 and Sh s (A) The entire constant region for Sh I°2 was sequenced, and a single nucleotide substitution was detected. The sequence depicted corresponds to nucleotides 1282-1320 in the cDNA H4 (Kamb et al., 1988; Schwarz et al., 1988). The arrow designates the location of the single G to A base change in the Sh ~°2 sequence. In Sh I°2, this results in the substitution of a stop codon for a tryptophan codon at amino acid 434 (deduced cDNA H4 sequence; Kamb et al., 1988). (B) The entire constant region for Sh s was sequenced, and a single nucleotide substitution was detected. The sequence depicted corresponds to nucleotides 1176-1233. The arrow designates the location of the single T to A base change in the Sh s sequence. In Sh s, this results in a single amino acid change from phenylalanine to isoleucine at amino acid 401. At this location, the K+ channel heptad leucine repeat overlaps the $5 segment (McCormack et al., 1989).
5 mM 4-aminopyridine (1-impe et al., 1988; Zagotta et al., 1989; Iverson and Rudy, 1990). The Sh 5 mutation alters voltage dependence of activation and voltage dependence of steady-state inactivation of the currents induced in Xenopus oocytes. Figure 4 shows the conductance-voltage relation and the voltage dependence of steady-state inactivation from pooled data for currents expressed from normal and mutant cDNA transcripts. The conductance-voltage curve for 294,Sh5 was shifted about 18 mV toward more positive potentials (Figure 4A, open circles). The voltage for half-maximal conductance was - 6 mV for 29-4 currents (n = 5) and +12 mV for 29-4,Sh5 currents (n = 6). The voltage dependence of steady-state inactivation for the macroscopic currents expressed from normal and mutant transcripts is shown in Figure 4B. The currents for 29-4,Sh5 were shifted about +5 mV in their voltage dependence of inactivation compared with the currents for 29-4. The voltage for half-maximal inactivation was -30 mV for 29-4 currents (n = 5) and -25 mV for 29-4,Sh5 currents (n = 5). With prepulses between -20 and -30 mV, the slope of the inactivation curve was less steep for 29-4,Sh5 currents than for 29-4 currents. These functional changes in the voltage dependence of activation and inactivation and in the slope of the inactivation curve are similar to those described previously for Sh s mutant muscle (Wu and Haugland, 1985). Thus, the single amino acid
change from phenylalanine to isoleucine is adequate to account for the Sh 5 phenotype. Discussion PCR amplification followed by nucleotide sequence analysis provides a powerful method for the molecular localization of mutations (Wong et al., 1987; Cross et al., 1988). They are a good alternative to genomic sequencing or construction of mutant cDNA libraries. PCR methods were particularly useful in localizing Sh 7°2 and Sh 5 mutations because of the large size of the gene and the low abundance and diversity of transcripts. These methods were not completely revealing in every case we examined. We found no alterations in nucleotide sequence in the constant regions of Sh E62and Sh Ks733.The alterations in these mutants are either elsewhere in the gene or within the primer sequences, since Taql polymerase is known to accommodate a mismatch in the primer region during PCR amplification (Lee et al., 1988). The absence of Sh M and 5h rK012° products using the oligonucleotide pair P1/P2 suggests that their defects may lie in the 5' portion of the constant region. For Sh u, this is consistent with the presence of a 2.2 kb insertion in the corresponding genomic region (Kamb et aL, 1987). A large insertion in the region could disable the amplification; alternatively, sequences corresponding to the
Neuron 70
A
START
sl $2 $3 $4 $5 phe
$6
A
SOP 1.8
START
ile
STOP
0.8
29-4,Sh5
0.6 0.4 0.2 0.0 -70
B
-so
v
-30
-10
10
30
so
70
V m (mV)
1.8
0.8 o
B
~
: -
~
0.6 0.,4 0.2
,400 n A
C
0.8
-6o
i
-so
-4o
.so
.zo
-1o
o
lO
PrelpUlsoVoltaoo (mV) Figure 4. Comparison of Current Properties for 29-4 and 29-4,Sh5 (A) Conductance-voltage curve for 29-4 (closed circles) and 29-
Figure 3. K+ Currents Expressed from 294 and 294,Sh5 RNA (A) Schematic representation of the 294 and 29-4,Sh5 cDNAs showing the approximate location of the Shs substitution within the $5 segment. RNA samples were transcribed in vitro from these cDNAs, denatured, and sized by electrophoresis on agarose-formaldehyde gels. (B and C) Ion currents recorded from oocytes injected with 29.4 and 294,Sh5 RNAs using a two-microelectrode voltage clamp. Membrane potentials were held at -90 mV, hyperpolarized to -120 mV for 2 s, and stepped to test potentials (90 ms duration) ranging from -80 to +70 mV in 10 mV increments. The time between trials was 5 s~
primers could be altered or deleted. Interestingly, whatever the defects are, they do not eliminate mutant transcripts completely, since amplifications using the oligonucleotide pair P3/P4 showed no sequence alteration in Shm and Sh rK0120for the 3' portion of the constant region. We are c o n t i n u i n g to determine the exact defects associated w i t h these four Sh mutant alleles. Sh 1°2 causes the substitution of a stop codon for a tryptophan codon in the coding region of Sh between the $5 and $6 segments. O u r results are in agreement w i t h an i n d e p e n d e n t identification of this mutation using genomic sequencing (Gisselmann et al., 1989). Introduction of a c D N A carrying Sh 7°2 into wild-type flies by germline transformation caused an antimor-
4,5h5 (open circles). Relative peak conductance was determined by dividing the conductance at the indicated potential (G) by the conductance at +80 mV, taken as GB=. Conductance was determined by the equation G = I(%/,. - Vk), assuming Vk = --80 mV. Means ± SD are plotted. For 29-4, n = 5; for 29-4,Sh5, n = 6. (B) Steady-state inactivation of currents for 29-4 (closed circles) and 29.4,Sh5(open circles). Membrane potential was held at -90 mV, prepulsed for I s to the indicated voltages, and stepped to a test pulse of +50 mV (80 ms duration). Peak current (I) was recorded. The ratio I/Io, was plotted as a function of the prepulse potential, where Io = peak current during the test pulse after a prepulse of -120 mV. The voltage of half-maximal inactivation was -30 mV for 294 and -25 mV for 29-4,Sh5.Means + SD are plotted. For 294, n = 5; for 29-4,Sh5, n = 5.
phic Sh defect. Thus, the antimorphic behavior of Sh 7°2 in genetic and electrophysiological analyses can now be explained by the apparent formation of truncated products that associate w i t h normal subunits in a channel complex and interfere w i t h their function (Gisselmann et al., 1989). Sh 5 has been one of the most interesting alleles of $h, behaving as a genetic neomorph. Behaviorally, $h 5 is a vigorous leg-shaker, but unlike other alleles, shaking tends to occur in bursts; Sh 5 also causes abnormal wing-scissoring (Tanouye and Ferrus, 1985). Sh 5 action potentials also differ from those of other Sh mutants: instead of having abnormally long durations (all other Sh mutants), Sh s action potentials display incomplete repolarization leading to multiple spikes. Thus, typical Sh s activity consists of a burst of four to five spikes following delivery of a single stimulus (Tanouye and Ferrus, 1985). In voltage-clamp experiments of larval muscle, embryonic myotubes, and pupal neurons, Shs fails to show an alteration in inac-
Structural Defects in Shaker Mutants 71
tivation kinetics, but changes the voltage dependence of activation by about +20 mV and inactivation by +5 to +10 mV (Wu and Haugland, 1985; Zagotta and Aldrich, 1990; Baker and Salkoff, 1990). In voltage-clamp experiments of pupal muscle, $ h 5 currents are altered in inactivation kinetics (Salkoff, 1983). We have identified a single nucleotide change in transcripts from Sh 5 flies. This changes the a m i n o acid sequence gly-leu-ile-phe-phe-leu to gly-leu-ileile-phe-leu at the beginning of the $5 segment in the central region (Figure 2B). This is a surprisingly subtle change of one neutral a m i n o acid residue for another. Nevertheless, this substitution is sufficient to account for at least some of the observed Sh 5 phenotypes. That is, transcripts from the 29-4,Sh5 construct, w h e n injected into Xenopus o0cytes , induced currents that were altered in voltage dependence for activation and inactivation, similar to the currents recorded from Sh s larval muscle, embryonic myotubes, and pupal neurons (Wu and Haugland, 1985; Zagotta and Aldrich, 1990; Baker and Salkoff, 1990). We suspect that this one change w o u l d also mimic other Sh s phenotypes if examined under the appropriate experimental conditions. Recently, two conserved sequence motifs in Na +, K+, and Ca2+ channels have been postulated to mediate voltage-dependent gating. The transmembrane $4 segment appears to be involved in voltage sensing (Stuhmer et al., 1989a; Papazian et al., 1989, Soc. Neurosci., abstract). A strongly conserved leucine heptad repeat or leucine zipper, which lies between the $4 and $5 segments and partially overlaps them, appears to be involved in the channel gate (McCormack et al., 1989; Auld et al., 1990). Our results on Sh s are consistent w i t h these ideas of channel gating: the mutation lies w i t h i n the region where the leucine repeat overlaps the $5 segment and alters channel voltage dependence. The percentage of a m i n o acid identities in the region containing $4, the leucine repeat, and $5 segments is remarkably high among $ h homologs of different species. In the " $ h class" channels (Stuhmer et al., 1989b; M a t h e w et al., 1989, Soc. Neurosci., abstract), o n l y 3 a m i n o acid positions appear to support any variation. This is in contrast to the larger variations in a m i n o acid sequence that are present in the regions immediately upstream of $4 and downstream of $5. An extrapolation from o u r results on Sh 5 is that many of these a m i n o acid identities are critical for channel gating, and their analysis may reveal underlying features of the gating mechanism. ExperimentalProcedures Fly Stocks and RNA Extraction The wild-type strain used was Oregon-R(OR).The Sh stocks have all been described previously: Shs, Sh rK°Tz°, Sh Ks733, and Sh 1°2 are ethyl methane sulfonate-induced alleles; Sh ~ is a spontaneous mutation associated with a 2.2 kb insertion (Tanouyeet al., 1981; Salkoff, 1983; Wu and Haugland, 1985; Kamb et al., 1987; ~mpe and Jan, 1987). Df(1)B55d/W32pis deleted for most of the coding region of Sh (Tanouyeet al., 1981;Kambet al., 1987; Schwarz et al., 1988). Fly RNA was prepared from 0.5 g of frozen
flies with guanidine isothiocyanate and subjected to ultracentrifugation (Maniatis et al., 1982). The RNA preparations were adequate for PCR without selection of poly(A)+ RNA. Oligonucleotides for PCR Two pairs of oligonucleotides were used as primers to amplify the entire constant region of Sh products (Figure 1). Amplifications were between P1 (183)(5:GTCTI-I'GCCCAAAI-I'GAGCAG-3"; sense) and P2 (769) (5'-CCTTGTAATGCTTAAATIC-3';antisense); and between P3(751)(5"GAATTI'AAGCATTACAAGG-3';sense)and P4 (1346) (5'-GTCATGTCACCATATCCAACG-3";antisense). PCR-amplified sequences were identified using two oligonucleotides as hybridization probes: P5 (268) (5:CCTCACGATCATGATI-rCTG-3';sense)and P6 (895)(5'-GGAACCTGACAGTIAGTT-3'; antisense). The position of the first nucleotide for each of the above sequences in the H4 cDNA (or ShB1)is designated by the number in parentheses(Kambet al., 1988; Schwarzet al., 1988). PCR Amplification and Identification of Amplified Products Reversetranscription was performed in a mix (20 Is.I)containing 5 I~g of total RNA, 100 ng of P2 or P4 oligonucleotides, 0.5 mM each dATP, dCTP, dGTP, and d-I-IP, 20 U of RNAase inhibitor (Boehringer Mannheim), 50 mM Tris-HCI (pH &3), 75 mM KCI, 10 mM dithiothreitol, 3 mM MgCI2, and 400 U of MMLV reverse transcriptase (Bethesda Research Laboratories). Reverse transcription reactions were incubated at 37°Cfor 3 hr. After incubation, the reversetranscription mix was used directly for PCR in a solution (50 ILl)containing 300 ng of I>1or P3 oligonucleotides, 200 ng of P2 or P4 oligonucleotides, 25 mM Tris-HCI (pH &3), 50 mM KCI, 2 mM MgCI2,0.006%gelatin, and 4 U of Taql polymerase (Cetus, Promega).PCR amplifications were for 40 cycles in a Thermal Cycler (Perkin-Elmer-Cetus) using annealing, extension, and denaturation conditions of 45°C (2 rain), 72°C (4 rain), and 94°C (1 rain), respectively.At the end of the run, reactions were incubated at 45°Cfor 2 rain and then at 72°C for 7 rain. Ampliflcation products were evident after electrophoresis through 1.5% agarose gels. Southern blots of amplified products were probed with 32p-labeledP5 or P6 oligonucleotides. The probes were prepared using a protocol provided in the TaqTrackDNA sequencing kit (Promega);hybridization conditions were as previously described (Meinkoth and Wahl, 1984). PCR-amplified sequences (100 ng) were prepared for cloning by incubation with 2 U of Klenow polymerase(BoehringerMannhelm) and 1 mM each dATE dCTP, dGTP, and dl-IP for 1 hr at 22°C. T4 DNA polymerase (1 U; Boehringer Mannheim) was added for the final 5 rain of incubation. Inserts were gel-purified, precipitated in ethanol, and resuspended in water (10 I~1). The inserts were then ligated into Sinai-cleavedBluescript vector (Stratagene), keeping an insert to vector molar ratio of 10:1. Sequencing of cloned inserts was done using Sequenase(US Biochemicals) and KS and SK primers (Stratagene).At least six isolares of amplified products from the 5' and 3' portions of the constant region were sequenced entirely for each mutant. A cDNA carrying the Shs mutation in Bluescript vector was constructed for electrophysiological analysis.The host cDNA for the mutation was a chimera composed of a 5' end from 1-129and a 3' end from H4 called 29-4 (Iverson and Rudy, 1990).29-4 is the best-expressing cDNA in our collection and is identical to the deduced amino acid sequence for Shl3and ShD1 (Pongset al., 1988; Schwarz et al., 1988). The resulting mutant cDNA was called 29-4,Sh5.The construction took advantageof the fact that the Shs mutation is flanked by two unique restriction enzyme sites, Bsml and Nsil (at nucleotides 1094and 1256, respectively, in 29-4). A 29-4 cDNA and a plasmid carrying the Shs insert were digested with Bsml and Nsil. The appropriate fragments were then gel-purified and ligated to generate 29-4,Sh5cDNA. Isolates of 29-4,Sh5cDNA were identified by restriction digests. Sequence analysis confirmed that there were no additional changes in this region of the construct. Preparation of RNA for Electrophysiology Full-length, capped transcripts were generated using 1"7 RNA polymerase (Promega)by in vitro transcription of linearized plas-
Neuron 72
mids (Krie8 and Melton, 1984; Iverson et al., 1988). Xenopus oocytes (stage V or stage VI) were microinjected with RNA (1-5 ng) and incubated for 2-3 days at 18°C in ND96 (96 mM NaCl, 2 mM KCl, 1.8 mM CaCI2, 1 mM MgCI2, 5 mM HEPES [pH 7.5]) supplemented with 100 U/ml penicillin, 100 p.8/ml streptomycin, and 2.5 mM sodium pyruvate (Krafte et al., 1988). Macroscopic currents were recorded with a standard two-microelectrode voltage clamp (Krafte et al., 1988). Bath solution was ND96; electrodes were filled with 3 M KCl and had a resistance of 0.7-1.5 MQ. All experiments were carried out at room temperature (20°C-22°C). Current signals were low-pass-filtered at 3 kHz. Voltage and current signals were digitized and analyzed using the pCLAMP package (Axon Instruments, Burlingame, CA) as previously described (Iverson et al., 1988). No significant K+ currents were observed in uninjected oocytes under these conditions. Leakage current was estimated from the average response to 10 mV steps from -90 mV in a voltage range in which time-dependent currents were not activated. Acknowledsments We thank K. McCormack, M. K. Mathew, and M. Ramaswami for valuable discussions and comments on the manuscript, R. McMahon for excellent technical assistance, and Dr. H. A. Lester for a generous gift of oocytes. This study was supported by USPHS grant NS 21327-06 to M. A. T. The Caltech Xenopus oocyte facility was supported by National Institutes of Health grant GM29836 to Dr. H. A. Lester. Received March 16, 1990; revised April 25, 1990.
References Auld, V.J., Goldin, A. L., Krafte, D.S., Catterall, W.A., Lester, H. A., Davidson, N., and Dunn, R. J. (1990). A neutral amino acid change in segment 1154dramatically alters the gating properties of the voltage-dependent sodium channel. Proc. Natl. Acad. Sci. USA 87, 323-327. Baker, K., and Salkoff, L. (1990). The Drosophila Shaker gene codes for a distinctive K+ current in a subset of neurons. Neuron 4, 129-140. Baumann, A., Krah-Jentgens, I., Mueller, R., Mueller-Holtkamp, F., Seidel, R., Kecskemethy, N., Casal, J., Ferrus, A., and Pongs, O. (1987). Molecular organization of the maternal effect region of the Shaker complex locus of Drosophila: characterization of an IA channel transcript with homology to vertebrate Na + channel. EMBO J. 6, 3419-3429. Cross, N. C. P., Tolan, D. R., and Cox, T. M. (1988). Catalytic deftciency of human aldolase B in hereditary fructose intolerance caused by a common missense mutation. Cell 53, 881-885. Gisselmann, G., Sewing, S., Madsen, B. W., Mallart, A., AngautPetit, D., Muller-Holtkamp, E, Ferrus, A., and Pongs, O. (1989). The interference of truncated with normal potassium channel subunits leads to abnormal behavior in transgenic Drosophila melanogaster. EMBO J. 8, 2359-2364. Haugland, F. N., and Wu, C.-E (1990). A voltage-clamp analysis of gene-dosage effects of the Shaker locus on larval muscle potassium currents in Drosophila. J. Neurosci. 10, 1357-~1371. Iverson, L.E., and Rudy, B. (1990). Both transient and nontransient potassium currents are expressed from RNAs derived from the Shaker gene of Drosophila. J. Neurosci., in press. Iverson, L. E., Tanouye, M. A., Lester, H.A., Davidson, N., and Rudy, B. (1988). Expression of A-type K+ channels from Shaker cDNAs. Proc. Natl. Acad. Sci. USA 85, 5723-5727. Jan, Y. N., Jan, L. Y., and Dennis, M. J. (1977). Two mutations of synaptic transmission in Drosophila. Proc. Roy. Soc. (Lond.) B 198, 87-108. Kamb, A., Iverson, L. E., and Tanouye, M. A. (1987). Molecular characterization of Shaker, a Drosophila gene that encodes a potassium channel. Cell 50, 405-413. Kamb, A., Tseng-Crank, J., and Tanouye, M. A. (1988). Multiple
products of the Drosophila Shaker gene may contribute to potassium channel diversity. Neuron 1, 421-430. Kaplan, W. D., and Trout, W. E., III. (1969). The behavior of four neurological mutants of Drosophila. Genetics 61, 399-409. Kraftet D. S., Snutch, T., Leonard, J., Davidson, N., and Lester, H. A. (1988). Evidence for the involvement of more than one mRNA species in controlling the inactivation process of rat and rabbit brain Na channels expressed in Xenopus oocytes. J. Neurosci. 8, 2859-2868. Krieg, R, and Melton, D. A. (1984). Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucl. Acids Res. 12, 7057-7070. Lee, C. C., Wu, X., Gibbs, R. A., Cook, R. G., Muzny, D. M., and Caskey, C.T. (1988). Generation of cDNA probes directed by amino acid sequence: cloning of urate oxidase. Science 239, 1288-1291. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982). Molecular Cloning: A Laboratory Manual (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory). McCormack, K., Campanelli, J.T., Ramaswami, M., Mathew, M. K., Tanouye, M. A., Iverson, L. E., and Rudy, B. (1989). Leucinezipper motif update. Nature 340, 103. Meinkoth, J., and Wahl, G. (1984). Hybridization of nucleic acids immobilized on solid supports. Anal. Biochem. 138, 267-284. Papazian, D. M., Schwarz, T. L., Tempel, B. L., Jan, Y. N., and Jan, L. Y. (1987). Cloning of genomic and complementary DNA from Shaker, a putative potassium channel gene from Drosophila. Science 237, 749-753. Pongs, 0., Kecskemethy, N., Muller, R., Krah-Jentgens, I., Baumann, A., Kiltz, H. H., Canal, I., Llamazares, S., and Ferrus, A. (1988). Shaker encodes a family of putative potassium channel proteins in the nervous system of Drosophila. EMBO J. 7, 10871096. Salkoff, L. (1983). Genetic and voltage clamp analysis of a Drosophila potassium channel. Cold Spring Harbor Symp. Quant. Biol. 48, 221-231. Salkoff, L., and Wyman, R.J. (1981). Genetic modification of potassium channels in Drosophila Shaker mutants. Nature 293, 228-230. Schwarz, T. L., Tempel, B. L., Papazian, D. M., Jan, Y. N., and Jan, L. Y. (1988). Multiple potassium channel components are produced by alternative splicing at the Shaker locus in Drosophila. Nature 331, 137-142. Stuhmer, W., Conti, F., Suzuki, H., Wang, X., Noda, M., Yahagi, N., Kubo, H., and Numa, S. (1989a). Structural parts involved in activation and inactivation of the sodium channel. Nature 339, 597-603. Stuhmer, W., Ruppersberg, J. P., Schroter, K. H., Sakmann, B., Stocker, M., Giese, K. R, Perschke, A., Baumann, A., and Pongs, O. (1989b). Molecular basis of functional diversity of voltagegated potassium channels in mammalian brain. EMBO J. 8, 3235-3244. Tanouye, M. A., and Ferrus, A. (1985). Action potentials in normal and Shaker mutant Drosophila. J. Neurogenet. 2, 253-271. Tanouye, M. A., Ferrus, A., and Fujita, S. C. (1981). Abnormal action potentials associated with the Shaker complex locus of Drosophila. Prec. Natl. Acad. Sci. USA 78, 6548-6552. Tanouye, M. A., Kamb, C. A., Iverson, L. E., and Salkoff, L. (1986). Genetics and molecular biology of ionic channels in Drosophila. Annu. Rev. Neurosci. 9, 255-276. Timpe, L. C., and Jan, L. Y. (1987). Gene dosage and complementation analysis of the Shaker locus in Drosophila. J. Neurosci. 7, 1307-1317. Timpe, L. C., Jan, Y. N., and Jan, L. Y. (1988). Four cDNA clones from the Shaker locus of Drosophila induce kinetically distinct A-type potassium currents in Xenopus oocytes. Neuron 1, 659667. Trout, W. E., III, and Kaplan, W. D. (1973). Genetic manipulation
Structural 73
Defects
of motor output 4, 495-512.
in Shaker
in Shaker
Mutants
mutants
of Drosophila.
Wong, C., Dowling, C. E., Saiki, R. K., Higuchi, H. A., and Kazazian, H. H. (1987). Characterization saemia mutations using direct genomic sequencing single copy DNA. Nature 330, 384-386
J. Neurobiol. R. C., Erlich, of B-thalasof amplified
Wu, C.-F., and Haugland, F. N. (1985). Voltage-clamp analysis of membrane currents in larval muscle fibers of Drosophila: alteration of potassium currents in Shaker mutants. 1. Neurosci. 5, 2626-2640. Wu, C.-F., Ganetzky, B., Haugland, F., and Liu, A.-X. (1983). Potassium currents in Drosophila: different components affected by mutations of two genes. Science 220, 1076-1078. Zagotta, W. N., and Aldrich, R. W. (1998). Alterations gating of single Shaker A-type potassium channels mutation. 1. Neurosci., in press.
in activation by the Sh5
Zagotta, W. N., Hoshi, 1, and Aldrich, R. W. (1989). Gating of single Shaker potassium channels in Drosophila muscle and in Xenopus oocytes injected with Shaker mRNA. Proc. Natl. Acad. Sci. USA 86, 7243-7247.
