Proc. Natl. Acad. Sci. USAVol. 92, pp. 6763-6767, July 1995Neurobiology

A potassium channel fi subunit related to the aldo-keto reductasesuperfamily is encoded by the Drosophila Hyperkinetic locus

(auxiliary subunit/modulation/Shaker)

SCOTT W. CHOUINARD, GISELA F. WILSON, A. KATHERINE SCHLIMGEN, AND BARRY GANETZKY*Laboratory of Genetics, 445 Henry Mall, University of Wisconsin, Madison, WI 53706

Communicated by James F. Crow, University of Wisconsin, Madison, WI, March 30, 1995 (received for review February 28, 1995)

ABSTRACT Genetic and physiological studies of the Dro-sophila Hyperkinetic (Hk) mutant revealed defects in thefunction or regulation of K+ channels encoded by the Shaker(Sh) locus. The Hk polypeptide, determined from analysis ofcDNA clones, is a homologue of mammalian K+ channel f3subunits (Kv.8). Coexpression ofHk with Sh inXenopus oocytesincreases current amplitudes and changes the voltage depen-dence and kinetics of activation and inactivation, consistentwith predicted functions of Hk in vivo. Sequence alignmentsshow that Hk, together with mammalian K4,3, represents anadditional branch of the aldo-keto reductase superfamily.These results are relevant to understanding the function andevolutionary origin of K,I3.

The signaling properties of neurons are largely determined bythe combination of K+ currents they express. Multiple genesencoding pore-forming K+ channel a subunits (Kva) and thedifferential splicing of a transcripts contribute to the molec-ular and functional diversity of K+ channels (1). Additionalcomplexity and regulation are conferred by two recentlyidentified 1 subunits that appear to associate in a 1:1 stoichi-ometry with the a subunits of mammalian K+ channels and, inone case, modify the inactivation properties of Kva (2, 3).Because outwardly rectifying Kva initially were identified andisolated using Drosophila mutations with distinctive behavioraland electrophysiological defects (4-8), mutations of other lociexhibiting similar defects might identify additional genes key toK+ channel function. One such candidate is the Hyperkinetic(Hk) locus, which was identified by mutations causing anether-sensitive leg-shaking phenotype (4). Studies of synaptictransmission in Hk larvae revealed a physiological phenotypesimilar to, but milder than, that of Sh (9). Moreover, Hk hasphenotypic interactions in double mutant combinations witheag and inebriated (ine) similar to those observed for Sh (9, 10).Because of the phenotypic similarities between Hk and Sh, aswell as the epistasis of Sh to Hk in double mutants, weproposed that Hk encoded a component of Sh channels oraffected their regulation (9). We show that Hkt encodes a K+channel 13 subunit (Kv13) with distinctive effects on Sh channelfunction and that Hk is a member of the aldo-keto reductasesuperfamily.

MATERIALS AND METHODSGerm-Line Transformation. Germ-line transformation (11)

was carried out using a 500 ,ug/ml solution of each constructinjected into embryos from the cross wHkl mf; P[ry+, A2-3] xw/w, TM3Sb/TM6BTb. The chromosomal markers are de-scribed elsewhere (12). Transformed Gl flies were identifiedby complementation of the w- phenotype. Multiple transgeniclines for both constructs were scored for the ability to com-plement the Hk leg-shaking phenotype in double-blind assays.

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

Nucleic Acid Manipulations. Three polymorphisms exist atthe amino acid level between the two Hk cDNAs used, HC208(oocyte expression) and HC206 (see Fig. 2). HC208 has anadditional serine and glutamine inserted at position 22 and anL -> V substitution at position 192. The polymorphisms areoutside the regions of overlap with mammalian Kv4 subunits.Hk HC208 (13) and ShH4 cDNAs (14) were subcloned into theXenopus expression vector pGH19 (provided by E. Goulding,Columbia University, New York), a modified pGEMHE (15).HkARV/Nru (HkA) was made by digesting HkpGH19 withEcoRV and Nru I and rejoining the two blunt ends with T4DNA ligase, which generated a construct with amino acids314-381 deleted, followed by 174 bp of non-Hk coding se-quence. Capped RNAs were transcribed in vitro in standardtranscription buffer (Promega). ShBA6-46 in the pSP72Bvector (16) was provided by R. W. Aldrich (Stanford Univer-sity). Except for an added valine between Leu-512 and Gly-513and the omission of Gln-558, ShH4 is identical to ShB (6, 14,17).

Electrophysiology. Oocytes were removed from anesthe-tized Xenopus laevis (Nasco, Ft. Atkinson, WI), and follicularcells were digested with collagenase (2 mg/ml, type 1A; Sigma)in Ca2+-free OR2 solution (82.5 mM NaCl/2.5 mM KCl/1 mMMgCl2/5 mM Hepes adjusted to pH 7.6 with NaOH) for 1-1.5hr at "20°C. Stage V-VI oocytes were injected with 13.8 nl ofmRNA solution containing either Sh RNA or Sh and Hk RNAat a 1:2 ratio (1-6 ng ofRNA per oocyte). ExcessHk RNA wasused to ensure assembly of subunits at the suggested 1:1stoichiometry (2). Tripling ShH4 RNA resulted in a tripling ofcurrent magnitudes. Oocytes were stored in L-15 medium[50% L-15 (GIBCO), 50% 2H20, 15 mM Hepes, 1 mML-glutamine, gentamycin at 50 mg/ml, and bovine serumalbumin (adjusted to pH 7.4 with NaOH)] at 18°C. Voltageclamp recordings (OC-725B, Warner Instruments, Hamden,CT), performed at "20°C, were digitized and analyzed usingPCLAMP 6 (Axon Instruments). Currents were filtered at 1-2kHz (-3 dB, 8 pole Bessel) and sampled at 5-10 kHz. Linearleak and capacitative currents were subtracted using P/Nmethods included in PCLAMP 6. For experiments medium wasreplaced by extracellular solution [140 mM NaCl, 2 mM KCl,6 mM MgCl2, and 10 mM Hepes (adjusted to pH 7.1 withNaOH)]. Pipettes (3 M KCl) had resistances of 0.5-2 Mfl.

RESULTS AND DISCUSSIONCytological examination of radiation-induced Hk alleles asso-ciated with inversion and translocation breakpoints, as well asof deletions uncoveringHk, placedHk at polytene bands 9B7-8(13). Cosmid clones spanning this region were obtained froma wild-type genomic library by chromosome walking beginning

Abbreviations: TEA, tetraethylammonium; KVa and Kvl3, K+ channela and f subunits.*To whom reprint requests should be addressed.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. U23545).

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6764 Neurobiology: Chouinard et al. Proc. Natl. Acad. Sci. USA 92 (1995)

with a probe derived from a neighboring P-element. Hkbreakpoints were located on the molecular map of the regionto pinpoint the locus (Fig. 1). Germ-line transformation witha 17-kb genomic fragment (17K/X194) encompassing theregion defined by the breakpoints resulted in almost completerescue of the Hk leg-shaking phenotype. A deletion constructmissing a central 600-bp BamHI fragment failed to rescue (Fig.1). These results demonstrated that most or all of the Hk locusis contained within 17K/X194.

Overlapping cDNAs corresponding to a single transcriptionunit were isolated from adult head libraries screened withgenomic probes derived from the 17K/X194 fragment. Thesize of the composite cDNA agrees with Northern blots thatdetect transcripts of -9 kb (13). Chromosomal in situ hybrid-ization and Southern blots demonstrate that these cDNAs arederived from the Hk region and span the lesions associatedwith at least three Hk mutations. Furthermore, transcriptsdetected by cDNA probes on Northern blots are missing oraltered in different Hk mutants, confirming that the cDNAsare derived from the Hk transcript (ref. 13; A.K.S., unpub-lished data).

Sequence analysis of Hk cDNAs reveals a single openreading frame encoding a deduced polypeptide of 546 aminoacids with a predicted molecular mass of about 60 kDa (Fig. 2).The presumptive start codon is preceded by a perfect match tothe Drosophila consensus for translation initiation (13). Com-puter searches of protein data bases revealed that the Hkpolypeptide shares significant amino acid identity with mam-malian Kv,l (3). Alignment of Hk with the Kvl3 begins atposition 132 of the Hk sequence and extends throughout theremainder of the polypeptide, sharing -42% amino acididentity with rat Kvp1 and -48% identity with rat and bovineKvI32 over this segment. The strongest similarity between Hkand the mammalian f3 subunits begins at position 204 of the Hksequence. A similar alignment pattern was reported for ratKv,3l and Kvf32. The similarity of Hk with mammalian Kv4,

A Df(l )Hk4El lDf(1 )A85;Hk+ Df(l )BX4;Hk+_

*710~ O !40 310 ;0*Il10,R

17K/X194 1--- 5' P(w+)9B7K/XAB BIB K

X BB K

B xProoter? K

FIG. 1. Molecular organization of the Hk region. (A) Breakpointsof mutant chromosomes identifying the Hk transcription unit (13) areshown at top. Centromere is to the right. Filled boxes represent deletedportions of the genome; open boxes indicate regions of uncertainty.Df(1)A85 (mapped by S. Schneuwly, personal communication) andDf(l)BX4 both complement Hk, defining the distal and proximallimits of the locus. Position of the P-element insertion used to initiatethe chromosomal walk (genomic library provided by J. Tamkun,University of California, Santa Cruz) and to generate additional Hkalleles (13) is shown above the molecular map (coordinates are in kb).Restriction enzymes: B, BamHI; K, Kpn I; R, EcoRI; X, Xba I.

Genomic DNA fragments used in the transgenic rescue analysis areshown below the map with the 5' end lying to the right. The locationof the "600-bp BamHI deletion is indicated by a gap in 17K/XAB194.Both constructs were subcloned into the transformation vectorCaSpeR194 (provided by M. Gorman, personal communication). (B)Schematic representation of the 17-kb Kpn I/Xba I (K/X) genomicfragment used in the transgenic rescue analysis. The transcription unitis represented by boxes; filled boxes indicate coding sequence. The 5'end has not been identified. Thin lines between boxes representintrons, whereas thin lines on either side of the transcription unitindicate flanking genomic DNA used in generating the rescuingconstruct. The relative positions of the BamHI restriction sites used increating the deletion construct are shown below the map.

Hk MSMALCNLNGDGSAAQSTSQSQSPAATAAAAPLLPHSBSHTLQ 43

Hk PESTPLLLGHEQSGSAAPEGSGGGDVADGAVTSIEMP TVVADD 86

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AGVPPLPLPLPQSTPQPLLMALPANLNFITGPTTQQLMIGNGA 129

MQVEIIACTEBXLKSRUGEMRLLSKQWSIAPNVVNV K 41MYPE TOGS .. .. 16

MGVIP TNDSNN >NNNNMNVMNTSDESNPVTIYRCR 172

TVAIIARSLGDFTGQHHISD5ESTAAQTXMK 84......... QHGSIMGMIYMTi2YMP RQLQFFA:XZtUd'@e:x§sS soCMEEFSGRSISLGSNPALPE H TPTPELRK IM 215

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K,pgll ovcx;paE........;..X-@3 2 5ICVP2 &M.......... : : :: 2 1Bk T s N RNAALSPQGSWGKDRID4 6 6

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FIG. 2. Amino acid sequence alignment for Hk (cDNA HC206)and the rat ,3 subunits KP/31 and K,I32 (3). Amino acid identities arehighlighted. The data base search was performed using BLAST. Se-quence alignments were generated using the Genetics ComputerGroup program PILEUP.

together with previous analysis of Hk mutations predicting adefect in K+ channel function or regulation, indicates that Hkencodes a X3 subunit of K+ channels.To assess the role ofHk in the function of Drosophila Sh K+

channels, we examined currents recorded in oocytes injectedwith ShH4 RNA (14), alone or together with Hk RNA.Oocytes injected only with Hk RNA did not differ detectablyfrom uninjected oocytes. Coexpression of ShH4 and Hk,however, resulted in currents that differed markedly fromthose observed when ShH4 was expressed alone. The mostsubstantial change was an "2-fold increase in ShH4 currentmagnitudes (Fig. 3A and B). A similar effect is seen when thea and /3 subunits of sodium and calcium channels are coex-pressed (18). Coexpression with Hk also resulted in ShH4currents that activated more rapidly (Fig. 4 Left). The effect onactivation kinetics was most dramatic at lower potentials (Fig.3E). For test pulses to -20 mV (holding potential, -100 mV),the time to peak for ShH4 currents decreased from a controlvalue of 26.4 ± 4.9 ms to 6.6 ± 0.4 ms in the presence of Hk.Similar results were obtained when measuring the time tohalf-activation (Table 1). In addition, normalized conduc-tance-voltage curves indicated that, in the presence of Hk, themidpoint of activation for ShH4 channels shifted by approxi-mately -8 mV (Fig. 3C). Alterations in activation wereaccompanied by parallel changes in inactivation (Fig. 3 D andF). In the presence of Hk both the fast and slow inactivationtime constants were decreased, and the midpoint of thesteady-state inactivation curve shifted by approximately -7mV. Hk also increased the time constant for recovery from 38to 56 ms. These changes in amplitude, voltage dependence, andkinetics of ShH4 currents were not observed when ShH4 wascoexpressed with the deletion construct HkA (see Materialsand Methods). Means ± SEM are given in Table 1.

Slow inactivation of Sh channels relies on domains distinctfrom the N-terminal domain governing fast inactivation (16,17). An interaction between fast and slow inactivation is

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FIG. 3. Comparison of voltage-dependent properties of ShH4 currents in Xenopus oocytes in the presence and absence of Hk. (A and B)Macroscopic currents recorded in two-electrode voltage clamp from oocytes in response to depolarizing steps from -60 to 80 mV in 20-mVincrements (holding potential, -100 mV; 6 s between steps). Scale bars apply to bothA and B. (C) Normalized average conductance-voltage curvesfor families of currents as shown inA and B except in 10-mV increments. Conductance was determined for individual oocytes using the relationG = Ip/(V - Vr), where Ip is the peak current at voltage V. The reversal potential, Vr, was determined for each oocyte; in a small number of cases,a V, of -80 mV was assumed. Conductance-voltage curves were normalized by dividing by the maximum conductance, typically at 70 or 80 mV(only the normalized conductance between -50 and +50 mV is shown). Solid curves are theoretical Boltzmann distributions generated using theaverage parameters obtained from best fits to data for individual oocytes. Examination of instantaneous currents for a subset of oocytes indicatedthat conductance was linear over the -60 to +60 mV range, and no difference in slope was detectable for ShH4 versus ShH4 + Hk. Note thatby providing a virtual ground in the bath, the Warner clamp eliminates the necessity of series resistance compensation. A series resistance error,if it were to exist, would shift the voltage dependence in a direction opposite to that seen here. (D) Normalized steady-state inactivation. Oocyteswere held at -100 mV, stepped for 500 ms to the indicated prepulse voltage, and then depolarized to 40 mV (10 s between episodes). Solid curvesare theoretical Boltzmann distributions generated by using the averaged parameters obtained from best fits to data for individual Qocytes I. (E)Time required to reach peak current levels as a function of test potential (holding potential, -100 mV). Note that the acceleration of activationseen with Hk is not accounted for by a change in voltage dependence. Shifting the time-to-peak curve by the amount suggested by the shift in voltagedependence does not result in a superimposition of time-to-peak curves, and Hk produces no change in voltage dependence for the ShBA6-46construct, although activation kinetics are altered (see Fig. 4). (F) Fast time constant of inactivation obtained by fitting the decay of current during100-ms pulses to the indicated potentials to a single exponential and a steady state. Solids curves in E and F were fit by eye.

suggested, however, by the observation that slow inactivationoccurs more rapidly from the fast inactivated state (17, 19).Thus, in our experiments, decreases in inactivation time con-stants may be a result of interactions ofHk with multiple ShH4domains, or the decrease in the slow time constant may be anindirect result of a single interaction of Hk with the ShN-terminal domain responsible for fast inactivation. Similarly,the acceleration of ShH4 activation observed with Hk may bean indirect result of the acceleration of fast inactivation. Toresolve between these alternatives, we coexpressed Hk withShBA6-46, a ShB mutant lacking the N-terminal inactivationdomain (16). Expression of Hk with ShBA6-46 increasedcurrent magnitudes (Table 1) and accelerated activation (Fig.4 Right) as previously observed. There was no reduction in theslow time constant. These results indicate that the acceleration

ShH4

+Hk -/-Hk

ShBA6-46

+Hk/ - Hk

5 ms

FIG. 4. Effect ofHk on the activation ofShH4 (Left) and ShBA6-46(Right) currents in Xenopus oocytes. Traces were normalized to thesame height and superimposed to allow comparison of activation in thepresence and absence of Hk. Currents were recorded in response todepolarizing steps to 0mV from a holding potential of -100 mV. Thescale bar applies to both sets of traces.

of activation is independent of the effect ofHk on inactivation.Acceleration of slow inactivation by Hk appears to occurindirectly, however, via the interaction between inactivationprocesses. In addition, because Hk did not restore fast inac-tivation, our experiments indicate that, in contrast to themammalian l31 subunit (3), Hk cannot act as a substitute forthe N-terminal inactivation "ball" of Sh channels. This resultis not unexpected given the lack of homology of Hk to the first34 aa of the ,B1 N-terminal sequence, which can substitute forthe Sh fast inactivation domain (3). Two observations suggestan interaction of Hk with the pore of Sh, however. ExpressionofHkwith ShH4 resulted in a decrease in the reversal potentialand an increase in sensitivity to block by 1 mM external TEA(Table 1). Because expression with Hk produced no changes inthe reversal potential or voltage dependence of activation ofShBA6-46, some aspects of the interaction of Hk may requirethe Sh N-terminal domain. Alternatively, these interactionsmay be influenced by the 2-aa difference in the sequences ofShH4 and ShB (see Materials and Methods).

Insight into the nature of the interaction between a and 13subunits of K+ channels may be provided by the identificationof a relationship between Hk and the aldo-keto reductasesuperfamily (20). A relationship between mammalian KVI3subunits and this superfamily has been reported independently(21). This gene family encompasses a structurally similar butfunctionally diverse group of cytosolic enzymes that utilizeNADPH as a cofactor. Members of this family include aldosereductase, prostaglandin F synthase, 2,5-diketogluconic acid

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Table 1. Properties of Sh channels expressed in Xenopus oocytes with and without the Drosophila 3 subunit Hk

Property ShH4 ShH4 + Hk ShH4 + HkA ShBA6-46 ShBA6-46 + Hk

Ipeak,* ,A at +40 mV 7.2 ± 0.8 (28) 12.8 ± 0.8t (31) 7.5 ± 0.9 (14) 2.6 ± 0.2 (12) 3.5 ± 0.2t (15)Reversal potential, mV -81.5 ± 2.2 (25) -71.1 ± 2.Ot (30) -84.1 ± 2.6 (10) -80.4 ± 7.3 (5) -83.6 ± 3.5 (11)TEA0 (1 mM), % current remaining 71 ± 2(6) 62 ± 2t (7) ND ND NDActivationTime to half activation, ms at 0 mV 4.0 ± 0.2 (27) 2.7 ± 0.1* (31) 3.6 ± 0.1 (14) 5.7 ± 0.5 (12) 3.9 ± 0.3t (15)Midpoint, mV 3.8 ± 1.3 (27) -3.8 ± 1.2t (31) 1.4 ± 1.6 (14) -17.0 ± 2.7 (12) -17.8 ± 1.9 (13)Slope, mV/e-fold 16.2 ± 0.6 (27) 18.5 ± 0.5t (31) 17.0 ± 0.7 (14) 7.4 ± 0.7 (12) 9.7 ± 0.6 (13)

Inactivationtaul, ms at +40 mV 7.9 ± 0.5 (18) 6.2 ± 0.3t (22) 7.2 ± 0.5 (12)tau2, ms at +40 mV 677 ± 31 (18) 505 ± 19* (22) 613 ± 24 (10) 5520 ± 386 (11) 6310 ± 599 (13)Midpoint, mV -31.4 ± 1.1 (23) -38.3 ± 1.1t (29) -32.1 ± 2.0 (10) ND NDSlope, mV/e-fold -4.1 ± 0.1 (23) -4.0 ± 0.4 (29) -5.3 ± 0.1t (10) ND NDFractional recovery at 22.5 ms 0.45 ± 0.01 (22) 0.36 ± 0.02t (27) 0.44 ± 0.02 (7) ND ND

Values given are the means ± SEM; the numbers in parentheses indicate the number of oocytes examined. Values were obtained as describedfor Fig. 3, except that time constants of inactivation were obtained by a two-exponential fit to the decay of current observed to pulses from - 100to 40 mV either 1.6 s (ShH4, ShH4 + Hk, and ShH4 + HkA) or 9 s (ShBA6-46 and ShBA6-46 + Hk) in duration. Recovery was measured usingtwo pulses to 40 mV (holding potential, -80 mV) with a variable interval (5-500 ms) separating pulses. Twenty seconds elapsed between episodes.Fractional recovery is the difference in the amplitude of the peak current and the current at the end of 50 ms divided by the corresponding valueduring the first pulse. To obtain reversal potentials, oocytes were stepped from - 100 to 40 mV for 10 ms and then to voltages ranging from - 120to 0 mV, and the difference between peak and steady-state tail currents was plotted as a function of potential. Tetraethylammonium (TEA) wasapplied using a gravity-driven perfusion system. % current remaining was calculated by dividing peak currents in TEA by the average of controland wash peak currents and multiplying by 100. Student's t-tests compared data obtained with Hk to data obtained for the same a subunit in theabsence of Hk. ND, Not determined.*Measurements are from four batches of oocytes; similar numbers of ShH4- and ShH4 + Hk-injected oocytes were studied per batch. Expressionwith Hk increased ShH4 peak current magnitudes in only three batches, whereas other variables were affected in all batches. Although the reasonfor the lack of an increase in current magnitude in the fourth batch is unknown, the expected increase with Hk may be closer to 2.3-fold.tp c 0.01.tP ' 0.001.

reductase, p-crystallin (22-25), and proteins of unknown func-tion.As shown in Fig. 5, Hk and mammalian KV,11 share clusters

of identical and conserved residues throughout the region ofoverlap with other members of the aldo-keto reductase super-family. Identity ranges from -18% to -22% for Hk andmammalian K,41 when compared with family members. Thisincreases to as much as -35% when conservative substitutionsare considered. In an alignment of Hk, Kv,81, and humanaldose reductase, all three proteins share identical amino acidsat 41 positions and conserved substitutions at another 65positions (S.W.C., unpublished data). Many of the conserva-tive substitutions are in regions where all family membersexhibit high levels of similarity (26). These conserved domainsmay contribute to the structural adaptability of the proteinfamily. Many of the residues involved in binding NADPH andthe hydrogen-transfer mechanism also are conserved in bothHk and mammalian Kv3 subunits (Fig. 5), whereas residuesresponsible for substrate specificity show significant diver-gence. These results suggest that the Kv4 subunits represent adistant branch of the aldo-keto reductase superfamily.

In addition to the evolutionary implications of the relation-ship, inferences about the three-dimensional structure of 1subunits based on homology with the aldo-keto reductasesuperfamily may provide clues about putative functions ofparticular domains. By aligning the primary sequence of Hkand the mammalian KvI3 subunits with the derived atomiccoordinates of aldose reductase and 3a-hydroxysteroid dehy-drogenase (27-30), defined topologies can be predicted forconserved domains in the 13 siubunits. In almost all instances,these conserved domains correspond to the 13 sheets present inthe a/13 barrel tertiary structure assumed by the aldo-ketoreductase family members.Hk mutants exhibit ether-sensitive leg-shaking, repetitive

nerve firing, and prolonged synaptic release similar to Shmutants (9). This phenotypic similarity may be explained bythe 50% reduction in Sh currents that, on the basis of ourobservations, would be expected in the absence of Hk in vivo.Compared with Sh homomultimers, our results predict that

Sh-Hk heteromeric channels would activate earlier in theaction potential and with greater magnitude and thereforewould be more efficient at repolarization. The physiologicaleffects seen when Hk is coexpressed with Sh are distinct fromthose reported for either Kv131 or KE,32. K,,41 can confer rapidinactivation, whereas KvP2 has no observed effect (3). Thesedifferences are not unexpected in view of the divergencebetween Drosophila and mammalian 1 subunits. In fact, thegreater evolutionary divergence of the core region of 13subunits in different species (<50% identity) relative to thatobserved for a subunits in the Sh family ('70% identity; ref.31) suggests that 13 subunits may play the greater role ingenerating K+ channel diversity. Experiments aimed at eluci-dating the physical interactions between subunits and theenzymatic properties of 13 subunits should provide new insightsinto K+ channel function and the mechanisms that generatedistinctive signaling properties of neurons. The availability ofmutations affecting both a and 13 subunits in Drosophila willoffer experimental opportunities to pursue these questions.

This paper is dedicated to the fond memory of Katie Schlimgen,colleague and friend, in recognition of her profound contributions tothe success of this work. We thank the members of our laboratory fordiscussions and S. Y. Chiu for comments on the manuscript. This workwas supported by a grant from the Muscular Dystrophy Association toS.W.C., a grant from the American Heart Association to G.F.W., andgrants from the National Institutes of Health (5T32 GMO 7131-15,5T32 GMO 7133, and NS15390). This is paper no. 3421 from theLaboratory of Genetics.

1. Hille, B. (1992) Ionic Channels in Excitable Membranes (Sinauer,Sunderland, MA), 2nd Ed.

2. Parcej, D. N., Scott, V. E. S. & Dolly, J. 0. (1992) Biochemistry31, 11084-11088.

3. Rettig, J., Heinemann, S. H., Wunder, F., Lorra, C., Parcej,D. N., Dolly, J. 0. & Pongs, 0. (1994) Nature (London) 369,289-294.

4. Kaplan, W. D. & Trout, W. E., III (1969) Genetics 61, 399-409.

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-CN LNQN.. .KIBSYCKsKDIpLV5YISVTW ELV KA HLQ.PMI LNKP GL Y YNIUPL TQE .. KEIQYCQSKGIV T!SPTWRIDAREAJVRSIHSJUJTEPM.L TLLIDETGV. . . VNS L FPQA ... ARAFHDEBHGIR TESjSP

0 00 0 0 00 * * * O O +O +O +

SMALSDTQNGDKLFLPKGSFKT.KSFSWTE ElNRNAALSPQGSWGKDRIDEGRRHCDRLRD A LGCfP TQiGIISGKYGNG ..VPESSRASLKCYQW RVS ... ............ SEEGRKQQNKLKD Pi EGC3PQAQL ............ LSEWVNSNNPVL P ........................... . A KQ P ALSHR ............ DRNWVDLSLPV

L.............................

N SAE

SP ............. DRPWARPEDPS PR ..... ...................... . N AQRS ............ E........ . L T .. . . .. .. ...........VYGV P TQ

00 0 0 0 00 0 0

LS S BE CELGATSAEQBHQSLQSLQLLP SS BEBEILE PVRPPMISTLALR|Lkpp& NEG5 sSVPGWS TPEQ EIE GAIQVLPK BHV EBINI- PYSKKDYRS|MAMYH RGV .]VLAF NKKR QVFDF. .E ED ADGL IRYYDFQKGIGHPEYPFSEEY

KG . .V4LAILKFTPA 4r4QLGVFEF. .E PED TE S HBYGPFREVKQHPEYPFHDEYKVRN ' MIR TPERJAE KVFDF. .E S QD T LSY RVCALLSCTSHKDYPFHEEFLGST. .PWP ISADPDRQREADVFGF.. DQ SGIE RLWDGDPDTHEEM

00 0 0 0 00 0

27114476767068

329204146146140120

404279218218212190

480339254254247216

546401323323316231

FIG. 5. Amino acid sequence alignment of Hk, Kv,I1, and representative members of the aldo-keto reductase superfamily. Pgfs, bovineprostaglandin F synthase (23); Frcrys, Rana catesbeiana lens p-crystallin (25); Ar2, human aldose reductase (22); 25dkg, Corynebacterium2,5-diketo-D-gluconate reductase (24). Black boxes represent amino acid identities in 5 out of 6 members while conservative substitutions in 5 outof 6 members are indicated with open boxes (the following groups of amino acids are considered to be conserved: M, I, L, and V; A and G; Kand R; F, W, and Y; D and E; C, S, and T. Invariant residues among 19 members of the aldo-keto reductase superfamily (26), Hk, and the threemammalian KvI3 subunits (3) are marked with filled circles beneath the alignment. Open circles represent positions where conservative substitutionsare present in at least 19 out of 23 family members. Positions that fall below the 5 out of 6 requirement or are not considered conservativesubstitutions, but are nonetheless highly conserved within the family, are represented by a +. Ar2 residues interacting with NADPH that are alsoconserved in Hk or 131 are indicated by a above the alignment. The data base search and sequence alignments were generated as in Fig. 2.

5. Kamb, A., Iverson, L. E. & Tanouye, M. A. (1987) Cell 50,405-413.

6. Schwarz, T. L., Tempel, B. L., Papazian, D. M., Jan, Y. N. & Jan,L. Y. (1988) Nature (London) 331, 137-142.

7. Atkinson, N. S., Robertson, G. A. & Ganetzky, B. (1991) Science253, 551-555.

8. Warmke, J., Drysdale, R. & Ganetzky, B. (1991) Science 252,1560-1562.

9. Stem, M. & Ganetzky, B. (1989) J. Neurogenet. 5, 215-228.10. Stem, M. & Ganetzky, B. (1992) J. Neurogenet. 8, 157-172.11. Robertson, H. M., Preston, C. R., Phillis, R. W., Johnson-Schlitz,

D. M., Benz, W. K. & Engels, W. R. (1988) Genetics 118, 461-470.

12. Lindsley, D. L. & Zimm, G. G. (1992) The Genome ofDrosophilamelanogaster (Academic, London).

13. Schlimgen, A. K. (1991) Ph.D. thesis, (Univ. of Wisconsin, Mad-ison).

14. Kamb, A., Tseng-Crank, J. C. L. & Tanouye, M. A. (1988) Neu-ron 1, 421-430.

15. Liman, E. R., Tytgat, J. & Hess, P. (1992) Neuron 9, 861-871.16. Hoshi, T., Zagotta, W. N. & Aldrich, R. W. (1990) Science 250,

533-538.17. Hoshi, T., Zagotta, W. N. & Aldrich, R. W. (1991) Neuron 7,

547-556.18. Isom, L. L., De Jongh, K S. & Catterall, W. A. (1994) Neuron 12,

1183-1194.19. Iverson, L. E. & Rudy, B. (1990) J. Neurosci. 10, 2903-2916.

20. Chouinard, S. W., Schlimgen, A. K & Ganetzky, B. (1993)Neurobiology of Drosophila (Cold Spring Harbor Lab. Press,Plainview, NY), p. 96 (abstr.).

21. McCormack, T. & McCormack, K (1994) Cell 79,1133-1135.22. Chung, S. & LaMendola, J. (1989) J. Biol. Chem. 264, 14775-

14777.23. Watanabe, K., Fujii, Y., Nakayama, K., Ohkubo, H., Kuramitsu,

S., Kagamiyama, H., Nakanishi, S. & Hayaishi, 0. (1988) Proc.Natl. Acad. Sci. USA 85, 11-15.

24. Grindley, J. F., Payton, M. A., Van De Pol, H. & Hardy, K. G.(1988) Appl. Environ. Microbiol 54, 1770-1775.

25. Fujii, Y., Watanabe, K., Hayashi, H., Urade, Y., Kuramitsu, S.,Kagamiyama, H. & Hayaishi, 0. (1990) J. Biol. Chem. 265,9914-9923.

26. Bruce, N. C., Willey, D. L., Coulson, A. F. W. & Jeffery, J. (1994)Biochem. J. 299, 805-811.

27. Wilson, D. K., Bohren, K. M., Gabbay, K. H. & Quiocho, F. A.(1992) Science 257, 81-84.

28. Borhani, D. W., Harter, T. M. & Petrash, J. M. (1992) J. Biol.Chem. 267, 24841-24847.

29. Rondeau, J.-M., Tete-Favier, F., Podjamy, A., Reymann, J.-M.,Barth, P., Biellmann, J.-F. & Moras, D. (1992) Nature (London)355, 469-472.

30. Hoog, S. S., Pawlowski, J. E., Alzari, P. M., Penning, T. M. &Lewis, M. (1994) Proc. Natl. Acad. Sci. USA 91, 2517-2521.

31. Salkoff, L., Baker, K., Butler, A., Covarrubias, M., Pak, M. D. &Wei, A. (1992) Trends Neurosci. 15, 161-166.

Hk

Kvf31Pgfs

FrcrysAr2

25dkg

HkKvp1Pgf a

FrcrysAr2

25dkg

HkKvpXPgfs

FrcrysAr2

2 5dkg

BkKv31Pgfa

Frc rysAr2

25dkg

HkKv3 1Pgfs

FrcrysAr2

25dkg

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