Shaker Mutants Research Articles

X-linked transmission of the shaker mutation in rats with hereditary Purkinje cell degeneration and ataxia

This study reports on the mode of inheritance of the shaker mutation and the development of an inbred strain of the shaker rat mutation from Sprague Dawley outbred stock onto a Wistar Furth background. Neuroanatomical and behavioral expression of the affected phenotype, through seven generations of backcross and intercross breeding, has confirmed the mode of inheritance to be X-linked. Behaviorally, affected mutants present with a wide-based ataxic gait and whole body tremor. In affected mutants calbindin immunostaining for surviving cerebellar Purkinje cells revealed widespread degeneration in the anterior lobe and in limited areas of the posterior lobe. Fast Fourier transform analysis of the tremor revealed a frequency of 3–5 Hz. As predicted by X-linked inheritance, female descendants of an affected male are carriers for the genotype and the phenotype is expressed in one-half of her male offspring. There was spatially random and limited degeneration of Purkinje cells in carrier females, but they did not display overt clinical signs of ataxia and tremor. These data provide further support for using the shaker mutant rat as an animal model for studies of mechanisms underlying human heredodegenerative diseases.

Deletion of the S3-S4 linker in the Shaker potassium channel reveals two quenching groups near the outside of S4.

When attached outside the voltage-sensing S4 segment of the Shaker potassium channel, the fluorescent probe tetramethylrhodamine (TMRM) undergoes voltage-dependent fluorescence changes (DeltaF) due to differential interaction with a pH-titratable external protein-lined vestibule (Cha, A., and F. Bezanilla. 1998. J. Gen. Physiol. 112:391-408.). We attached TMRM at the same sites [corresponding to M356C and A359C in the wild-type (wt) channel] in a deletion mutant of Shaker where all but the five amino acids closest to S4 had been removed from the S3-S4 linker. In the deletion mutant, the maximal DeltaF/F seen was diminished 10-fold, and the DeltaF at M356C became pH independent, suggesting that the protein-lined vestibule is made up in large part by the S3-S4 linker. The residual DeltaF showed that the probe still interacted with two putative quenching groups near the S4 segment. One group was detected by M356C-TMRM (located outside of S3 in the deletion mutant) and reported on deactivation gating charge movement when applying hyperpolarizing voltage steps from a holding potential of 0 mV. During activating voltage steps from a holding potential of -90 mV, the fluorescence lagged considerably behind the movement of gating charge over a range of potentials. Another putative quenching group was seen by probes attached closer to the S4 and caused a DeltaF at extreme hyperpolarizations (more negative than -90 mV) only. A signal from the interaction with this group in the wt S3-S4 linker channel (at L361C) correlated with gating charge moving in the hyperpolarized part of the Q-V curve. Probe attached at A359C in the deletion mutant and at L361C in wt channel showed a biphasic DeltaF as the probe oscillated between the two groups, revealing that there is a transient state of the voltage sensor in between, where the probe has maximal fluorescence. We conclude that the voltage sensor undergoes two distinct conformational changes as seen from probes attached outside the S4 segment.

Voltage-dependent membrane displacements measured by atomic force microscopy.

Cells use polar molecules in the membrane to sense changes in the transmembrane potential. The opening of voltage-gated ion channels and membrane bending due to the inverse flexoelectric effect are two examples of such electromechanical coupling. We have looked for membrane motions in an electric field using atomic (or scanning) force microscopy (AFM) with the intent of studying voltage-dependent conformational changes of ion channels. Voltage-clamped HEK293 cells were either untransfected controls or transfected with Shaker K+ channels. Using a +/- 10-mV peak-peak AC carrier stimulus, untransfected cells moved 0.5-15 nm normal to the plane of the membrane. These movements tracked the voltage at frequencies >1 kHz with a phase lead of 60-120 degrees, as expected of a displacement current. The movement was outward with depolarization, but the holding potential only weakly influenced the amplitude of the movement. In contrast, cells transfected with a noninactivating mutant of Shaker K+channels showed similar movements, but these were sensitive to the holding potential; decreasing with depolarization between -80 and 0 mV. Searching for artifactual origins of these movements, we used open or sealed pipettes and AFM cantilever placements just above the cells. These results were negative, suggesting that the observed movements were produced by the cell membrane rather than by movement of the patch pipette, or by acoustic or electrical interactions of the membrane with the AFM tip. In control cells, the electrical motor may arise from the flexoelectric effect, where changes in potential induce changes in curvature. In transfected cells, it appears that channel-specific movements also occurred. These experiments demonstrate that the AFM may be able to exploit voltage-dependent movements as a source of contrast for imaging membrane components. The electrically induced motility will cause twitching during action potentials, and may have physiological consequences.

Blockade of the Delayed Rectifier Potassium Current in Drosophila by Quinidine and Related Compounds

Quinidine is a potent blocker of the delayed rectifier K+ channels (IK). Although it has been used for understanding the physiology of K+ channels in many organisms and for treating cardiac arrhythmia in humans, mechanisms of its interaction with the channel molecule are not well understood. As a first step in understanding these mechanisms, we used the Shaker mutant of Drosophila in which the delayed rectifier can be resolved in complete isolation from other currents and determined the importance of the major groups of quinidine (methoxy, quinoline, quinuclide and the bridge groups) in the blockade of IK. It appears that the quinoline moiety, while possessing little channel-blocking activity by itself, may provide a template for positioning the groups that may be important for affinity and blockade. These groups, in the order of importance in imparting inhibitory activity to quinoline, seemed to be quinuclide > methylene bridge > 6-methoxy group. In particular, the quinoline ring and the quinuclide group, when linked together by a hydroxymethylene bridge, might be responsible for a major part of the IK blocking activity of quinidine. Action of quinidine was not affected by either quinuclidine, which did not block IK, or by quinoline.

Pore mutations in Shaker K+ channels distinguish between the sites of tetraethylammonium blockade and C-type inactivation.

1. We have studied the effect of external K+ and tetraethylammonium (TEA) on several mutants of Shaker B K+ channels with amino acid substitutions in the pore which alter TEA affinity and the rate of C-type inactivation. In all channels studied high external K+ makes C-type inactivation slower. 2. In the wild-type channel, TEA blockade is voltage dependent and produces slowing of the inactivation time course. However, in the double mutant channel (T449Y, D447E) TEA blockade, although of higher affinity, is voltage independent and does not affect the rate of C-type inactivation. 3. Mutants with a charged amino acid at position 449 (T449K and T449E) are resistant to TEA block. In these channels, C-type inactivation is also unaffected by TEA. 4. These results indicate that the sites where TEA blocks and competes with C-type inactivation can be segregated. To modulate inactivation, TEA must enter deeply into the channel mouth. These results suggest that C-type inactivation is not due to a large molecular rearrangement in the outer channel vestibule, but it is essentially produced by a conformational change restricted to a local site in the pore.

Genetic Dissection of Structural and Functional Components of Synaptic Plasticity. II. Fasciclin II Controls Presynaptic Structural Plasticity

Increased neuronal activity ( eag Shaker mutants) and cAMP concentration ( dunce mutants) lead to increased synaptic structure and function at the Drosophila neuromuscular junction. Here, we show that the increase in synaptic growth is accompanied by an ∼50% decrease in synaptic levels of the cell adhesion molecule Fasciclin II (Fas II). This decrease in Fas II is both necessary and sufficient for presynaptic sprouting; FasII mutants that decrease Fas II levels by ∼50% lead to sprouting similar to eag Shaker and dunce, while transgenes that maintain synaptic Fas II levels suppress sprouting in eag Shaker and dunce. However, FasII mutants that cause a 50% increase in bouton number do not alter synaptic strength; rather, evoked release from single boutons has a reduced quantal content, suggesting that the wild-type amount of release machinery is distributed throughout more boutons.

A behavioral study of the development of hereditary cerebellar ataxia in the shaker rat mutant

shaker mutant rats were first identified by their abnormal motor behaviors and degeneration of cerebellar Purkinje cells and brainstem inferior olivary neurons. After 6 generations of inbreeding 77% of shaker rat mutants are mildly ataxic (identified as mild shaker mutants) and 23% are ataxic and exhibit a whole body tremor (strong shaker mutants) by 3 months of age. This study of shaker mutants from birth to 3 months of age was designed to: (1) compare the somatic and motor development of shaker mutants with age matched normal rats; (2) identify the temporal onset of motor deficits; and (3) correlate qualitative differences in Purkinje cell degeneration between 3-month-old mild and strong shaker rat mutants shaker mutants rats consistently weighed less than age-matched control animals. Analysis of motor development using the hindlimb splay test demonstrated the distance between hindpaws was significantly greater in shaker mutant rats than in controls starting at 42 postnatal days (PND) of age. Hindlimb stride width was greater for shaker than control rats at 42 PNDs. However, after 42 PNDS shaker mutant average hindlimb width was narrower than controls. Forelimb stride width was consistently narrower in shaker mutants than in normal rats. Hindlimb placement was impaired in shaker rat mutants after 15 PND. Forelimb placement, cliff avoidance and surface righting were only transiently impaired in shaker mutants. Mid-air righting, performance of a geotaxic response, and climbing and jumping postural reactions were similar in shaker and normal rats. The spatial extent of Purkinje cell survival/degeneration correlated with differences in abnormal motor activity seen in 3-month-old mild and strong shaker mutants. In mild shaker rat mutants, Purkinje cells appeared to have degenerated randomly throughout the cortex. In strong shaker mutants most Purkinje cells in the anterior lobe had degenerated. In the posterior lobe Purkinje cell degeneration appeared to be numerically significant, but many surviving cells were present. Although Purkinje cell loss was not numerically quantified in this study, a strong association between the extent and type of spatial loss of Purkinje cells, and the severity of clinical signs, appears to exist.

Spatial and temporal pattern of Purkinje cell degeneration in shaker mutant rats with hereditary cerebellar ataxia.

Temporal-spatial patterns of surviving Purkinje cells were studied quantitatively in a rat mutant (shaker) with differential hereditary cerebellar ataxia and Purkinje cell degeneration. Shaker rat mutants are characterized behaviorally as mild if they are ataxic or as strong if they have ataxia and tremor. Purkinje cells degenerate in both mild and strong shaker mutants, but the temporal and spatial patterns of cell death are strikingly different. In mild shaker mutants, Purkinje cell death is temporally restricted, with 31-46% of the Purkinje cells in lobules I-IX dying by 3 months of age. Very few Purkinje cells degenerate after this age. Purkinje cell death is spatially random. In lobules I-IX, every second, third, or fourth Purkinje cell degenerates. Purkinje cells in lobule X do not degenerate. In strong shaker mutants, Purkinje cell degeneration is temporally protracted and spatially restricted. By 3 months of age, most Purkinje cells in lobules I-VIa, -b, and -d have degenerated. Numerous Purkinje cells in the paravermis of lobules VIIb-VIII have also degenerated. Surviving Purkinje cells in the vermis and lateral hemisphere of lobules VIIb-VIII are aligned in parasagittally oriented stripes or transversely oriented bands. Purkinje cells continue to degenerate in localized areas of the posterior lobe such that, by 18 months of age, surviving Purkinje cells are limited primarily to lobules VIc, VIIa, IXd, and X. Quantitative analysis indicates that none of the Purkinje cells in these lobules degenerate.

Induction of leg-shaking, knock-down and killing responses by gamma-ray irradiation in Shaker mutants of Drosophila melanogaster.

Induction of membrane-associated responses, a leg-shaking, a knock-down and a killing, by gamma-ray irradiation was investigated in Shaker (Sh) mutants of Drosophila melanogaster in which the gene cords for the A-current K+ channel. Sh mutants were more sensitive in the knock-down response after gamma-ray irradiation than wild types. There were a great amount of sex difference in the knock-down response, males being more sensitive than females, but not in the killing response. The sex difference was larger than gene dosage effect on X chromosome in females. Genetical analysis revealed that the sensitivity of the knock-down response is an incompletely dominant character without maternal effects. The leg-shaking response, which had previously been reported to be induced by ether treatment, was demonstrated in the head-removed flies of Sh mutants. It was found to be the most sensitive among the responses tested, and may involve changes in K+ channel. The knock-down response may be related to expansion of the leg-shaking response. The killing response should have causes different from the leg-shaking and the knock-down responses judging from the lack of correlation with them.

Mutations in the P-Region of a Mammalian Potassium Channel (RCK1): A Comparison with the Shaker Potassium Channel

Deletion mutants of Shaker K channels lacking the middlemost 2 residues of the amino acid sequence GYGD in their P-region lose K-selectivity and become functionally similar to voltage-activated Ca channels. Ca channel characteristics are also conferred on voltage-activated Na channels when residues K in domain III and/or A in domain IV in the P-region of the Na channel are replaced by E residues. In this study, we have investigated a possible evolutionary connection between voltage-activated K and Na channels. Mutant monomeric and multi-heteromeric RCK1 K channel cDNAs were made to match the residues at equivalent positions in the P-region of the Na channel. We found that in contrast to mutant Shaker K channels, the same mutants in RCK1 K channels did not yield functional expression. Therefore possible Na channel-like ion conduction properties conferred on K channels could not be demonstrated. However, our results show that the same mutations in highly homologous channels can produce different effects and point to hitherto unknown structural differences in tile P-region of these homologous K channels. Therefore we conclude that extrapolation of the structural and functional importance of residues should be done with caution, even when ion channels belong to the same family.

Shaker Mutants Lack Post‐tetanic Potentiation at Motor End‐plates

The two-electrode voltage clamp technique was employed to measure end-plate currents in larval neuromuscular junctions of wild-type (Canton-S) and of three different Drosophila Shaker mutants: ShakerKS133, Shaker102 and f5Shaker5. In the Shaker mutants, nerve-evoked end-plate currents (neepc) were 4-5-fold larger than those measured in Canton-S. Shaker motor end-plates were found to lack post-tetanic potentiation (PTP), but could undergo facilitation. Moreover, PTP but not facilitation was lost in wild-type larvae if the neuromuscular junction was exposed to 4-aminopyridine (4-AP), a blocker of Shaker A-type K+ currents. End-plate currents were depressed by Ca2+ channel blockers like Mg2+, at millimolar concentrations, and Co2+ and Cd2+, at micromolar concentrations, but not by nifedipine (100 nM) and verapamil (100 nM). After exposure to Ca2+ channel blockers, Shaker end-plates exhibited PTP. In particular, Cd2+ was most effective in depressing neepcs and in restoring PTP in all Shaker mutants. The results obtained indicate the abnormal function of Shaker K+ channels at motor nerves specifically abolishes PTP in Drosophila larval neuromuscular junctions.

Irregular activity in the giant neurons from Shaker mutants suggests that the Shaker locus may encode non-A-type K+ channel subunits in Drosophila.

Annals of the New York Academy of SciencesVolume 707, Issue 1 p. 392-395 Irregular Activity in the Giant Neurons from Shaker Mutants Suggests that the Shaker Locus May Encode Non-A-Type K+ Channel Subunits in Drosophila M. SAITO, M. SAITO Department of Biology University of Iowa Iowa City, Iowa 52242Search for more papers by this authorM.-L. ZHAO, M.-L. ZHAO Department of Biology University of Iowa Iowa City, Iowa 52242Search for more papers by this authorC.-F. WU, C.-F. WU Department of Biology University of Iowa Iowa City, Iowa 52242Search for more papers by this author M. SAITO, M. SAITO Department of Biology University of Iowa Iowa City, Iowa 52242Search for more papers by this authorM.-L. ZHAO, M.-L. ZHAO Department of Biology University of Iowa Iowa City, Iowa 52242Search for more papers by this authorC.-F. WU, C.-F. WU Department of Biology University of Iowa Iowa City, Iowa 52242Search for more papers by this author First published: December 1993 https://doi.org/10.1111/j.1749-6632.1993.tb38079.xCitations: 3AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume707, Issue1Molecular Basis of Ion Channels and Receptors Involved in Nerve Excitation, Synaptic Transmission, and Muscle ContractionDecember 1993Pages 392-395 RelatedInformation

Development of larval muscle properties in the embryonic myotubes of Drosophila melanogaster

The entire developmental history of muscle membrane electrogenesis can be observed in the embryonic myotubes of Drosophila. We have examined the development of ionic currents and muscle properties using whole-cell patch-clamp techniques throughout embryonic myogenesis. In the early stages of myogenesis, from myoblast fusion through to establishing epidermal insertions, the myotubes are electrically inert and are electrically and dye coupled to adjacent myotubes. Membrane electrogenesis begins in the mid-embryonic stages (early stage 16), when the myotubes abruptly uncouple, revealing the first of five prominent extrajunctional currents: a small, inward, voltage-gated calcium current (ICa). The uncoupling of the embryonic myotubes heralds the onset of extremely rapid electrogenesis; within several minutes both the fast, inactivating (IA; Shaker) and delayed, noninactivating (IK) outward potassium currents, the stretch-activated outward potassium current, and the junctional glutamate-gated inward current all appear and begin to develop in a current-specific manner. Very late in embryogenesis (late stage 17), the calcium-dependent, outward potassium currents [rapid, inactivating (ICF; slowpoke) then delayed, noninactivating (ICS)] develop, completing the complement of macroscopic currents in the mature larval muscle. Hence, the voltage-gated currents (ICa, IA, and IK, respectively) appear relatively early, and the calcium-dependent currents (ICF, ICS) appear only very late during myogenesis. This developmental progression of current maturation is reflected in dynamic changes in the voltage responses of the embryonic membrane, from wholly passive response to current injection in the early, coupled myotubes to regenerating, overshooting action potentials in the mature embryonic muscle. The earliest embryonic IA current has a midpoint of inactivation 40 mV more negative than the IA current in the mature embryo. As myogenesis proceeds, the inactivation curve develops a biphasic character, suggesting that a low-inactivation IA channel is present in early development and progressively replaced by the mature form as development proceeds. The current at all stages can be completely eliminated in Shaker mutants (ShKS133). These findings suggest that an embryonic form of the Shaker IA channel is present during early myogenesis. The prominent IA current present in early development is almost entirely inactivated at the physiological resting potential; the significance and mechanism of this developmental shift are unclear.

Ultrastructural Findings in the Inner Ear of Jackson Shaker Mice

The ultrastructural characteristics of the inner ear of Jackson shaker mice were analyzed. We used 12 Jackson shaker mutants (js/js) with ages ranging from 10 to 47 days and 10 heterozygotes of the Jackson shaker (js/+) with ages ranging from 10 to 30 days. The most striking findings observed were incomplete differentiation of the stereocilia of the outer hair cells and the maculae, although outer and macular hair cell cytoplasm, including the nerve terminals, became fully developed. Most outer hair cells did not show regular W-shaped configuration of the stereocilia throughout the entire turns of the cochlea except for a few hair cells. In many hair cells of the utricular and saccular maculae, the classical pipe organ configuration of the stereocilia was not observed. The Jackson shaker mice have been reported to have a gene abnormality on chromosome 11, and its gene locus was close to that of our new-mutant mice which showed deranged stereocilia of the outer and macular hair cells. Therefore, future studies can provide additional information on the cytodifferentiation of the stereocilia as a function of the gene on chromosome 11.

Analysis of Anesthetic Action on the Potassium Channels of the Shaker Mutant of Drosophila

Recent evidence suggest that exposure to volatile anesthetic agents causes a change in conductance through an undelineated potassium channel. With recently developed genetic and molecular techniques the Drosophila melanogaster (D.m.) genome can be manipulated to study the role that potassium ion channel function plays in anesthetic action. The IA potassium channel is encoded by the Shaker (Sh) locus on the X chromosome of D.m. Because this channel may be one of those involved in volatile anesthetic action, we tested the sensitivity to isoflurane in three Shaker strains with different degrees of dysfunctional IA conductance (Shnull greater than ShKS133 greater than Sh5). Anesthetic sensitivity was also examined in mutant strains of D.m. which express abnormalities either in other potassium channel conductances (eag, slo) or other ion conductances (para). The normally conducting wild type served as the control. Two-day-old adult D.m. were stimulated with a heat source during exposure to the volatile anesthetic isoflurane, and the number moving in response to the noxious stimulus was noted. After testing the Shaker and control strains at no fewer than 13 concentrations, the IC50S (isoflurane concentration in percent vol/vol at which 50% of the flies did not respond to the heat stimulus) were derived. The IC50 values for Sh 5 (0.89), Sh133 (1.29), and Shnull (1.37) were significantly different from the wild type (0.56). The rank order of insensitivity of these Shaker mutants corresponded to the extent of the alteration in IA conductance as established by previous studies in these mutants. Neither eag (0.66), para (0.48), nor slo (0.63) differed significantly from the wild type. These data suggest that the IA potassium channel plays a role in volatile anesthetic action.

Novel potassium channels encoded by the Shaker locus in drosophila photoreceptors

The Shaker gene, responsible for A-type potassium channels in Drosophila muscle, encodes a large family of transcripts capable of generating a variety of kinetically distinct A channels when expressed in oocytes. We describe a distinct class of A channel encoded by the Shaker gene in a novel preparation of dissociated Drosophila photoreceptors. Whole-cell recordings reveal a rapidly inactivating A current that is absent in Shaker mutants and that can be readily isolated in cell-attached patches. Although very similar to their muscle counterparts, the photoreceptor A channels show a striking 40–50 mV negative shift in their voltage-operating range. Two mutations ( Sh E62 and T(1;Y)W32), which exclude only certain classes of Shaker transcripts, were used to show that photoreceptor A channels are encoded by multiple transcripts distinct from those encoding muscle A channels, while PCR techniques identified four transcripts ( ShAl, ShA2, ShG1, and ShG2) in mRNA from dissected retina.

Molecular basis of altered excitability in Shaker mutants of Drosophila melanogaster.

Mutations in the Shaker (Sh) locus of Drosophila melanogaster have differing effects on action potential duration and repolarization in neurons as well as on A-type K+ channels (IA) in muscle. The molecular basis of three exemplary Sh alleles (ShKS133, ShE62 and Sh5) has been identified. They are point mutations in the Sh transcription unit expressing aberrant voltage-gated A-type K+ channels. Replicas of each mutation have been introduced by in vitro mutagenesis into Sh cDNA. The expression of in vitro transcribed mutant Sh cRNA in Xenopus laevis oocytes reproduced the specific phenotypic traits of each Sh allele. The lack of IA in ShKS133 is due to a missense mutation within a sequence motif occurring in all hitherto characterized voltage-gated K+ channel forming proteins. The reduction of IA in ShE62 is due to a mutation in an AG acceptor site. The intervening sequence between exons 19 and 20 is not spliced in ShE62 RNA. As a consequence, ShE62 flies do not contain the full complement of Sh K+ forming proteins. Finally, the Sh5 mutation leads to an altered voltage dependence of K+ channel activation and inactivation as well as to an accelerated rate of recovery from inactivation. This is due to a missense mutation altering the amino acid sequence of the proposed transmembrane segment S5 of the Sh K+ channels. Segment S5 is located adjacently to the presumed voltage sensor of voltage-gated ion channels. The results explain the altered properties of excitable cells in Sh mutants and provide a general model for the possible role of A-type K+ channels in modulating action potential profiles.

Properties of ShB AType potassium channels expressed in Shaker mutant drosophil by germline transformation

We have used P element-mediated germline transformation to express ShB channels in Shaker mutant Drosophila and have examined their properties by patchclamp of embryonic myotubes. The transformed ShB cDNA was placed under the transcriptional control of a heat shock promoter ( hsp70). Northern blots revealed that transformed DNA is efficiently transcribed in response to heat shock. Cultured myotubes from the transformants produced large A-type potassium currents in response to heat shock. Although qualitatively similar to native Shaker A-currrents in wild-type myotubes, transformant A-currrent inactivates more rapidly and recovers from inactivation more rapidly, similar to ShB channels expressed in Xenopus oocytes. Unlike the channels in oocytes, however, the transformant A-current is insensitive to 50 nM charybdotoxin.

The interference of truncated with normal potassium channel subunits leads to abnormal behaviour in transgenic Drosophila melanogaster.

The Shaker locus of Drosophila melanogaster encodes a family of A-type potassium channel subunits. Shaker mutants behave as antimorphs in gene dosage tests. This behaviour is due to the production of truncated A-channel subunits. We propose that they interfere with the function of their normal counterpart by forming multimeric A-channel structures. This hypothesis was tested by constructing transgenic flies carrying a heat-inducible gene encoding a truncated A-type potassium channel subunit together with a normal wild type doses of A-type potassium channel subunits. The altered subunit leads at larval, pupal or adult stages to the transformation of wild type into Shaker flies. The transformed flies exhibited a heat-inducible abnormal leg shaking behaviour and a heat-inducible facilitated neurotransmitter release at larval neuromuscular junctions. By the overexpression of an aberrant A-channel subunit the normal behaviour of transgenic D. melanogaster can be altered in a predictable way.

Cloning of Genomic and Complementary DNA from Shaker , a Putative Potassium Channel Gene from Drosophila

On the basis of electrophysiological analysis of Shaker mutants, the Shaker locus of Drosophila melanogaster has been proposed to encode a structural component of a voltage-dependent potassium channel, the A channel. Unlike sodium channels, acetylcholine receptors, and calcium channels, K+ channels have not been purified biochemically. To facilitate biochemical studies of a K+ channel, genomic DNA from the Shaker locus has been cloned. Rearrangements in five Shaker mutants have been mapped to a 60-kilobase segment of the genome. Four complementary DNA clones have been analyzed. These clones indicate that the Shaker gene contains multiple exons distributed over at least 65 kilobases of genomic DNA in the region where the mutations mapped. Furthermore, the gene may produce several classes of alternatively spliced transcripts. Two of the complementary DNA clones have been sequenced and their sequences support the hypothesis that Shaker encodes a component of a K+ channel.