Drug-resistant Drosophila indicate glutamate-gated chloride channels are targets for the antiparasitics nodulisporic acid and ivermectin.

The fruit fly Drosophila melanogaster was used to examine the mode of action of the novel insecticide and acaricide nodulisporic acid. Flies resistant to nodulisporic acid were selected by stepwise increasing the dose of drug in the culture media. The resistant strain, glc(1), is at least 20-fold resistant to nodulisporic acid and 3-fold cross-resistant to the parasiticide ivermectin, and exhibited decreased brood size, decreased locomotion, and bang sensitivity. Binding assays using glc(1) head membranes showed a marked decrease in the affinity for nodulisporic acid and ivermectin. A combination of genetics and sequencing identified a proline to serine mutation (P299S) in the gene coding for the glutamate-gated chloride channel subunit DmGluClalpha. To examine the effect of this mutation on the biophysical properties of DmGluClalpha channels, it was introduced into a recombinant DmGluClalpha, and RNA encoding wild-type and mutant subunits was injected into Xenopus oocytes. Nodulisporic acid directly activated wild-type and mutant DmGluClalpha channels. However, mutant channels were approximately 10-fold less sensitive to activation by nodulisporic acid, as well as ivermectin and the endogenous ligand glutamate, providing direct evidence that nodulisporic acid and ivermectin act on DmGluClalpha channels.

Activation of Drosophila sodium channels promotes modification by deltamethrin. Reductions in affinity caused by knock-down resistance mutations.

kdr and super-kdr are mutations in houseflies and other insects that confer 30- and 500-fold resistance to the pyrethroid deltamethrin. They correspond to single (L1014F) and double (L1014F+M918T) mutations in segment IIS6 and linker II(S4-S5) of Na channels. We expressed Drosophila para Na channels with and without these mutations and characterized their modification by deltamethrin. All wild-type channels can be modified by <10 nM deltamethrin, but high affinity binding requires channel opening: (a) modification is promoted more by trains of brief depolarizations than by a single long depolarization, (b) the voltage dependence of modification parallels that of channel opening, and (c) modification is promoted by toxin II from Anemonia sulcata, which slows inactivation. The mutations reduce channel opening by enhancing closed-state inactivation. In addition, these mutations reduce the affinity for open channels by 20- and 100-fold, respectively. Deltamethrin inhibits channel closing and the mutations reduce the time that channels remain open once drug has bound. The super-kdr mutations effectively reduce the number of deltamethrin binding sites per channel from two to one. Thus, the mutations reduce both the potency and efficacy of insecticide action.

The pharmacological flexibility of the insect voltage gated sodium channel: toxicity of AaIT to knockdown resistant (kdr) flies.

AaIT is an insect selective neurotoxic polypeptide shown to affect insect neuronal sodium conductance by binding to excitable sodium channels. In the present study the paralytic potency of AaIT to wild type and various mutant strains of houseflies (Musca domestica) and fruitflies (Drosophila melanogaster) was examined and it has been shown that: On the basis of body weight when compared to published data on Sarcophaga falculata blowflies, the Musca and Drosophila flies reveal at least two orders of magnitude decreased susceptibility to the AaIT. When compared to wild type flies the toxicity of AaIT is greatly altered in knockdown resistant fly strains which are mutated in their para gene encoding the voltage gated sodium channel. Several strains, with genetically mapped para mutations conferring pyrethroid resistance, exhibited opposing response to AaIT. The para ts2 Drosophila strain, with a point of mutation in domain I of the para gene conferring a 6-fold resistance to deltamethrin also showed about 15-fold tolerance to AaIT. On the other hand the Musca kdr and super-kdr flies, with a single or a double point mutation, respectively in domain II of the para gene, are about 9- and 14-fold more susceptible to AaIT, respectively. The above data are interpreted in terms of the pharmacological diversity and flexibility ("allosteric coupling") of voltage gated sodium channels and their implications for the management of pesticide resistance are discussed.

Functional expression of Drosophila para sodium channels. Modulation by the membrane protein TipE and toxin pharmacology.

The Drosophila para sodium channel alpha subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (Kd congruent with 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.

The Drosophila erg K+ channel polypeptide is encoded by the seizure locus.

The eag family of K+ channels contains three known subtypes: eag, elk, and erg. Genes representing the first two subtypes have been identified in flies and mammals, whereas the third subtype has been defined only by the human HERG gene, which encodes an inwardly rectifying channel that is mutated in some cardiac arrhythmias. To establish the predicted existence of a Drosophila gene in the erg subfamily and to learn more about the structure and biological function of channels within this subfamily, we undertook a search for the Drosophila counterpart of HERG. Here we report the isolation and characterization of the Drosophila erg gene. We show that it corresponds with the previously identified seizure (sei) locus, mutations of which cause a temperature-sensitive paralytic phenotype associated with hyperactivity in the flight motor pathway. These results yield new insights into the structure and evolution of the eag family of channels, provide a molecular explanation for the sei mutant phenotype, and demonstrate the important physiological roles of erg-type channels from invertebrates to mammals.

Potassium currents expressed from Drosophila and mouse eag cDNAs in Xenopus oocytes.

The ether-a-go-go (eag) gene family encodes a set of related ion channel polypeptides expressed in the excitable cells of organisms ranging from invertebrates to mammals. Earlier studies demonstrated that eag mutations in Drosophila cause an increase in membrane excitability in the nervous system. Mutations in the human eag-related gene (HERG) have been implicated in cardiac arrhythmia, and recent studies show that HERG subunits contribute to the channels mediating IKr and the terminal repolarization of the cardiac action potential. A physiological role for M-EAG, the mouse counterpart to Drosophila eag, has not been determined. Here, we describe basic properties of Eag and M-EAG channels expressed in frog oocytes, using two-electrode voltage clamp and patch clamp techniques. Both Eag and M-EAG channels are voltage-dependent, outwardly rectifying and highly selective for K+ over Na+ over Na+ ions. In contrast to previous reports, we found no evidence for Ca2+ flux through Eag channels. The most notable difference between these closely related channels is that Eag currents exhibit partial inactivation, whereas M-EAG currents are sustained for the duration of an activating voltage command. In addition, Eag currents run down more rapidly than do M-EAG currents in excised macropatches. Rundown is reversible by inserting the patch into the interior of the oocyte, indicating that a cytosolic factor regulates channel activity or stability. These studies should facilitate identification of currents mediated by Eag and M-EAG channels in vivo.

Characterization of tissue-expressed alpha subunits of the high conductance Ca(2+)-activated K+ channel.

Purified high conductance calcium-activated potassium (maxi-K) channels from tracheal smooth muscle have been shown to consist of a 60-70-kDa alpha subunit, encoded by the slo gene, and a 31-kDa beta subunit. Although the size of the beta subunit is that expected for the product of the gene encoding this protein, the size of the alpha subunit is smaller than that predicted from the slo coding region. To determine the basis for this discrepancy, sequence-directed antibodies have been raised against slo. These antibodies specifically precipitate the in vitro translation product of mslo, which yields an alpha subunit of the expected molecular mass (135 kDa). Immunostaining experiments employing smooth muscle sarcolemma, skeletal muscle T-tubules, as well as membranes derived from GH3 cells reveal the presence of an alpha subunit with an apparent molecular mass of 125 kDa. The difference in size of the alpha subunit as expressed in these membranes and the purified preparations is due to a highly reproducible proteolytic decay that occurs mostly at an advanced stage of the maxi-K channel purification. In the purified maxi-K channel preparations investigated, the full-length alpha subunit, an intermediate size product of 90 kDa, and the 65-kDa polypeptide, as well as other smaller fragments can be detected using appropriate antibodies. Proteolysis occurs exclusively at two distinct positions within the long C-terminal tail of slo. In addition, evidence for the tissue expression of distinct splice variants in membrane-bound as well as purified maxi-K channels is presented.

HERG, a human inward rectifier in the voltage-gated potassium channel family.

In contrast to other members of the Eag family of voltage-gated, outwardly rectifying potassium channels, the human eag-related gene (HERG) has now been shown to encode an inwardly rectifying potassium channel. The properties of HERG channels are consistent with the gating properties of Eag-related and other outwardly rectifying, S4-containing potassium channels, but with the addition of an inactivation mechanism that attenuates potassium efflux during depolarization. Because mutations in HERG cause a form of long-QT syndrome, these properties of HERG channel function may be critical to the maintenance of normal cardiac rhythmicity.

A family of potassium channel genes related to eag in Drosophila and mammals.

We have identified a conserved family of genes related to Drosophila eag, which encodes a distinct type of voltage-activated K+ channel. Three related genes were recovered in screens of cDNA libraries from Drosophila, mouse, and human tissues. One gene is the mouse counterpart of eag; the other two represent additional subfamilies. The human gene maps to chromosome 7. Family members share at least 47% amino acid identity in their hydrophobic cores and all contain a segment homologous to a cyclic nucleotide-binding domain. Sequence comparisons indicate that members of this family are most closely related to vertebrate cyclic nucleotide-gated cation channels and plant inward-rectifying K+ channels. The existence of another family of K+ channel structural genes further extends the known diversity of K+ channels and has important implications for the structure, function, and evolution of the superfamily of voltage-sensitive ion channels.

Myosin light chain-2 mutation affects flight, wing beat frequency, and indirect flight muscle contraction kinetics in Drosophila.

We have used a combination of classical genetic, molecular genetic, histological, biochemical, and biophysical techniques to identify and characterize a null mutation of the myosin light chain-2 (MLC-2) locus of Drosophila melanogaster. Mlc2E38 is a null mutation of the MLC-2 gene resulting from a nonsense mutation at the tenth codon position. Mlc2E38 confers dominant flightless behavior that is associated with reduced wing beat frequency. Mlc2E38 heterozygotes exhibit a 50% reduction of MLC-2 mRNA concentration in adult thoracic musculature, which results in a commensurate reduction of MLC-2 protein in the indirect flight muscles. Indirect flight muscle myofibrils from Mlc2E38 heterozygotes are aberrant, exhibiting myofilaments in disarray at the periphery. Calcium-activated Triton X-100-treated single fiber segments exhibit slower contraction kinetics than wild type. Introduction of a transformed copy of the wild type MLC-2 gene rescues the dominant flightless behavior of Mlc2E38 heterozygotes. Wing beat frequency and single fiber contraction kinetics of a representative rescued line are not significantly different from those of wild type. Together, these results indicate that wild type MLC-2 stoichiometry is required for normal indirect flight muscle assembly and function. Furthermore, these results suggest that the reduced wing beat frequency and possibly the flightless behavior conferred by Mlc2E38 is due in part to slower contraction kinetics of sarcomeric regions devoid or partly deficient in MLC-2.

A distinct potassium channel polypeptide encoded by the Drosophila eag locus.

Many of the signaling properties of neurons and other electrically excitable cells are determined by a diverse family of potassium channels. A number of genes that encode potassium channel polypeptides have been cloned from various organisms on the basis of their sequence similarity to the Drosophila Shaker (Sh) locus. As an alternative strategy, a molecular analysis of other Drosophila genes that were defined by mutations that perturb potassium channel function was undertaken. Sequence analysis of complementary DNA from the ether à go-go (eag) locus revealed that it encodes a structural component of potassium channels that is related to but is distinct from all identified potassium channel polypeptides.

Molecular characterization of eag: a gene affecting potassium channels in Drosophila melanogaster.

Genes encoding proteins involved in the function of the nervous system can be identified via mutations causing behavioral abnormalities. An example is ether à go-go (eag) in Drosophila melanogaster, which was identified originally as an X-linked mutation that displayed ether-induced leg-shaking behavior. Electrophysiological and genetic evidence suggests that the product of the eag locus is intimately involved in the normal functioning of one or more types of voltage-gated potassium channels. To initiate a molecular analysis of eag we first generated a collection of deletions to pinpoint its cytological location. On the basis of this location, we identified an existing inversion, In(1)sc29, with one breakpoint at the eag locus and the other in the scute (sc) complex. A genomic library was prepared from In(1)sc29 and screened with a genomic DNA fragment that spanned the sc breakpoint to isolate DNA from the eag region. Beginning from this starting point over 85 kb of DNA were isolated by chromosome walking. Three additional eag alleles, including two dysgenesis-induced insertion mutations and a gamma-ray-induced insertional translocation, were located on the molecular map of the eag locus by Southern blot analysis. The molecular defects associated with these alleles encompass a total of 27 kb within the chromosome walk. A 10-kb transcript derived from this region, which is expressed most abundantly in heads, was identified on Northern blots. Two different eag mutations separated by over 20 kb interrupt the same transcript identifying it as the likely eag message. cDNAs representing a portion of this transcript have been isolated.(ABSTRACT TRUNCATED AT 250 WORDS)

Co-localization to chromosome bands 99E1-3 of the Drosophila melanogaster myosin light chain-2 gene and a haplo-insufficient locus that affects flight behavior.

Using overlapping synthetic deficiencies, we find that a haplo-insufficient locus affecting flight behavior and the myosin light chain-2 gene co-map to the Drosophila melanogaster polytene chromosome interval 99D9-E1 to 99E2-3. From screening over 9000 EMS-treated chromosomes, we obtained alleles of two complementation groups that map to this same interval. One of these complementation groups lfm(3)99Eb, exhibits dominant flightless behavior; thus, flightless behavior of the deficiency is in all likelihood due to hemizygosity of this single locus. Rescue of flightless behavior by a duplication indicates that the single allele, E38, of the Ifm(3)99Eb complementation group is a hypomorph. Based upon its map position and a reduction in concentration of myosin light chain-2 mRNA in heterozygotes, we propose that Ifm(3)Eb(E38) is a mutant allele of the myosin light chain-2 gene. Our genetic analysis also resulted in the identification of four dominant flightless alleles of an unlinked locus, l(3)nc99Eb, that exhibits dominant lethal synergism with Ifm(3)99Eb.