Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica).

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.

Actions of the pyrethroid insecticides cismethrin and cypermethrin on house fly Vssc1 sodium channels expressed in Xenopus oocytes.

Voltage-sensitive sodium channels encoded by the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1/tipE sodium channels expressed in oocytes were highly sensitive to tetrodotoxin; half-maximal inhibition of sodium currents by tetrodotoxin was obtained at a concentration of 2.4 nM. Cismethrin, a pyrethroid that produces Type I effects on intact nerve, slowed the inactivation of sodium currents carried by Vssc1/tipE channels during a depolarizing pulse and induced a tail current after repolarization that decayed with a first-order time constant of approximately 650 ms. The voltage dependence of activation and steady-state inactivation of cismethrin-modified channels were shifted to more negative potentials. Cypermethrin, a pyrethroid with Type II effects on intact nerve, also prolonged the inactivation of Vssc1/tipE sodium channels and induced a tail current. However, the cypermethrin-induced tail current was extremely persistent, decaying with a first-order time constant of approximately 42 s. Unlike cismethrin, the effect of cypermethrin was use dependent, requiring repeated depolarizing pulses for the full development of modified sodium currents. The divergent effects of cismethrin and cypermethrin on Vssc1/tipE sodium channels expressed in oocytes are consistent with the actions of these and related compounds on sodium channels in invertebrate and vertebrate nerve preparations and provide insight into the mechanisms underlying the production of Type I and II effects on neuronal excitability.

The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids.

Voltage-sensitive sodium channels encoded by a full-length cDNA corresponding to the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes either alone or in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1 cRNA alone produced very small (50-150 nA) sodium currents, whereas the combination of Vssc1 and tipE cRNAs produced robust (0.5-3 microA), rapidly inactivating sodium currents. The pyrethroid insecticide cismethrin prolonged the sodium current carried by Vssc1/tipE sodium channels during a depolarizing pulse and induced a tail current after repolarization. The Vssc1 cDNA was specifically mutated to substitute phenylalanine for leucine at position 1014 of the inferred amino acid sequence (L1014F), a polymorphism shown previously to be associated with the kdr (knockdown resistance) trait of the house fly. The L1014F substitution reduced the sensitivity of expressed house fly sodium channels to cismethrin at least 10-fold and increased the rate of decay of pyrethroid-induced sodium tail currents. These results demonstrate that the resistance-associated L1014F mutation confers a reduction in the sensitivity of house fly sodium channels to pyrethroids that is sufficient to account for the kdr resistance trait.

Characterization of voltage-sensitive sodium channel gene coding sequences from insecticide-susceptible and knockdown-resistant house fly strains.

The kdr insecticide resistance trait of the house fly (Musca domestica .L.), which confers reduced neuronal sensitivity to DDT and pyrethroid insecticides, was previously shown to exhibit tight genetic linkage to restriction fragment length polymorphism markers lying within a voltage-sensitive sodium channel gene that is homologous to the para gene of Drosophila melanogaster. In the present study, the 6315 nucleotide coding sequences of this voltage-sensitive sodium channel gene from insecticide-susceptible (NAIDM strain) and kdr (538ge strain) house flies were determined by automated direct DNA sequencing of PCR fragments obtained by amplification on first strand cDNA from adult heads. The deduced 2105-residue amino acid sequence from each strain exhibited overall structure and organization typical of sodium channel alpha subunit genes and was 90.0% identical to that of the D. melanogaster para gene product. We did not detect any splice variants among voltage-sensitive sodium channel cDNAs obtained from adult house fly head preparations. Comparison of the coding sequence of the voltage-sensitive sodium channel gene of the kdr house fly strain to that of the NAIDM strain revealed 12 amino acid differences in the 538ge strain. The significance of these polymorphisms as candidate resistance-conferring mutations is discussed.

A spontaneous red clover necrotic mosaic virus mutant with a truncated movement protein.

A spontaneous red clover necrotic mosaic virus mutant, TpM-341, was isolated by multiple passage of Czechoslovakian isolate TpM-34 in beans, followed by three cycles of single lesion isolation in cowpea and in Chenopodium quinoa. The symptoms induced in cowpea by TpM-34 and TpM-341 differed. TpM-34 gave rise to chlorotic lesions which expanded with time, often becoming confluent with adjacent lesions, and developed necrotic margins; the plants became systemically infected. TpM-341 induced necrotic lesions which, once developed, did not expand further; plants did not become systemically infected. Analysis of pseudorecombinants formed between the RNAs of TpM-34 and TpM-341 showed that RNA 2 determined the difference in symptoms. Comparison of the nucleotide sequence of the open reading frames (ORFs) encoding the P2 movement proteins of the two isolates revealed only one difference, a deletion of an A residue in a sequence of four A residues (nucleotides 790 to 793). Construction of full-length TpM-34 and TpM-341 RNA 2 cDNA clones, from which infectious RNA 2 could be transcribed in vitro, and in vitro mutagenesis of a cDNA clone of TpM-341, confirmed that the difference in the symptoms induced by TpM-341 was caused by the loss of this A residue. This single base deletion was predicted to cause a translational frame-shift in the P2 ORF causing the loss of 88 amino acids at the C terminus which were replaced by a sequence of 34 different amino acids, producing a truncated P2 protein. In vitro translation of RNA 2 transcribed from cDNA clones showed that RNA with four or three A residues starting at nucleotide 790 produced proteins of Mr 36K and 30K respectively, in agreement with the predictions based on the nucleotide sequence.